CAT QCA - INSERTS en r010522 v7.0

Una empresa al servicio del biodiagnóstico - A company in the service of biodiagnostics - Une entreprise au service du bio-diagnostique.
Desde 1976 - Since 1976 - Depuis 1976
r07.0
CE Mark products: our products are
CE Marked. Those products intended
for IVD testing with samples of human
origin comply with the Directive
98/79CE – all of them are of the auto Some of our references as general purpose
evaluation kind- . All of our products are reagents, Water test kits, Ancillary, washing
for professional use only and they are solutions etc.. are not CE marked ; they are not
not intended for autodiagnostic. involved in human diagnostics.
ENZYMES
Acid Phosphatase��10
Adenosin Deaminase�� 11
Adenosin Deaminase - Controls Set��12
Alkaline Phosphatase Liquid��13
Alkaline Phosphatase P-NPP��14
Amylase Liquid��15
Cholinesterase��16
CK-MB��17
CK-MB Liquid��18
CK-NAC Liquid��19
Gamma-GT Liquid��20
GOT/AST and GPT/ALT Colourimetric��21
GOT/AST U.V.��22
GOT/AST U.V. Liquid��23
GPT/ALT U.V.��24
GPT/ALT U.V. Liquid��25
LDH Liquid��26
Lipase��27
SUBSTRATES
Albumin��28
Bilirubin��29
Bilirubin Direct��30
Bilirubin Total��31
Bilirubin Direct DPD��32
Bilirubin Total DPD��33
Cholesterol Liquid��34
Creatinine��35
Creatinine DMSO��36
HDL-Cholesterol��37
HDL-Cholesterol Direct��38
Hemoglobin��39
LDL-Cholesterol��40
LDL-Cholesterol Direct��41
Glucose Liquid��42
Glycohemoglobin (HbA1)��43
Glycohemoglobin, Control Set��44
Automatic Glycohemoglobin (HbA1c)��45
Automatic Glycohemoglobin - Calibrator Set��46
Automatic Glycohemoglobin - Control Set��47
Total Protein��48
Triglycerides Liquid��49
Urea Enzymatic��50
Urea U.V.��51
Urea U.V. Liquid��52
Urea Acid Liquid��53
Urine / CSF Proteins - Benzethonium Chloride Method��54
Urine / CSF Proteins - Pyrogallol Red Method��55
Calcium��56
Calcium-Arsenazo III��57
ISO 9001 / ISO 13485
QUIMICA CLINICA APLICADA S.A.
A 7 Km 1081 – P.O. Box 20 - E43870 AMPOSTA / SPAIN
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Chloride��58
Copper��59
Inorganic Phosphorus U.V.��60
Inorganic Phosphorus Visible��61
Iron - CAB��62
Iron - Ferrozine��63
Magnesium��64
Magnesium - Calmagite��65
Potassium��66
TIBC��67
Zinc��68
CONTROLS AND CALIBRATORS FOR CLINICAL CHEMISTRY
Calibrator for Autoanalyzers��70
Seriscann Abnormal��72
Seriscann Normal��74
IMMUNOLOGY: AUTO-IMMUNE DISEASES
ANTI-n-DNA Latex��76
IMMUNOLOGY: RHEUMA LINE SLIDE TEST
AS Direct Latex��77
CRP Direct Latex��78
RF Direct Latex��79
IMMUNOLOGY: TURBIDIMETRIC RHEUMA LINE
AS Turbidimetric Latex 1:5��80
CRP Turbidimetric Latex 1:5��81
RF Turbidimetric Latex 1:5��82
Rheuma Line Control High Polyvalent��83
Rheuma Line Control Low Polyvalent��84
INFECTIOUS SEROLOGY
Bengal Rose��85
Febrile Antigens��86
Infectious Mononucleosis��87
RPR Carbon��88
IMMUNOLOGY: PLASMA PROTEINS
alpha-1-Acid glycoprotein��89
alpha-1-Antitrypsin��90
Apolipoprotein A1��91
Apolipoprotein B��92
Apolipoprotein Calibrator��93
Apolipoprotein Control��94
C3 Complement��95
C4 Complement��96
Ferritin��97
Ferritin Calibrator��98
Ferritin Control��99
Immunoglobulin A (IgA)��100
Immunoglobulin G (IgG)��101
Immunoglobulin M (IgM)��102
Microalbumin��103
ISO 9001 / ISO 13485

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Transferrin�� 104
Proteins Positive Control�� 105
Protein Calibrator�� 106
HAEMOSTASIS REAGENTS
Antithrombin III�� 107
Coagulation Controls�� 108
Fibrinogen�� 109
Hemoscann (APTT)�� 110
Plasmascann (PT)��111
Plasmascann Liquid (PT)�� 112
Plasmascann HS (PT)�� 114
Anticoagulants. Ancillary Products for Coagulation Test�� 116
GRAM STAINING
Gram PVP Kit�� 118
Gram Staining�� 119
ZIEHL-NEELSEN STAINING
TB Ziehl - Neelsen Kit�� 120
TB Ziehl - Neelsen Tensoactive�� 121
KINYOUN STAINING
TB Kinyoun Kit�� 122
FLUORESCENT STAINING
TB Auramine (Morse) Kit�� 123
TB Auramine - Rhodamine (Truant)�� 124
FUNGI STAINING
Lactophenol Blue�� 125
ENDOSPORE STAINING
Malachite Green�� 126
UREASE FAST TEST FOR HELICOBACTER
Helicobacter�� 127
HAEMATOLOGY STAINS
Brilliant Cresyl Blue�� 128
Giemsa�� 129
Hayem�� 130
May-Grünwald�� 131
Quick Panoptic�� 132
Quick Panoptic Concentrated 1:10�� 133
Türk�� 134
Wright�� 135
SPERM STAINING
Eosin-Nigrosin�� 136
HISTOPATHOLOGY STAINS
Alcian Blue�� 137
Masson’s Trichrome Stain�� 138
Myeloperoxidase�� 139
Perl’s Staining�� 140
Shorr’s Stain�� 141
Sudan Black�� 142
ISO 9001 / ISO 13485

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Hematoxylin Solutions for Nuclear Staining��143
Solutions for Cytoplasm Staining��144
DEVICES
QCA-Bio 100 Autoanalyzer��146
QCA-PLUS II / BT 1500 Autoanalyzer��149
BT 3500 Autoanalyzer��150
BT 4500 Autoanalyzer��151
QCA Coag I��152
QCA Coag II��153
QCA Coag Auto��154
QCA-MICRO Automatic Staining System��155
QCA-STAINER Automatic Staining System��156
REAGENTS FOR SPECIFIC ELECTRODES
I.S.E / Targa��157
I.S.E / Targa Plus��158
WASHING SOLUTIONS
Extra Wash Kit��159
Washing Solutioni PS-200��160
LABWARE
Staining Glass Jars and Spare Parts��161
SOLVENTS, FIXATIVES, DECALCIFIERS, PARAFIN
Ancillary Products��162
Bone Marrow Biopsy Kit��163
QCA-PBS��164
Tissue Fixatives��165
Tissue Decalcifiers��166
WATER TEST KITS
Water: Chlorine��167
Water: Chlorine / pH��168
DEXTROSE SOLUTION
Dextrose Solution��169
ISO 9001 / ISO 13485

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QCA is a registered trademark. The manufacturer reserves the right to make changes or
modifications it deems appropriate without notice, for the benefit of the final product and
the customer.
All data presented in this catalog are correct except for typographical errors. In case of
doubt or discrepancy, the information sheet located inside the kit.
Revision 07
ISO 9001 / ISO 13485

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Enzymes
ISO 9001 / ISO 13485

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ACID PHOSPHATASE
HILLMANN METHOD
For “in vitro” determination of total and prostatic acid phosphatase in serum or plasma
PRINCIPLE
In acid medium, acid phosphatase hydrolyzes the substrate producing α-naphtol that reacts with the diazonium salt forming an azo dye and changing the Abs of the reagent.
In optimum conditions, the ΔAbs/min is directly related to the concentration of acid phosphatase of the sample.
acid phosphatases
α-naphthyl phosphate + H2O phosphate + α-naphtol
α-naphtol + Fast Red TR* salt azo dye
* 4-chloro-2-methylphenyl diazonium salt.
DIAGNOSTIC USE PROCEDURE

Acid phosphatase, especially the tartrate labile fraction or prostatic phosphatase (PAP), is elevated Bring reagents and the analyzer to working temperature (30ºC,37ºC).
in cases of metastatic prostate cancer. The concentration does not increase in the absence of
metastasis or it slightly does. a) Method for TOTAL Acid phosphatase
High values are also found in multiple myeloma, in some lymphoblastic leukemias and osteolytic
bone metastases. Technique Macro (mL) Semimicro (mL)
Working reagent Aa 2.0 1.0
Single test result can not be used to make a clinical diagnosis. It should integrate clinical and
laboratory data. Incubate 2 - 3 min at the desired assay temperature (30ºC, 37ºC).
REAGENTS Sample 0.2 0.1

Kit 18 x 2 mL. (Ref. 99 00 16). Contents: Mix and start the stopwatch. Transfer to the measuring cuvette and incubate for 5 min at 30ºC/37ºC.
A. 18 x 2 mL Substrate (powder) Ref. 99 01 05
Read the absorbance after 1, 2 and 3 min.
B. 1 x 50 mL Buffer solution Ref. 99 02 02
C. 1 x 6 mL Sodium tartrate.135 mM Ref. 99 02 07
b) Method for NON PROSTATIC Acid phosphatse
D. 1 x 6 mL Acetic acid 3.3 N Ref. 99 92 35
Technique Macro (mL) Semimicro (mL)
Kit 10 x 10 mL. (Ref. 99 00 22). Contents:
A. 10 x 10 mL Substrate (powder) Ref. 99 01 23 Working reagent Ab 2.0 1.0
Incubate 2 - 3 min. at the desired assay temperature (30º, 37ºC).
C. 1 x 6 mL Sodium tartrate. 135 mM Ref. 99 02 07
D. 1 x 6 mL Acetic acid 3.3 N Ref. 99 92 35 Sample 0.2 0.1
WORKING REAGENT PREPARATION Mix and start the stopwatch. Transfer to the measuring cuvette and incubate for 5 min.at 30ºC/
Dissolve one vial of substrate A with the appropriate volume of buffer B, as follows:
37ºC. Read the absorbance after 1, 2 and 3 min.
(Aa) For total acid phosphatase determination
Vial 2 mL: 2 mL of buffer (B) + 250 μL deionized water. Find the ΔAbs/min of each reading and calculate the mean value.
Vial 10 mL: 9 mL of buffer (B) + 1 mL deionized water.
Reading
(Ab) For non-prostatic acid phosphatase determination Wavelength: 405 nm
Vial 2 mL: 2 mL of buffer (B) + 250 μL tartrate (C). Blank: Water
Vial 10 mL: 9 mL of buffer (B) + 1 mL tartrate (C). Cuvette: Thermostatized 1 cm light path
WORKING REAGENT CONCENTRATIONS CALCULATIONS

The concentrations in the reagent solution are: Total acid phosphatase
Citrate buffer pH 4.9 140 mM Calculate the mean ΔAbs /min. obtained in the determination of total acid phosphatase. Reagent Aa.
α-naphthyl phosphate 10 mM
U/L (Total)= 743 x ΔAbs /min
Fast Red TR salt* 1.6 mM
Non reactive stabilizers Prostatic Acid phosphatase
Calculate the mean ΔAbs /min. obtained in the determination of Non Prostatic acid phosphatase.
STORAGE AND STABILITY Reagent Ab
The components of the kit, stored at 2-8ºC, will remain stable until the expiration date stated on the U/L (Prostatic) = U/L (Total) – U/L (No Prostatic)
label. Once the substrate vial has been dissolved, the reagent solution is stable 10 days at 2-8º C
U/L (Prostatic) = 743 x [ΔAbs /min using Reagent Aa - ΔAbs /min using Reagent Ab.]
and 2 days at room temperature (≤ 25ºC), protected from the light.
REFERENCE VALUES
Signs of reagent deterioration:
Total acid phosphatase
Turbidity or visible particles.
30º 37º
Men up to 4.3 U/L up to 5.4 U/L
ADDITIONAL EQUIPMENT
Women up to 3.1 U/L up to 4.2 U/L
General laboratory equipment.
Spectrophotometer or photometer thermostatable at 37ºC. Cuvette: 1 cm light-path. Prostatic phosphatase
up to 1.5 U/L up to 1.7 U/L
CAUTION
The safety statements are on the label. Handle the reagent with care. The stated values are for guidance. Each particular laboratory should establish its own normal
It is advisable to read the SDS before the reagent manipulation. range, using its own instrumentation, blood collection methods and test procedures.
The disposal of the residues has to be made according to local regulations.
PERFORMANCE CHARACTERISTICS
SAMPLE Performance of the reagent depends on the reagent itself and also depends on the method and
Serum or plasma. Specimens must be absolutely free from hemolysis. Serum or plasma should be the analyzer used.
separated as soon as possible and perform the test quickly. The results indicated below were obtained using a manual method.
Serum samples can be stored for up to 3 days at 2-8º C and 24h at room temperature (≤ 25ºC) by
adding one drop (40 μL) of acetic acid (D) per 1 mL of serum. Sensitivity, as detection limit: 0.4 U/L
Linearity: Up to 75 U/L. For higher values, it is recommended to dilute the sample 1/3 in saline
QUALITY CONTROL (NaCl 0.9%), (0.2 mL sample + 0.4 mL saline) and assay once again. Multiply the final result by 3.
Accuracy: 97.4%
Control serum, Seriscann Normal (Ref. 99 41 48) and Seriscann Anormal (Ref. 99 46 85)
Repetitivity as Coefficient of Variation: 2.4 %
should be included in each test series. Each particular laboratory should establish its own
Reproducibility as Coefficient of Variation: 2.8 %
control program. Trueness: Results obtained with this reagent did not show systematic differences when
compared with the reference reagent.
AUTOANALYZERS
Details of the performance studies are available on request.
Adaptations to different autoanalyzers are available on request.
INTERFERENCES
Hemolyzed samples will interfere with the assay. bilirubin concentrations higher than 3 mg/dL
interfere with the assay too.
REFERENCES
Hillmann, G., Clin. Chem. Clin. Biochem. (1971) 9, 273-274.
Seiler, D., Nagel, D., Tritschler, W., Looser, S., J. Clin. Chem. Clin. Biochem. (1983) 21, 519-
525.
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Enzymes
ADENOSINE DEAMINASE
METHOD GLDH / UV
For “in vitro” determination of ADA in serum, cerebrospinal or pleural fluid
PRINCIPLE
The hydrolysis of the adenosine present in the sample is catalyzed by adenosine deaminase (ADA), producing ammonium ions and inosine. Ammonium ions react with α-Ketoglutarate by the action
of glutamate dehydrogenase, oxidizing the NADPH to NADP .
The Adenosine concentration present in the sample is proportional to the decrease in the concentration of NADPH in the reaction.
ADA
Adenosine+ 2H2O → 2NH4+ + Inosine
GLDH
α- Ketoglutarate + NH4+ + NADPH → Glutamate + NADP+ + H2O

ADA is one of the enzymes of purine metabolism present in microorganisms, plants and animals. Let the reagents reach the room temperature before use.
The enzyme is widely distributed in human tissues, especially in lymphocytes. Elevated serum
ADA activity has been observed in patients with liver diseases. Increased ADA activity in pleural
fluid was also observed in patients with tuberculosis effusions. Technique Monoreagent BL SA CAL
Single test result can not be used to make a clinical diagnosis. It should integrate clinical and mL mL mL
laboratory data.
Calibrator -- -- 0.05
REAGENTS
Kit 1 x 50 mL (Ref. 99 32 30) Contents: Sample -- 0.05 --
A. 1 x 40 mL. Reagent A Ref. 99 03 84
B. 1 x 10 mL. Reagent B Ref. 99 04 44 Working reagent 1.00 1.00 1.00
C. 1 x 1 mL Calibrator. Lyophilized Ref. 99 08 64
The concentration is stated on the vial label.
Technique Bireagent BL SA CAL
WORKING REAGENT PREPARATION
Reagent A and B are ready to use. If a Monoreagent procedure is preferred, then the reagents mL mL mL
must be mixed in the ratio: 4 parts of Reagent A (Buffer solution) + 1 part of Reagent B (Substrate Reagent A 0.80 0.80 0.80
solution).
Calibrator: Rehydrate the vial with 1 mL of deionized water. Mix gently and let stand at room Calibrator -- -- 0.05
temperature for 15 min prior to use.
Sample -- 0.05 --
WORKING REAGENT COMPOSITION Reagent B 0.20 0.20 0.20
The concentrations in the reagent solution are:
Buffer Tris-HCl pH 7.5 100 mM
Adenosine 12 mM Mix well and transfer to the measuring cuvette. Read the absorbance after 4 minutes of reaction
Sodium α-Ketoglutarate 15 mM (Abs1) and, after 5 more minutes, read again (Abs2)
ADP 2.5 mM
NADPH 0.3mM Reading
GLDH ≥ 800 U/L Wavelength: 340 nm.
Stabilizers and preservatives Blank: Deionised water.
Cuvette: 1 cm light-path.
Calibrator: Pool of human serum with stabilizers containing known ADA concentration. This
calibrator is traceable against the reference material BCR-647. CALCULATIONS
Determine ΔAbs/min for each sample and the calibrator:
STORAGE AND STABILITY ΔAbs/min = Abs2/min - Abs1/min
The components of the kit, stored at 2-8ºC, will remain stable until the expiration date stated on
the label. The monoreagent is stable 2 weeks at 2-8ºC and 2 days at room temperature (≤ 25ºC). ΔAbs/min SA
Once rehydrated, the calibrator is stable for 5 days, when stored tightly capped at 2-8ºC or 2 x CAL Conc. = U/L Adenosine deaminase
months at -20ºC. Freeze only once. ΔAbs/min CAL
Presence of particles or turbidity in the reagent. Working reagent blank < 1.00 Where:
ΔAbs/min SA: Absorbance increase of sample
ΔAbs/min CAL: Absorbance increase of calibrator
ADDITIONAL EQUIPMENT CAL Conc.: Calibrator concentration, stated in the label on the vial.
Spectrophotometer or photometer thermostatable at 37ºC. Cuvette: 1 cm light-path.
REFERENCE VALUES
Serum: up to 18 U/L
Pleural liquid: up to 35 U/L
CAUTION
CSF: Not available reference values in literature
The reagents contain sodium azide at 0.09%. Handle with care.
The stated values are for guidance. It is advisable that each laboratory determines its own
The safety statements are on the label. It is advisable to look at the SDS before using the reagent.
reference values.
The calibrator must be considered as a human sample, and, thus, potentially infectious. Use
adequate protection. PERFORMANCE CHARACTERISTICS
The disposal of the residues has to be made according to local regulations. Performance of the reagent depends on the reagent itself and also depends on method and
analyzer used.
SAMPLE
Serum, CSF or pleural fluid. ADA enzyme will remain stable in the sample 7 days at 2-8ºC. Do not use Sensitivity, as detection limit: 3.69 UI/L
hemolyzed samples, because of the high ADA concentration in the blood cells. Linearity: Up to 200 UI/L
Accuracy: 105 %
QUALITY CONTROL Within-run Precision as Coefficient of Variation: 3.24%
It is recommended to include control sera, (ADA, Controls QCA Ref. 99 32 40) in each test series Run-to-run Precision as Coefficient of Variation: 6.53%
for assuring the results. Calibration should be made in each test run as long as whenever the lot of Trueness: Results obtained with this reagent did not show systematic differences when compared
the reagent and/or calibrator is changed. with reference reagent
Each particular laboratory should establish its own control program as well as the procedures to Details of the performance studies are available on request
correct the deviations of the measurements.
INTERFERENCES
Bilirubin (up to 30 mg/dL), intralipid (up to 300 mg/dL), hemoglobin (up to 500mg/dL), ascorbic acid
AUTOANALYZERS
(up to 30mg/dL) do not interfere.
REFERENCES
Tietz, NW., Textbook of Clinical Chemistry 5th Edition, W.B. Saunders, Philadelphia (2012).
CLSI Guidelines and Standards, CLSI, Wayne, P.A
Young D.S., Effect of drugs on Clinical Lab. Test, 5th Ed. AACC Press (2000).
Martinek R.G., Micromethod for estimation of serum Adenosine deaminase. Clin Chem 9:620-625
(1963)
Delia S. et al. Adenosine Deaminase Activity and AIDS. Clin Chem 33: 1675 (1987)
Collazos J, España P, Mayo J, Martínez E, Izquierdo F. Sequential evaluation of serum adenosine
deaminase in patients treated for tuberculosis. CHEST 114:432-435(1998)
Ungerer JPJ, Oosthuizen HM, Bissbort SH, et al. Serum Adenosine deaminase isoenzymes in
tuberculous effusions. Clin Chem 38:1322-1326(1992)
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ADENOSIN DEAMINASE CONTROLS
For “in vitro” diagnostics
PRODUCT DESCRIPTION
Pool of human sera intended to be used for the quality control in the determination
of Adenosine Deaminase concentration in biological samples.
CONTROLS
Kit 2 x 1 mL. (Ref.99 32 40) Content:
A. 1 x 1 mL Control Level 1 Ref. 99 08 69
B. 1 x 1 mL Control Level 2 Ref. 99 08 63
The concentration is stated on the vial label.
REAGENT PREPARATION
Rehydrate the vial with 1 mL of deionized water. Mix gently and let stand at room
temperature for 15 min.
REAGENT COMPOSITION
Pool of human sera containing known quantities of ADA with stabilizers. These
controls are traceable against reference material BCR-647.
Volumetric pipettes.
STORAGE AND STABILITY

The controls, when stored at 2 - 8ºC, will remain stable until the expiration date
stated on the label. Once opened, are stable for 5 days, when kept tightly capped
at 2 -8ºC and two months at -20ºC.
ASSIGNED VALUES
The concentration of each component is stated on the vial label as well as on the
certificate of analysis of the corresponding lot (www.qca.es).
The assigned concentrations are lot specific.
To determine these values the QCA ADA reagent (Ref. 993230) in different
autoanalyzers was used. The expected range for each level is derived from
interlaboratory data and they are given for orientation only; each laboratory
should establish its own range of acceptance.
CAUTION
The safety statements are on the label. It is advised to read SDS before reagent
manipulation. Waste products must be handled as per local regulations.
PROCEDURE
The reagent shall be processed as any other sample, according to the test
instructions (ADA test).
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Enzymes
ALKALINE PHOSPHATASE LIQUID
IFCC METHOD
For “in vitro” determination of alkaline phosphatase in serum or plasma
PRINCIPLE
Determination of p-Nitrophenol produced by the interaction of Alkaline Phosphatase with the substrate p – Nitrophenylphosphate allows the measurement of enzymatic activity.
In optimum conditions the ΔAbs/min value is directly related to the Alkaline Phosphatase concentraction of the sample.
alk. phosphatase
p-nitrophenylphosphate + H2O → p-nitrophenol + phosphate
Alkaline phosphatase is an enzyme whose concentration in serum may be altered by various Bring the reagents and the analyzer to 37ºC.
diseases.
Its main clinical application is related to cases of obstructive liver disease, with significant elevations Monoreagent technique 37ºC
of basal level, especially in extrahepatic obstruction.
Working reagent 1.0 mL
In metabolic bone diseases the increase of alkaline phosphatase level is associated to increased
osteoblastic activity. Sample 0.02 mL
In bone metastasis the increase appears only in the case of osteosclerotic alterations.
Bireagent technique 37ºC
Single test result can not be used to make a clinical diagnosis. It should integrate clinical and Buffer solution (A) 1.0 mL
laboratory data.
Sample 0.02 mL
REAGENTS
Mix, incubate for approx. 1 minute
A. 1 x 40 mL Buffer solution Ref. 99 71 04 Sustrate (B) 0.25 mL
B. 1 x 10 mL Substrate Ref. 99 84 39
Mix, transfer to the reading cuvette and start the stopwatch. Read the absorbance after 1, 2, 3
Kit 1 x 125 mL. (Ref. 99 55 15). Contents: and 4 min.
A. 1 x 100 mL Buffer solution Ref. 99 49 13
B. 1 x 25 mL Substrate Ref. 99 67 43 Reading
Wavelength: 405 nm
Blank: Water
WORKING REAGENT PREPARATION Cuvette: Thermostatized 1 cm light-path
Reagents A and B are ready-to-use. If a Monoreagent procedure is preferred, then the
reagents must be mixed in the ratio: 4 parts of A (Buffer solution) + 1 part of B (Substrate). CALCULATIONS
The formula indicated is used to obtain the factor to calculate the U/L
WORKING REAGENT COMPOSITION
The concentrations in the reagent solution are: Vt x 106
2-Amino-2-methyl-1-propanol buffer pH 10.4 0.70 M ΔAbs/min x = U/L
p-nitrophenylphosphate 12 mM Є x l x Vs
HEDTA 1.55 mM Where:
Magnesium Acetate 1.50 mM Vt: Total volume of reaction
Zinc sulphate 0.95 mM Vs: Sample volume
Preservatives and stabilizers l: Cuvette light path
Є: Extinction Coefficient of p-nitrophenol in alkaline medium, at 405 nm: 18500
The components of the kit, stored at 2-8ºC, will remain stable until the expiration date stated on Monoreagent 405 nm Factor: 2760
the label. Bireagent 405 nm Factor: 3432
The Monoreagent is stable for 5 weeks at 2-8ºC and for 3 week at room temperature (≤ 25ºC),
when protected from the sunlight. U/L = ΔAbs/ min x Factor
Presence of particles or turbidity in the reagent. Working reagent blank ≥ 1.500.
REFERENCE VALUES
General laboratory equipment. Men Women
Spectrophotometer or photometer thermostatable at 37ºC. Cuvette: 1 cm light-path. Adults 53 – 128 U/L 42 – 141 U/L (*)
Under 18 years: Changeable levels between 42 – 383 U/L
SAMPLE
Serum or heparinized plasma. Use samples free from hemolysis. Sera kept at 2-8ºC will remain
(*)In pregnant women the alkaline phosphatase value can be double than that in non pregnant
stable for 7 days.
ones.
CAUTION The stated values are for guidance. Each particular laboratory should establish its own normal
The reagents contain sodium azide at 0.09%. Handle with care. PERFORMANCE CHARACTERISTICS
The safety statements are on the label. It is advisable to look at the SDS before using the reagent. The performance characteristics depend on the method used. It is recommended to calculate
The calibrator must be considered as a human sample, and, thus, potentially infectious. Use these data for each particular test protocol.
adequate protection. These results have been obtained using a manual method.
Sensitivity, as detection limit: 5 U/L
QUALITY CONTROL (NaCl 0.9%) and assay once again. Multiply the final result by 10.
Control serum, Seriscann Normal (Ref. 99 41 48) and Seriscann Anormal (Ref. 99 46 85) should Accuracy: 98.5 %
be included in each test series. Each particular laboratory should establish its own control program. Repetitivity, as Coefficient of Variation: 2.1%
Reproducibility, as Coefficient of Variation: 2.2%
Trueness: Results obtained with this reagent did not show systematic differences when compared
AUTOANALYZERS with reference reagent.
Adaptations to different autoanalyzers are available on request. Details of the performance studies are available on request.
INTERFERENCES
Hemolysis and lipemia will interfere with the assay.
Anticoagulants such as EDTA, oxalate or citrate which chelate divalent cations should not be
used since they would result in enzyme inhibition.
REFERENCES
Szasz, G., Rautenburg, H.W. (1971). Z. Kinderheilk., 111, 233 - 239.
George N.,Bowers Jr, and Rober B., (1975). Clin. Chem., vol 21; Nº 13. Measurement of Total
Alkaline phosphatase activity in human serum.
Tietz N.W., Rinker D., Shaw L.M. (1983). IFCC methods for the measurement of catalytic
concentrations of enzymes. Part 5: IFCC method for Alkaline phosphatase. J. Clin. Chem. Clin.
Biochem.; 21, 731 - 748.
Soldin J.S., Brugnara, C., Wong, E.C., (2003). Pediatric references ranges. Washington AACC
Press; p.10.
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ALKALINE PHOSPHATASE P-NPP
DGKC METHOD
For “in vitro” determination of alkaline phosphatase in serum or plasma
PRINCIPLE
Determination of p-Nitrophenol produced by the interaction of Alkaline Phosphatase with the substrate p – Nitrophenylphosphate allows the measurement of enzymatic activity.
In optimum conditions the ΔAbs/min value is directly related to the Alkaline Phosphatase concentraction of the sample.
alk. phosphatase
p-nitrophenylphosphate + H2O → p-nitrophenol + phosphate

Alkaline phosphatase is an enzyme whose concentration in serum may be altered by various Bring the reagents and the analyzer to 37ºC.
diseases.
Its main clinical application is related to cases of obstructive liver disease, with significant elevations Monoreagent technique 37ºC
of basal level, especially in extrahepatic obstruction.
Working reagent 1.0 mL
In metabolic bone diseases the increase of alkaline phosphatase level is associated to increased
osteoblastic activity. Sample 0.02 mL
In bone metastasis the increase appears only in the case of osteosclerotic alterations.
Bireagent technique 37ºC
Buffer solution (A) 1.0 mL
laboratory data.
Sample 0.02 mL
REAGENTS Mix, incubate for approx. 1 minute
A. 1 x 100 mL Buffer solution Ref. 99 73 12 Sustrate (B) 0.25 mL
WORKING REAGENT PREPARATION Mix, transfer to the reading cuvette and start the stopwatch. Read the absorbance after 1, 2, 3
Reagents A and B are ready-to-use. If a Monoreagent procedure is preferred, then the and 4 min.
reagents must be mixed in the ratio: 4 parts of A (Buffer solution) + 1 part of B (Substrate).
Reading
Wavelength: 405 nm
Blank: Water
Cuvette: Thermostatized 1 cm light-path
Diethanolamine buffer pH 9.8 1.2 M
p-nitrophenylphosphate 12 mM CALCULATIONS
Preservatives and stabilizers The formula indicated is used to obtain the factor to calculate the U/L
STORAGE AND STABILITY Vt x 106

The components of the kit, stored at 2-8ºC, will remain stable until the expiration date stated on ΔAbs/min x = U/L
the label. Є x l x Vs
The Monoreagent is stable for 4 weeks at 2-8ºC and for 3 week at room temperature (≤ 25ºC), Where:
when protected from the sunlight. Vt: Total volume of reaction
Signs of reagent deterioration: Vs: Sample volume
Presence of particles or turbidity in the reagent. Working reagent blank ≥ 1.500. l: Cuvette light path
ADDITIONAL EQUIPMENT Є: Extinction Coefficient of p-nitrophenol in alkaline medium, at 405 nm: 18500
Spectrophotometer or photometer thermostatable at 37ºC. Cuvette: 1 cm light-path. Monoreagent 405 nm Factor: 2760
SAMPLE Bireagent 405 nm Factor: 3432
Serum or heparinized plasma. Use samples free from hemolysis. Sera kept at 2-8ºC will remain U/L = ΔAbs/ min x Factor
stable for 7 days.
REFERENCE VALUES
CAUTION
The safety statements are on the label. It is advisable to look at the SDS before using the reagent. Adults Children 3-15 years
adequate protection. 37ºC 98-279 U/L 250-775 U/L
The stated values are for guidance. Each particular laboratory should establish its own normal
range, using its own instrumentation, blood collection methods and test procedures.
QUALITY CONTROL
Control serum, Seriscann Normal (Ref. 99 41 48) and Seriscann Anormal (Ref. 99 46 85) should PERFORMANCE CHARACTERISTICS
be included in each test series. Each particular laboratory should establish its own control program. The performance characteristics depend on the method used. It is recommended to calculate
these data for each particular test protocol.
These results have been obtained using a manual method.
AUTOANALYZERS
Adaptations to different autoanalyzers are available on request. Sensitivity, as detection limit: 6 U/L
(NaCl 0.9%) and assay once again. Multiply the final result by 10.
Accuracy: 102%
Repetitivity, as Coefficient of Variation: 0.98%
with reference reagent.
INTERFERENCES
Bilirubin (up to 30 mg/dL), intralipid (up to 5000 mg/dL), hemoglobin (up to 1000mg/dL), do not
interfere.
Anticoagulants such as EDTA, oxalate or citrate which chelate divalent cations should not be
used since they would result in enzyme inhibition.
REFERENCES
Szasz, G., Rautenburg, H.W. (1971). Z. Kinderheilk., 111, 233 - 239.
Hausamen, T.U., Helger, R., Gross, W. (1967). Clin. Chim. Acta, 15, 241 - 245.
Recommendations of the German Society for Clinical Chemistry. (1970). Z. Klin. Chem. u Klin.
Biochem., 8, 658 -660.
ISO 9001 / ISO 13485

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Enzymes
AMYLASE LIQUID
IFCC METHOD
For “in vitro” determination of α-amylase
PRINCIPLE
The p-NP group is bonded to a maltose oligosaccharide with a blocked end by ethylidene. α-amylase reacts with the substrate producing lower chain oligosaccharides that, after the action of
α-glucosidase, liberate the p-NP coloured group.
The Abs at 405 nm is directly related to α-amylase activity of the sample.
α-amilase
5 ethylidene-G PNP* + 5 H O → ethylidene-G + G PNP + 2 ethylidene-G + 2 G PNP + 2 ethylidene-G + 2 G PNP
7 2 3 4 4 3 5 2
α-glucosidase
2 G PNP + 2 G PNP + G PNP + 14 H O → 5 PNP + 14 glucose
2 3 4 2
* 4,6-ethylidene(G7)-p-nitrophenyl(G1)-α,D maltoheptaoside;
* PNP = p-Nitrophenol
Determination of amylase activity, in sera or urines, is used for assessing the possibility of chronic Bring the working reagent and the analyzer to 37ºC.
or acute pancreatitis.
An 80% of patients suffering from acute pancreatitis have elevated values during the first 24 hours.
Serum amylase values may be elevated in patients with parotitis, intestinal obstruction, cholecystitis, Monoreagent Bireagent
Technique
diabetic ketoacidosis or peritonitis and in chronic alcoholics. Some lung or ovarian tumours can mL mL
give elevated values of amylase. Monoreagent (A+B) 3.00 ---
laboratory data. Reagent A --- 2.50
REAGENTS Sample 0.10 0.10

Kit 1 x 60 mL. (Ref. 99 60 66). Contents: Reagent B --- 0.50
A. 1 x 50 mL Buffer / Enzyme Ref. 99 60 88
B. 1 x 10 mL Buffer / Substrate Ref. 99 60 90
Mix well and incubate for 3 min. Read the absorbance and start the stopwatch at the same time.
Kit 1 x 120 mL. (Ref. 99 26 40). Contents: Read the absorbance again, after exactly 1, 2 and 3 min.
A. 1 x 100 mL Buffer / Enzyme Ref. 99 26 45
B. 1 x 20 mL Buffer / Substrate Ref. 99 26 50 Determine the average ΔAbs/min of the three readings.
Reading
WORKING REAGENT PREPARATION Wavelength: 405 nm (405 – 420 nm)
Reagents A and B are ready-to-use. If a Monoreagent procedure is preferred, then the reagents Blank: against air
must be mixed in the ratio: 5 parts of A (Buffer/Enzyme) + 1 part of B (Buffer / Substrate). Cuvette: Thermostated 1 cm light-path.
WORKING REAGENT CONCENTRATIONS Semimicromethod

The concentrations in the reagent solution are: It can be carried out with half of the volumes as those in the macro method.
Hepes buffer pH 7.1 65 mM
NaCl 95 mM CALCULATIONS
MgCl2 15 mM Use the stated formula for factor calculation:
Ethylidene-G7PNP 17 mM
α-glucosidase ≥ 4500 U/L Vt x 106
Stabilizers and preservatives ΔAbs/ min x = U/L
Є x l x Vs
When stored at 2-8ºC the components of the kit will remain stable until the expiration date stated Vt: Reaction total volume
on the label. The Monoreagent is stable for 1 week at 2-8ºC and for 3 days at room temperature (≤ Vs: Sample volume
25ºC), when protected from the sunlight. l: Cuvette light path
Є: Extinction Coefficient of p-nitrophenol, 10.600
Reagent turbid or with visible particles. Reagent blank: > 0.300. A slight yellowing of the reagent Determine the mean ΔAbs/min value and multiply by the factor:
solution does not affect the determination.
ADDITIONAL EQUIPMENT U / L (37ºC) = 2930 x ΔAbs 405 nm./min.
General laboratory equipment. µkal / L (37ºC) = 48.8 x ΔAbs 405 nm/min.
REFERENCE VALUES
Serum / plasma: 28 - 100 U/L (0,47- 1.67 µkal/L)
SAMPLE
Urine: ≤ 460 U/L (≤ 7.67 µkal/L)
Serum or plasma with heparin or urine. Samples free from hemolysis should be used.
Each particular laboratory should establish its own normal range, obtained from samples of a
In absence of bacterial contamination the α-amylase activity in serum or plasma will remain stable
representative population, using its own instrumentation, blood collection methods and assaying
for 6 days when stored at 2-8ºC. α-amylase activity in urine will remain stable for 10 days at 2-8ºC,
procedures.
and for 2 days at room temperature (< 25ºC).
CAUTION
PERFORMANCE. CARACTÉRISTIQUES DE FONCTIONNEMENT
The reagents contain sodium azide at 0.09%. Handle with care. Le fonctionnement du produit dépend tant du réactif que du système de lecture manuel ou
The safety statements are on the label. It is advisable to look at the SDS before using the reagent. automatique utilisé. Les résultats suivants ont été obtenues avec une methode manuelle:
adequate protection. Sensibilité comme limite de détection: 12 U/L
The disposal of the residues has to be made according to local regulations. Linéarité: L’essai est linéaire jusqu’à 1500 U/L. Pour des concentrations plus élevées, diluer
l’échantillon 1/10 avec une solution saline (NaCl 0,9%). Multipliez le résultat par 10.
QUALITY CONTROL Exactitude: le pourcentage de récupération est de 98,8 %.
Control serum, Seriscann Normal (Ref. 99 41 48) and Seriscann Anormal (Ref. 99 46 85) should Coefficient de variation dans la série: 1,56 %
be included in each test series. Each particular laboratory should establish its own control program. Coefficient de variation entre les séries: 1,98 %
Justesse. Les résultats obtenus avec le réactif ne sont pas significativement différents par rapport
AUTOANALYZERS au réactif de référence considéré.
Adaptations to different autoanalyzers are available on request. L’étude détaillée de la performance du réactif sont disponibles sur demande.
INTERFÉRENCES
Aucune interférence n’a été observée avec la bilirubine jusqu’à 15 mg/dL ni avec l’hémoglobine
jusqu’à 450 mg/dL.
NOTES
Sweat and saliva contamination in the glassware will alter the final results
REFERENCES
Junge, W., Troge, B., Klein, G., Poppe, W., Gerber, H. (1989). Clin. Biochem. 22, 109 – 114.
Lorentz, K., (2000). Clin.Chem., 46, 644-649.
ISO 9001 / ISO 13485

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CHOLINESTERASE
BUTYRYLTHIOCHOLINE SUBSTRATE
For “in vitro” determination of cholinesterase in serum or plasma
PRINCIPLE
Cholinesterase hydrolyzes butyrylthiocholine to thiocholine, that reacts with DTNB *producing a yellow compound measured at 405 nm. At optimum reaction conditions, the ΔAbs is directly related to
cholinesterase activity of the sample
* DTNB: dithiobis-nitrobenzoate. cholinesterase
butyrylthiocholine + H O thiocholine + butyrate
2
thiocholine + DTNB* 2-nitro-mercapto-benzoate

Cholinesterase values are decreased in liver disease, in processes that occur with hypoalbuminemia Bring the working reagent and the analyzer to the reaction temperature (25º, 30º, 37º C).
and in poisoning by organophosphates.
Increases in activity occur in patients suffering from hipelipoproteinemia type IV, in myasthenia
gravis and nephrosis. 1. -Temperature 25º-30ºC.
Macro Semimicro
laboratory data. mL mL
REAGENTS Reagent A 3.00 1.50

Kit 1 x 50 mL.(Ref. 99 08 90). Contents: Sample 0.02 0.01
A. 1 x 50 mL. Buffer / chromogen. Ref. 99 08 28
B. 2 x 1 mL. Freeze-dried substrate. Ref. 99 05 62 Reagent B 0.10 0.05
Kit 5 x 25 mL.(Ref. 99 08 92). Contents: Mix, transfer to the measuring cuvette. Read initial absorbance, and start the stopwatch.
A. 5 x 25 mL. Buffer / chromogen. Ref. 99 08 08 Read again after 30, 60 and 90 sec.
B. 5 x 1 mL. Freeze-dried substrate. Ref. 99 05 62
2. -Temperature 37ºC.
Reagent A: Ready-to-use. Macro Semimicro
Reagent B: Rehydrate one vial of substrate with 1 mL. of deionized water.
mL mL
To work as a mono-reactive (in the case of analyzers), reagents A and B can be proportionally
Reagent A 3.00 1.50
mixed.
Sample (dil 1/2 with saline) 0.02 0.01
WORKING REAGENT CONCENTRATIONS
Concentrations in the working reagent are: Reagent B 0.10 0.05
Phosphate buffer pH 7.4 50 mM
DTNB* 0.26 mM Mix and put into the reading cuvette. Read initial Abs and read Abs again at 30, 60, 90 sec. exactly
Butyrylthiocholine iodide 6 mM
Stabilizers and preservatives Reading
Wavelength: 405 nm
STORAGE AND STABILITY Blank: air
When stored at 2-8ºC the components of the kit will remain stable until the expiration date stated Cuvette: Thermostatized 1 cm light path
on the label.
Reagent B, once rehydrated, is stable 6 weeks at 2-8ºC, protected from light CALCULATIONS
Determine the ΔAbs/30 sec obtained in each read and calculate the mean value.
Once mixed A and B solutions, the monoreagent is stable for 2 hours at room temperature (≤25ºC).
U/L (25º/30ºC) = 23460 x ΔAbs 405 nm/30 sec
Signs of reagents deterioration: U/L (37ºC) = 46920 x ΔAbs 405 nm/30 sec
REFERENCE VALUES
ADDITIONAL EQUIPMENT 1.- Children, men of all ages and women ≥40 years old.
General laboratory equipment. 25ºC 30ºC 37ºC
Spectrophotometer or photometer thermostatable at 37ºC. Cuvette: 1 cm light-path. 3500-8500 U/L 4300-10500 U/L 5400-13200 U/L
2.-Women 18 -39 years old, non pregnant and not taking oral contraceptives
SAMPLE 25ºC 30ºC 37ºC
Serum, heparinized plasma or EDTA plasma 2800-7400 U/L 3500-9200 U/L 4300-11500 U/L
The Cholinesterase in serum is stable for 7 days at 2 - 8º C.
3.- Women 18-39 years old, pregnant or taking oral contraceptive
CAUTION 25ºC 30ºC 37ºC
The reagents contain sodium azide at 0.09%. Handle with care. 2400-6000 U/L 3000-7000 U/L 3700 - 9300 U/L
The safety statements are on the label. It is advisable to look at the SDS before using
the reagent. The calibrator must be considered as a human sample, and, thus, potentially The stated values are for guidance. Each particular laboratory should establish its own normal
range, using its own instrumentation, blood collection methods and test procedures.
infectious. Use adequate protection.
The disposal of the residues has to be made according to local regulations. PERFORMANCE CHARACTERISTICS
Performance of the reagent depends on the reagent itself and also depends on method and
QUALITY CONTROL analyzer used.
The results indicated are obtained using a manual method.
control program. Linearity: Up to 22500 U/L. For higher values, it is recommended to dilute the sample 1/5 in saline
Accuracy, as recovery %: 98.1%
AUTOANALYZERS Repetitivity, as Coefficient of Variation: 2.19 %
Adaptations to different autoanalyzers are available on request. Reproducibility as Coefficient of Variation: 2.61%
INTERFERENCES
Relevant interferences are not known.
REFERENCES
Kendel, M., Bottger, R., (1967). Klin. Wschr., 45, 325-327.
Den Blawen, D.H.,Poppe, W.A., Trischler, W., (1983). J.Clin. Chem. Biochem., 21, 381-386.
ISO 9001 / ISO 13485

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Enzymes
CK - MB
IMMUNOLOGICAL METHOD
For “in vitro” determination of CK – MB fraction in serum or plasma
PRINCIPLE
CK-MB is composed of two kind of subunits: M and B. A specific antibody inhibits the M subunit of the CK-MB and CK-MM not affecting the activity of the B subunit of the CK-MB and CK-BB isoenzymes.
The remaining CK-MB activity, corresponding to the CK-B, is determined by the CK-NAC activated method.
CK- B
phosphocreatine + ADP creatine + ATP
HK
ATP + glucose glucose-6-phosphate + ADP
G-6-PDH
glucose-6-phosphate + NADP+ 6-phosphogluconate + NADPH + H+
The phosphate released from phosphocreatine by CK-B binds to ADP forming ATP, that reacts with glucose by the action of hexokinase producing glucose-6-phosphate and ADP.
The G-6-PDH degrades this product to phosphogluconate at the same time that NADP+ reduces to NADPH. This last reaction is measured by the variation of the Abs at 340 nm. In optimal conditions, the ΔAbs/min is
proportional to the creatinkinase activity in the sample.
DIAGNOSTIC USE
The CK-MB increases take place in myopathic diseases and inflammatory diseases of the heart. Technique
In myocardial infarction, CK-MB activity increases rapidly. When serial determinations are performed it is Bring reagents and the analyzer to the desired assay temperature (25º, 30º, 37ºC).
observed that the maximum point is quickly reached with an also very quick decrease.
Technique mL
Single test result can not be used to make a clinical diagnosis. It should integrate clinical and laboratory
Working reagent 1.0
data.
Incubate 2-3 min at the desired assay temperature
REAGENTS
Kit 20 x 2.5 mL.(Ref. 99 91 11). Contents Sample 0.04
A. 20 x 2.5 mL Substrate / antibody Ref. 99 60 52
B. 1 x 50 mL Buffer solution Ref. 99 43 35 Mix well and let stand 10 min. at the assay temperature. Then add the mixture to a cuvette and read
C. 1 x 2 mL Control Ref. 99 30 56 Abs1, and then read Abs2 exactly 5 min. later. Determine: ΔAbs = Abs2 - Abs1
Concentration is stated on the label
Reading
PREPARATION OF WORKING REAGENT AND CONTROLS Wavelength: 365 nm; 340 nm; 334 nm
Reagent: Rehydrate 1 vial of freeze-dried substrate/antibody with the volume of buffer solution stated on Blank: water
the label. Mix gently until completely dissolved. Cuvette: thermostated 1 cm light path
Control: Reconstitute the contents of the vial with the volume of deionised water stated on the label. Leave
to stand for 15 min and dissolve completely by mixing gently. CALCULATIONS
Using the stated formula for factor and U/L calculation:
WORKING REAGENT COMPOSITION Vt x 106
ΔAbs x = U/L
The concentrations in the reagent solution are: Є x l x Vs x t
Imidazole/Acetic acid buffer pH 6.8 100 mM Vt: Total volume of reaction mixture;
Glucose 20 mM Vs: Sample volume
Creatine phosphate 30 mM l: Cuvette light path
Mg2+ 10 mM Є: Extinction coefficient of NADPH:
ADP 2 mM 365 nm: 3.53 x 103
AMP 5 mM 340 nm: 6.31 x 103
di-adenosine pentaphosphate 10 μM 334 nm: 6.17 x 103
N-acetyl cysteine 20 mM t: Time of reading
NADP+ 2 mM To calculate the U/L with the values of ΔAbs
Glucose-6-phosphate dehydrogenase ≥ 1,500 U/L CK-B CK-MB
Hexokinase ≥ 2,500 U/L 365 nm 1486 x ΔAbs 2972 x ΔAbs
Antibody to CK-M ≥ 2,000 U/L 340 nm 825 x ΔAbs 1651 x ΔAbs
Stabilizers and preservatives 334 nm 841 x ΔAbs 1683 x ΔAbs
As the activity tested is the one due to CK-B subunit, to retrieve the value of the total activity of the
Control: Pool of human sera with a known CK-MB enzymatic activity, added with stabilizers and isoenzyme CK-MB the U/L obtained are to be multiplied by 2. This is why the factor used to calculate CK-
preservatives MB activity is double.
REFERENCE VALUES
The components of the kit, stored at 2-8ºC, will remain stable until the expiration date stated on the label.
There will be suspicion of myocardial infarction, provided the following three conditions are given:
The rehydrated reagent is stable for 12 days at 2-8ºC and for 3 days at room temperature (≤ 25ºC).
The reconstituted control is stable for 8 hours at room temperature and 5 days at a 2-8ºC. If deep frozen,
25ºC 30ºC 37ºC
at -20ºC it will remain stable for 4 weeks. Freeze and thaw only once.
CK men > 80 U/L > 130 U/L > 190 U/L
CK women > 70 U/L > 110 U/L > 170 U/L
Presence of particles or turbidity in the reagent. Working reagent blank ≥ 0.800. CK - MB > 10 U/L > 16 U/L > 25 U/L
ADDITIONAL EQUIPMENT CK-MB activity

General laboratory equipment. CK-MB index x 100: 6 - 25%
Spectrophotometer; automated analyzer or photometer with thermostated cuvette, 1 cm light path. CK total activity
SAMPLE The stated values are for guidance. Each particular laboratory should establish its own normal range, using
Serum or plasma, with heparin. its own instrumentation, blood collection methods and test procedures
When the sample is stored at 2º-8º C, the CK - MB activity remains stable for one week..
CAUTION The performance characteristics depend on the method used. It is recommended to calculate these data
The reagent contains sodium azide at 0.09%. Handle with care. for each particular test protocol. These results have been obtained using an automated method at 37ºC
The safety statements are on the label. It is advisable to look at the SDS before using the reagent. and 340nm.
QUALITY CONTROL Linearity: For values higher than 600 U/L, it is recommended to dilute the sample 1/10 in saline (NaCl
It is advisable to include the CK-MB control (Ref. 99 30 56) along with other sera, Seriscann Normal (Ref. 0.9%) and assay once again. Multiply the final result by 10.
99 41 48) and Seriscann Anormal (Ref. 99 46 85) in each test series for results verification. Repetitivity, as Coefficient of Variation: 2.19%
Each particular laboratory should establish its own control program. Reproducibility, as Coefficient of Variation: 2.52%
Trueness: Results obtained with this reagent did not show systematic differences when compared with
AUTOANALYZERS
reference reagent.
PROCEDURE
Prior to assay the CK-MB, the CK total activity has to be determined (QCA Ref. 99 44 10).or CK-NAC INTERFERENCES
Liquid (Ref. 99 05 24 or 99 79 74). If the CK total activity ≥ 1000 U/L, dilute the sample 1/10 with saline Highly haemolysed sera will interfere with the reaction. Glucose up to 600 mg/dL, ascorbic acid up to 40
(NaCl 0.9%), and then proceed with the CK-MB test. mg/dL and haemoglobin up to 500 mg/dL, do not interfere the assay.
REFERENCES
Lang, H, Würzburg, U. (1982) Clin. Chem., 28, 1439-1447.
Szasz, G., Gruber, W., Bernt, E. (1976). Clin. Chem. 22, 650-655.
Stein, W. (1985). Med. Weit., 36, 572 - 577.
Gerhardt W., Waldenström, J. (1979), Clin. Chem., 25, 1274-1280.
ISO 9001 / ISO 13485

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CK - MB LIQUID
IMMUNOLOGICAL METHOD
For “in vitro” determination of CK – MB fraction in serum or plasma
PRINCIPLE
CK-MB is composed of two kind of subunits: M and B. A specific antibody inhibits the M subunit of the CK-MB and the CK-MM subunit not affecting the activity of the B subunit of the CK-MB and CK-BB isoenzymes.
The remaining CK-MB activity, corresponding to the CK-B, is determined by the CK-NAC activated method.
CK-B
creatine phosphate+ ADP → creatine + ATP
HK
ATP + glucose → glucose-6-phosphate + ADP
G-6-PDH
glucose-6-phosphate + NADP+ → 6-phosphogluconate + NADPH + H+
The phosphate released from phosphocreatine by CK-B binds to ADP forming ATP, that reacts with glucose by the action of hexokinase producing glucose-6-phosphate and ADP. The glucose-6-phosphate dehydrogenase
degrades this product to phosphogluconate at the same time that NADP+ reduces to NADPH. This last reaction is measured by the Abs change at 340 nm. In optimal conditions, the ΔAbs/min is proportional to the
creatinkinase activity in the sample.
DIAGNOSTIC USE Mix well and let stand 10 min. at the assay temperature. Then add the mixture to a cuvette and read
The CK-MB increases take place in myopathic diseases and inflammatory diseases of the heart.
In myocardial infarction, CK-MB activity increases rapidly. When serial determinations are performed it is Abs1, and then read Abs2 exactly 5 min. later.
observed that the maximum point is quickly reached with an also very quick decrease.
Determine: ΔAbs = Abs2 - Abs1
A single test result can not be used to make a clinical diagnosis. It should integrate clinical and laboratory
data. Reading
Wavelength: 365 nm; 340 nm; 334 nm
REAGENTS Blank: water
Kit 1 x 50 mL. (Ref. 99 44 03). Contents: Cuvette: thermostated 1 cm light path
B. 1 x 10 mL Substrate Ref. 99 04 68 CALCULATIONS
C. 1 x 2 mL Control. Lyophilized Ref. 99 30 56 Using the stated formula for factor and U/L calculation:
The concentration is stated on the vial label. Vt x 106
ΔAbs x = U/L, where
PREPARATION OF WORKING REAGENT AND CONTROLS Є x l x Vs x t
Reagent A and B are ready to use. If a monoreagent procedure is preferred, then the reagents must be Vt: Total volume of reaction mixture;
mixed in the ratio: 4 parts of Reagent A (Buffer solution) + 1 part of Reagent B (Substrate solution). Vs: Sample volume
Control: Reconstitute the contents of the vial with the volume of deionised water stated on the label. Let l: Cuvette light path
stand for 15 min and dissolve completely with a gentle mixing. Є: Extinction coefficient of NADPH:
365 nm: 3.53 x 103
WORKING REAGENT COMPOSITION 340 nm: 6.31 x 103
The concentrations in the reagent solution are: 334 nm: 6.17 x 103
t: Time of reading
Imidazole/acetic acid buffer pH 6.1 120 mM To calculate the U/L with the values of ΔAbs
Glucose 23 mM CK-B CK-MB
Creatine phosphate 22 mM 365 nm 1486 x ΔAbs 2972 x ΔAbs
Mg2+ 12 mM 340 nm 825 x ΔAbs 1651 x ΔAbs
ADP 3 mM 334 nm 841 x ΔAbs 1683 x ΔAbs
AMP 7 mM As the activity tested is the one due to CK-B subunit, to retrieve the value of the total activity of the
N-acetyl cysteine 21 mM isoenzyme CK-MB, the U/L obtained are to be multiplied by 2. This is why the factor used to calculate the
NADP+ 2.4 mM CK-MB activity is double.
Glucose-6-phosphate dehydrogenase ≥ 1500 U/L
Hexokinase ≥ 2500 U/L REFERENCE VALUES
Antibody to CK-M ≥ 2000 U/L There will be suspicion of myocardial infarction, provided the following three conditions take place:
Stabilizers and preservatives
25ºC 30ºC 37ºC
Control: Pool of human sera with a known CK-MB enzymatic activity, added with stabilizers and
preservatives. CK men > 80 U/L > 130 U/L > 190 U/L
STORAGE AND STABILITY CK women > 70 U/L > 110 U/L > 170 U/L
The components of the kit, stored at 2-8ºC, will remain stable until the expiration date stated on the label.
The working reagent is stable for 3 weeks at 2-8ºC and for 1 week at room temperature (≤ 25ºC). CK-MB > 10 U/L > 16 U/L > 25 U/L
The reconstituted control is stable for 8 hours at room temperature and 5 days at 2 -8ºC.
If deep frozen, at -20ºC it will stable for 4 weeks. Freeze and thaw only once.
CK-MB activity
Presence of particles or turbidity in the reagent. Working reagent blank > 0.250 CK-MB index = -------------------------- x 100 : 6-25%
CK total activity
General laboratory equipment. The stated values are for guidance. Each particular laboratory should establish its own normal range, using
Spectrophotometer or photometer thermostatable at 37ºC. Cuvette: 1 cm light-path. its own instrumentation, blood collection methods and test procedures
SAMPLE PERFORMANCE CHARACTERISTICS
Serum or plasma, with heparin. The performance characteristics depend on the method used. It is recommended to calculate these data
When the sample is stored at 2-8ºC, the CK-MB activity remains stable for one week. for each particular test protocol. These results have been obtained using an automated method at 37ºC
and 340nm.
CAUTION
The reagents contain sodium azide at 0.09%. Handle with care. Sensitivity, as detection limit: 4 U/L
The safety statements are on the label. It is advisable to look at the SDS before using the reagent. Linearity: 945 U/L
The calibrator must be considered as a human sample, and, thus, potentially infectious. Use adequate Within-run Precision, as Coefficient of Variation: 4.13%
protection. Run-to-run Precision, as Coefficient of Variation: 5.93%
The disposal of the residues has to be made according to local regulations. Trueness: Results obtained with this reagent did not show systematic differences when compared with
reference reagent
QUALITY CONTROL Details of the performance studies are available on request
It is advisable to include the CK-MB control (Ref. 99 30 56) along with other control sera, Seriscann Normal
(Ref. 99 41 48) and Seriscann Anormal (Ref. 99 46 85), in each test series for results verification. INTERFERENCES
Each particular laboratory should establish its own control program. Highly haemolysed sera will interfere with the reaction. No interferences were observed with bilirubin up to
30 mg/dL and lipemia (Intralipid) up to 300 mg/dL.
PROCEDURE
Prior to assay the CK-MB, the CK total activity has to be determined (QCA Ref. 99 44 10) or CK-NAC Liquid AUTOANALYZERS
(Ref. 99 05 24 or Ref. 99 79 74). If the CK total activity ≥ 1000 U/L, dilute the sample 1/10 with saline (NaCl Adaptations to different autoanalyzers are available on request.
0.9%) and then proceed with the CK-MB test. REFERENCES
Technique Tietz, NW., Textbook of Clinical Chemistry 6th Edition, W.B. Saunders, Philadelphia (2018).
Bring reagents and the analyzer to the desired assay temperature (25ºC, 30ºC, 37ºC). CLSI Guidelines and Standards, CLSI, Wayne, P.A
Technique mL
Lang, H, Würzburg, U. (1982) Clin. Chem., 28, 1439-1447.
Working reagent 1 Szasz, G., Gruber, W., Bernt, E. (1976). Clin. Chem. 22, 650-655.
Stein, W. (1985). Med. Weit., 36, 572 - 577.
Incubate 2-3 minutes at the desired assay temperature Gerhardt W., Waldenström, J. (1979), Clin.Chem., 25, 1274-1280.
Sample 0.04
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Enzymes
CK - NAC LIQUID
IFCC METHOD
For “in vitro” determination of creatine kinase in serum or plasma
PRINCIPLE
Creatine kinase (CK) releases creatine from phosphocreatine with the aid of ADP that is transformed in ATP. The glucose of reaction media reacts with ATP by the action of hexokinase, forming glucose-
6-phosphate. This degrades to 6-phosphogluconate in the presence of glucose-6-phosphate dehydrogenase and NADP+. The reaction reduces the NADP+ to NADH and there is an Abs change.
When the reaction conditions are optimum, the ΔAbs/min is directly related to the CK activity of the sample.
CK
creatine phosphate+ ADP → creatine + ATP
HK
ATP + glucose → glucose-6-phosphate + ADP
G-6-PDH
glucose-6-phosphate + NADP+ → 6-phosphogluconate + NADPH + H+

Mostly, the increase of CK values is related to diseases of skeletal muscle or the heart. Bring reagents and the analyzer to working temperature.
Values higher than usual are observed after trauma, surgery, myopathic disorders, myocardial
infarction, prolonged hypothermia or hypothyroidism. Also, elevated values are described in cases Monoreagent Bireagent
Technique
of Reye syndrome or Duchenne disease. 37ºC (µL) 37ºC (µL)
Results lower than the reference values may reflect a sedentary lifestyle or the presence of low Working reagent 1000 ---
muscle mass.
Single test result can not be used to make a clinical diagnosis. It should integrate clinical and Buffer Solution (A) --- 1000
laboratory data.
Sample 40 50
REAGENTS --- Mix, incubate for 1 min
A. 1 x 40 mL Buffer solution Ref. 99 46 27 Substrate (B) --- 250
Mix, read the Abs. after 3 min. and start the stopwatch.
Read again the Abs. after 1, 2, 3 and 4 min.
Determine the mean of Abs/min of different lectures.
A. 1 x 100 mL Buffer solution. Ref. 99 49 10
B. 1 x 25 mL Substrate. Ref. 99 69 18
Reading
Wavelength: 365 nm; 340 nm; 334 nm
WORKING REAGENT PREPARATION Blank: water
Reagents A and B are ready-to-use. Cuvette: thermostatized 1 cm light-path
If a Monoreagent procedure is preferred, then the reagents must be mixed in the ratio: 4 parts of A
(Buffer solution) + 1 part of B (Liquid substrate).
CALCULATIONS
To calculate the U/L, the following formula shall be used:
WORKING REAGENT CONCENTRATIONS
Vt x 106
The concentrations in the reagent solution are: ΔAbs/min x = U/L
Imidazole/Acetic acid buffer pH 6.7 120 mM Є x l x Vs
Glucose 23 mM Vt: Total volume
Creatine phosphate 36 mM Vs: Sample volume
Mg2+ 12 mM l: Cuvette light path
ADP 3 mM Є: Extinction Coefficient NADPH:
AMP 7 mM 365 nm: 3.53 x 103
N-acetyl cysteine 21 mM 340 nm: 6.31 x 103
NADP+ 2.2 mM 334 nm: 6.17 x 103
Glucose-6-phosphate dehydrogenase ≥ 3500 U/l Find the mean ΔAbs/min value and multiply by the factor:
Hexokinase ≥ 7500 U/l
U/L = (ΔAbs365 nm/min) x 7429
U/L = (ΔAbs340 nm/min) x 4127
STORAGE AND STABILITY U/L = (ΔAbs334 nm/min) x 4207
The components of the kit, stored at 2-8ºC, will remain stable until the expiration date stated
on the label. The Monoreagent is stable for 2 weeks at 2-8ºC and at room temperature (≤ 25ºC), REFERENCE VALUES
when protected from the sunlight.
Reaction Tª Men Women
Presence of particles or turbidity in the reagent. Working reagent blank ≥0.800. 25ºC 19-70 U/L 14-60 U/L
30ºC 29-108 U/L 21-91 U/L
ADDITIONAL EQUIPMENT 37ºC 46-171 U/L 34-145 U/L
General laboratory equipment. The stated values are for guidance. Each particular laboratory should establish its own normal
Spectrophotometer or photometer thermostatable at 37ºC. Cuvette: 1 cm light-path. range, using its own instrumentation, blood collection methods and test procedures
Conversion to SI Units: Creatine Kinase (U/L)x 0.017= Creatine Kinase (μkat/L)
Serum or plasma with EDTA or heparin. A light haemolysis will not interfere with the assay. The performance characteristics depend on the method used. It is recommended to calculate
Samples kept in the refrigerator at 2-8ºC are stable for one week. these data for each particular test protocol. These results have been obtained using a automated
method at 37ºC and 340 nm.
CAUTION
The reagents contain sodium azide at 0.09%. Handle with care. Sensitivity, as detection limit: 2 U/L
The safety statements are on the label. It is advisable to look at the SDS before using the reagent. Linearity: Up to 1500 U/L. For higher values, it is recommended to dilute the sample 1/10 in saline
The calibrator must be considered as a human sample, and, thus, potentially infectious. Use (NaCl 0.9%) and assay once again. Multiply the final result by 10.
adequate protection. Accuracy: 103 %
The disposal of the residues has to be made according to local regulations. Repetitivity, as Coefficient of Variation: 1.8%
QUALITY CONTROL Trueness: Results obtained with this reagent did not show systematic differences when compared
Control serum, Seriscann Normal (Ref. 99 41 48) and Seriscann Anormal (Ref. 99 46 85) should with reference reagent.
be included in each test series. Each particular laboratory should establish its own control program. Details of the performance studies are available on request
AUTOANALYZERS INTERFERENCES
Adaptations to different autoanalyzers are available on request. Highly haemolysed sera will interfere with the reaction.
Glucose concentrations higher than 600 mg/dL, ascorbic acid higher than 40 mg/dL and
haemoglobin higher than 500 mg/dL will interfere with the reaction.
REFERENCES
Gunzer, G., Kretzschmar, F., Wrynn, K., USP 6306617, 2001.
IFCC, (2002), Clin. Chem. Lad. Med., 40, 635-642.
Matheu, M., et.al., (1982), Ann. Biol. Clin, 40, 87-164.
Szasz, G., Kinne, E., (1979), J. Clin. Chem. Clin. Biochem., 17, 689-691.
ISO 9001 / ISO 13485

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GAMMA - GT LIQUID
SZASZ MODIFIED METHOD
For “in vitro” determination of gamma-GT in serum or plasma
PRINCIPLE
Serum γ-glutamyl transferase (γ-GT) transfer the glutamiy group to cosubstrate glycyl-glycyne producing 5-amino-2-nitrobenzoate. In optimum conditions enzymatc activity is directly
related to amino-2-nitrobenzoate produced that is measured by spectrophotometry.
gamma - GT
L-γ-glutamyl-3-carboxy-p-nitroanilide + glycyl-glycine → L-γ-glutamyl-glycyl-glycine + 5-amino-2-nitrobenzoate

In 90% of liver diseases there is a rise of γ-GT levels. The values are moderate in cirrhosis Bring the working reagent and the analyzer to the working temperature (25ºC, 30ºC, 37ºC).
and infectious mononucleosis, while significant increases were found in obstructive liver
disease, intrahepatic cholestasis or primary biliary cirrhosis.
Monoreagent technique 25ºC/30ºC/37ºC
Single test result could not be used to make a clinical diagnosis. It should integrate clinical Working reagent 1.0 mL
and laboratory data.
Sample 0.1 mL
REAGENTS Bireagent technique 25ºC/30ºC/37ºC
Kit 1 x 50 mL. Ref. 99 35 61. Contents:
A. 1 x 40 mL Buffer solution Ref. 99 92 48 Buffer sol.(A) 1.0 mL
B. 1 x 10 mL Liquid substrate Ref. 99 38 43
Sample 0.1 mL
Kit 1 x 125 mL. Ref. 99 10 56. Contents: Mix, incubate for approx. 1 minute
B. 1 x 25 mL Liquid substrate Ref. 99 10 81 Substrate (B) 0.25 mL
Kit 1 x 250 mL. Ref. 99 10 54. Contents: Mix, read the absorbance after 1 min. and start the stopwatch. Read again the absorbance
A. 2 x 100 mL Buffer solution. Ref. 99 10 68 after 1, 2 and 3 min.
Reagents A and B are ready-to-use. If a monoreagent procedure is preferred, then Reading
the reagents must be mixed in the ratio: 4 parts of A (Buffer solution) + 1 part of B (Liquid Wavelength: 405 nm
substrate). Blank: Water
Cuvette: Thermostatized 1 cm light path
The concentrations in the reagent solution are: CALCULATIONS
Tris buffer pH 8.4 90 mM The formula indicated is used to obtain the factor to calculate the U/L
L-gamma-glutamyl-3-carboxy-p-nitroanilide 6 mM Vt x 106
ΔAbs/min x = U/L
Glycyl-glycine 100 mM Є x l x Vs
Stabilizers and preservatives Vt: Total reaction volume
Vs: Sample volume
l: Cuvette light path
The components of the kit, stored at 2-8ºC, will remain stable until the expiration date
Є: Extinction coefficient of 5-amino-2-nitrobenzoate at 405 nm: 9500
stated on the label.
The monoreagent is stable 5 weeks at 2-8ºC and 3 weeks at room temperature (≤ 25ºC),
For the calculation of the U/L determine the average Abs/min and multiply by the factor:
when protected from the sunlight.
Monoreagent method U/L = (ΔAbs405 nm / min.) x 1158
Bireagent method U/L = (ΔAbs405 nm / min.) x 1420
ADDITIONAL EQUIPMENT REFERENCE VALUES
General laboratory equipment. Assay temp. Men Women
Spectrophotometer or photometer thermostatable at 37ºC. Cuvette: 1 cm light-path. 25 ºC 6-28 U/L 4-18 U/L
30 ºC 8-38 U/L 5-25 U/L
37 ºC 11-50 U/L 7-32 U/L
CAUTION
The reagents contain sodium azide at 0.09%. Handle with care. The stated values are for guidance. Each particular laboratory should establish its own
The safety statements are on the label. It is advisable to look at the SDS before using normal range, using its own instrumentation, blood collection methods and test procedures.
the reagent. The calibrator must be considered as a human sample, and, thus, potentially
infectious. Use adequate protection. PERFORMANCE CHARACTERISTICS
The disposal of the residues has to be made according to local regulations. The performance characteristics depend on the method used. It is recommended to
calculate these data for each particular test protocol. These results have been obtained
SAMPLE using a manual method.
Serum or plasma. Samples free from hemolysis should be used. The gamma-GT-asic
activity will remain stable for 10 days if the sample is stored at 2-8ºC. Sensitivity, as detection limit: 4 U/mL
Linearity: Up to 600 U/L. For higher values, it is recommended to dilute the sample 1/10 in
QUALITY CONTROL saline (NaCl 0.9%) and assay once again. Multiply the final result by 10.
Control serum, Seriscann Normal (Ref. 99 41 48) and Seriscann Anormal (Ref. 99 46 85) Accuracy: 98.4%
should be included in each test series. Each particular laboratory should establish its own Repetitivity, as Coefficient of Variation: 1.77%
control program. Reproducibility, as Coefficient of Variation: 2.29%
Trueness: Results obtained with this reagent did not show systematic differences when
AUTOANALYZERS
compared with reference reagent.
INTERFERENCES
Hemolyzed samples will give false results. Fluoride, EDTA, citrate and oxalate inhibit the
enzyme activity.
REFERENCES
Szasz, G., (1969) Clin. Chem., 15, 124 - 136.
ISO 9001 / ISO 13485

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Enzymes
COLOURIMETRIC GOT/AST AND GPT/ALT
REITMAN-FRANKEL METHOD
For “in vitro” determination of GOT and GPT in serum or plasma
PRINCIPLE
GOT
L-aspartic acid + α-ketoglutaric acid oxalacetic acid + L-glutamic acid
GPT
L-alanine + α-ketoglutaric acid pyruvic acid + L-g lutamic acid
The ketonics acids produced (oxalacetic acid, pyruvic acid) will form, by reaction with 2,4-dinitrophenylhydrazine, the corresponding coloured hydrazones.

Increases in GOT and GPT activity were observed in cases of liver damage: hepatitis of various types,
necrosis or damage in hepatocytes, cholestatic icteria. High levels are also seen in diseases affecting the Technique GOT (mL) GPT (mL)
heart muscle.
In alcoholic hepatitis and acute myocardial infarction, the increase of GOT activity is greater than the GOT Substrate 0.5 --
activity of GPT. Although, in viral hepatitis GPT increase is always greater than that of GOT.
GPT Substrate -- 0.5
The increase of GPT enzymatic activity is more specific for liver damage than ratio GOT / GPT.
Incubate 5 minutes at 37º C
Single test result could not be used to make a clinical diagnosis. It should integrate clinical and laboratory
data. Sample 0.1 0.1
REAGENTS Incubate at 37ºC 60 min. 30 min.

GOT/AST
Colour developer 0.5 0.5
Kit (Ref. 99 87 20). Contents:
A. 1 x 100 mL Substrate solution Ref. 99 85 78 Let stand 20 min at room temp. (20-25ºC)
B. 1 x 100 mL Colour developer Ref. 99 35 35
C. 1 x 100 mL NaOH 4N Ref. 99 67 52 NaOH 0,4N 5.0 5.0
D. 1 x 10 mL Standard Ref. 99 43 30
Let stand 15 min at room temp. (20-25ºC)
GPT/ALT
Kit (Ref. 99 76 10). Contents: Reading
A. 1 x 100 mL Substrate solution Ref. 99 73 01 Wavelength: 505 nm
B. 1 x 100 mL Colour developer Ref. 99 35 35 Blank: Water
C. 1 x 100 mL NaOH 4N Ref. 99 67 52 Colour stability: a minimum of 1 hour
CALCULATIONS
WORKING REAGENT PREPARATION To find out the GOT or GPT activity value, the Abs obtained shall be interpolated in the calibration curve.
The substrate, coloured solutions and standard are ready to used. The NaOH solution should be diluted Results are expressed in WU/mL.
1/10 with deionized water prior to use.
Calibration curve
WORKING REAGENT COMPOSITION Prepare the followings tubes:
The concentrations in the reagent solution are: TUBE 1 2 3 4 5 6
GOT substrate solution: Water 0.10 0.10 0.10 0.10 0.10 0.10
Phosphate buffer pH 7.4 100 mM GOT/GPT Subs. 0.50 0.45 0.40 0.35 0.30 0.25
α-ketoglutaric acid 2 mM Standard 0.00 0.05 0.10 0.15 0.20 0.25
L-aspartic acid 100 mM
GOT(WU/mL) 0 22 55 95 150 215
GPT substrate solution: GPT(WU/mL) 0 25 50 83 126 ---
Phosphate buffer pH 7.4 100 mM
α-ketoglutaric acid 2 mM Add 0.5 mL of colour reagent (DNPH) to each tube. Mix and let stand at room temp. (≤ 25ºC) for 20 min.
L-alanine 200 mM Finally, add 5 mL of NaOH 0.4N and read the Absorbance after 15 min, against water blank.
Colour developer: Plot the Absorbance in the ordinates and WU/mL in the abscissa.
2,4-dinitrophenylhydrazine (DNPH) 1 mM
Sera with GOT activity higher than 215 WU/mL or with GPT activity higher than 126 WU/mL should be
Standard: Aqueous solution of sodium piruvate 1.4mmol/L diluted 1/10 in saline (NaCl 0.9%) and determined once again. Multiply the final result by 10.
STORAGE AND STABILITY Note

When kept at 2-8ºC, the components of the kit will remain stable until the expiration date stated on the Whenever GOT and GPT activities are simultaneously determined, it is recommended to initiate first the
label. GOT substrate incubation (30 minutes before) in order to end both reactions at the same time.
The diluted NaOH can be stored at room temp. (≤ 25ºC) provided it is well stoppered in a polyethylene
botlle to avoid undesirable carbonation. SI Units
To convert colorimetric units WU/mL into IU/L the following factor shall be used: UI/L = (WU/mL) x 0,483
REFERENCE VALUES
Presence of particles or turbidity in the reagent. Working reagent blank > 0.350.
GOT: 8 - 40 WU/mL (3,5 - 19 UI/L)
GPT: 5 - 30 WU/mL (2,5 - 15 UI/L)
The values shown are for guidance. It is recommended that each laboratory establish its own reference
Spectrophotometer; automated analyzer or photometer with thermostated cuvette at 37ºC. 1cm light path
values.
CAUTION
The reagent contains Sodium azide at 0.09%. Handle with care.
The performance characteristics depend on the method used. It is recommended to calculate these data
The security statements are on the label. It is advisable to look at the MSDS before using the reagent. The
for each particular test protocol. These results have been obtained using a manual method.
disposal of the residues has to be made according to legal local regulations.
Reaction range: GOT: up to 215 WU/mL; GPT up to 126 WU/mL. For higher values, it is recommended to
SAMPLE
dilute the sample 1/10 in saline (NaCl 0.9%) and assay once again. Multiply the final result by 10.
Serum or plasma with EDTA or heparin. Samples free from hemolysis should be used. Sera kept at 2-8ºC
Accuracy: GOT: 97.0%; GPT: 105.2%
looses ca. 10% of its activity after 3 days.
Repetitivity, as Variation Coefficient: GOT: 4.5 %; GPT: 5.0
Reproducibility, as Variation Coefficient: GOT: 6.6 %; GPT: 6.8
QUALITY CONTROL
Control serum, Seriscann Normal (Ref. 99 41 48) and Seriscann Anormal (Ref. 99 46 85) should be
reference reagent.
included in each test series. Each particular laboratory should establish its own control program.
Details of the performance studies are available on request
AUTOANALYZERS
INTERFERENCES
Highly haemolysed sera will interfere with the reaction.
REFERENCES
Methods of enzymatic analysis, 2a. ed, vol.II, 735-739, Editado por H.U. Bergemeyer Verlag Chemie.
ISO 9001 / ISO 13485

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GOT / AST U.V.
IFCC METHOD
For “in vitro” determination of Aspartate aminotransferase GOT/AST in serum or plasma
PRINCIPLE
The glutamic-oxalacetic transaminase (GOT), actually AST, catalyzes the reaction between L-aspartic acid and α-ketoglutaric acid. The oxalacetic acid formed is reduced by NADH with the aid of the auxiliary enzyme
MDH.
The change of NADH to NAD+ produces a change of Abs. The reaction media contains, also, LDH to remove endogenous pyruvate to avoid possible interferences.
In optimum reaction conditions the ΔAbs/min is directly related to GOT concentration in the sample.
GOT
α-ketoglutaric acid + L-aspartic acid L-glutamic acid + oxalacetic acid
MDH
oxalacetic acid + NADH + H+ L-malic acid + NAD+

Increments in GOT activity are observed in cases of liver damage: hepatitis of various types, necrosis or The previously described method is the one proposed by the International Federation of Clinical Chemistry
damage in hepatocytes, cholestatic icteria. (IFCC) in its modified version without pyridoxal phosphate pre-incubation, using pH 7.8 Tris-HCl buffer and
High levels are also seen in heart muscle diseases. In alcoholic hepatitis and acute myocardial infarction, not taking into account the correction of the blank.
the increase of GOT activity is greater than that of GPT.
Bring reagents and the analyzer to the working temperature (25º, 30º, 37ºC).
Single test result can not be used to make a clinical diagnosis. It should integrate clinical and laboratory
data.
Technique Macro (mL) Semimicro (µL)
REAGENTS
Working reagent 1.0 500
A. 20 vials of Freeze-dried substrate Ref. 99 30 27 Incubate 2-3 min at working temperature.
Sample 0.1 50
A. 12 vials of Freeze-dried substrate. Ref. 99 34 11
B. 2 x 100 mL Buffer solution. Ref. 99 75 35 Mix and start the stopwatch. Transfer to the measuring cuvette. Read the absorbance after 1,2,3 and 4 min.
Find the Abs/min mean value of the different readings.
WORKING REAGENT PREPARATION Reading

Rehydrate one vial of freeze-dried substrate with the volume of buffer solution stated on the label. Wavelength: 334 nm; 340 nm; 365 nm
Mix gently until completely dissolved. Blank: Water
Cuvette: Thermostatized 1 cm light-path.
The formula indicated is used to obtain the factor to calculate the U/L
Tris-HCl buffer pH 7.8 80 mM Vt x 106
L-aspartic acid 240 mM ΔAbs/min x = U/L
α-ketoglutaric acid 12 mM Є x l x Vs
NADH 0.18 mM
MDH ≥ 500 U/L Vt: Total reaction volume
LDH ≥ 1,200 U/L Vs: Sample volume
Stabilizers and preservatives l: Cuvette light path
Є: Extinction coefficient of NADH:
STORAGE AND STABILITY 365 nm: 3.40 x 103
The components of the kit, stored at 2-8ºC, will remain stable until the expiration date stated on the label. 340 nm: 6.31 x 103
The rehydrated reagent is stable for 4 weeks at 2-8ºC and for 1 week at room temperature (≤ 25º C). 334 nm: 6.17 x 103

To find the U/L value:
Presence of particles or turbidity in the reagent. Working reagent blank ≤ 1.0
U/L = ΔAbs/min. x Factor
334 nm: ΔAbs/min x 1780=U/L
Spectrophotometer; automated analyzer or photometer with thermostated cuvette, 1 cm light path
SAMPLE
Serum or plasma with EDTA or heparin. Samples free from hemolysis should be used. Sera kept at 2-8ºC REFERENCE VALUES
lose ca. 10% of their activity after 3 days. Temperature Men Women
25ºC ≤ 18 U/L ≤ 15 U/L
CAUTION 30ºC ≤ 25 U/L ≤ 21 U/L
The reagent contains sodium azide at 0.09%. Handle with care. 37ºC ≤ 37 U/L ≤ 31 U/L
The disposal of the residues has to be made according to local regulations. The stated values are for guidance. Each particular laboratory should establish its own normal range, using
its own instrumentation, blood collection methods and test procedures
QUALITY CONTROL
Control serum, Seriscann Normal (Ref. 99 41 48) and Seriscann Anormal (Ref. 99 46 85) should be PERFORMANCE CHARACTERISTICS
included in each test series. Each particular laboratory should establish its own control program. The performance characteristics depend on the method used. It is recommended to calculate these data
for each particular test protocol. These results have been obtained using a manual method at 37ºC and
AUTOANALYZERS 340 nm.
Linearity: Up to 430 U/L. For higher values, it is recommended to dilute the sample 1/10 in saline (NaCl
0.9%) and assay once again. Multiply the final result by 10.
Accuracy: 97.5 %
Trueness: Results obtained with this reagent did not show systematic differences when compared with the
reference reagent.
INTERFERENCES
Highly haemolysed sera will interfere with the reaction.
When assaying high activity samples, a very low initial absorbance can be found which is mainly due to
the rapid conversion of the NADH at the early stage of the reaction. In such a case, dilute the sample with
saline (NaCl 0.9%) 1/10 and assay it once again.Multiply the final result by 10.
REFERENCES
Bergmeyer, H.U., Scheibe, P., Wahlefeld, A.W. (1978). Clin. Chem., 24, 58 - 73.
IFCC, (2002), Clin. Chem. Lab. Met., 40, 631-634.
Bergmeyer, H.U., et. al (1986). J. Clin. Chem. Clin. Biochem., 24,497.
Isherwood, D., (1979), Med. Lab. Sci., 36, 211-235
ISO 9001 / ISO 13485

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Enzymes
GOT / AST U.V. LIQUID
IFCC METHOD
For “in vitro” determination of Aspartate aminotransferase GOT/AST in serum or plasma
PRINCIPLE
The glutamic-oxalacetic transaminase (GOT), actually AST, catalyzes the reaction between L-aspartic acid and α-ketoglutaric acid. The oxalacetic acid formed is reduced by NADH with the aid of auxiliary enzyme MDH.
The change of NADH to NAD+ produces a change of Abs. The reaction media contains, also, LDH to remove endogenous pyruvate to avoid possible interferences.
In optimum reaction conditions the ΔAbs/min is directly related to GOT concentration in the sample.
GOT
α-ketoglutaric acid + L-aspartic acid → L-glutamic acid + oxalacetic acid
MDH
oxalacetic acid + NADH + H
+ → L-malic acid + NAD+

Increases in GOT activity were observed in cases of liver damage: hepatitis of various types, necrosis or The described method is the one proposed by the International Federation of Clinical Chemistry (IFCC).
damage in hepatocytes, cholestatic icteria.
Bring reagents and the analyzer to working temperature.
High levels are also seen in diseases affecting the heart muscle. In alcoholic hepatitis and acute myocardial
infarction, the increase of GOT activity is greater than the activity of GPT. Monoreagent technique 25/30ºC 37ºC
Single test result can not be used to make a clinical diagnosis. It should integrate clinical and laboratory Working reagent 1.0 mL 1.0 mL
data.
Sample 0.2 mL 0.1 mL
REAGENTS
Kit 1 x 50 mL. (Ref. 99 80 03). Contents: Bireagent technique 25/30ºC 37ºC
A. 1 x 40 mL Enzymes solution Ref. 99 61 07 Enzymes sol.(A) 1.0 mL 1.0 mL
Sample 0.2 mL 0.1 mL
Kit 1 x 250 ml. (Ref. 99 95 00). Contents:
A. 2 x 100 mL Enzymes solution Ref. 99 95 20 Mix, incubate for approx. 1 min
Substrate (B) 0.25 mL 0.25 mL
A. 3 x 250 mL Enzymes solution Ref. 99 04 02 Mix and start the stopwatch. Read the absorbance after 1, 2 and 3 min
B. 1 x 190 mL Liquid substrate Ref. 99 04 11 Determine the mean of Abs/min of different lectures.

Reagents A and B are ready-to-use. If a monoreagent procedure is preferred, then the reagents must be Wavelength: 334 nm; 340 nm; 365 nm
mixed in the ratio: 4 parts of A (enzymes solution) + 1 part of B (liquid substrate). Blank: Water.
Cuvette: Thermostated, 1 cm light-path
Tris-HCl buffer pH 7.8 80 mM The formula indicated is used to obtain the factor to calculate the U/L
L-aspartic acid 240 mM Vt x 106
α-ketoglutaric acid 12 mM ΔAbs/min x = U/L
NADH 0.18 mM Є x l x Vs
MDH ≥ 600 U/L
LDH ≥ 800 U/L Vt: Total reaction volume
Stabilizers and preservatives Vs: Sample volume
STORAGE AND STABILITY Є: Extinction coefficient of NADH:
The components of the kit, stored at 2-8ºC, will remain stable until the expiration date stated on the label. 365 nm: 3.40 x 103
The Monoreagent is stable for 4 weeks at 2-8ºC and for 1 week at room temperature (≤ 25ºC), when 340 nm: 6.31 x 103
protected from the sunlight. 334 nm: 6.17 x 103
Monoreagent procedure
25/30ºC 37ºC
Presence of particles or turbidity in the reagent. Working reagent blank ≤ 1.0 334 nm ΔAbs/min x 970=U/L ΔAbs/min x 1780=U/L
340 nm ΔAbs/min x 950=U/L ΔAbs/min x 1745=U/L
ADDITIONAL EQUIPMENT 365 nm ΔAbs/min x 1765=U/L ΔAbs/min x 3235=U/L
Spectrophotometer or photometer thermostatable at 37ºC. Cuvette: 1 cm light-path. Bireagent procedure
25/30ºC 37ºC
SAMPLE 334 nm ΔAbs/min x 1175=U/L ΔAbs/min x 2185=U/L
Serum or plasma with EDTA or heparin. Samples free from hemolysis should be used. Sera kept at 2-8ºC 340 nm ΔAbs/min x 1150=U/L ΔAbs/min x 2140=U/L
looses ca. 10% of its activity after 3 days. 365 nm ΔAbs/min x 2130=U/L ΔAbs/min x 3970=U/L
CAUTION
REFERENCE VALUES
Temperature Men Women
25 ºC ≤ 18 U/L ≤ 15 U/L
The calibrator must be considered as a human sample, and, thus, potentially infectious. Use adequate
30 ºC ≤ 25 U/L ≤ 21 U/L
protection.
37 ºC ≤ 37 U/L ≤ 31 U/L
The stated values are for guidance. Each particular laboratory should establish its own normal range, using
its own instrumentation, blood collection methods and test procedures
QUALITY CONTROL
Control serum, Seriscann Normal (Ref. 99 41 48) and Seriscann Anormal (Ref. 99 46 85) should be
included in each test series. Each particular laboratory should establish its own control program.
The performance characteristics depend on the method used. It is recommended to calculate these data
340nm.
AUTOANALYZERS
Adaptations to different autoanalyzers are available on request. Sensitivity, as detection limit: 2 UI/ml
Accuracy: 97.9 %
reference reagent.
INTERFERENCES
Highly haemolysed sera will interfere with the reaction. When assaying high activity samples, a very low
initial absorbance can be found which is mainly due to the rapid conversion of the NADH at the early stage
of the reaction. In such a case, dilute the sample with saline (NaCl 0.9%) 1/10 and assay it once again.
Multiply the final result by 10.
REFERENCES
Bergmeyer, H.U., et. al (1986). J. Clin. Chem. Clin. Biochem., 24,497.
Isherwood, D., (1979), Med. Lab. Sci., 36, 211-235.
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GPT / ALT U.V.
IFCC METHOD
For “in vitro” determination of Transaminase GPT/ALT in serum or plasma
PRINCIPLE
The glutamic-pyruvate transaminase (GPT), actually ALT, catalyzes the reaction between L-alanine and α-ketoglutaric acid. The pyruvic acid formed is reduced by NADH with the aid of the auxiliary
enzyme LDH. The change of NADH to NAD+ produces a change in the Abs.
In optimum reaction conditions the ΔAbs/min is directly related to GPT concentration in the sample.
GPT
α-ketoglutaric acid + L-alanine L-glutamic acid + pyruvic acid
LDH
pyruvic acid + NADH + H +
L-lactic acid + NAD+

High values of GPT activity are found in hepatocyte necrosis, cirrhosis or obstructive jaundice. The previously described method is the one proposed by the International Federation of Clinical
The increase of GPT enzymatic activity is more specific for liver damage than the ratio GOT / GPT. Chemistry (IFCC).
In viral hepatitis GPT increase is always greater than that of GOT. High levels of enzyme activity
were detected in cases of myocarditis, myocardial infarction or hemolytic diseases. Bring working reagent and the instrument to the working temperature (25ºC,30ºC or 37ºC).
Single test result can not be used to make a clinical diagnosis. It should integrate clinical and Technique Macro (mL) Semimicro (μL)
laboratory data. Working reagent 1.0 500
REAGENTS Incubate 2-3 min. at the desired assay temperature (25ºC, 30ºC or 37º C).
Kit 20 x 3 mL. (Ref. 99 84 37). Contents: Sample 0.1 50
A. 20 vials of freeze-dried substrate. Ref. 99 57 46
B. 1 x 60 mL. Buffer solution. Ref. 99 00 76 Mix and start the stopwatch.
Kit 12 x 16 mL. (Ref. 99 36 15). Contents: Transfer to the measuring cuvette. Read the absorbance after 1,2,3 and 4
A. 12 vials of freeze-dried substrate. Ref. 99 09 77 min.
B. 2 x 100 mL. Buffer solution. Ref. 99 60 19 Determine the average value of ΔAbs / min.

Rehydrate one vial of freeze-dried substrate with the volume of buffer solution stated on the label. Wavelength: 334 nm; 340 nm; 365 nm.
Mix gently until completely dissolved. Blank: Water.
Cuvette: Thermostatized 1 cm light-path.
The formula indicated below is the one used to obtain the factor to calculate the U/L
Tris-HCl buffer pH 7.8 100 mM Vt x 106
L-alanine 500 mM ΔAbs/min x = U/L
α-ketoglutaric acid 15 mM Є x l x Vs
NADH 0.18 mM Vt: Total reaction volume
NaHCO3 10 mM Vs: Sample volume
LDH ≥ 1428 U/L l: Cuvette light path
Stabilizers and preservatives Є: Extinction coefficient of NADH:
365 nm: 3.40 x 103
STORAGE AND STABILITY 340 nm: 6.31 x 103
The components of the kit, stored at 2-8ºC, will remain stable until the expiration date stated on 334 nm: 6.17 x 103
the label. The rehydrated reagent is stable for 4 weeks at 2-8ºC and for 1 week at room temperature Determine the average value of ΔAbs / min and then:
(< 25ºC), when protected from the sunlight.
U/L = ΔAbs/min. x Factor
Factors
25/30ºC 37ºC
Presence of particles or turbidity in the reagent. Working reagent blank ≤ 1.0.
334 nm ΔAbs/min x 970=U/L ΔAbs/min x1780=U/L
340 nm ΔAbs/min x 950=U/L ΔAbs/min x1745=U/L
365 nm ΔAbs/min x1765=U/L ΔAbs/min x3235=U/Lº
Spectrophotometer; automated analyzer or photometer with thermostated cuvette.
REFERENCE VALUES
Temperature Men Women
SAMPLE
25 ºC ≤22 U/L ≤17 U/L
Serum or plasma with EDTA or heparin. Samples free from hemolysis should be used. Sera kept
30 ºC ≤29 U/L ≤22 U/L
in the refrigerator at 2-8ºC loses 10% of activity after 3 days.
37 ºC ≤45 U/L ≤34 U/L
CAUTION Each particular laboratory should establish its own normal range, obtained from samples of a
The reagent contains Sodium azide at 0.09%. Handle with care. representative population, using its own instrumentation, blood collection methods and assaying
The safety statements are on the label. It is advisable to look at the SDS before using the reagent. procedures.
QUALITY CONTROL The performance characteristics depend on the method used. It is recommended to calculate
Control serum, Seriscann Normal (Ref. 99 41 48) and Seriscann Anormal (Ref. 99 46 85) should these data for each particular test protocol. These results have been obtained using a manual
be included in each test series. Each particular laboratory should establish its own control program. method at 37ºC and 340nm.
Sensitivity, as detection limit: 4 IU/L
AUTOANALYZERS Linearity: Up to 350 U/L. For higher values, it is recommended to dilute the sample 1/10 in saline
Adaptations to different autoanalyzers are available on request. (NaCl 0.9%) and assay once again. Multiply the final result by 10.
Accuracy: 98.3 %.
with the reference reagent.
INTERFERENCES
Hemolyzed samples will give false results. When assaying high activity samples, a very low initial
absorbance can be found which is mainly due to the rapid conversion of the NADH at the early
stage of the reaction. In such a case, dilute the sample 1/10 with saline (NaCl 0.9%) and assay it
once again. Multiply the final result by 10.
REFERENCES
International Federation of Clinical Chemistry; Recommendations of IFCC Methods for the
measurement of catalytic concentrations of Enzymes, (1978) Clin. Chem. 24, 58-73.
Bergmeyer, H.U., Scheibe, P., Wahlefeld, A.W. (1978) Clin. Chem. 24, 58-73.
Isherwood,D. (1979 ) Med. Lab. Sci., 36, 211-235.
Tietz, NW., Textbook of Clinical Chemistry 5th Edition, W.B. Saunders, Philadelphia (2012)
ISO 9001 / ISO 13485

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Enzymes
GPT / ALT U.V. LIQUID
IFCC METHOD
For “in vitro” determination of Alanine aminotransferase GPT/ALT in serum or plasma
PRINCIPLE
The glutamic-pyruvate transaminase (GPT), actually ALT, catalyzes the reaction between L-alanine and α-ketoglutaric acid. The pyruvic acid formed is reduced by NADH with the aid of auxiliary enzyme LDH. The change
of NADH to NAD+ produces a change of Abs.
In optimum reaction conditions the ΔAbs/min is directly related to GPT concentration in the sample.
GPT
α-ketoglutaric acid + L-alanine → L-glutamic acid + pyruvic acid
LDH
+ →
pyruvic acid + NADH + H L-lactic acid + NAD+

High values of GPT activity are found in hepatocyte necrosis, cirrhosis or obstructive jaundice. The above described method is the one proposed by the International Federation of Clinical Chemistry
The increase of GPT enzymatic activity is more specific for liver damage than ratio GOT/GPT. (IFCC). Bring reagents and the analyzer to working temperature.
In viral hepatitis GPT increase is always greater than that of GOT. Also, higher values found in myocarditis,
myocardial infarction or hemolytic diseases. Monoreagent technique 25ºC/30ºC (mL) 37ºC (mL)
data. Working reagent 1.0 1.0
Sample 0.2 0.1

REAGENTS
Kit 1 x 50 mL. (Ref. 99 15 16). Contents: Bireagent technique 25ºC/30ºC (mL) 37ºC (mL)
A. 1 x 40 mL Enzymes solution Ref. 99 51 78
B. 1 x 10 mL Liquid substrate Ref. 99 13 45 Enzymes sol.(A) 1.0 1.0
Kit 1 x 250 mL. (Ref. 99 92 00). Contents: Sample 0.2 0.1

A. 2 x 100 mL Enzymes solution Ref. 99 92 20 Mix, incubate for approx. 1 minute
Substrate (B) 0.25 0.25
A. 3 x 250 mL Enzymes solution Ref. 99 04 26 Mix, read the absorbance after 1 min. and start the stopwatch. Read again the absorbance after 1, 2 and
B. 1 x 190 mL Liquid substrate Ref. 99 04 28 3 min.
Reagents A and B are ready-to-use. If a monoreagent procedure is preferred, then the reagents must be Reading
mixed in the ratio: 4 parts of A (Enzymes solution) + 1 part of B (Liquid substrate). Wavelength: 334 nm; 340 nm; 365 nm
Blank: Water
WORKING REAGENT COMPOSITION Cuvette: Thermostatized, 1 cm light-path
Tris-HCl buffer pH 7.8 90 mM CALCULATIONS
L-alanine 500 mM The formula indicated is used to obtain the factor to calculate the U/L
α-ketoglutaric acid 17 mM Vt x 106
NADH 0.18 mM ΔAbs/min x = U/L
LDH ≥ 800 U/L Є x l x Vs
Stabilizers and preservatives Vt: Total reaction volume
Vs: Sample volume
STORAGE AND STABILITY l: Cuvette light path
The components of the kit, stored at 2-8ºC, will remain stable until the expiration date stated on the label. Є: Extinction coefficient of NADH:
The monoreagent is stable for 4 weeks at 2-8ºC and for 1 week at room temperature (≤25ºC), when 365 nm: 3.40 x 103
protected from the sunlight. 340 nm: 6.31 x 103
334 nm: 6.17 x 103
Signs of reagent deterioration: Monoreagent procedure
Presence of particles or turbidity in the reagent. Working reagent blank ≤ 1.0 25/30º C 37º C
334 nm ΔAbs/min.x 970=U/L ΔAbs/min x 1780=U/L
Bireagent procedure
25/30º C 37º C
CAUTION
The calibrator must be considered as a human sample, and, thus, potentially infectious. Use adequate
protection. REFERENCE VALUES
The disposal of the residues has to be made according to local regulations. Temperature Men Women
25 ºC ≤ 22 U/L ≤ 17 U/L
SAMPLE
30 ºC ≤ 29 U/L ≤ 22 U/L
Serum or plasma with EDTA or heparin. Samples free from hemolysis should be used. Sera kept at 2-8ºC
37 ºC ≤ 45 U/L ≤ 34 U/L
loses ca. 10% of its activity after 3 days.
Each particular laboratory should establish its own normal range, obtained from samples of a representative
population, using its own instrumentation, blood collection methods and assaying procedures.
QUALITY CONTROL
Control serum, Seriscann Normal (Ref. 99 41 48) and Seriscann Anormal (Ref. 99 46 85) should be PERFORMANCE CHARACTERISTICS
included in each test series. Each particular laboratory should establish its own control program. The performance characteristics depend on the method used. It is recommended to calculate these data
AUTOANALYZERS 340nm.
Sensitivity, as detection limit: 5 UI/mL.
Accuracy: 98.1 %
reference reagent.
INTERFERENCES
Highly haemolysed sera will interfere with the reaction. When assaying high activity samples, a very low
initial absorbance can be found which is mainly due to the rapid conversion of the NADH at the early stage
of the reaction. In such a case, dilute the sample with saline (NaCl 0.9%) 1/10 and assay it once again.
REFERENCES
Bergmeyer, H.U.,et al. (1986). J. Clin. Chem. Clin. Biochem., 24, 497.
Isherwood, D., (1979), Med. Lab. Sci., 36, 211-235.
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LDH LIQUID
SFBC METHOD
For “in vitro” determination of lactate dehydrogenase in serum or plasma
PRINCIPLE
The lactate dehydrogenase catalyzes the reduction of pyruvate with the aid of NADH as cofactor, which is oxidized to NAD+, giving an absorbance change.
In optimum reaction conditions the ΔAbs/min is directly related to LDH concentration in the sample.
LDH
pyruvate + NADH + H+ → L - lactate + NAD+

Increased levels of LDH in serum are found in patients suffering from hepatitis, infectious The method described is the one proposed by the French Society of Clinical Biology.
mononucleosis, malignant tumours, pancreatitis, cirrhosis, or myocardial infarction, due to release Bring the working reagent and the instrument to the working temperature. (30º / 37ºC)
of enzyme from the tissue. Values also increase in cases of muscular dystrophy or anemias.
Values below the usual are not clinically significant.
Monoreagent technique 30º / 37ºC
Single test result could not be used to make a clinical diagnosis. It should integrate clinical and
Working Reagent 1.0 mL
laboratory data.
Sample 0.02 mL
REAGENTS
Kit 1 x 50 mL. Ref. 99 18 18. Contents: Bireagent technique 30º / 37ºC
Buffer solution (A) 1.0 mL
B. 1 x 10 mL Buffered NADH Ref. 99 32 19
Sample 0.02 mL
Kit 1 x 125 mL. Ref. 99 00 35. Contents:
A. 1 x 100 mL Buffer solution Ref. 99 00 40 Mix, incubate for approx. 1 min
B. 1 x 25 mL Buffered NADH Ref. 99 00 45 NADH (B) 0.25 mL
WORKING REAGENT PREPARATION Mix, read the absorbance after 1 min and start the stopwatch.
Reagents A and B are ready-to-use. If a monoreagent procedure is preferred, then the reagents Read again the absorbance after 1, 2 and 3 min.
must be mixed in the ratio: 4 parts of A (buffer solution) + 1 part of B (buffered NADH).
Determine the average value of ΔAbs / min
Tris buffer pH 7.2 100 mM Reading
sodium pyruvate 1.6 mM Wavelength: 334 nm; 340 nm; 365 nm
NaCl 200 mM Blank: Water
NADH 0.20 mM Cuvette: Thermostatized; 1cm light-path
CALCULATIONS
STORAGE AND STABILITY The formula indicated is used to obtain the factor to calculate the U/L
ΔAbs/min x Vt x 10 = U/L
6
the label. The monoreagent is stable for 9 weeks at 2-8ºC and for 3 weeks at room temperature (≤
25ºC), when protected from the sunlight. Є x l x Vs
Where:
Vt: Total volume of reaction mixture;
Vs: Sample volume
Presence of particles or turbidity in the reagent. Working reagent blank ≤ 1.0
ADDITIONAL EQUIPMENT Є: Extinction coefficient of NADH:
General laboratory equipment. 365 nm: 3.40 x 103
Spectrophotometer or photometer thermostatable at 37ºC. Cuvette: 1 cm light-path. 340 nm: 6.31 x 103
334 nm: 6.17 x 103
Determine the average value of ΔAbs / min.
SAMPLE
Serum, heparinized plasma. Samples free from hemolysis should be used. The enzyme in serum U/L = ΔAbs/min x Factor
is stable for 2 days at 2-8ºC. Frozen samples are rapidly inactivated. Factors
Monoreagent Bireagent
CAUTION 334 nm 8252 10275
The reagents contain sodium azide at 0.09%. Handle with care. 340 nm 8095 10080
The safety statements are on the label. It is advisable to look at the SDS before using the reagent. 365 nm 15000 18675
adequate protection. REFERENCE VALUES
The disposal of the residues has to be made according to local regulations. Adults (37ºC) 200 - 400 U/L
The stated values are for guidance. Each particular laboratory should establish its own normal
range, using its own instrumentation, blood collection methods and test procedures
QUALITY CONTROL
Control serum, Seriscann Normal (Ref. 99 41 48) and Seriscann Anormal (Ref. 99 46 85) should PERFORMANCE CHARACTERISTICS
be included in each test series. Each particular laboratory should establish its own control program. The performance characteristics depend on the method used. It is recommended to calculate
these data for each particular test protocol. These results have been obtained using a manual
method at 37ºC and 340 nm.
AUTOANALYZERS
Adaptations to different autoanalyzers are available on request. Sensitivity, as detection limit: 10 U/L
Accuracy: 98.8 %
INTERFERENCES
It is recommended to use heparin as anticoagulant. Others, like citrate, oxalate, or fluoride may
interfere with the test. Hemolized samples will give false results.
When assaying high activity samples, a very low initial absorbance can be found, which is mainly
due to the rapid consumption of the NADH at the early stage of the reaction. In such a case, dilute
the sample with saline (NaCl 0.9 %) and assay it once again.
REFERENCES
Gay, R.J., McComb, R.B., Bowers, G.N. (1968) Clin. Chem., 14, 740 - 753.
Comission Enzymologie de la Societé Française de Biologie Clinique. (1982) Ann. Biol. Clin., 40,
123 - 128.
Methods of Enzymatic Analysis. (1983) 3rd edition, VIII, 118 – 126, Editado por H.U.
Bergmeyer Verlag Chemie.
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Enzymes
LIPASE
COLORIMETRIC METHOD
For the “in vitro” determination of lipase in serum or plasma
PRINCIPLE PROCEDURE
At alkaline pH the lipase substrate 1,2-O-dilauryl-rac-glycero-3- glutaric acid-(6- Bring reagents and the analyzer to 37ºC.
methylresorufin)-esther is cleaved by the catalytic action of pancreatic lipase to form
1,2-O-dilauryl-rac-glycerol and unstable compound glutaric acid-(6-methylresorufin)-esther.
BL SA ST
In that alkaline solution, this compound degrades to glutaric acid and methyl-resorufin. The Technique mL mL mL
color intensity of the red dye formed is directly proportional to the lipase activity. This activity
can be measured at 578nm. Water 0.01 --- ---
DIAGNOSTIC USE Sample --- 0.01 ---

The lipase is increased in pancreatitis from any source, and these values remain high Standard --- --- 0.01
longer than amylase values.
In chronic disorders of the pancreas the Lipase value is high and the same applies in cases Reagent A 1.00 1.00 1.00
of primary biliary cirrhosis, chronic renal failure and hemodialysis patients.
Single test result could not be used to make a clinical diagnosis. It should integrate clinical Mix and incubate 5min, at 37ºC.
and laboratory data. Then add Reagent B
Reagent B 0.60 0.60 0.60
REAGENTS
Kit 1 x 80 mL. (Ref. 99 11 15). Contains: Mix well. After 60s read (Abs1). After 90s more, read again (Abs2)
B. 1 x 30 mL Substrate Ref. 99 12 15 Reading
C. 1 x 1mL Freeze-dried Standard Ref. 99 13 07 Wavelength: 578nm
The concentration is stated on the label of the vial. Blank: deionised water
Cuvette thermostatized: 1cm light-path
Reagents are ready to use. CALCULATION
Rehydrate the standard with 1 mL of deionised water. Determine ΔAbs for each sample and the standard:
ΔAbs = Abs2 – Abs1
REAGENT COMPOSITION
The concentrations in the reagents solutions are: ΔAbs SA - ΔAbs BL
A. Buffer solution x Conc. ST (U/L) = U/L
ΔAbs ST - ΔAbs BL
Bicin buffer pH 8.0 50 mM
Colipase ≥ 1 mg/L
Results as μkat/L
sodium deoxycholate 1.8 mM
U/L x 0.0167 = μkat/L
calcium chloride 12 mM
REFERENCE VALUES
B. Substrate Serum, plasma: < 38 U/L.
Tartrate buffer pH 4.0 12 mM Each particular laboratory should establish its own normal range, obtained from samples
1,2-O-dilauryl-rac-glycero-3-glutaric acid- of a representative population, using its own instrumentation, blood collection methods and
-(6-methylresorufin) - esther 0.27 mM assaying procedures.
Taurodeoxy-cholate 9.0 mM
Surfactants and preservatives
The performance characteristics depend on the method used. It is recommended to
When stored at 2º-8ºC, the components of the kit will remain stable until the expiration date
using a manual method.
The standard, once rehydrated, is stable for 15 days at 2-8ºC.
Sensitivity, as detection limit: 6.9 U/L
Signs of reagent deterioration: Linearity: Up to 300 U/L. For higher values, it is recommended to dilute the sample 1/5 in
Presence of particles or turbidity in the reagent. Working reagent blank ≥0.500. saline (NaCl 0.9%) and assay once again. Multiply the final result by 5.
Accuracy: 104%
ADDITIONAL EQUIPMENT Within-run Precision as Coefficient of Variation: 3.01%
General laboratory equipment. Run-to-run Precision as Coefficient of Variation: 3.65%
Spectrophotometer or photometer thermostatable at 37ºC. Cuvette: 1 cm light-path. Trueness: Results obtained with this reagent did not show systematic differences when
SAMPLE
Serum or heparinized plasma. Use samples free from hemolysis. Do not use plasma with INTERFERENCES
EDTA. Hemolysis interferes with the assay.
The lipase activity in serum is stable for 4 days at 2-8ºC and for 24h at room temperature Concentrations of hemoglobin higher than 125 mg/dL , of bilirubin higher than 20 mg/dL and
(≤ 25ºC). triglycerides higher than 625 mg/dL can interfere with the assay.
CAUTION To perform this test, the use of disposable labware is highly recommended.
The safety statements are on the label. It is advisable to look at the SDS before using QUALITY CONTROL
the reagent. The calibrator must be considered as a human sample, and, thus, potentially Control serum, Seriscann Normal (Ref. 99 41 48) and Seriscann Anormal (Ref. 99 46 85)
infectious. Use adequate protection. should be included in each test series. Each particular laboratory should establish its own
The disposal of the residues has to be made according to local regulations. control program.
REFERENCES
Rick, W., (1969), Zeittschrift Clin. Chem. Clin. Biochem., 7, 530 – 536.
Ziegenhorn, J. et al., (1979), Clin. Chem., 25, 1067.
Neumann, U., Kaspar, P., Ziegenhorn, J., (1984) Meth. Enz. Anal (3rd edition), 26 – 34.
Lott, J. A. et al., (1986). Clin. Chem., 32, 1290 – 1302.
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ALBUMIN
BROMOCRESOL GREEN METHOD
For “in vitro” determination of albumin in serum or plasma
PRINCIPLE PROCEDURE
At acidic pH, albumin specifically combined with bromocresol green (BCG) to form Bring the working reagent and the analyzer to room temperature.
a colored complex that is determined photometrically. The color produced in the
reaction is proportional to the concentration of albumin in the sample under optimal
Technique BL SA ST
assay conditions.
mL mL mL
DIAGNOSTIC USE Sample -- 0.01 --
Albumin is the most abundant protein in human plasma. It’s principal funtions are
source of endogenous amino acids, non-specific transport vehicle for nonpolar Standard -- -- 0.01
compounds and specially it contributes towards maintaining the colloid oncotic
pressure. Albumin values decrease in severe infections, rheumatic fever, liver Reagent 2.50 2.50 2.50
diseases (cirrhosis or chronic active hepatitis), dermatitis or severe burns, ascites,
bad absorption processes or intestinal obstruction and in cases of renal diseases Mix well and let stand for 5 minutes at room temperature (20-25ºC). Read
(nephrotic syndrome, lupus, diabetes mellitus, glomerulonephritis). immediately.
The albumin levels are high in dehydration processes.
Single test result could not be used to make a clinical diagnosis. It should integrate Reading
clinical and laboratory data. Wavelength: 630 nm
Blank: BL contents
Colour stability: See INTERFERENCE
REAGENTS
CALCULATION
SA Abs.
A. 2 x 250 mL Bromcresol green Ref. 99 01 62 x 5 = g albumin/dL
B. 1 x 5 mL Standard Ref. 99 02 46 ST Abs.
Kit 3 x 100 mL. (Ref. 99 72 58). Contents: Where:

A. 3 x 100 mL Bromcresol green Ref. 99 94 82 SA Abs: Sample Absorption
B. 1 x 5 mL Standard Ref. 99 02 46 ST Abs: Standard Absorption
SI Units
WORKING REAGENT PREPARATION (g/dL) x 10 = g/L
The components of the kit are ready to use.
REFERENCE VALUES
REAGENT COMPOSITION 0-4 days: 2.8-4.4 g/dL
Concentration in the reagent solution are: 4 days-14 years: 3.8-5.4 g/dL
Succinate buffer pH 4.2 50 mM Adult (20-60 years): 3.5 - 5.2 g/dL
Bromcresol green 0.75 g/L >60 years: 3.2-4.6 g/dL
Surfactants
Preservatives and stabilizers The stated values are for guidance. Each particular laboratory should establish its
own normal range.
Standard: Aqueous solution, equivalent to 5 g/dL of Albumin (50 g/L).
When stored at 2-8ºC, the reagent will remain stable until the expiration date stated PERFORMANCE CHARACTERISTICS
on the label. The performance characteristics depend on the method used. It is recommended
to calculate these data for each particular test protocol. These results have been
obtained using a manual method.
Presence of particles or turbidity in the reagent. Working reagent blank ≥0.500.
ADDITIONAL EQUIPMENT Sensitivity, as detection limit: 0.05 g/dL
General laboratory equipment. Linearity: up to 6 g of Albumin/dL
Spectrophotometer or photometer thermostatable at 37ºC. Cuvette: 1 cm light-path. Accuracy: 99.1%
CAUTION Repetitivity, as Variation Coefficient: 0.77%
The reagents contain sodium azide at 0.09%. Handle with care. Reproducibility, as Variation Coefficient: 0.98%
The safety statements are on the label. It is advisable to look at the SDS before Trueness: Results obtained with this reagent did not show systematic differences
using the reagent. The calibrator must be considered as a human sample, and, thus, when compared with reference reagent.
potentially infectious. Use adequate protection.
The disposal of the residues has to be made according to local regulations. Details of the performance studies are available on request.
SAMPLE
Serum or plasma. Sample may be stored for 2 weeks at 2-8ºC, or 4 months at -20ºC. INTERFERENCE
While the colour change upon binding of albumin to bromcresol green is almost
instantaneous, the colour change from binding with other protein fractions occurs
continuously with time, so it is recommended not to delay the absorbance reading.
QUALITY CONTROL
Control serum, Seriscann Normal (Ref. 99 41 48) and Seriscann Anormal (Ref. 99 46 AUTOANALYZERS
85) should be included in each test series. Each particular laboratory should establish Adaptations to different autoanalyzers are available on request.
its own control program.
REFERENCES
Doumas, B.T., Watson, W.A., Biggs, H.G. (1971). Clin. Chim. Acta, 31, 87-96.
Tietz, NW., Textbook of Clinical Chemistry 5th Edition, W.B. Saunders, Philadelphia
(2012).
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BILIRUBIN
JENDRASSIK - GROF METHOD
For “in vitro” determination of direct and total bilirubin in serum or plasma
PRINCIPLE PROCEDURE
Total bilirubin is determined by reaction with diazotized sulfanilic acid, in the presence of TOTAL BILIRUBIN
caffeine, with the final production of an azopigment. The same reaction, but in the absence
of caffeine, is used to measure direct bilirubin. Technique BL mL SA mL
Substrates
Sulphanilic acid 0.2 0.2
DIAGNOSTIC USE
Bilirubin is a metabolic hemoglobin product which is transported to the liver, where is Sodium nitrite -- 1 gota
conjugated with glucuronic acid and it is excreted through bile. This “conjugated” bilirubin is Caffeine 1.0 1.0
called direct bilirubin, while unconjugated bilirubin is called indirect bilirubin. The total serum
bilirubin is direct bilirubin plus indirect bilirubin. Sample 0.2 0.2
Direct bilirubin increases in biliary obstruction, cirrhosis, and hepatitis. High level of indirect Mix and let stand 10 min. at room temperature (20 - 25ºC).
or total bilirubin may indicate hemolytic diseases, physiological neonatal jaundice, Gilbert’s
disease or fructose intolerance. Tartrate 1.0 1.0
Single test result could not be used to make a clinical diagnosis. It should integrate clinical
and laboratory data. Mix and let stand for 5 min at room temperature (20 - 25ºC).
REAGENTS Reading
Kit 1 x 245 mL. (Ref. 99 20 93). Contents: Wavelength: 578 nm.
A. 1 x 40 mL Sulphanilic acid solution. Ref. 99 29 04 Blank: BL contents
B. 1 x 100 mL Caffeine solution. Ref. 99 23 82 Cuvette: 1 cm light-path.
C. 1 x 100 mL Tartrate solution. Ref. 99 29 46 Color stability: a minimum of 1 hour.
D. 1 x 5 mL Sodium nitrite solution. Ref. 99 25 25
DIRECT BILIRUBIN
WORKING REAGENT PREPARATION Technique BL SA
All reagents are ready to use.
Sulphanilic acid 0.2 mL 0.2 mL
REAGENTS COMPOSITION Sodium nitrite -- 1 drop
The concentrations in the reagents solution are:
A. Sulphanilic acid 30 mM Saline 2.0 mL 2.0 mL
HCl 0.17 M Sample 0.2 mL 0.2 mL
B. Caffeine 0.25 M Mix and let stand for 5 min at room temperature (20 - 25ºC).
Sodium benzoate 0.50 M Read after exactly 5 min.
C. Potassium tartrate 0.80 M Reading

NaOH 1.85 M Wavelength:546 nm.
Blank: BL contents
D. Sodium nitrite 30 mM Cuvette: 1 cm light-path.
STORAGE AND STABILITY CALCULATIONS

When kept at room temperature (≤ 25ºC), the components of the kit will remain stable until Abs. SA x 10.8 = mg total bilirubin / dL.
the expiration date stated on the label. Abs. SA x 14.4 = mg direct bilirubin / dL.
Signs of reagent deterioration: Where: Abs SA= Sample absorption

Presence of particles or turbidity in the reagent. Working reagent blank > 0,100.
SI Units
(mg/dL) x 17.1 = μmol/L.
NOTE: In case of small-volume samples, please use 50 µL, in place of the 0,2 mL as herein
indicated, but keeping the same reagent volumes, and multiply the results by the following
CAUTION factors:
The reagents contain sodium azide at 0.09%. Handle with care. F= 43.2 (total bilirubin)
The safety statements are on the label. It is advisable to look at the SDS before using F= 57.6 (direct bilirubin)
infectious. Use adequate protection. PERFORMANCE CHARACTERISTICS
The disposal of the residues has to be made according to local regulations. The performance characteristics depend on the method used. It is recommended to
calculate these data for each particular test protocol. The indicated results have been
SAMPLE obtained using a manual method.
Serum or plasma free from hemolysis. Serum bilirubin will decrease 50% in one hour if
stored at room temperature (≤ 25ºC) and at direct sunlight. Samples stored in the dark at Sensitivity, as detection limit: 0.1 mg/dL
2 - 8ºC will remain stable for up to 3 days. Linearity: Up to 25 mg/dL. Concentrations higher than 15 mg/dL may produce an
absorbance value (Abs > to 1.500) that can hardly be read in non-digital photometers. It is
QUALITY CONTROL recommended in such a case, to carry out a 1/10 dilution of the sample with saline, (NaCl
Control serum, Seriscann Normal (Ref. 99 41 48) and Seriscann Anormal (Ref. 99 46 85) 0,9%) and then to multiply the final result by 10.
should be included in each test series. Each particular laboratory should establish its own Accuracy: 96.3 %
control program. Repetitivity, as Variation Coefficient: 1.64%
Reproducibility, as Variation Coefficient: 1.98%
REFERENCE VALUES Trueness: Results obtained with this reagent did not show systematic differences when
Total bilirubin. Adults: up to 1.2 mg/dL compared with reference reagent.
Pediatric:
< 14 days 0.19 - 16.6 mg/dL Details of the performance studies are available on request.
< 15 days-1 year 0.05 - 0.68 mg/dL
1-3 years 0.05- 0.40 mg/dL INTERFERENCES
The stated values are for guidance. Ea Hemolized samples could give false results.
Direct bilirubin Adults: up to 0.4 mg/dl
REFERENCES
The stated values are for guidance. Each particular laboratory should establish its own Jendrassik, L., Grof, P.(1938). Biochem. Z., 297, 81-89.
normal range. Doumas, B. T., Perry, B. W., Sasse, E. A., Straumfjord Jr., J.V.(1973). Clin.Chem., 19, 984-
993.
Tiezt, NW., Textbook of Clinical Chemistry 5th Edition, W.B. Saunders, Philadelphia (2012).
ISO 9001 / ISO 13485

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DIRECT BILIRUBIN
JENDRASSIK – GROF METHOD
For “in vitro” determination of direct bilirubin in serum or plasma
PRINCIPLE PROCEDURE
Total bilirubin is determined by reaction with diazotized sulfanilic acid, in the
presence of caffeine, with the final production of an azopigment. The same
Technique BL S SA
reaction, but in the absence of caffeine, is used to measure direct bilirubin. mL mL
DIAGNOSTIC USE Saline 1.0 1.0

Bilirubin is a metabolic hemoglobin product which is transported to the liver, Diazo reagent -- 0.1
where there is conjugated with glucuronic acid and it is secreted through bile.
Saline 0.1 --
This conjugated bilirubin is the direct bilirubin, while unconjugated bilirubin is
called indirect bilirubin. The total serum bilirubin is direct bilirubin plus indirect Sample 0.1 0.1
bilirubin.
Direct bilirubin increases in biliary obstruction, cirrhosis and hepatitis. High Mix and let stand at room temperature (20 - 25ºC)
level of indirect or total bilirubin may indicate hemolytic diseases, physiological Read exactly at 5 minutes
neonatal jaundice, Gilbert’s disease or fructose intolerance.
Reading
Single test result could not be used to make a clinical diagnosis. It should Wavelength: 546 nm
integrate clinical and laboratory data. Blank: H2O
Cuvette: 1 cm light-path
REAGENTS
Kit 1 x 250 mL. (Ref. 99 02 08). Contents: CALCULATIONS
A. 1 x 50 mL Sulfanilic Acid. Ref. 99 90 11 Δ Abs = Abs SA - Abs BL S
B. 2 x 100 mL Saline Solution. Ref. 99 01 99
C. 1 x 2 mL Sodium Nitrite. Ref. 99 90 94 Where:
Abs SA: Sample Absorption
WORKING REAGENT PREPARATION Abs BLS: Sample blank Absorption
Preparation of the diazo reagent: Mix 1 part of solution C with 50 parts of solution A.
It is advisable to use single use material for working reagent preparation. Once prepared, it Δ Abs x 12.8 = mg direct bilirubin / dL
is introduced into the empty container supplied with the kit.
SI Units
REAGENTS COMPOSITION
The concentrations in the reagent solutions are: mg / dL x 17.1 = μmol / L
A. Sulfanilic acid 6 mM REFERENCE VALUES

HCl 0.17 M Adults: up to 0.4 mg/dl direct bilirubin
B NaCl 0,9% The stated values are for guidance. Each particular laboratory should establish
its own normal range, using its own instrumentation, blood collection methods
C. Sodium nitrite 0.4 M
and test procedures
The components of the kit, when stored at room temperature (≤ 25ºC), will remain stable PERFORMANCE CHARACTERISTICS
until the expiration date stated on the label. The performance characteristics depend on the method used. It is recommended
Diazo reagent is stable for 2 days at room temperature (≤ 25ºC). to calculate these data for each particular test protocol. These results have been
obtained using an automated analyzer.
Presence of particles or turbidity in the reagent. Working reagent blank >0.100 Sensitivity, as detection limit: 0.1 mg/dL
ADDITIONAL EQUIPMENT Reaction range: Up to 15 mg/dL. Concentrations higher than 15 mg/dL may
General laboratory equipment. produce an absorbance value (ΔAbs > to 1.500) that can hardly be read in non-
Spectrophotometer or photometer thermostatable at 37ºC. Cuvette: 1 cm light-path. digital photometers. It is recommended in such a case, to carry out a 1/10 dilution
of the sample with saline, (NaCl 0.9%) and then to multiply the final result by 10.
SAMPLE
Accuracy: 96.3%
Serum or plasma free from haemolysis.
Repetitivity, as CV%: 1.8%
Serum bilirubin will decrease 50% in 1 hour if kept at room temperature (≤ 25ºC),
Reproducibility, as CV%: 2.7%
and at direct sunlight. Samples kept in the dark at 2-8ºC will remain stables for
Trueness: Results obtained with this reagent did not show systematic differences
up to 3 days. Do not use frozen samples.
when compared with reference reagent.
Details of the comparison experiments are available on request.
CAUTION
INTERFERENCES
The safety statements are on the label. It is advisable to look at the SDS before
To avoid possible errors, it is very important, when assaying for direct bilirubin,
using the reagent. The calibrator must be considered as a human sample, and,
to read the corresponding absorbance exactly 5 min. after the reagents have
thus, potentially infectious. Use adequate protection.
been added.
Avoid hemolysed samples.
QUALITY CONTROL
Control serum, Seriscann Normal (Ref. 99 41 48) and Seriscann Anormal (Ref.
99 46 85) should be included in each test series. Each particular laboratory
should establish its own control program.
AUTOANALYZERS
REFERENCES
Jendrassik, L., Gróf, P. (1938). Biochem Z., 297, 81-89.
Doumas, B.T., Perry, B.W., Sasse,E.A., Straumjord Jr. J.V. (1973). Clin.Chem.,
19, 984-993.
ISO 9001 / ISO 13485

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TOTAL BILIRUBIN
JENDRASSIK – GROF METHOD
For “in vitro” determination of total bilirubin in serum or plasma
PRINCIPLE PROCEDURE
Total bilirubin is determined by reaction with diazotized sulphanilic acid, in the
presence of caffeine, with the final production of an azopigment. The same
Technique BL S (mL) SA (mL)
reaction, but in the absence of caffeine, is used to measure direct bilirubin.
Substrates
Reagent B (Caffeine) 1.0 1.0
DIAGNOSTIC USE
Diazo reagent -- 0.1
Bilirubin is a metabolic hemoglobin product which is transported to the liver,
where there is conjugated with glucuronic acid and it is secreted through bile. Saline 0.1 --
This conjugated bilirubin is called direct bilirubin, while unconjugated bilirubin is Sample 0.1 0.1
called indirect bilirubin. The total serum bilirubin is direct bilirubin plus indirect
bilirubin.
Mix and let stand at room tem (20 - 25ºC). Read exactly at 10 minutes.
Direct bilirubin increases in biliary obstruction, cirrhosis and hepatitis. High
level of indirect or total bilirubin may indicate hemolytic diseases, physiological
Reading
neonatal jaundice, Gilbert’s disease or fructose intolerance.
Wavelength: 546 nm
Single test result could not be used to make a clinical diagnosis. It should
Blank: H2O
integrate clinical and laboratory data.
Cuvette: 1 cm light-path
REAGENTS
Kit 1 x 250 mL. (Ref. 99 27 14). Contents: CALCULATIONS
A. 1 x 50 mL Sulphanilic acid. Ref. 99 90 11 Δ Abs = Abs SA - Abs BL S
B. 2 x 100 mL Caffeine. Ref. 99 23 91
C. 1 x 2 mL Sodium nitrite. Ref. 99 90 94 Where:
Abs BLS: Sample blank Absorption
Preparation of the diazo reagent: Mix 1 part of solution C with 50 parts of solution Δ Abs x 12.8 = mg total bilirubin / dL
A. It is advisable to use single use material for working reagent preparation.
Once prepared, it is introduced into the empty container supplied with the kit. SI Units
mg / dL x 17.1 = μmol / L
The concentrations in the reagent solutions are: REFERENCE VALUES
Adults: up to 1.2 mg/dL
A. Sulphanilic acid 6 mM Pediatric:
HCl 0.17 M < 14 days 0.19 - 16.6 mg/dL
< 15 days-1 year 0.05 - 0.68 mg/dL
B. Caffeine 0.25 M 1-3 years 0.05- 0.40 mg/dL
Sodium benzoate 0.50 M The stated values are for guidance. Each particular laboratory should establish
its own normal range.
C. Sodium nitrite 0.4 M PERFORMANCE CHARACTERISTICS
The performance characteristics depend on the method used. It is recommended
STORAGE AND STABILITY to calculate these data for each particular test protocol. These results have been
The components of the kit, when stored at room temperature (≤ 25ºC), will obtained using an automated analyzer.
remain stable until the expiration date stated on the label.
Diazo reagent is stable for 10 days at room temperature (≤ 25ºC). Sensitivity, as detection limit: 0.1 mg/dL
Linearity: Up to 25 mg/dL. Concentrations higher than 25 mg/dL may produce
Signs of reagent deterioration: an absorbance value (ΔAbs > to 1.500) that can hardly be read in non-digital
Presence of particles or turbidity in the reagent. Working reagent blank >0.100 photometers. It is recommended in such a case, to carry out a 1/10 dilution of
ADDITIONAL EQUIPMENT the sample with saline, (NaCl 0.9%) and then to multiply the final result by 10.
General laboratory equipment. Accuracy: 97.8%
Spectrophotometer or photometer thermostatable at 37ºC. Cuvette: 1 cm light- Repetitivity, as CV%: 1.3%
path. Reproducibility, as CV%: 1.7%
SAMPLE when compared with reference reagent.
Serum or plasma free from haemolysis. Serum bilirubin will decrease 50% in one Details of the comparison experiments are available on request.
hour if kept at room temperature (≤ 25ºC), and at direct sunlight. Samples kept
in the dark at 2-8ºC will remain stables for up to 3 days. INTERFERENCES
CAUTION Avoid hemolysed samples.
QUALITY CONTROL
The safety statements are on the label. It is advisable to look at the SDS before
using the reagent. The calibrator must be considered as a human sample, and,
thus, potentially infectious. Use adequate protection.
AUTOANALYZERS REFERENCES
Adaptations to different autoanalyzers are available on request. Jendrassik, L., Gróf, P. (1938). Biochem Z., 297, 81-89.
Doumas, B.T., Perry, B.W., Sasse,E.A., Straumjord Jr. J.V. (1973). Clin.Chem.,
19, 984-993.
Tietz, NW., Textbook of Clinical Chemistry 5th Edition, W.B. Saunders,
Philadelphia (2012).
ISO 9001 / ISO 13485

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DIRECT BILIRUBIN DPD
DPD METHOD
PRINCIPLE PROCEDURE
Conjugated bilirubin, in acid media, forms red color azobilirubin complex by
reaction with 3,5-dichlorophenyldiazonium salt. The color produced in the
Technique BL S SA
reaction is proportional to the concentration of bilirubin in the sample and it is mL mL
determined photometrically.
Buffer (A) 0.8 0.8
DIAGNOSTIC USE Saline 0.2 --
where there is conjugated with glucuronic acid and it is secreted through bile. Substrate (B) -- 0.2
This conjugated bilirubin is the direct bilirubin, while unconjugated bilirubin is Sample 0.08 0.08
bilirubin. Mix and let stand at reaction temperature (37ºC)
Direct bilirubin increases in biliary obstruction, cirrhosis and hepatitis. Read exactly at 5 minutes
Single test result could not be used to make a clinical diagnosis. It should Reading
integrate clinical and laboratory data. Wavelength: 546 nm
REAGENTS Blank: H2O
Kit 1 x 300 mL. (Ref. 99 05 58 ). Contents: Cuvette: 1 cm light-path
A. 1 x 240 mL Buffer. Ref. 99 07 07
B. 1 x 60 mL Substrate Ref. 99 07 35 CALCULATIONS
Δ Abs = Abs SA - Abs BL S
Reagents are ready to use. Where:
REAGENTS COMPOSITION Abs BLS: Sample blank Absorption
The concentrations in the reagent solutions are:
Δ Abs x 13.5 = mg direct bilirubin / dL
A. 3,5-DPD 0.25 mM
HCl 0.5 M SI Units
mg / dL x 17.1 = μmol / L
B Ac. Sulfámico 0.1 M
REFERENCE VALUES
Adults: up to 0.4 mg/dl direct bilirubin
The components of the kit, when stored at 2-8ºC, will remain stable until the
The stated values are for guidance. Each particular laboratory should establish
expiration date stated on the label.
and test procedures
Presence of particles or turbidity in the reagent. Working reagent blank >0.100 PERFORMANCE CHARACTERISTICS
ADDITIONAL EQUIPMENT The performance characteristics depend on the method used. It is recommended
General laboratory equipment. to calculate these data for each particular test protocol. These results have been
Spectrophotometer or photometer thermostatable at 37ºC. Cuvette: 1 cm light- obtained using an automated analyzer.
path.
SAMPLE Sensitivity, as detection limit: 0.1 mg/dL
Serum or plasma free from haemolysis. Reaction range: Up to 10 mg/dL. Concentrations higher than 10 mg/dL, it is
Serum bilirubin will decrease 50% in 1 hour if kept at room temperature (≤ 25ºC), recommended in such a case, to carry out a 1/10 dilution of the sample with
and at direct sunlight. Samples kept in the dark at 2-8ºC will remain stables for saline, (NaCl 0.9%) and then to multiply the final result by 10.
up to 3 days. Do not use frozen samples. Accuracy: 98.7%
CAUTION
The safety statements are on the label. Handle the reagent with care.
It is advisable to read the SDS before the reagent manipulation.
QUALITY CONTROL INTERFERENCES
Control serum, Seriscann Normal (Ref. 99 41 48) and Seriscann Anormal (Ref. Haemoglobin concentrations up to 30 mg/dL, ascorbic acid up to 6 mg/dL and
99 46 85) should be included in each test series. Each particular laboratory indican up to 4 mg/dL do not interfere with the assay. Lipidic samples could
should establish its own control program. interfere.
To avoid possible errors, it is very important, when assaying for direct bilirubin,
AUTOANALYZERS to read the corresponding absorbance exactly 5 min. after the reagents have
Adaptations to different autoanalyzers are available on request. been added.
REFERENCES
Doumas, B. T.; et al., (1987) Clin. Chem., 33, 769-774.
Malloy, H. et.al., (1937), 119, 481-490.
Colantonio, D:A., et al., (2012) Clin. Chem., 58, 854–868.
ISO 9001 / ISO 13485

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TOTAL BILIRUBIN DPD
DPD METHOD
PRINCIPLE PROCEDURE
Conjugated bilirubin, in acid media and suitable detergents, forms red color
azobilirubin complex by reaction with 3,5-dichlorophenyldiazonium salt. The
Substrates
Technique BL S SA
color produced in the reaction is proportional to the concentration of total bilirubin mL mL
in the sample and it is determined photometrically.
Buffer (A) 0.8 0.8
DIAGNOSTIC USE Saline 0.2 --
Substrate (B) -- 0.2
where there is conjugated with glucuronic acid and it is secreted through bile.
This conjugated bilirubin is the direct bilirubin, while unconjugated bilirubin is Sample 0.08 0.08
bilirubin. Mix and let stand at reaction temperature (37ºC)
High level of indirect or total bilirubin may indicate hemolytic diseases, Read exactly at 5 minutes
physiological neonatal jaundice, Gilbert’s disease or fructose intolerance.
Reading
Single test result could not be used to make a clinical diagnosis. It should Wavelength: 546 nm
integrate clinical and laboratory data. Blank: H2O
REAGENTS Cuvette: 1 cm light-path
Kit 1 x 300 mL. (Ref. 99 05 39 ). Contents:
A. 1 x 240 mL Buffer. Ref. 99 07 49 CALCULATIONS
B. 1 x 60 mL Substrate Ref. 99 07 89 Δ Abs = Abs SA - Abs BL S
Reagents are ready to use. The presence of tubidity in buffer reagent (A) do not Where:
affect on the reaction. Abs SA: Sample Absorption
Abs BLS: Sample blank Absorption
The concentrations in the reagent solutions are: Δ Abs x 19.5 = mg direct bilirubin / dL
A. 3,5-DPD 2 mM SI Units
HCl 50 mM mg / dL x 17.1 = μmol / L
REFERENCE VALUES
B HCl 150 mM Adults: up to 1.2 mg/dL
Detergents and preservatives Pediatric:
< 14 days 0.19 - 16.6 mg/dL
STORAGE AND STABILITY < 15 days-1 year 0.05 - 0.68 mg/dL
The components of the kit, when stored at 2-8ºC, will remain stable until the 1-3 years 0.05- 0.40 mg/dL
expiration date stated on the label. The stated values are for guidance. Each particular laboratory should establish
its own normal range.
Presence of particles in the reagent. Working reagent blank >0.300 PERFORMANCE CHARACTERISTICS
ADDITIONAL EQUIPMENT The performance characteristics depend on the method used. It is recommended
General laboratory equipment. to calculate these data for each particular test protocol. These results have been
Spectrophotometer or photometer thermostatable at 37ºC. Cuvette: 1 cm light- obtained using an automated analyzer.
path.
Sensitivity, as detection limit: 0.12 mg/dL
SAMPLE Reaction range: Up to 30 mg/dL. Concentrations higher than 30 mg/dL, it is
Serum or plasma free from haemolysis. recommended in such a case, to carry out a 1/10 dilution of the sample with
Serum bilirubin will decrease 50% in 1 hour if kept at room temperature (≤ 25ºC), saline, (NaCl 0.9%) and then to multiply the final result by 10.
and at direct sunlight. Samples kept in the dark at 2-8ºC will remain stables for Accuracy: 93.7%
up to 3 days. Do not use frozen samples. Repetitivity, as CV%: 2.0%
CAUTION Trueness: Results obtained with this reagent did not show systematic differences
The safety statements are on the label. Handle the reagent with care. when compared with reference reagent.
It is advisable to read the SDS before the reagent manipulation. Details of the comparison experiments are available on request.
INTERFERENCES
QUALITY CONTROL Haemoglobin concentrations up to 60 mg/dL, ascorbic acid up to 50 mg/dL and
Control serum, Seriscann Normal (Ref. 99 41 48) and Seriscann Anormal (Ref. indican up to 3 mg/dL do not interfere with the assay. Lipidic samples could
99 46 85) should be included in each test series. Each particular laboratory interfere.
should establish its own control program. To avoid possible errors, it is very important, when assaying for direct bilirubin,
to read the corresponding absorbance exactly 5 min. after the reagents have
AUTOANALYZERS been added.
REFERENCES
Doumas, B. T.; et al. (1987) Clin. Chem., 33, 769-774.
Malloy, H. et.al, (1937), 119, 481-490.
ISO 9001 / ISO 13485

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CHOLESTEROL LIQUID
CHOD - POD METHOD
For “in vitro” determination of cholesterol in serum or plasma.
PRINCIPLE
Cholesterol present in serum or plasma, originates a coloured complex, according to the following reactions, which can be quantified by spectrophotometry.
chol. esterase
cholesterol esters+ H2O → cholesterol + fatty acids
chol. oxidase
cholesterol + H2O + O2 → cholestenone + H2O2
POD
H2O2 + 4-aminoantipyrine + 3,5 – dichlorophenol → coloured quinonic derivative + 4 H2O
The cholesterol in serum or plasma can be of exogenous origin, ingested with the diet,
or endogenous synthesized primarily in the liver. It is transported by lipoproteins and
it is excreted in the bile.
Procedure BL SA ST
mL mL mL
The study of the serum cholesterol level allows the detection and classification of the
hyperlipidemia and risk assessment for heart disease. Sample -- 0.01 --
Cholesterol levels are low in hypolipoproteinemia, hyperthyroidism and in some
anemia. Standard -- -- 0.01
Single test result cannot be used to make a clinical diagnosis. It should integrate Working reagent 1.00 1.00 1.00
clinical and laboratory data.
Mix well and let stand for 5 min at 37ºC, or 10 min at room temperature (20 - 25ºC).
REAGENTS
Kit 1 x 100 mL. (Ref. 99 52 82). Contents: Reading
A. 1 x 100 mL Reagent Ref. 99 52 20 Wavelength: 546 nm; 505 nm
B. 1 x 5 mL Standard Ref. 99 02 48 Blank: the contents of BL
Colour stability: 1 hour (when protected from direct sunlight).
A. 3 x 100 mL Reagent Ref. 99 52 20 CALCULATIONS
B. 1 x 5 mL Standard Ref. 99 02 48 SA Abs.
x 200 = mg Cholesterol/dL
ST Abs.
A. 2 x 250 mL Reagent Ref. 99 01 59 Where:
B. 1 x 5 mL Standard Ref. 99 02 48 SA Abs: Sample Absorbance
ST Abs: Standard Absorbance
SI Units
(mg/100 dL) x 0.0259 = mmol/L
Concentration in the reagent solution is: REFERENCE VALUES
Mes buffer pH 6.5 75 mM Coronary heart disease risk:
Phenol 6 mM < 200 mg/dL Desirable
2,4-dichlorophenol 0.2 mM 200 - 239 mg/dL Borderline high
4-aminoantipyrine 0.5 mM > 239 mg/dL High
Cholesterol esterase ≥ 500 kU/L
Cholesterol oxidase ≥ 300 kU/L
Peroxidase ≥ 1200 kU/L
Non reactive Stabilizers
Standard: Solution of cholesterol in isopropanol/water equivalent to 200 mg/dL (5.18
mmol/L). Sensitivity, as detection limit: 2 mg/dL
STORAGE AND STABILITY Linearity: 700 mg/dL. For higher concentrations dilute the sample 1/2 with saline
The components of the kit, when stored at 2-8ºC, will remain stable until the expiration (NaCl 0.9%). Multiply the final result by 2.
date stated on the label. Accuracy: 98.6%
Signs of reagent deterioration: Reproducibility, as CV%: 1.44%
Presence of particles or turbidity in the reagent. Trueness: Results obtained with this reagent did not show systematic differences
Working reagent blank >0.500 when compared with reference reagent.
General laboratory equipment. INTERFERENCES
Spectrophotometer or photometer thermostatable at 37ºC. Cuvette: 1 cm light-path. There are no interferences by hemoglobin up to 200 mg/dL, as well as by bilirubin
up to 15 mg/dL.
SAMPLE
Do not pipette directly from the bottle of reagent, to avoid undesirable contamination.
Serum, or plasma. Samples are stable 1 week at 2-8ºC, and up to three months at
-20ºC. REFERENCES
Fiedewald, W., Levy, R., Fredrickson, D.S. (1972), Clin.Chem, 18, 499-502.
Allain,.C.C.,Poon, L.S.,Chan,C.S.G.,Richmond,W., Fu,P.C. (1974) Clin. Chem., 20,
CAUTION 470-475.
The reagent contains phenol. Handle with care. The safety statements are on the Zoppi, F., Fellini,D. (1976), Clin. Chem., 22, 690-691.
label. It is advised to read SDS before reagent handling. Waste products must be Tietz, NW., Textbook of Clinical Chemistry 5th Edition, W.B. Saunders, Philadelphia
handled as per local regulations. (2012).
QUALITY CONTROL Young D.S., Effect of drugs on Clinical Lab. Test, 5th Ed. AACC Press (2000).
Control serum, Seriscann Normal (Ref. 99 41 48) and Seriscann Anormal (Ref. 99 46 National Cholesterol Educarion Program Expert Panel. Third report of the National
85) should be included in each test series. Each particular laboratory should establish Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation,
its own control program. and Treatment of High Blood Cholesterol in Adults (ATP III), (2001) NIH Publication.
Bethesda: National Heart, Lung, and Blood Institute.
AUTOANALYZERS Zapico E. y Ordóñez J. (2002),Clin Invest Ar ter ioscl;14(5):272-6.
ISO 9001 / ISO 13485

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CREATININE
MODIFIED JAFFE METHOD
For “in vitro” determination of creatinine in serum, plasma or urine
PRINCIPLE PROCEDURE
In alkaline medium, creatinine with picric acid form a colored compound, alkaline creatinine Bring the reagent to working temperature
picrate which is determined photometrically. The color produced in the reaction is
proportional to the concentration of creatinine in the sample under optimal assay conditions.
Substrates
Technique BL SA ST
DIAGNOSTIC USE
mL mL mL
Creatinine in serum increases in case of renal failure, acute or chronic, hyperthyroidism,
acromegaly and gigantism. Standard --- --- 0.10
Increasing creatinine in urine occurs in diabetes mellitus, and infection. Also, exercise
promotes increased excretion in the urine. Sample --- 0.10 ---
During pregnancy or decreased muscle mass, serum creatinine concentration is reduced, Working reagent 1.00 1.00 1.00
while creatinine in urine is decreased in renal failure, myopathy, anemia or hypothyroidism.
Mix and start the stopwatch. Transfer to the measuring cuvette.
One single test result could not be used to make a clinical diagnosis. It should integrate Read the absorbances at 20 and 80 seconds
Reading
REAGENTS Wavelength: 546 nm; 510 nm
Kit 2 x 100 mL. (Ref. 99 88 91). Contents: Blank: the contents of BL
A. 1 x 100 mL Picric acid solution Ref. 99 90 06
B. 1 x 100 mL Alkaline solution Ref. 99 56 35 CALCULATIONS
C. 1 x 5 mL Standard Ref. 99 83 99 a) Creatinine concentration:
Determine the ΔAbs. for sample and standard.
ΔAbs. = Abs 80 sec - Abs 20 sec
B. 2 x 250 mL Alkaline solution Ref. 99 01 08
ΔAbs. SA
C. 1 x 5 mL Standard Ref. 99 83 99 x 2 = mg creatinine/dL
ΔAbs. ST
b) To calculate the Clearance:
The components of the kit are ready to use. To prepare working reagent, mix equal
parts of each reagent (A and B) prior to assay.
(mg creatinine/dL urine) x mL urine/24 h.
= mL / min
WORKING REAGENT COMPOSITION (mg creatinine/dL serum) x 1440
The reagent composition is as follows:
Picric acid 24 mM Where:
Sodium carbonate 50 mM mg/dL urine and serum: values obtained in a)
NaOH 0.40 M mL urine/24h: excreted urine volume in 24h
Preservatives and stabilizers 1440: conversion factor (24 hours to minutes)
Standard. Aqueous solution equivalent to 2 mg/dL, (176.8 μmol/L). Ready-to-use.
S.I. Units
(mg/dL) x 88.4 = μmol/L
The components of the kit, when stored at room temperature (≤ 25ºC), will remain stable
until the expiration date stated on the label. REFERENCES VALUES
Working reagent is stable for up to 15 days at room temperature (≤ 25ºC). Store in the dark.
Serum, plasma Urine (*)
Signs of reagent deterioration: Men: 0.6-1.1 mg/dL 21-26 mg/Kg/24 h.
Presence of particles or turbidity in the reagent. Women: 0.5-0.9 mg/dL 6-22 mg/Kg/24 h.
Working reagent blank ≥ 0,300
Clearance
ADDITIONAL EQUIPMENT Men: 97-137 mL/min
General laboratory equipment. Women: 88-128 mL/min
(*)The values are indicated as amount of creatinine excreted per Kg of weight in 24h.
SAMPLE
Serum or plasma with heparin and urine. Serum or plasma samples are stable for 24 hours Each particular laboratory should establish its own normal range, obtained from samples
at 2 - 8ºC. of a representative population, using its own instrumentation, blood collection methods and
To assay urinary creatinine, dilute the sample 1/20 in deionized water. Multiply the final assaying procedures.
result by 20.
CAUTION Performance of the reagent depends on the reagent itself and also depends on the method
The reagents contain sodium azide at 0.09%. Handle with care. and the analyzer used.
The safety statements are on the label. It is advisable to look at the SDS before using The results indicated have been obtained using a manual method.
infectious. Use adequate protection. Sensitivity, as detection limit: 0.16 mg/dL
The disposal of the residues has to be made according to local regulations. Linearity: Up to 15 mg/dL of Creatinine. For higher values, dilute the sample 1:1 in deionized
water and assay once again. Multiply the final result by 2.
Accuracy: 105%
QUALITY CONTROL
Repetitivity as Variation Coefficient: 1.18%
Trueness: Results obtained with this reagent did not show sensible differences when
control program.
AUTOANALYZERS Details of the performance studies are available on request

INTERFERENCES
No interference was observed by haemoglobin up to 1000 mg/dL, by lipemic up to 1250
mg/dL and by bilirubin up to 5 mg/dL. Other drugs and substances may interfere in the test
(see references).
REFERENCES
Blass, K.G., Thibert, R.J., Lam, L.K.(1974) Z. Clin. Chem. Clin. Biochem. 12, 336 - 343.
Spierto, F.W., McNeil, M.L., Burtis, C.A. (1979), Clin. Biochem. 12, 18 - 21.
Clinical Diagnosis and Management, Todd – Sanford - Davidsohn, Ed. J. B. Henry 6ª
Edition 1979. W. B. Saunders, Philadelphia, 262 - 263.
Young DS. Effects of drugs on clinical laboratory tests, 5th ed. AACC Press, 2000.
ISO 9001 / ISO 13485

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CREATININE DMSO
MODIFIED JAFFE METHOD
For “in vitro” determination of creatinine in serum, plasma or urine
PRINCIPLE PROCEDURE
In alkaline medium, creatinine with picric acid form a coloured compound, alkaline creatinine Bring the reagent to working temperature
picrate which is determined photometrically. The colour produced in the reaction is
proportional to the concentration of creatinine in the sample under optimal assay conditions.
Technique BL SA ST
DIAGNOSTIC USE mL mL mL
Creatinine in serum increases in case of renal failure, acute or chronic, hyperthyroidism,
acromegaly and gigantism. Standard --- --- 0.10
Increasing creatinine in urine occurs in diabetes mellitus, and infection. Also, exercise
promotes increased excretion in the urine. Sample --- 0.10 ---
During pregnancy or decreased muscle mass, serum creatinine concentration is reduced, Working reagent 1.00 1.00 1.00
while creatinine in urine is decreased in renal failure, myopathy, anemia or hypothyroidism.
Mix and start the stopwatch. Transfer to the measuring cuvette.
One single test result could not be used to make a clinical diagnosis. It should integrate Read the absorbances at 20 and 80 sec
Reading
REAGENTS
Wavelength: 546 nm; 510 nm
Blank: the contents of BL
B. 1 x 100 mL Alkaline solution Ref. 99 03 15 CALCULATIONS
C. 1 x 5 mL Standard Ref. 99 83 99 a) Creatinine concentration:
Determine the ΔAbs for sample and standard.
Mix equal parts of each reagent (A and B) prior to assay. ΔAbs. = Abs 80 sec - Abs 20 sec
WORKING REAGENT COMPOSITION ΔAbs. SA

x 2 = mg creatinine/dL
The reagent composition is as follows: ΔAbs. ST
Picric acid 55 mM
DMSO 2.8 mM b) To calculate the Clearance:
NaOH 0.50 M
Preservatives and stabilizers (mg creatinine/dL urine) x mL urine/24 h.
= mL / min
Standard. Aqueous solution equivalent to 2 mg/dL, (176.8 μmol/L). Ready-to-use. (mg creatinine/dL serum) x 1440
STORAGE AND STABILITY Where:

The components of the kit, when stored at room temperature (≤ 25ºC), will remain stable mg/dL urine and serum: values obtained in a)
until the expiration date stated on the label. mL urine/24h: excreted urine volume in 24h
Working reagent is stable for up to 21 days at room temperature (≤ 25ºC). Store in the dark. 1440: factor to change hours to minutes (minutes in 24 hours)
Signs of reagent deterioration: S.I. Units

Presence of particles or turbidity in the reagent. (mg/dL) x 88.4 = μmol/L
Working reagent blank ≥ 0,300.
REFERENCES VALUES
General laboratory equipment. Serum, plasma Urine (*)
Spectrophotometer or photometer thermostatable at 37ºC. Cuvette: 1 cm light-path Men: 0.6-1.1 mg/dL 21-26 mg/Kg/24 h.
Women: 0.5-0.9 mg/dL 6-22 mg/Kg/24 h.
SAMPLE
Serum or plasma with heparin and urine. Serum or plasma samples are stable for 24 hours Clearance
at 2 - 8ºC. Men: 97-137 mL/min
To assay urinary creatinine, dilute the sample 1/20 in deionized water. Multiply the final Women: 88-128 mL/min
result by 20.
(*)The values are indicated as amount of creatinine excreted per Kg of weight in 24h.
CAUTION
The reagent contains sodium azide at 0.09%. Handle with care. Each particular laboratory should establish its own normal range, obtained from samples
The security statements are on the label. It is advisable to look at the SDS before using the of a representative population, using its own instrumentation, blood collection methods and
reagent. assaying procedures.
QUALITY CONTROL
Control serum, Seriscann Normal (Ref. 99 41 48) and Seriscann Anormal (Ref. 99 46 85) should Performance of the reagent depends on the reagent itself and also depends on the method
be included in each test series. Each particular laboratory should establish its own control program. and the analyzer used.
The results indicated have been obtained using a manual method.

AUTOANALYZERS Linearity: Up to 15 mg/dL of creatinine. For higher values, dilute the sample 1:1 in deionized
Adaptations to different autoanalyzers are available on request. water and assay once again. Multiply the final result by 2.
Accuracy: 97.1%
Trueness: Results obtained with this reagent did not show sensible differences when
INTERFERENCES
Lipemic and hemolyzed sera will interfere with the assay.
Several medicinal substances, eventually present in the sample, such as ascorbic acid or
levodopa, can interfere with the final result.
At the described assay conditions, protein and glucose interferences are minimized.
REFERENCES
Blass, K.G., Thibert, R.J., Lam, L.K.(1974) Z. Clin. Chem. Clin. Biochem. 12, 336 - 343.
Spierto, F.W., McNeil, M.L., Burtis, C.A. (1979), Clin. Biochem. 12, 18 - 21.
Clinical Diagnosis and Management, Todd – Sanford - Davidsohn, Ed. J. B. Henry 6ª
Edition 1979. W. B. Saunders, Philadelphia, 262 - 263.
ISO 9001 / ISO 13485

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HDL - CHOLESTEROL
DEXTRAN SULPHATE - Mg (II) METHOD
For “in vitro” determination of HDL - cholesterol in serum
PRINCIPLE PROCEDURE
LDL and VLDL (low and very low density lipoproteins) are precipitated from serum by
the action of a sulfated polysaccharide, in the presence of divalent cations. Then, the 1. Precipitation reaction:
cholesterol bound to high density lipoproteins (HDL- cholesterol) present in the supernatant, Sample 0.3 mL
Substrates
is determined. Precipitant solution 1 drop
Mix and let stand for 15 min at room temperature (20 - 25ºC).
DIAGNOSTIC USE Centrifuge at 2000 x g (1500 -2300 rpm) / 15 min or
The fraction of the cholesterol bound to high density lipoprotein is an indicator of the risk of Centrifuge 10000 x g (8000 - 12000 rpm) / 2 min
coronary heart disease. High levels of HDL-cholesterol appear to act as a protective factor, Determine, in the supernatant, the concentration of cholesterol.
while low values are one of the major risk factors.
Determination of HDL- cholesterol, along with comprehensive study of the lipid profile of the 2.- Determination of cholesterol (1)
patient, allows assessing the risk of coronary heart disease.
Low levels of HDL- cholesterol are found in cases of unbalanced diet, sedentary lifestyle,
Technique BL (mL) SA (mL) ST (mL)
alcoholism or smoking.
Supernatant -- 0.01 --
Single test result could not be used to make a clinical diagnosis. It should integrate Standard -- -- 0.01
clinical and laboratory data. Working reagent 1.00 1.00 1.00
REAGENTS Mix well and let stand for 5 min at 37ºC or 10 min at room temperature.
1 x 4 mL Precipitant solution Ref. 99 38 85 Reading
Dropper for a minimum of 100 tests Wavelength: 546 nm; 505 nm
WORKING REAGENT PREPARATION Colour stability: 1 hour (when protected from direct light)
Reagent is ready to use
WORKING REAGENT COMPOSITION CALCULATIONS

Concentrations in the reagent solution are: Abs. SA
x 200 x 1.13 = mg HDL- cholesterol / dL
Dextran sulphate 10 g/L Abs. ST
Magnesium acetate 1M
Stabilizers Where:
Abs SA: Sample Absortion
STORAGE AND STABILITY Abs ST: Standard Absortion
When kept at 2 - 8ºC, the reagent will remain stable until the expiration date stated on the
label. S.I. Units: mg/dL x 0.0259 = mmol / L
Signs of reagent deterioration: Note: When the precipitant reagent is added to the sample, the latter is diluted by a
Presence of particles or turbidity in the reagent. factor of 1.13. Therefore, the cholesterol value should be multiplied by such a factor.
(1)With QCA cholesterol reagent
General laboratory equipment. REFERENCE VALUES
Spectrophotometer or photometer thermostable at 37ºC. Coronary heart disease risk:
Centrifuge. < 40 mg/dL High
≥ 60 mg/dL Low
Reagent for cholesterol determination and standard
It is advisable to use QCA reagents: The concentrations vary considerably with age and sex.
Kit 1 x 100 mL 99 52 82
Kit 3 x 100 mL 99 52 80 Performance characteristics
Kit 2 x 250 mL 99 50 12 The performance characteristics depend on the precipitant reagent, the reagent to determine
cholesterol and the method used. These results have been obtained by manual method.
Standard is included in the kits The cholesterol in the supernatant has been determinate with the reagent cholesterol Liquid
from QCA.
CAUTION
The safety statements are on the label. Sensitivity, as detection limit: 3 mg/dL
We advise to read MSDS before reagent handling. Waste products must be handled as per Accuracy: 97.4%
local regulations. Repetitivity, as CV%: 1.87%
SAMPLE Trueness: Results obtained with this reagent did not show systematic differences when
Serum. The separation of the HDL fraction has to be carried out as soon as possible; compared with reference reagent.
otherwise, it is recommended to freeze the sample at - 15ºC. Stored in this way, it will Details of the performance studies are available on request.
remain stable for up to 1 week.
INTERFERENCES
QUALITY CONTROL Neither aged nor haemolysed samples shall be assayed. Bilirubin, at concentrations higher
Control serum, Seriscann Normal (Ref. 99 41 48) and Seriscann Anormal (Ref. 99 46 than 9 mg/dL, interferes in the precipitation reaction.
85) should be included in each test series. Each particular laboratory should establish Sera with triglycerides concentration higher than 350 mg/dL will be diluted 1/2 in saline
its own control program. (NaCl 0.9%) prior to precipitation.
BIBLIOGRAFÍA
AUTOANALYZERS
Albers,J.J., Warmick,G.R., Cheng,M.C. (1978). Lipids, 13, 926 - 932.
Benzie,I. (1979). Med. Lab. Sci., 36, 289 - 291.
Wieland, H. Seidel, D. (1981). Ärtztl. Lab.,27, 141 - 154.
A policy statement of the European Atherosclerosis Society, European Heart Journal
8,(1987) 77 - 88.
(2012).
National Cholesterol Educarion Program Expert Panel. Third report of the National
Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation,
and Treatment of High Blood Cholesterol in Adults (ATP III), (2001) NIH Publication.
Bethesda: National Heart, Lung, and Blood Institute.
Zapico E. y Ordóñez J. (2002),Clin Invest Ar ter ioscl;14(5):272-6.
ISO 9001 / ISO 13485

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HDL - CHOLESTEROL DIRECT
COLOURIMETRIC METHOD
For “in vitro” determination of HDL - cholesterol in serum or plasma
PRINCIPLE PROCEDURE
HDL-cholesterol direct is used for the determination of this cholesterol fraction, with no
previous treatments, that is to say, without precipitation nor centrifugation. Technique SA CAL
The method is based on the property of a detergent to liberate the HDL fraction by μL μL
solubilization, which then reacts with the chromogen, cholesterol esterase and oxidase to Sample 4.0 --
give rise to a measurable colour at 600 nm. The use of a polyanion stabilizes the lipoproteins
Calibrator -- 4.0
(VLDL, LDL and chylomicrons) by adsorption, and therefore cannot react with the enzymatic
complex. Reagent A 300 300
Mix and incubate 5 min/37ºC. Read (Abs1) for the sample
DIAGNOSTIC USE and the calibrator.
The fraction of the cholesterol bound to high density lipoprotein is an indicator of the risk of
coronary heart disease. High levels of HDL-cholesterol appear to act as a protective factor, Reagent B 100 100
while low values are one of the major risk factors.
Mix and incubate 5 min/37ºC. Read (Abs2) for the sample
Determination of HDL- cholesterol, along with comprehensive study of the lipid profile of the
and the calibrator..
patient, allows assessing the risk of coronary heart disease.
Low levels of HDL-cholesterol are found in cases of unbalanced diet, sedentary lifestyle, Reading
alcoholism or smoking. Wavelength: 600 nm (546 nm - 640 nm)
Blank: read against air
Single test result could not be used to make a clinical diagnosis. It should integrate clinical Cuvette: thermostized. 1 cm light-path
CALCULATIONS
REAGENTS (Abs2 - Abs1) sample
Kit 1 x 40 mL. (Ref. 99 48 58). Contents: x [CAL] * = mg/dL
A. 1 x 30 mL Reagent (A) Ref. 99 48 60 (Abs2 - Abs1) calibrator
B. 1 x 10 mL Reagent (B) Ref. 99 48 62
C. 1 x 1 mL Freeze-dried HDL-calibrator Ref. 99 03 72 Where:
(Abs2 - Abs1) Sample: Reading at 10 min – reading at 5 min of the sample
Kit 1 x 80 mL. (Ref. 99 80 58). Contents: (Abs2 - Abs1) calibrator: Reading at 10 min – reading at 5 min of the calibrator
A.1 x 60 mL Reagent (A) Ref. 99 80 60 (* ) The calibrator concentration is stated on the vial label
B.1 x 20 mL Reagent (B) Ref. 99 80 62
C.1 x 1 mL Freeze-dried HDL-calibrator Ref. 99 03 72 S.I. Units
mg/dL x 0.0259 = mmol/L.
Kit 1 x 400 mL. (Ref. 99 57 88). Contents: (*). See concentration on the vial label.
A.3 x 100 mL Reagent (A) Ref. 99 33 47
B.1 x 100 mL Reagent (B) Ref. 99 49 40 REFERENCE VALUES
C. 1 x 1 mL Freeze-dried HDL-calibrator Ref. 99 03 72 Coronary heart disease risk:
< 40 mg/dL High
≥ 60 mg/dL Low
Reagents (A) and (B) are ready-to-use.
The calibrator should be rehydrated with 1 mL of deionised water and let stand for 20 min
The concentrations vary considerably with age and sex.
prior to be used. Its concentration is stated on the vial label.
REAGENT COMPOSITION PERFORMANCE CHARACTERISTICS

Composition of reagents is: The performance characteristics depend on the method used. It is recommended to
Reagent (A) calculate these data for each particular test protocol. These results have been obtained
N,N-bis(4-sulphobutyl)-3-methylaniline 1.2 mM using a BT Autoanalyzer.
Polyanion/polymer 0.8 mM
Stabilizers and preservatives Sensitivity, as detection limit: 2 mg/dL
Reagent (B) Linearity: 200 mg/dL. For higher concentrations dilute the sample 1/2 with saline (NaCl
Cholesterol esterase ≥ 550 kU/L 0.9%). Multiply the final result by 2.
Cholesterol oxidase ≥ 300 kU/L Accuracy: 97.4%
POD ≥ 1500 kU/L Repetitivity, as CV%: 1.4%
4-aminoantipyrine 0.95 mM Reproducibility, as CV%: 2.2%
Stabilizers and preservatives Trueness: Results obtained with this reagent did not show systematic differences when
STORAGE AND STABILITY Details of the performance studies are available on request
label. Reagents (A) and (B), once opened are stable 2 months when stored between 2º - 8º INTERFERENCES
C and protected from direct sunlight. Bilirubin concentrations up to 40 mg/dL, ascorbic acid up to 10 mM, haemoglobin up to 1000
Once rehydrated, the calibrator will remain stable for 21 days when stored between 2º-8º C. mg/dL and triglyceride up to 1000 mg/dL will not interfere with the assay.
AUTOANALYZERS
Presence of particles or turbidity in the reagent. Working reagent blank >0.100
REFERENCES
Dahlen, G.H., Guyton, J.R., Attar, M., Farmer, J.A., Kautz, J.A., Gotto, A.M. (1986),
Circulation, 74, 758-765.
SAMPLE Sugiuchi, H., et.al.(1995). Clin.Chem., 41, 717-723.
Fresh serum or plasma (EDTA, citrate or heparin). Fasting and non-fasting samples can Tietz, NW., Textbook of Clinical Chemistry 6th Edition, W.B. Saunders, Philadelphia (2018).
be used. CLSI Guidelines and Standards, CLSI, Wayne, P.A
CAUTION National Cholesterol Educarion Program Expert Panel. Third report of the National
The safety statements are on the label. Handle the reagent with care. Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and
It is advisable to read the SDS before the reagent manipulation. Treatment of High Blood Cholesterol in Adults (ATP III), (2001) NIH Publication. Bethesda:
The disposal of the residues has to be made according to local regulations. National Heart, Lung, and Blood Institute.
QUALITY CONTROL
control program.
ISO 9001 / ISO 13485

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HEMOGLOBIN
CYANMETHAEMOGLOBIN METHOD
For “in vitro” determination of Hemoglobin in whole blood
PRINCIPLE
Ferricyanide oxidizes the Fe(II) present in hemoglobin, oxyhemoglobin and carboxyhemoglobin, into Fe(III), giving rise to methaemoglobin which, in the presence of cyanide ion, produces
cyanmethaemoglobin, a stable red compound that is photometrically determined.
Substrates
HbFe(ΙΙ) + Fe(ΙΙΙ)(CN)63- HbFe(ΙΙΙ) + Fe(ΙΙ)(CN)64-
HbFe(ΙΙΙ) + CN- HbFe(ΙΙΙ)CN

Hemoglobin is a blood protein with quaternary structure, red-colored and has a heme group
and iron inside. Its main function is to transport oxygen from the respiratory organs to the
tissues, carbon dioxide from the tissues to the lungs to remove it and also participates in
Technique BL mL SA mL
the regulation of blood pH. Sample - 0.010
A hemoglobin level below normal values may be due to anemia, cancer, kidney disease,
bleeding, hemolysis or bone marrow damage. Reagent 2.5 2.5
High hemoglobin values may be indicative of dehydration, renal and lung chronic disease, Mix and allow to stand 10 min at room temperature (20-25ºC). Read the absorbance.
tumors or cardiopathies.
Single test result could not be used to make a clinical diagnosis. It should integrate clinical Reading
and laboratory data. Wavelength:546 nm; 540 nm
REAGENTS Colour stability: a minimum of 8 hours
1 x 100 mL. (Ref. 99 60 23). Contents:
1 x 100 mL Concentrated (1:10) Drabkin’s reagent Ref. 99 03 11 CALCULATIONS
With factor
Kit 3 x 100 mL. (Ref. 99 60 30). Contents: Abs sample x 37 = g Haemoglobin / dL
3 x 100 mL Concentrated (1:10) Drabkin’s reagent Ref. 99 03 11
The value of the Calculation Factor is obtained by:
WORKING REAGENT PREPARATION 20g/dL / 0,545 540nm
To prepare working reagent is necessary to dilute 1:10 the concentrated Drabkin’s reagent
with deionized water. The value of Abs. 0.545 is the result that you could obtain after processing a hemoglobin
Add 900 mL of deionized water to 100 mL of concentrated reagent. sample, under the conditions indicated in the procedure, with a concentration of 20 g/dL
hemoglobin.
Concentrations in the reagent solution are: S.I. Units
Potassium ferricyanide 0.5 mM (g/dL) x 0.155 = mmol/L
Potassium cyanide 0.8 mM
Preservatives and stabilizers REFERENCES VALUES
Men: 13-18 g/dL
STORAGE AND STABILITY Women:11-16g/dL
The components of the kit, stored at room temperature (≤ 25ºC), will remain stable until the The stated values are for guidance. It is advisable that each laboratory determines its own
expiration date stated on the label. reference values.
Signs of reagent deterioration: CONVERSION OF HEMOGLOBIN VALUES

Presence of particles or turbidity in the reagent. Working reagent blank > 0.1 100 % Hemoglobin = 16 g/dL
ADDITIONAL EQUIPMENT g/dL Hb % Hemoglobin g/dL Hb % Hemoglobin

General laboratory equipment. 18.0 112.5 12.8 80.0
Spectrophotometer or photometer thermostatable at 37ºC. Cuvette: 1 cm light-path. 17.6 110.0 12.4 77.5
17.2 107.5 12.0 75.0
SAMPLE 16.8 105.0 11.6 72.5
Whole blood with Heparin or EDTA. 16.4 102.5 11.2 70.0
Hemoglobin is stable1 week when stored at 2-8ºC. 16.0 100.0 10.8 67.5
15.6 97.5 10.4 65.0
15.2 95.0 10.0 62.5
CAUTION 14.8 92.5 9.6 60.0
The safety statements are on the label. Handle the reagent with care. 14.4 90.0 9.2 57.5
It is advisable to read the SDS before the reagent manipulation. 14.0 87.5 8.8 55.0
The disposal of the residues has to be made according to local regulations. 13.6 85.0 8.4 52.5
13.2 82.5 8.0 50.0
QUALITY CONTROL PERFORMANCE CHARACTERISTICS

Control serum, Seriscann Normal (Ref. 99 41 48) and Seriscann Anormal (Ref. 99 46 85) Performance of the reagent depends on the reagent itself and also depends on method and
should be included in each test series. Each particular laboratory should establish its own analyzer used.
control program. The results indicated are obtained using a manual method.
Sensitivity, as detection limit: 2.0 g/dL

AUTOANALYZERS
Linearity: Up to 25 g of hemoglobin/dL
Accuracy: 95.2 %
INTERFERENCES
No interferences are known.
The use of disposable equipment is recommended to prevent unwanted contamination.
REFERENCES
Van Kampen, E.J., Zijlstra, W.G.(1961). Clin. Chim. Acta, 6, 538-544.
International Committee for Standarization in Haematology (1978), J.Clin. Path., 31, 139-
143.
ISO 9001 / ISO 13485

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LDL - CHOLESTEROL
POLYVINYL SULPHATE METHOD
For “in vitro” determination of LDL - cholesterol in serum
PRINCIPLE PROCEDURE
LDL-cholesterol can be determined as the difference between total cholesterol and the 1. Precipitation reaction
cholesterol content of the supernatant after precipitation of the LDL fraction by polyvinyl Precipitant solution 0.1mL (3 drops)
sulphate (PVS) in the presence of polyethylene-glycol monomethyl ether. Sample 0.2 mL
Mix and let stand for 15 min at room temperature (20 – 25 ºC).
DIAGNOSTIC USE Centrifuge at 2000 x g (1500 -2300 rpm)/ 15 min or
LDL proteins are low density lipoproteins that transport cholesterol, triglycerides and lipids
Centrifuge at 10000 x g (8000 -12000 rpm)/ 2 min
in the blood. A high level of cholesterol bound to the LDL fraction, LDL cholesterol, is
Determine cholesterol concentration in the supernatant.
associated with myocardial infarction and cardiovascular disease.
Other diseases that present high levels of LDL cholesterol are: nephrosis, diabetes,
hypothyroidism or obesity. 2.- Determination of cholesterol
Single test result could not be used to make a clinical diagnosis. It should integrate clinical Samples: Total serum (SA) and supernatant (SP)
and laboratory data. Technique BL mL SA mL SP mL ST mL
Serum -- 0.01 0.01 --
REAGENTS Supernatant -- -- -- --
1 x 10 mL Precipitant solution Ref. 99 06 10 Standard -- -- -- 0.01
Dropper for 100 tests Working reagent 1.00 1.00 1.00 1.00
Mix well and let stand for 5 min at 37 ºC or 10 min at room temperature.
Reading
Reagent is ready to use.
Colour stability: 1 hour( when protected from direct light)
Concentrations in the reagent solution are: CALCULATIONS
Polyvinyl sulphate 0.7 g/L A. Cholesterol in supernatant
EDTA Na2 5.0 mM Abs SA
Polyethyleneglycol monomethyl ether 170 g/L x 200 x 1.5 = mg supernatant cholesterol / dL
Stabilizers Abs ST
B. Cholesterol in total serum

STORAGE AND STABILITY Abs SP
When kept at 2 - 8ºC, the reagent will remain stable until the expiration date stated on the x 200 = mg total cholesterol / dL
label. Abs ST
Where:
Signs of reagent deterioration: Abs SP: Serum sample Absortion
Presence of particles. Non-homogeneous appearance. Abs SA: Supernatant Absortion
Abs ST: Standard Absortion
ADDITIONAL EQUIPMENT C. LDL- cholesterol

General laboratory equipment. LDL- cholesterol (mg/dL) = total cholesterol in the sample (mg/dL) - supernatant cholesterol
Spectrophotometer or photometer thermostable at 37ºC. (mg/dL)
Centrifuge.
SI Units: mg/dL x 0.0259 = mmol/L
Reagent for cholesterol determination and standard
Note: When the precipitant reagent is added to the sample, the latter is diluted
It is advisable to use QCA reagents:
by a factor of 1.5. Therefore, the cholesterol value should be multiplied by such
Kit 1 x 100 mL 99 52 82 a factor.
Kit 3 x 100 mL 99 52 80
Kit 2 x 250 mL 99 50 12 REFERENCE VALUES
Standard is included in the kits Coronary heart disease risk:
< 100 mg/dL Optimal
130 - 159 mg/dL Borderline high
CAUTION 160 - 189 mg/dL High
The safety statements are on the label. It is recommended to read the MSDS before the > 189 mg/dL Very high
reagent handling.
Waste products must be handled as per local regulations. PERFORMANCE CHARACTERISTICS
The performance characteristics depend on the precipitant reagent, the reagent to
SAMPLE determine cholesterol and the method used. It is recommended to calculate these data
Serum. The serum may be stored at 2 - 8°C for 1 day. Do not freeze. for each particular test protocol. These results have been obtained by manual method. The
cholesterol has been determinate with the reagent cholesterol liquid from QCA.
QUALITY CONTROL Sensitivity, as detection limit: 6 mg/dL
Control serum, Seriscann Normal (Ref. 99 41 48) and Seriscann Anormal (Ref. 99 46 Accuracy: 96.4%
85) should be included in each test series. Each particular laboratory should establish Repetitivity, as CV%: 2.87%
its own control program. Reproducibility, as CV%: 2.96%
AUTOANALYZERS Details of the comparison experiments are available on request.
REFERENCES
INTERFERENCES Methods of Enzymatic Analysis, 3rd Edition, Volume VIII, Edited by H. U. Bergmenyer.
Sera with Triglyceride higher than 400 mg/dL could interfere. In such a case the sample (1985), 154 - 160.
shall be diluted 1/2 with saline (NaCl 0.9 %) prior to precipitation. Multiply the final result Assmann, G., Jabs, H. U., Kohnert, U., Nolte, W., Schriewer, H., (1984), Clin. Chim. Acta.,
by 2. 140, 77 - 83.
Neither aged nor haemolysed sera will be assayed Demacker, P. N., et al. (1984), Clin. Chem., 30, 1797 -1800.
National Cholesterol Educarion Program Expert Panel. Third report of the National
Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and
Treatment of High Blood Cholesterol in Adults (ATP III), (2001) NIH Publication. Bethesda:
National Heart, Lung, and Blood Institute.
ISO 9001 / ISO 13485

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LDL - CHOLESTEROL DIRECT
COLORIMETRIC METHOD
For “in vitro” determination of LDL - Cholesterol in serum
PRINCIPLE PROCEDURE
The LDL-cholesterol reagent allows measuring the LDL-cholesterol fraction levels in serum
or plasma directly, without the need for any sample pretreatment or centrifugation.
The method consists of two specific steps. In the first one, chylomicron, VLDL and HDL are Procedure BL SA CAL
Substrates
selectively eliminated by means of specific oxidation reactions with cholesterol esterase μL μL μL
and cholesterol oxidase, and also by a specific degradation reaction wiht catalase, forming Deionized water 5.00 -- --
cholestenone and H2O2. Sample -- 5.00 --
In the second step the remainder of LDL-cholesterol can be spectrophotometrically Calibrator -- -- 5.00
measured when a color is formed as it is transformed (quinone complex) by the presence Reagent (A) 300 300 300
of specific surfactants.
Mix well and incubate at 37ºC / 5 min Read the absorbance
DIAGNOSTIC USE (Abs1 PR o Abs1 CAL) of the sample and of the calibrator.
LDL are low density lipoprotein, they transport cholesterol, triglycerides and lipids to cells. Reagent (B) 100 100 100
Even within the normal range of total cholesterol concentrations, an increase in LDL
cholesterol can occur with an associated increased risk for coronary heart disease. High Mix well and incubate at 37ºC / 5 min Read (against the
levels of LDL could be associated with obesity, diabetes and nephrosis. reagent blank), the absorbance of the sample (Abs2 PR) and of
Single test result could not be used to make a clinical diagnosis. It should integrate clinical the calibrator (Abs2 CAL).
Reading:
REAGENTS Wavelenght: 600 nm
Kit 1 x 40 mL. (Ref. 99 18 70). Contents: Blank: the contents of BL
A. 1 x 30 mL Reagent (A) Ref. 99 18 72 Cuvette: 1 cm. light-path
C. 1 x 2 mL Freeze-dried LDL-cholesterol calibrator Ref. 99 18 78 CALCULATIONS
(Abs2 PR – Abs1 PR)
Kit 1 x 80 mL. (Ref. 99 18 80). Contents: x conc. of calibrator = LDL-cholesterol (mg/dL)
A. 1 x 60 mL Reagent (A) Ref. 99 06 13 (Abs2 CAL – Abs1 CAL)
C. 1 x 2 mL Freeze-dried LDL-cholesterol calibrator Ref. 99 18 78
S.I. Units
Kit 1 x 400 mL. (Ref. 99 07 04). Contents: mg/dL x 0.0259 = mmol/L
A. 3 x 100 mL Reagent (A) Ref. 99 91 17
REFERENCES VALUES
Coronary heart disease risk:
C. 1 x 2 mL Freeze-dried LDL-cholesterol calibrator Ref. 99 18 78
< 100 mg/dL Optimal
130 - 159 mg/dL Borderline high
WORKING REAGENT PREPARATION 160 - 189 mg/dL High
Reagents (A) and (B) are ready-to-use. > 189 mg/dL Very high
The calibrator, should be rehydrated with 2 mL of deionized water and let stand for 20 min
prior to be used. Its concentration is stated on the vial label. PERFORMANCE CHARACTERISTICS
REAGENT COMPOSITION calculate these data for each particular test protocol. These results have been obtained
The concentrations in the reagents are: using an autoanalyzer.
Reagent (A)
Good´s buffer pH 7.0 100 mM Sensitivity, as detection limit: 4 mg/dL
N-(2-hydroxy-3-sulphopropyl)-3.5-dimethoxyaniline 0.45 mM Linearity: 500 mg/dL. Samples that give higher concentration should be diluted in saline
Catalase ≥ 225 U/L NaCl 0.9% (1+1) and the final result have to be multiplied per 2.
Cholesterol estearase ≥ 600 kU/L Accuracy: 98.5%
Cholesterol oxidase ≥ 380 kU/L Repetitivity, as CV%: 0.68%
Non reactive stabilizers Reproducibility, as CV%: 0.96%
Reagent (B) compared with reference reagent.
Good´s buffer pH 7.0 100 mM Details of the comparison experiments are available on request.
4-aminoantipyrine 1.2 mM
Peroxidase ≥ 1200 kU/L INTERFERENCES
Non reactive stabilizers Concentrations of ascorbic acid higher than 50 mg/dL, hemoglobin higher than 500 mg/dL
and of bilirubin higher than 20 mg/dL can interfere with the assay.
STORAGE AND STABILITY Discard aged, hemolyzed or lipemic sera.
label. QUALITY CONTROL
Reagents (A) and (B), once opened are stable for 2 months when stored at 2º - 8ºC and Control serum, Seriscann Normal (Ref. 99 41 48) and Seriscann Anormal (Ref. 99 46 85)
protected from direct sunlight. should be included in each test series. Each particular laboratory should establish its own
Once rehydrated, the calibrator will remain stable for 10 days when stored at 2º - 8ºC. control program.
AUTOANALYZERS
Presence of particles or turbidity in the reagent. Working reagent blank > 0.100
General laboratory equipment. REFERENCES
Spectrophotometer or photometer thermostatable at 37ºC. Cuvette: 1 cm light-path. Freedman, D., et al., (2004), Clin. Chem., 50, 1189-1200.
Methods of Enzymatic Analysis, 3rd Edition, VIII, Edited by H. U. Bergmenyer. (1985),
154 - 160.
CAUTION Bachorick, P.S., et al. (1995) Clin. Chem., 41, Nº 10.
The reagents contain sodium azide at 0.09%. Handle with care. Young D.S., Effect of drugs on Clinical Lab. Test, 5th Ed. AACC Press (2000).
The safety statements are on the label. It is advisable to look at the SDS before using National Cholesterol Educarion Program Expert Panel. Third report of the National
the reagent. The calibrator must be considered as a human sample, and, thus, potentially Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and
infectious. Use adequate protection. Treatment of High Blood Cholesterol in Adults (ATP III), (2001) NIH Publication. Bethesda:
The disposal of the residues has to be made according to local regulations. National Heart, Lung, and Blood Institute.
SAMPLE
Serum. The serum samples can be kept at 2 - 8ºC for 1 day. Do not freeze.
ISO 9001 / ISO 13485

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GLUCOSE LIQUID
GOD – POD METHOD
For “in vitro” determination of Glucose in serum, plasma or CSF
PRINCIPLE
The oxidation of glucose to gluconic acid is catalyzed by glucose oxidase producing hydrogen peroxide.
The hydrogen peroxide reacts with 4-aminoantipyrine and p-hydroxybenzoic acid in the presence of peroxidase to give a quinone derivative, whose coloration is proportional to the glucose concentration
in the sample.
GOD
D- Glucose + H2O + O2 → gluconic acid + H2O2
POD
2 H2O2 + 4-aminoantipyrine + p-hydroxybenzoic acid → coloured quinonic derivative + 4 H2O

The determination of glucose in serum or urine is used for the evaluation of disorders of the Bring the reagent and the analyzer to the working temperature.
metabolism of carbohydrates.
Glucose is the major source of energy for body cells. Insulin produced in the pancreatic cells , Technique BL SA ST
facilitates the entry of glucose into cells of tissues. mL mL mL
Increased blood glucose is associated with a decrease in insulin activity or with its deficiency. Standard -- -- 0.01
In serum or plasma it is found elevated glucose values mainly in patients with diabetes mellitus but
also acute pancreatitis , Cushing syndrome, acromegaly and gigantism. Sample -- 0.01 --
Hypoglycemia can occur in response to fasting, or may be due to drugs , poisons or inborn errors Working reagent 1.0 1.0 1.0
of metabolism.
The presence of glucose in urine with the individual not suffering from diabetes is usually a sign of
disease in the renal tubules. Mix well and incubate 5 - 10 min. at 37ºC or 20-25 min. at 20 – 25ºC.
Glucose determination in CSF is primarily of interest in case of bacterial meningitis, when its
concentration is low or not detected. Reading
Wavelength: 505 nm.
Single test result cannot be used to make a clinical diagnosis. It should integrate clinical and Blank: the contents of BL.
laboratory data. Colour stability: a minimum of 1 hour, when protected from direct sunlight.
CALCULATIONS
REAGENTS SA Abs.
Kit 1 x 100 mL. (Ref. 99 82 25) Contents: x 100 = mg glucose / dL
A. 1 x 100 mL Reagent Ref. 99 82 84 ST Abs.
B. 1 x 5 mL Standard Ref. 99 02 93 Where:
SA Abs: Sample Absorption
Kit 3 x 100 mL. (Ref. 99 82 82) Contents: ST Abs: Standard Absorption
A. 3 x 100 mL Reagent Ref. 99 82 84
B. 1 x 5 mL Standard Ref. 99 02 93 S.I. Units
(mg/dL) x 0.0555 = mmol/L
Kit 4 x 250 mL. (Ref. 99 86 60) Contents:
REFERENCES VALUES
B. 1 x 5 mL Standard Ref. 99 02 93
Serum, plasma (fasting):
Adult: 74 - 115 mg/dL (4.1-6.4 mmol/L)
WORKING REAGENT PREPARATION Child: 60 - 100 mg/dL (3.3-5.6 mmol/L)
The components of the kit are ready to use. Neonate : 30 - 80 mg/dL (1.7-4.5 mmol/L)
Neonate premature: 20 - 60 mg/dL (1.1-3.3 mmol/L)
REAGENT COMPOSITION CSF:
Concentration in the reagent solution is: Adult: 40 - 70 mg/dL (2.2-3.9 mmol/L)
Phosphate buffer pH 6.8 100 mM Child: 60 - 80 mg/dL (3.3-4.5 mmol/L)
p-hydroxybenzoic acid 39.5 mM Urine: 1 - 15 mg/dL (0.1-0.8 mmol/L)
4-aminoantipyrine 0.8 mM Each particular laboratory should establish its own normal range, obtained from samples of a
Phenol 4.5 mM representative population, using its own instrumentation, blood collection methods and assaying
Glucose oxidase ≥ 18 kU/l procedures.
Peroxidase ≥ 1.1 kU/l
Preservatives and stabilizers PERFORMANCE CHARACTERISTICS
Standard: Aqueous solution equivalent to 100 mg/dL (5.55 mmol/L). The performance characteristics depend on the method used. It is recommended to calculate
STORAGE AND STABILITY these data for each particular test protocol. These results have been obtained using a manual
The components of the kit, stored at 2-8ºC, will remain stable until the expiration date stated on method.
the label. Sensitivity, as detection limit: 2.0 mg/dL
Linearity: Up to 500 mg/dL. For higher values, it is recommended to dilute the sample 1/2 in saline
Signs of reagent deterioration: (NaCl 0.9%) and assay once again. Multiply the final result by 2.
Presence of particles or turbidity in the reagent. Working reagent blank >0.400. Accuracy: 98.9 %.
Repetitivity, as Variation Coefficient: 0.79%
ADDITIONAL EQUIPMENT Trueness: Results obtained with this reagent did not show systematic differences when compared
General laboratory equipment. with reference reagent.
Spectrophotometer or photometer thermostatable at 37ºC. Cuvette: 1 cm light-path. Details of the performance studies are available on request
SAMPLE
Serum, plasma or CSF. Serum or plasma glucose (but not whole blood glucose, due to glycolytic INTERFERENCES
processes) can be stored during 2 - 3 days, when refrigerated at 2-8ºC. Haemoglobin, higher than 200 mg/dL; Bilirubin, higher than 20 mg/dL; Uric acid, higher than 20
The CSF must be clear and without debris. In these conditions the glucose is stable 48 hours at mg/dL; Creatinine, higher than 15 mg/dL.
2-8°C. Interferences caused by the anticoagulants of current use such as Heparin, EDTA or oxalate have
CAUTION not been described.
QUALITY CONTROL
Control serum, Seriscann Normal (Ref. 99 41 48) and Seriscann Anormal (Ref. 99 46 85) should
be included in each test series. Each particular laboratory should establish its own control program.
adequate protection.
AUTOANALYZERS
REFERENCES
Trinder, P. (1969). Ann. Clin. Chem. 6, 24-27.
ISO 9001 / ISO 13485

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GLYCOHEMOGLOBINA(HbA1)
IONIC EXCHANGE RESIN METHOD
FOR “IN VITRO” DETERMINATION OF GLYCOHEMOGLOBIN IN BLOOD
BASIS OF THE TEST
In healthy individuals glucose circulates in the blood. Red blood cells are highly permeable to glucose. The concentration of glucose in red blood cells and plasma is approximately the
same. The amount of glucose in the red blood cells increases proportionately as plasma glucose levels increase. Glucose may react with the haemoglobin molecules of red blood cells
to form glycohaemoglobin. This reaction depends entirely on the concentration of glucose in the red blood cell and consists of two stages: one which is quick and reversible in which
Substrates
aldimines are formed, and a slow and relatively irreversible stage in which stable glycohaemoglobin (ketoamine) is formed, which will last throughout the useful life span of the red blood
cell (approximately 120 days):
glucose + haemoglobin aldimine (labile HbA1) stable HbA1
In this ion exchange resin method (“batch” technique), the first step is to haemolyse the whole blood with the lysing reagent. The next step is to mix the lysed blood with the resin. At a
certain pH value and conductivity, the fraction of non-glycated haemoglobin (HbA0) binds to the resin, while the HbA1 fraction remains free in the supernatant. Separation by centrifugation
of both stages (resin and supernatant) enables the evaluation of the relative proportion of the HbA1 fraction in relation to the total haemoglobin which is measured separately. The results
are calculated by comparing them with a standard or control of a known concentration.
PRINCIPLE PROCEDURE
At a certain pH value, the HbA0 hemoglobin fraction is retained by the ion exchange resin, A. Sample hemolysis
while the HbA1 fraction (glycosated hemoglobin) has a net charge that will make it to 1. Dispense 0.5 ml of the lysing reagent into a test tube.
remain in the supernatant. The rapid phase separation of resin from the supernatant by 2. Add 0.1 ml of blood sample, standard or control.
centrifugation, will allow a quick evaluation about the relative proportion of HbA1 with regard Mix and incubate for 5 min. at room temperature (20-25ºC).
to the total hemoglobin (Batch technique).
B. HbA1 separation
1. Mix thoroughly the resin suspension to guarantee total homogeneity and dispense 3 ml.
REAGENTS into a test tube. It is very important to homogenize the resin suspension before each
Kit for 20 tests. (Ref. 99 83 90). Contents: dispensation. Otherwise, the amount dispensed will be variable and the separation
A. 1 x 60 mL Buffered resin. Ref. 99 00 54 will be erroneous.
B. 1 x 10 mL Lysing reagent. Ref. 99 07 90 2. Add 0.1 ml of the hemolyzed sample (A.2.)
C. 1 x 1 mL Standard. Ref. 99 03 01 3. Mix the resin suspension and the hemolyzed sample for 5 min. (hematological mixer,
vortex...). Continuous resin mixing is recommended so as to avoid its sedimentation as well
Kit for 100 tests. (Ref. 99 50 84). Contents: as to let the reaction to proceed under proper conditions.
A. 3 x 100 mL Buffered resin. Ref. 99 80 00 4. Centrifuge x 580 g (2000 rpm aprox.) for 10 min. Take a certain volume of the super-
B. 1 x 50 mL Lysing reagent. Ref. 99 76 26 natant out of the tube, with special care of not aspirating the pellet of resin, and read the
C. 1 x 1 mL Standard. Ref. 99 03 01 absorbance (Abs1).
Optionally: C. Total hemoglobin
Set of Controls (Ref. 99 66 36).Contents: 1. Dispense 20 μl of the hemolyzed sample (A.2) into a test tube.
1 x 1 mL Control low level. Ref. 99 41 06 2. Add 5 ml of deionized water and mix vigorously. Read the absorbance (AbsT).
1 x 1 mL Control high level. Ref. 99 88 07
READING
Wavelength: 415 nm.
WORKING REAGENT PREPARATION: Blank: Water.
Reagents A and B are ready to use. Stability: 1 hour.
Standard and Control: Rehydrate one vial with 1 mL of deionized water. Let stand at room
temp. (≤ 25 ºC), for 30 min. with occasional mixing until rehydration is complete.
CALCULATIONS
Determine the value of the ratio Csample= (Abs1/AbsT) C standard= (Abs1/AbsT)
REAGENT COMPOSITION
% HbA1 sample = (Csample/Cst) x %HbA1 ST
A. Buffered resin: Buffered ionic exchange resin 0.8%; pH 6.85
B. Lysing reagent: Potassium cyanide 8 mM and surfactant. (%HbA1ST is stated on the label of the standard vial).
C. Standard: Freeze-dried erythrocytes. The concentration value is stated on the label.
Set of Controls: Freeze-dried erythrocytes. The concentration values (two levels) are stated REFERENCE VALUES
on the label. Non-diabetics patients: 6.0-8.3%
Uncontrolled diabetics: values can be higher than 10%.
STORAGE AND STABILITY The stated values are for guidance. It is advisable that each laboratory determines its own
The buffered resin and the lysing reagent, stored at 2-8ºC, will remain stable until the reference values.
Standard and Controls should be kept at 2-8ºC, protected from light. Once rehydrated PERFORMANCE CHARACTERISTICS
these are stable for two weeks stored at 2-8ºC, or 8 weeks if aliquoted and frozen (-20 ºC). The performance characteristics depend on the method used. It is recommended to calcu-
late these data for each particular test protocol. These results have been obtained using a
Signs of reagent deterioration: manual method.
Reagent A blank < 0.065 Linearity: Up to 25%. For higher values, it is recommended to dilute the sample 1/2 in saline
Presence of particles or turbidity in the reagent B, Standard and Controls (NaCl 0.9%) and assay once again. Multiply the final result by 2.
Repetitivity, as %CV: 1.54%
Reproducibility, as %CV: 5.95%
ADDITIONAL EQUIPMENT Trueness: Results obtained with this reagent did not show systematic differences when
General laboratory equipment. compared with reference reagent.
Centrifug
Spectrophotometer or photometer with thermostated cuvette (37 ºC). Light path cuvette: INTERFERENCES
1 cm High faetal hemoglobin concentrations (HbF) will give abnormally high HbA1 values.
Likewise, abnormally low values can be obtained with samples with a high abnormal
hemoglobin content (HbC,HbS). The unstable fraction (aldimine) is eliminated during resin
SAMPLE mixing and does not contribute to the glycohemoglobin value.
Whole blood with EDTA as anticoagulant. Stable 1 week at 2-8º C. The method is independent of the working temperature, provided the assay is carried
out within a reasonable temperature range, 20 and 30 ºC. If the temperature during the
assay is far from this range the results can be erroneous.
CAUTION
Lysing reagent contains cyanide. Do not mix with acids. Wash hands after handling. REFERENCES
Trivelli, L.A., Ranney, H.M., Lai, H.(1971) The New Engl. J. Med., 284, 353-357.
The disposal of the residues has to be made according to local regulations. Gabbay, K. H., Hasty, K., Breslow, J. L., Ellison, R.C., Bunn, H. F., Gallop, P. M. (1977). J. Clin. Endocrinol.
Metabol., 44, 859-864.
Nathan, D.M., Singer, D.E., Hurxthal, K., Goodson, J.D., (1984) New Engl. J. Med.,310, 341-346.
QUALITY CONTROL Johnson, M.W., Dobrea, G.M., Bendezu, R., Wieland, R.G. (1980), Clin. Chim. Acta, 104, 319-328.
Control serum should be included in each test series. Each particular laboratory should establish its own Flückiger, R., Woodti, T., (1985) Clin. Chem., 31, 114-117.
control program. Tietz, NW., Textbook of Clinical Chemistry 5th Edition, W.B. Saunders, Philadelphia (2012).
ISO 9001 / ISO 13485

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GLYCOHEMOGLOBIN, CONTROL SET
For “in vitro” determination of HbA1 in blood
PRODUCT DESCRIPTION
The HbA1 control is a hemolysate prepared from packed erythrocytes. For the
quality control in the determination of glycohemoglobine in whole blood.
REAGENTS
Set of Controls Ref. 99 66 36
Contents:
A. 1 x 1 mL Freeze-dried Control. Low level Ref. 99 41 06
B. 1x 1 mL Freeze-dried Control. High level Ref. 99 88 07
REAGENT PREPARATION
Rehydrate the vial with 1mL of deionized water. Mix gently and let stand at room
temperature for 30 min. Swirl gently prior to each test, for homogenization.
REAGENT COMPOSITION
Freeze-dried hemolysate prepared from packed erythrocytes. Stabilizers and
preservatives are added to mantain HbA1 concentration. The concentration is
Spectrophotometer or photometer thermostatable at 37ºC. Cuvette: 1 cm light-
path.

The Controls, when stored at 2-8ºC, will remain stable until the expiration date
stated on the label. Once rehydrated, the control is stable for 2 weeks at 2-8ºC
or 8 weeks at -20ºC, when protected from the sunlight.
ASSIGNED VALUES
The concentration of each component is stated on the vial label as well as in the
To determine these values QCA glycohemoglobin reagent was used. The
expected range for each level is derived from interlaboratory data and they are
given for orientation only; each laboratory should establish its own range of
acceptance.
CAUTION
PROCEDURE
The freeze-dried HbA1 controls should be assayed in the same manner as blood
specimens including the hemolysate procedure of glycohemoglobin.
ISO 9001 / ISO 13485

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AUTOMATIC GLYCOHEMOGLOBIN (HbA1c)
IMMUNOTURBIDIMETRIC METHOD
For “in vitro” determination of HbA1c in blood
PRINCIPLE PROCEURE
Total haemoglobin and HbA1c have the same unspecific absortion rate to latex To determine HbA1c, prepare a hemolysate for every patient sample, calibrator and
particles. When mouse antihuman HbA1c monoclonal antibody is added, a latex- control.
HbA1c-mouse antihuman HbA1c antibody complex is formed. Agglutination is formed 1. Dispense 1 mL of Hemolysis reagent into labeled tubes.
Substrates
when goat anti-IgG polyclonal antibody interacts with the monoclonal antibody. The 2. Dispense 20 µL of whole blood (properly mixed), calibrator or control, into the
extent of agglutination is proportional to the amount of HbA1c absorbed on the surface corresponding labeled tubes with Hemolysis reagent.
of the latex particles. 3. Let stand for 5 min (lysis should be complete).
The method is based on latex - enhanced immunoturbidimetric analysis. Competitive Use the hemolysate as the sample. Hemolysates may be stored up to 10 days at
binding is included in a first reaction step that allows the %HbA1c to be directly 2-8ºC.
determined from the turbidity that develops during the second reaction.
Reagents must be used in clinical chemistry analyzers.
DIAGNOSTIC USE Use Reagent A and Reagent B directly in the analyzer.
The HbA1c determination is useful in monitoring and control of diabetic patients. Reagent A: 180µL
The concentration of this red blood cell protein is conditioned by the mean blood Reagent B: 60µL
glucose level, for a period of 4-8 weeks, for what it turns out as a test not influenced Sample: 5 µl
by occasional sudden fluctuations in the serum glucose concentration. Incubation time: Reagent A + sample, 300s
Single test result could not be used to make a clinical diagnosis. It should integrate Reaction time: Reagent A+ sample + Reagent B, 300s
Reading
REAGENTS Wavelenght: 660 nm
Kit 40 mL. (Ref. 99 50 25) Contents:
CALIBRATION
A. 1 x 30 mL. Latex reagent. Ref. 99 50 30 Blank reagent must be performed everyrun. Calibration should be performed at least every 5 days,
B. 1 x 10 mL. Buffer / Antibody reagent. Ref. 99 50 35 after reagent lot changing, or when quality control precedure requires it.
C. 2 x 70 mL. Hemolysis reagent. Ref. 99 50 45 Calibration. Calibration curve non lineal.
Preparing the calibration curve with the 4 calibrators of the kit, reference 99 97 20;
Optionally: each concentration values are indicated on the label. For the value of 0% use saline
Kit HbA1c Calibrator 4 x 0.5 mL Ref. 99 97 20 solution.
The HbA1c calibrator is a hemolysate prepared from erythrocytes. Stabilizers are
added to maintain HbA1c concentration. CALCULATION
The concentration of each level is stated on the vial label. Results are obtained from the calibration curve (Abs/%), prepared with the
corresponding calibrators.
Kit HbA1c Control 2 x 0.5 mL Ref. 99 90 96
The HbA1c control is a hemolysate prepared from erythrocytes. Stabilizers are added REFERENCE VALUES
to mantain HbA1c concentration. Less than 6% for a non-diabetic. Less than 7% for glicemic control of a patient with
The concentration is stated on the label. diabetes. There is a 3-4 week time lag before HbA1c reflects changes in blood
glucose level.
WORKING REAGENT PREPARATION Each particular laboratory should establish its own normal range, obtained from
Reagents, control and calibrator are ready to use. Mix gently before use. samples of a representative population, using its own instrumentation, blood
collection methods and assaying procedures.
Reagent A.
Performance of the reagent depends on the reagent itself and also depends on
Glycine buffer 22 mM
method and analyzer used.
Latex 0.13%
The results indicated are obtained using a automatic method.
Reagent B.
Mouse anti-human HbA1c monoclonal antibody 0.05 mg/mL
Sensitivity, as detection limit: 0.2%
Goat anti-mouse IgG polyclonal antibody 0.08 mg/dL
Reaction range: 2.0 – 16.0%
Buffer, surfactants and stabilizers.
Accuracy: 99.95%
Reagent C
Hemolysis agents and stabilizers in aqueous media.
The components of the kit, stored at 2-8ºC, will remain stable until the expiration date
Once opened, they will remain stable for al least one month at 2-8ºC.
The Calibrator and Control are stable up to the expiry date indicated on the label when
INTERFERENCES
kept at 2-8ºC. Reconstituted controls and calibrator are stable 1 month at 2-8 ºC.
No interference was observed by Ascorbic acid (50 mg/dL), Bilirubin (50 mg/dL), and
Lipids (2000 mg/dL).
Samples from patients with opiate addiction, lead poisoning, alcoholism or that ingest
Presence of particles or turbidity in the reagent B or C. Quality control values outside
large doses of aspirin, may give inconsistent results.
acceptation range.
Elevated levels of HbF may lead to underestimation of HbA1c: Hemoglobin variants
ADDITIONAL EQUIPMENT HbA2, HbC y HbS do not interfere.
Spectrophotometer or photometer thermostatable at 37ºC. Cuvette: 1 cm light-path. QUALITY CONTROL
Positive control of kit Control (99 90 96) should be included in each test series. Each
particular laboratory should establish its own control program.
SAMPLE
Venous blood with EDTA. HbA1c in whole EDTA blood is stable for one week at 2-8ºC.
AUTOANALYZERS
CAUTION
Red cells used in the controls and calibrators preparation have been found negative
in the reaction to HBsAg and HIV I/II. However they should always be handLed with REFERENCES
care. Avoid contact with skin and mucous membranes. Trivell,L.A., Ranney,H.M. and Lai,H.T., (1971) New Eng. J. Med. 284, 353.
The security statements are on the label. It is advisable to look at the MSDS before Nathan,D.M. et al, Clin. Chem. (1983), 29, 466-469.
using the reagent. Goldstein, D.E., et al, Clin. Chem. (1986) 32, B64-B70.
The disposal of the residues has to be made according to legal local regulations. Engbaek,F. Et al, Clin. Chem. (1989), 35, 93-97.
American Diabetes Association: Clinical Practice Recommendations. Diabetes Care
30 (Suppl. 1): S4-S41 (2007).
ISO 9001 / ISO 13485

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AUTOMATIC GLYCOHEMOGLOBIN
CALIBRATOR SET
PRODUCT DESCRIPTION
The HbA1c calibrator is a hemolysate prepared from packed erythrocytes for
the preparation of the standard curve for glycohemoglobin determination by
turbidimetry.
REAGENTS
Calibrator Set HbA1c Ref. 99 97 20
Contents:
A. 1 x 0.5 mL Calibrator 1 Ref. 99 97 22
B. 1 x 0.5 mL Calibrator 2 Ref. 99 97 24
C. 1 x 0.5 mL Calibrator 3 Ref. 99 97 26
D. 1 x 0.5 mL Calibrator 4 Ref. 99 97 28
REAGENT PREPARATION
Rehydrate the vial with 0.5 mL of deionized water. Mix gently and let stand at
room temperature for 10 min. Swirl gently prior to each test for homogenization.
REAGENT COMPOSITION
preservatives are added to mantain HbA1c concentration. The concentration of
the Calibrators is stated on the label.
path.
The Calibrators, when stored at 2-8ºC, will remain stable until the expiration date
stated on the label. Once rehydrated, the calibrator is stable for 1 month, when
kept at 2-8ºC.
ASSIGNED VALUES
CAUTION
PROCEDURE
The freeze-dried HbA1c calibrator should be assayed in the same manner
as blood specimens including the hemolysate procedure of Automatic
Glycohemoglobin.
ISO 9001 / ISO 13485

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AUTOMATIC GLYCOHEMOGLOBIN
CONTROL SET
PRODUCT DESCRIPTION
The HbA1c control is a hemolysate prepared from packed erythrocytes. For the
quality control in the determination of glycohemoglobin in whole blood.
Substrates
REAGENTS
HbA1c Control set Ref. 99 90 96
Contents:
A. 1 x 0.5mL Freeze-dried Control. Low level. Ref. 99 90 97
B. 1 x 0.5mL Freeze-dried Control. High level. Ref. 99 90 99
REAGENT PREPARATION
Rehydrate the vial with 0.5mL of deionized water.
Mix gently and let stand at room temperature for 10 min. Swirl gently prior to
each test, for homogenization.
REAGENT COMPOSITION
preservatives are added to maintain HbA1c concentration. The concentration is
path.
The controls, when stored at 2-8ºC, will remain stable until the expiration date
stated on the label. Once rehydrated, the control is stable for 1 month, when
kept at 2-8ºC.
ASSIGNED VALUES
To determine these values QCA Automatic Glycohemoglobin reagent was used.
The expected range for each level is derived from interlaboratory data and they
are given for orientation only; each laboratory should establish its own range of
acceptance.
CAUTION
PROCEDURE
The freeze-dried HbA1c controls should be assayed in the same manner as blood
specimens including the hemolysate procedure of Automatic Glycohemoglobin.
ISO 9001 / ISO 13485

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TOTAL PROTEIN
BIURET METHOD
For “in vitro” determination of total protein in serum or plasma
PRINCIPLE PROCEDURE
In alkaline solution, the proteins form with copper(II) ions a colored complex,
highly stable, which is spectrophotometrically measurable and proportional to the
Technique BL ST SA
concentration of protein in the sample.
mL mL mL
DIAGNOSTIC USE
The determination of total protein in serum is used in the study of the nutritional status Sample --- --- 0.02
or edematous processes.
Total protein levels are high (> 9.0g/dL) in: Hyperimmunoglobulinaemia, mono or Standard --- 0.02 ---
polyclonal gammopathy, dehydration, chronic liver disease and and neoplasms,
especially myeloma Reagent 1.00 1.00 1.00
Values lower than 6.0 g/dL are associated with protein loss in cases of
gastroenteropathies, burns, nephrotic syndrome or due to decreased protein Mix well and let stand for 10 min at room temperature (20-25ºC).
synthesis in chronic liver disease, malabsorption syndrome, agammaglobulinemia or Reading
malnutrition. Wavelength: 540 nm
In cases of pregnancy, administration of intravenous fluids, chronic alcoholism, heart Blank: BL contents
failure or hyperthyroidism, the blood protein values are also lower than normal. Colour stability: a minimum of 3 hours
CALCULATIONS
SA Abs
REAGENTS x 5 = g of protein / dL
ST Abs
A. 3 x 100 mL Biuret reagent Ref. 99 96 02
Where:
SA Abs: Sample absorbance
WORKING REAGENT PREPARATION ST Abs: Standard absorbance
SI Units
REAGENT COMPOSITION (g/dL) x 10 = g/L
NaOH 0.47 M REFERENCES VALUES

Potassium iodide 23.3 mM Adults: 6.4-8.3 g/dL
Copper (II) sulphate 6.5 mM Children
Sodium-potassium tartrate 22.1 mM Newborns: 4.6-7.0 g/dL
Preservatives and stabilizers < 1 year: 5.1 - 7.3 g/dL
Standard: Aqueous solution of protein equivalent to 5 g/dL (50 g/L). 1 - 2 years: 5.6 - 7.5 g/dL
> 3 years: 6,0 - 8.0 g/dL
The components of the kit, when kept at the temperature stated on the label, will The stated values are for guidance. Each particular laboratory should establish its
remain stable until the expiration date stated on the one. own reference range.
Signs of reagent deterioration: Performance of the reagent depends on the reagent itself and also depends on
Presence of particles or turbidity in the reagent. method and analyzer used.
Working reagent blank >0.300 The results indicated are obtained using a manual method.
General laboratory equipment. Sensitivity, as detection limit: 0.10 g/dL
Spectrophotometer or photometer thermostatable at 37ºC. Cuvette: 1 cm light-path. Linearity: Up to 12g/dL. For higher values, it is recommended to dilute the sample
with saline (NaCl 0.9%) and assay once again. Multiply the final result by dilution
factor.
CAUTION
Accuracy: 98.7 %.
It is advisable to read the SDS before the reagent manipulation. Reproducibility, as Variation Coefficient: 1.13%
The disposal of the residues has to be made according to local regulations. Trueness: Results obtained with this reagent did not show systematic differences
SAMPLE when compared with reference reagent.
Serum or plasma, free from haemolysis. The sample will remain stable for up to 5 Details of the performance studies are available on request
days, when kept at 2-8ºC.
When using other corporal fluids as sample (amniotic liquid, urine, exudates, etc) it INTERFERENCES
is advisable to take into account the margin values to test and the sensitivity of the No interferences are known
reagent. See Performance characteristics.
The use of disposable equipment is recommended to prevent unwanted contamination.
QUALITY CONTROL
Control serum, Seriscann Normal (Ref. 99 41 48) and Seriscann Anormal (Ref. 99 46
85) should be included in each test series. Each particular laboratory should establish
AUTOANALYZERS
REFERENCES
Wiechselbaum, T.E. (1946). Am. J. Clin. Pathol., 16, 40 - 49.
Gornall, A.G., Bardawill, C.J., David, M.M. (1948). Biol. Chem., 177, 751-766.
Peters, T. (1968). Clin. Chem., 14, 1147-1159.
(2012).
ISO 9001 / ISO 13485

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TRIGLYCERIDES LIQUID
GPO METHOD
For “in vitro” determination of triglycerides in serum plasma
PRINCIPLE
Triglycerides present in the sample are enzymatically hydrolysed by the action of lipases leading to glycerol and fatty acids. In presence of glycerol kinase (GK), the ATP phosphorylates glycerol to give glycerol-3-
phosphate and the corresponding ADP. By glycerophosphate oxidase (GPO), glycerol -3 - phosphate is oxidized to dihydroxyacetone phosphate and hydrogen peroxide.
In the last stage, with the peroxidase as a catalyst, hydrogen peroxide reacts with 4-aminoantipyrine and 4-chlorophenol to give quinoneimine. The intensity of the red colour is proportional to the amount of triglycerides
Substrates
present in the sample.
lipases
triglycerides → glycerol + fatty acids
GK
glycerol + ATP → glycerol-3 - phosphate + ADP
GPO
glycerol-3-phosphate + O2 → dihydroxyacetone-phosphate + H2O2
POD
2 H2O2 + 4-aminoantipyrine + 4-chlorophenol → quinoneimine + HCl + 4 H2O

The increased blood triglyceride level is a risk factor in the development of coronary heart disease. About
50% of the lipids of atheromatous lesions occurring in the coronary arteries are triglycerides, making it Bring the reagent and the analyzer to working temperature.
possible to relate to the pathogenesis of coronary arteriosclerosis.
The determination of triglycerides allows assessing the early risk of developing coronary atherosclerosis
The TGL may be increased by chylomicronemia (Fredrickson types I and V ) , type IV hypertriglyceridemia, Technique BL SA ST
diabetes mellitus, insulin resistance, obesity, hypothyroidism, pancreatitis, glycogen storage disease,
mL mL mL
nephrotic syndrome, hypertriglyceridemia sensitive to carbohydrates, Tangier’s disease , von Gierke’s
disease, pernicious anemia, acute pancreatitis , Down’s syndrome, biliary cirrhosis, septicemia. Standard --- --- 0.01
Single test result cannot be used to make a clinical diagnosis. The results are to be evaluated in the context Sample --- 0.01 ---
of all clinical and laboratory data obtained.
Reagent 1.00 1.00 1.00
REAGENTS
Kit 1 x 100 mL. (Ref. 99 23 30) Contents: Mix well and let stand 5 min at 37ºC or 10 min. at room temperature (15 - 25ºC)
B. 1 x 5 mL Standard Ref. 99 03 17 Reading
Kit 3 x 100 mL. (Ref. 99 23 20). Contents: Wavelenght: 546 nm; 505 nm
A. 3 x 100 mL Reagent Ref. 99 23 25 Blank: the contents of BL
B. 1 x 5 mL Standard Ref. 99 03 17 Colour stability: 1 hour, when protected from direct sunlight
A. 2x 250 mL Reagent Ref. 99 01 53 CALCULATIONS
B. 1 x 5 mL Standard Ref. 99 03 17 SA Abs
x 200 = mg of triglycerides/dL
WORKING REAGENT PREPARATION ST Abs
Where:
REAGENT COMPOSITION SA Abs: Sample absorbance
Concentration in the reagent is: ST Abs: Standard absorbance
Pipes buffer pH 6,8 50 mM
4-chlorophenol 4.2 mM S.I. Units
4-aminoantipyrine 0.35 mM (mg/dL ) x 0.01143 = mmol/L
ATP 2 mM
Magnesium aspartate 40 mM REFERENCES VALUES
Glycerol kinase ≥ 800 U/l According to the recommendations of the European Atherosclerosis Society, Europea Society of Cardiology
Glycerol-3-phosphate oxidase ≥ 2000 U/l and NCEP:
Peroxidase ≥ 500 U/l Normal: <150 mg/dL (<1,7 mmol/L)
Lipases ≥ 9000 U/l Borderline: 150 - 199 mg/dL (1,7 - 2,25 mmol/L)
Non reactive stabilizers High: 200 - 499 mg/dL (2,26 - 5,64 mmol/L)
Very high: >500 mg/dL (>5,64 mmol/L)
Standard: Solution of glycerol in water equivalent to 200 mg/dL (2.29 mmol/L)
STORAGE AND STABILITY Performance of the reagent depends on the reagent itself and also depends on method and analyzer used.
The components of the kit, stored at 2-8ºC, will remain stable until the expiration date stated on the label. The results indicated are obtained using a manual method.
Signs of reagent deterioration: Sensitivity, as detection limit: 3.0 mg/dL

Presence of particles or turbidity in the reagent. Working reagent blank >0.400 Linearity: Up to 1000 mg of Triglycerides/dl. Samples with a higher concentrations shall be diluted 1/10 with
ADDITIONAL EQUIPMENT NaCl 0.9% and assayed once again. Multiply the final result by 10.
General laboratory equipment. Accuracy: 98.5 %.
Spectrophotometer or photometer thermostatable at 37ºC. Cuvette: 1 cm light-path. Repetitivity as Variation Coefficient: 0.89%
Reproducibility as Variation Coefficient: 1.52%
SAMPLE
Serum, or plasma with EDTA or heparin. Triglycerides are stable 4 days at 2-8ºC, and up to three months
reference reagent.
at -20ºC
CAUTION
The reagent contains phenol derivatives. Handle with care. INTERFERENCES
The safety statements are on the label. It is advisable to look at the SDS before using the reagent. The assay is not interfered by hemoglobin (up to 150 mg/dL) nor bilirubin (up to 20mg/dL).
The disposal of the residues has to be made according to local regulations. The use of disposable equipment is recommended to prevent unwanted contamination.
QUALITY CONTROL REFERENCES
Control serum, Seriscann Normal (Ref. 99 41 48) and Seriscann Anormal (Ref. 99 46 85) should be Jacobs,N.J., VanDemark,P.J. (1960). J. Bacteriol.,79, 532 - 538.
included in each test series. Each particular laboratory should establish its own control program. Trinder,P. (1969). Ann.Clin. Biochem.,6, 24 - 27.
A policy statement of the European Atherosclerosis Society, European Heart Journal 8,(1987) 77 - 88.
AUTOANALYZERS Tietz, NW., Textbook of Clinical Chemistry 5th Edition, W.B. Saunders, Philadelphia (2012).
Adaptations to different autoanalyzers are available on request. CLSI Guidelines and Standards, CLSI, Wayne, P.A
National Cholesterol Educarion Program Expert Panel. Third report of the National Cholesterol Education
Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults
(ATP III), (2001) NIH Publication. Bethesda: National Heart, Lung, and Blood Institute.
Guidelines for the management of dyslipidaemias. (2016). European Heart Journal, 37, 2999-3058.
ISO 9001 / ISO 13485

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UREA ENZYMATIC
MODIFIED BERTHELOT – SEARCY METHOD
For “in vitro” determination of urea in serum, plasma or urine
PRINCIPLE
The hydrolysis of the urea present in the sample is catalyzed by urease producing ammonium and carbonate ions. In the presence of nitroprusside, the ammonium ions formed react with
salicylate and hypochlorite in basic medium, producing an indophenol derivative. Color intensity is proportional to the urea concentration in the sample.
urease
urea + H2O 2 NH4+ + CO32-
2 NH4+ + salicylate + hypochlorite+ nitroprusiate indophenol derivative
DIAGNOSTIC USE
Urea is the end product of protein metabolism. It is produced in the liver and eliminated QUALITY CONTROL
from the blood in the kidneys Urea determination, together with that of creatinine, allows Control serum, Seriscann Normal (Ref. 99 41 48) and Seriscann Anormal (Ref. 99 46 85) should
assessing kidney function.
Usually the elevated levels of blood urea reflect some alteration of the excretory function of
the kidney. This increase may also be due to poor liver function, cardiac failure or diet itself. AUTOANALYZERS
Dehydration and bleeding can raise levels of blood urea. Adaptations to different autoanalyzers are available on request.
and laboratory data. PROCEDURE
REAGENTS
Kit 5 x 100 mL. (Ref.99 36 48). Contents: Techique BL mL SA mL ST mL
A. 5 x 100 mL Urease/salicylate Ref. 99 21 04
B. 1 x 15 mL Alkaline hypochlorite Ref. 99 14 75 Standard -- -- 0.01
C. 1 x 5 mL Standard Ref. 99 02 41 Sample -- 0.01 --
D. White polyethylene bottle 100mL Ref. 99 70 73
Kit 2 x 100 mL. (Ref.99 70 71). Contents: Reagent A 1.00 1.00 1.00
A. 2 x 50 mL Urease/salicylate Ref. 99 70 72
Mix well and incubate 3 min/37ºC or 5 min at room temperature
B. 1 x 3 mL Alkaline hypochlorite Ref. 99 70 74
(20-25ºC).
C. 1 x 5 mL Standard Ref. 99 02 41
D. White polyethylene bottle 100mL Ref. 99 70 73 Reagent B 1.00 1.00 1.00
Mix well and incubate again 3 min/37ºC or 5 min at room temperature
(20-25ºC).
Ref 99 36 48:
Vial A. Pour the contents of the urease/salicylate vial into the bottle. Add while softly stirring,
Lecture
100 mL of deionized water.
Vial B. Dilute the contents of the alkaline hypochlorite vial up to 500 mL with deionized
Blank: The contents of the BL tube
water.
Colour stability: 4 hours
Vial C. Standard is ready to use.
Ref 99 70 71: CALCULATIONS
Vial A. Add 50mL of deionized water softly stirring in each vial until completely dissolution. SA Abs
Vial B. Pour the content of the alkalyne hypochlorite vial into the plastic bottle and add x 40 = mg urea/dL
deionized water up to 100mL. ST Abs
Vial C. Standard is ready to use.
Where:
ST Abs: Standard absorbance
The concentrations in the reagent A solution are: S.I. Units
Phosphate buffer pH 6.8 20 mM (mg/dL) x 0.1665 = mmol/L
Sodium salicylate 61 mM REFERENCES VALUES
Sodium nitroprusiate 3.4 mM Serum, plasma: 6 - 20 mg/dL urea
EDTA-Na2 1.34 mM >60 years: 8 - 23 mg/dL urea
Urease ≥ 23 U/mL Urine: 10 - 20 g/24h urea.
Stabilizers
Serum concentartions tend to be slightly higher in males than in females. The stated values
Concentrations of reagent B solution are: are for guidance. It is advisable that each laboratory determines its own reference values.
Alkaline hypochlorite 7.5 mM
NaOH 160 mM Results as BUN (Blood Urea Nitrogen)
mg/dL urea x 0.467 = mg/dL BUN
Standard: Aqueous solution of urea equivalent to 40 mg/dL (6.6 mmol/L)
STORAGE AND STABILITY Performance of the reagent depends on the reagent itself and also depends on method
The components of the kit, stored at 2-8ºC, will remain stable until the expiration date stated and analyzer used.
on the label. Once the urease/salicylate vial has been dissolved, will remain stable for 3 The results indicated are obtained using a manual method.
weeks, if stored protected from light at 2-8ºC. The alkaline hypochlorite solution will remain
stable for 8 months, if stored in the same way. Sensitivity, as detection limit: 2.0 mg/dL
Linearity: Up to 400 mg/dL of Urea. For higher values, dilute the sample 1/2 in deionised
Signs of reagent deterioration: water and assay once again. Multiply the final result by 2.
Presence of particles or turbidity in the reagent. Working reagent blank >0.500 Accuracy: 97.9 %.
ADDITIONAL EQUIPMENT Reproducibility, as Variation Coefficient: 2.05%
General laboratory equipment. Trueness: Results obtained with this reagent did not show systematic differences when
Spectrophotometer or photometer thermostatable at 37ºC. Cuvette: 1cm light-path compared with reference reagent.
SAMPLE
Serum, plasma and urine. Urea will remain stable in serum for at least 1 day at room
temperature (≤ 25ºC), 5 days at 2-8ºC and 6 months when frozen (-20º C). In urine, urea will INTERFERENCES
remain stable, when kept at 2-8ºC, for 5 days, provided that the pH value be lower than 4. Any glassware contamination by ammonium salts or ammonia should be avoided.
If a urine sample is to be assayed, it should be previously diluted 1/100 with deionised Serum samples should be free from haemolysis and turbidity. Fluoride as well as ammonium
water. Multiply the final result by 100. heparinate inhibit the reaction.
CAUTION The use of disposable equipment is recommended to prevent unwanted contamination.
Reagent B: In case of contact with the skin, mucose or eyes, wash thoroughly with water REFERENCES
and ask the physician. Foster, L.B., Hochholzer, J.M. (1971), Clin. Chem., 17, 921-925.
The reagent A contains sodium azide at 0.09%. Handle with care. Wilcox, A., Wallace, E.C., Sterling, R.E., David, H.A., Ware, A.G. (1966), Clin. Chem. 12,
The safety statements are on the label. It is advisable to look at the MSDS before using 151-157.
the reagent. Sampson E.J., Baird M.A.,Burtis C.A., Smith E.M., Witte D.L.,Bayse D.D. (1980), Clin.
The disposal of the residues has to be made according to legal local regulations. Chem. 26,816-826.
ISO 9001 / ISO 13485

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UREA U.V.
UREASE – GLDH METHOD
For “in vitro” determination of urea in serum, plasma or urine
PRINCIPLE
The hydrolysis of the urea present in the sample is catalyzed by urease producing ammonium and carbonate ions. Ammonium ions react with α-ketoglutarate by the action of
glutamate dehydrogenase, oxidizing the NADH to NAD +
The urea concentration present in the sample is proportional to the decrease in the concentration of NADH in the reaction.
Substrates
urease
Urea + 2H2O 2 NH4+ + CO32-
GLDH
2 α-ketoglutarate + 2 NH4+ + 2 NADH 2 glutamate + 2 NAD+ + 2H2O

Urea is the end product of protein metabolism; it is produced in the liver and eliminated
from the blood in the kidneys. The serum level of urea is used in conjunction with Let stand the reagents for 2-3 min.at 37ºC.
creatinine, for the evaluation of renal function. Technique ST mL SA mL
Usually the elevated levels of blood urea reflect some alteration of the excretory
function of the kidney. This increase may also be due to poor liver function, cardiac Sample -- 0.01
failure or diet itself. Dehydration and bleeding can raise levels of blood urea. Standard 0.01 --
Single test result could not be used to make a clinical diagnosis. It should integrate Working reagent 1.00 1.00
clinical and laboratory data. Mix well and transfer to the measuring cuvette. Read the absorbance at 30s (Abs1)
and after 1 min, read again (Abs2).
REAGENTS
A. 12 vials of Freeze-dried enzymes Ref. 99 16 00 Wavelength: 340 nm
B. 2 x 100 mL Buffer solution Ref. 99 27 11 Blank: Deionized water
C. 1 x 5 mL Standard Ref. 99 02 41 Cuvette: 1 cm light-path
WORKING REAGENT PREPARATION CALCULATIONS

Dissolve the contents of the enzymes vial A with the volume of buffer B stated on the Determine ΔAbs for each sample and the standard:
label. Mix gently. ΔAbs = Abs1 - Abs2.
Standard is ready-to-use.
ΔAbs SA
WORKING REAGENT COMPOSITION x 40 = mg urea / dL
ΔAbs ST
Tris-HCl buffer pH 7.8 100 mM
Where:
Sodium α-ketoglutarate 6 mM ΔAbs SA: Absorbance increase of sample
ADP 2 mM ΔAbs ST: Absorbance increase of standard
NADH 0.20 mM
EDTA 4 mM S.I. Units
Urease ≥ 4,000 U/L (mg/dL Urea) x 0.1665 = mmol/L Urea
GLDH ≥ 9,000 U/L
Stabilizers and preservatives REFERENCES VALUES
Standard: Aqueous solution of Urea equivalent to 40 mg/dL. (6.6 mmol/L). Serum: 10 - 50 mg/dl Urea; 1.7 - 8.3 mmol/L Urea
Urine: 20 - 35 g Urea/24 h.; 333 - 583 mmol Urea /24 h
The components of the kit, stored at 2-8ºC, will remain stable until the expiration date Each particular laboratory should establish its own normal range, obtained from
stated on the label. The rehydrated reagent is stable up to 6 weeks at 2-8ºC and 10 samples of a representative population, using its own instrumentation, blood collection
days at room temperature (≤ 25ºC). methods and assaying procedures.
Signs of reagent deterioration: Results as BUN (Blood Urea Nitrogen)

Presence of particles or turbidity. Working reagent blank < 1.00 Mg/dL Urea x 0.467 = mg/dL BUN
ADDITIONAL EQUIPMENT PERFORMANCE CHARACTERISTICS

General laboratory equipment. Performance of the reagent depends on the reagent itself and also depends on method
and analyzer used. The results indicated are obtained using a manual method.
SAMPLE Sensitivity, as detection limit: 2.0 mg/dL

Serum, plasma or urine. Urea will remain stable in serum for at least 1 day at room Linearity: Up to 300 mg/dL of Urea.
temperature (≤ 25º C), 4-5 days at 2-8ºC and 6 months when frozen (-20ºC). In urine, Accuracy: 98.6 %.
urea will remain stable, 4-5 days when kept at 2-8ºC, provided that the pH value be Repetitivity as Variation Coefficient: 1.46%
lower than 4. Reproducibility, as Variation Coefficient: 1.65%
If a urine sample is to be assayed, it should be previously diluted 1/100 with deionized Trueness: Results obtained with this reagent did not show systematic differences
water. Multiply the final result by 100. when compared with reference reagent.
CAUTION
The reagents contain sodium azide at 0.09%. Handle with care. INTERFERENCES
The safety statements are on the label. It is advisable to look at the SDS before using the reagent. Fluoride as well as ammonium heparinate inhibit the reaction. Serum samples should
The calibrator must be considered as a human sample, and, thus, potentially infectious. Use be free from hemolysis and turbidity.
adequate protection.
The use of disposable labware is recommended to prevent unwanted contamination.
REFERENCES
QUALITY CONTROL Christian, G.D., Knobloch, E., Purdy, W.C. (1965) Clin. Chem, 11, 700-707.
Control serum, Seriscann Normal (Ref. 99 41 48) and Seriscann Anormal (Ref. 99 46 85) should
Gutman, I., Bergmeyer, H. U.; (1974). Methods of Enzymatic Analysis, Ed. H. U.
Bergmeyer, Verlag Chemie, Academic Press. 2ª edición, 4, 1794-1798.
Sampson, E.J., Baird, M.A., Burtis, C.A., Smith, E.M., Witte, D. L. y Bayse, D. D.,
(1980) Clin. Chem., 26, 816-826
AUTOANALYZERS
ISO 9001 / ISO 13485

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UREA U.V. LIQUID
UREASE – GLDH METHOD
For “in vitro” determination of Urea in serum, plasma or urine
PRINCIPLE
The hydrolysis of the urea present in the sample is catalyzed by urease producing ammonium and carbonate ions. Ammonium ions react with α-ketoglutarate by the action of glutamate dehydrogenase,
oxidizing the NADH to NAD +
The urea concentration present in the sample is proportional to the decrease in the concentration of NADH in the reaction.
urease
urea + 2H2O → 2 NH4+ + CO32-
GLDH
2 α-ketoglutarate + 2 NH4+ + 2 NADH → 2 glutamate + 2 NAD+ + 2H2O

Urea is the end product of protein metabolism; it is produced in the liver and eliminated from Let stand the reagents for 2-3 min at 37ºC.
the blood in the kidneys. The serum level of urea is used in conjunction with creatinine, for the
evaluation of renal function. Birreagent technique ST SA
Usually the elevated levels of blood urea reflect some alteration of the excretory function of mL mL
the kidney. This increase may also be due to poor liver function, cardiac failure or diet itself.
Dehydration and bleeding can raise levels of blood urea. Buffer solution (A) 1.50 1.50
Sample -- 0.02
Single test result cannot be used to make a clinical diagnosis. It should integrate clinical and
laboratory data. Standard 0.02 --
Enzymes solution (B) 0.50 0.50
REAGENTS
A. 1 x 60 mL Buffer solution Ref. 99 56 72 Monorreagent technique ST SA
B. 1 x 20 mL Enzymes solution Ref. 99 71 20 mL mL
Sample -- 0.01
A. 3 x 50 mL Buffer solution Ref. 99 41 25 Standard 0.01 --
B. 1 x 50 mL Enzymes solution Ref. 99 41 30 Working reagent 1.00 1.00
Mix well and transfer to the measuring cuvette. Read the absorbance at 30 sec (Abs1)
and after 60 sec, read again (Abs2)
B. 1 x 100 mL Enzymes solution Ref. 99 60 80
Reading
Wavelenght: 340 nm
Blank: Deionized water
WORKING REAGENT PREPARATION Cuvette: 1 cm light-path
The components of the kit are ready-to-use. If a Monoreagent procedure is preferred, then the
reagents must be mixed in the ratio: 3 parts of reagent A (buffer solution) + 1 part of reagent B CALCULATIONS
(enzymes solution). Determine ΔAbs for each sample and the standard:
ΔAbs = Abs1 - Abs2
The concentrations in the reagent solution are: ΔAbs SA
Tris-HCl buffer pH 7,6 100 mM x 40 = mg urea/dL
Sodium α-ketoglutarate 9 mM ΔAbs ST
ADP 0.7 mM
NADH 0.18 mM Where:
Urease ≥ 7.000 U/L ΔAbs SA: Absorbance increase of sample
GLDH ≥ 2.000 U/L ΔAbs ST: Absorbance increase of standard
Standard: Aqueous solution of urea equivalent to 40 mg/dL (6.6 mmol/L). S.I. Units
(mg/dL urea) x 0.1665 = mmol/L urea
Results as BUN (Blood Urea Nitrogen)
the label. The monoreagent is stable 5 weeks at 2-8ºC and 4 week room temperature (≤25ºC).
mg/dL urea x 0.467 = mg/dL BUN
Signs of reagent deterioration: REFERENCES VALUES
Presence of particles or turbidity in the reagent. Serum: 10 - 50 mg/dL urea; 1.7 - 8.3 mmol/L urea
Working reagent blank < 1.00 Urine: 20 - 35 g urea/24 h; 333 - 583 mmol urea /24 h
Each particular laboratory should establish its own normal range, obtained from samples of a
representative population, using its own instrumentation, blood collection methods and assaying
procedures.
CAUTION PERFORMANCE CHARACTERISTICS
The reagents contain sodium azide at 0.09%. Handle with care. Performance of the reagent depends on the reagent itself and also depends on method and
The safety statements are on the label. It is advisable to look at the SDS before using the reagent. analyzer used. The results indicated are obtained using a manual method.
adequate protection. Sensitivity, as detection limit: 2.0 mg/dL
The disposal of the residues has to be made according to local regulations. Linearity: Up to 300 mg/dL of Urea.
Accuracy: 98.2 %.
SAMPLE
Serum, plasma or urine. Urea will remain stable in serum for at least 1 day at room temperature
(≤25ºC), 4-5 days at 2-8ºC and 6 months when frozen (-20ºC).
In urine, urea will remain stable, 4-5 days when kept at 2-8ºC, provided that the pH value be lower
than 4.
If a urine sample is to be assayed, it should be previously diluted 1/100 with deionised water.
INTERFERENCES
Fluoride as well as ammonium heparinate inhibit the reaction. Serum samples should be free from
QUALITY CONTROL hemolysis and turbidity.
Control serum, Seriscann Normal (Ref. 99 41 48) and Seriscann Anormal (Ref. 99 46 85) should The use of disposable equipment is recommended to prevent unwanted contamination.
REFERENCES
AUTOANALYZERS Christian, G.D., Knobloch, E., Purdy, W.C. (1965) Clin. Chem, 11, 700-707.
Adaptations to different autoanalyzers are available on request. Gutman, I., Bergmeyer, H. U.; (1974). Methods of Enzymatic Analysis, Ed. H. U. Bergmeyer,
Verlag Chemie, Academic Press. 2ª edición, 4, 1794-1798.
ISO 9001 / ISO 13485

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URIC ACID LIQUID
URICASE – POD METHOD
For “in vitro” determination of uric acid in serum, plasma or urine.
PRINCIPLE
The uric acid of the sample is degraded by the action of uricase to allantoin with the release of hydrogen peroxide. Quantification of hydrogen peroxide released is performed by Trinder
reaction, wherein a colored quinone compound is formed by reacting with 4-aminoantipyrine and the chromogen 3,5-dichloro-2-hydroxysulfonate in the presence of peroxidase (POD) .
The color produced in the reaction is proportional to the concentration of uric acid of the sample under optimal assay conditions.
Substrates
uricase
uric acid + H2O + O2 → allantoin + CO2 + H2O2
POD
2 H2O2 + 4-aminoantipyrine + 3,5-dichloro-2-hydroxy-sulfonate → colored quinonic derivative + 4 H2O

Elevated uric acid values are found in kidney disease, gout, hyperthyroidism, malignancies Bring the working reagent and the analyzer to 37ºC.
and myeloproliferative diseases.
Lower values than those usual ones are found in congenital disorders of metabolism as
xanthinuria and in cases of poor purine intake. BL ST SA
Technique
The usual range of values for healthy subjects is large, depending on the feed, age, sex, and mL mL mL
also, it seems to be related to inherited genetic variations. Sample -- -- 0.02
Single test result cannot be used to make a clinical diagnosis. It should integrate clinical Standard -- 0.02 --
Working Reagent 1.00 1.00 1.00
REAGENTS
Kit 1 x 100 mL. (Ref. 99 40 22). Contents: Mix well and let stand for 10 min at 37ºC, read absorbance.
A. 1 x 100 mL Reagent. Ref. 99 40 25 Reading:
B. 1 x 5 mL Standard. Ref. 99 02 63 Wavelength: 546, 505 nm.
Blank: the contents of BL.
Kit 3 x 100 mL. (Ref. 99 40 20). Contents: Colour stability: 30 min (When protected from direct sunlight)
A. 3 x 100 mL Reagent. Ref. 99 40 25
B. 1 x 5 mL Standard. Ref. 99 02 63 CALCULATIONS
SA Abs.
Kit 2 x 250 mL. (Ref. 99 40 15). Contents: x 5 = mg of Uric acid / dL
A. 2 x 250 mL Reagent. Ref. 99 01 48 ST Abs.
SI Units
(mg/dL) x 59.5 = μmol/L
The components of the kit are ready to use. REFERENCE VALUES
Serum and plasma: Men: 3,5 - 7,2 mg/dL; Women: 2,6-6 mg/dL; Children: 2-5 mg/dL
REAGENT COMPOSITION Urine 24 hours: 250 - 750 mg/dL.
Pipes buffer pH 7.0 100 mM Each particular laboratory should establish its own normal range, obtained from samples
3,5-dichloro-2-hydroxy-sulphonate 3.2 mM of a representative population, using its own instrumentation, blood collection methods and
4-aminoantipyrine 0.4 mM assaying procedures.
EDTA Na2·H2O 0.6 mM
K3Fe(CN)6 0.1 mM PERFORMANCE CHARACTERISTICS
Uricase ≥ 350 U/L The performance characteristics depend on the method used. It is recommended to
Peroxidase ≥ 1300 U/L calculate these data for each particular test protocol. These results have been obtained
Non reactive stabilizers using a manual method.
Standard: Aqueous solution of uric acid equivalent to 5 mg/dL. (297.5 μmol/L). Sensitivity, as detection limit: 0.04 mg/dL
Linearity: Up to 25 mg/dL. For higher values, it is recommended to dilute the sample 1/2 in
STORAGE AND STABILITY saline (NaCl 0.9%) and assay once again. Multiply the final result by 2.
The components of the kit, stored at 2-8ºC, will remain stable until the expiration date stated Accuracy: 105 %.
on the label. Protect from sunlight. Repetitivity, as Variation Coefficient: 0.7%
Signs of reagent deterioration: Reagent turbid or with visible particles. Trueness: Results obtained with this reagent did not show systematic differences when
Reagent blank: ≥ 0.400 compared with reference reagent.
ADDITIONAL EQUIPMENT Details of the performance studies are available on request
Spectrophotometer or photometer thermostatable at 37ºC. Cuvette: 1 cm light-path. INTERFERENCES
SAMPLE Haemoglobin concentrations higher than 100 mg/dL interfere the assay, as well as bilirubin
Serum, plasma or urine samples are stable 4 days at 2-8ºC. concentrations higher than 15 mg/dL.
Urine samples should be diluted 1/10 with deionised water prior to assay. The final result
should be multiplied by 10. QUALITY CONTROL
CAUTION control program.
AUTOANALYZERS
REFERENCES
Trinder,P. (1969). Ann.Clin.Biochem., 6, 24 - 27.
Trivedi, R., Rebar, L., Berta, E., Stong, L., (1978), Clin. Chem., 24, 1908-1911.
Fossati, P., Prencipe, L., Berti, G., (1980). Clin. Chem., 26, 227 - 231.
Klose, S., Stoltz, M., Munz, E., Portenhauser, R., (1978), Clin.Chem, 24, 250-255.
Desideri G., (2014), Europ. Rev. for Med. and Pharmac Scien.,18, 1295-1306.
ISO 9001 / ISO 13485

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URINE / CSF PROTEINS
BENZETHONIUM CHLORIDE METHOD
For “in vitro” determination of proteins in urine and CSF
PRINCIPLE PROCEDURE
At alkaline pH solution, the sample is incubated with EDTA, which denatures the proteins and
basically eliminates the possible interferences from magnesium ions. Then, benzethonium
chloride is added and produces with the protein turbidity at 505 nm. Technique BL SA ST
This turbidity is proportional to the protein concentration and it can be measured mL mL mL
spectrophotometrically. Sample -- 0.1 --
DIAGNOSTIC USE Standard -- -- 0.1

Cerebrospinal fluid (CSF) protein: Protein concentration in CSF allows evaluating the Reagent A 2.0 2.0 2.0
integrity of the blood brain barrier; high concentrations are generally associated with
inflammatory processes. Mix and incubate 5 min, at 37ºC. Read Abs1
High values are found in patients suffering from: bacterial meningitis, tuberculosis, brain
Reagent B 1.0 1.0 1.0
abscess, stroke, encephalitis and other degenerative processes that cause neurological
disease. Mix and incubate 1 min, at 37ºC. Read Abs2
Urine Protein: Protein concentrations in urine are high in case of kidney damage, as in Reading
cases of nephrotic syndrome, glomerulonephritis, renal failure and malignant renal tumours. Wavelength: 505nm
Blank: BL contents
and laboratory data. Semi-micro method
It can be carried out with half of the volumes above indicated
REAGENTS
Kit 1 x 150 mL. (Ref.99 30 30). Contents: CALCULATION
A. 1 x 100 mL Alkaline solution Ref. 99 30 32 ∆Abs = Abs2 – Abs1
B. 1 x 50 mL Benzethonium chloride Ref. 99 30 34
C. 1 x 5 mL Standard Ref. 99 23 05 SA ∆Abs
D. 1 x 5 mL Protein Control Ref. 99 58 95 x 100 = mg prot/dL
ST ∆Abs
The components of the kit are ready to use. Where:
SA ∆Abs: Sample absorbance
REAGENT COMPOSITION ST ∆Abs: Standard absorbance
Concentrations in the reagents solution are:
Reagent A To calculate mg prot./24 h: mg/L x urine liters 24 h = mg/24 h
NaOH 550 mM To express the results in µg/min, multiply the mg/ 24h by the factor 0.6944:
EDTA 76 mM (mg / 24 h) x 0.6944 = μg / min
Reagent B
Benzethonium chloride 35 mM NOTE: For a better accuracy, it is recommended to build a calibration curve, by preparing
Preservatives and surfactants dilutions from the standard of 100 mg/dL included in the kit, with the following points: 100,
50, 25, 12.5 mg/dL.
Standard: Aqueous protein solution equivalent to 100 mg of proteins/dL (1 g/L).
Protein control: Aqueous protein solution. See the concentration on the vial label. REFERENCES VALUES
CSF adults: 15 - 45 mg/dL children: 10 - 40 mg/dL
STORAGE AND STABILITY Urine < 100 mg/24h
When stored at 2 -8ºC, the components of the kit will remain stable until the expiration date
stated on the label. The stated values are for guidance. It is advisable that each laboratory determines its own
reference values.
Presence of particles or turbidity in the reagent. Working reagent blank >0.200 PERFORMANCE CHARACTERISTICS
Performance of the reagent depends on the reagent itself and also depends on method and
analyzer used.
The results indicated are obtained using a manual method.
Linearity: Up to 200 mg/dL. For higher concentration, sample should be diluted in saline
SAMPLE
solution and the final result has to be multiplied by the dilution factor used.
Urine collected for 24 h using the established procedure. Can be stored 8 days when
Accuracy: 96.7 %.
refrigerated at 2-8ºC.
Cerebrospinal fluid (CSF) is stable 4 days at 2-8ºC.
CAUTION Trueness: Results obtained with this reagent did not show systematic differences when
The reagents contain sodium azide at 0.09%. Handle with care. compared with reference reagent.
the reagent. The calibrator must be considered as a human sample, and, thus, potentially Details of the performance studies are available on request
The disposal of the residues has to be made according to local regulations. INTERFERENCES
Concentrations of haemoglobin up to 50 mg/dL, as well as bilirubin up to 45 mg/dL do not
QUALITY CONTROL interfere with the assay.
It is recommended to include controls, as the control included in the kit, in each test series Traces of detergent may alter the final result. Thus, it is recommended to work with very
for assuring the obtained values . clean material or plasticware to avoid any undiserable contamination.
Each particular laboratory should establish its own quality control program and measures Protect from direct sunlight.
for avoiding results deviations.
REFERENCES
AUTOANALYZERS
Iwata, J., Nishikaza, O., (1979), Clin. Chem., 25, 1317 – 1319.
Luxton, R.W., Patel, P., Keir, G., Thompson, E.J., (1989), Clin. Chem. 35, 1731 – 1734.
ISO 9001 / ISO 13485

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URINE / CSF PROTEINS
PYROGALLOL RED METHOD.
For the “in vitro” determination of proteins in urine and CSF
PRINCIPLE PROCEDURE
At pH 2.5 the formation of the complex between the pyrogallol red - molybdate and the
proteins produces a change in the absorbance at 600 nm. This change is proportional to the
protein concentration and can be measured spectrophotometrically. Technique BL SA ST
Substrates
mL mL mL
DIAGNOSTIC USE Standard -- -- 0.02
Protein concentration measures commonly CSF-brain barrier integrity. Higher concentrations
are associated with inflammatory processes. These values are increased in bacterial Sample -- 0.02 --
meningitis, tuberculosis, brain abscess, stroke, encephalomyelitis and other degenerative Reagent 1.00 1.00 1.00
processes that cause neurological disease.
High protein concentrations are found in urine when there is renal injury, such as nephrotic
syndrome, glomerulonephritis, renal failure and malignant renal tumors. High sensitivity assay
Technique BL SA ST
Single test result could not be used to make a clinical diagnosis. It should integrate clinical mL mL mL
Standard -- -- 0.01
REAGENTS
Kit 2 x 100 mL. (Ref. 99 21 00). Contents: Sample -- 0.01 --
A. 2 x 100 mL Pyrogallol red reagent Ref. 99 22 08
Reagent 1.00 1.00 1.00
C. 1 x 5 mL Protein control Ref. 99 58 95
Mix well and let stand for 10 min, at room temperature (20-25ºC). Read.
Reagent, control and standard are ready to use Reading
Wavelength: 600 nm (580 - 620 nm)
REAGENT COMPOSITION Blank: BL contents
Concentration in the reagent solution is: Colour stability: 30 minutes
Succinate buffer pH 2.5 60 mM
Pyrogallol red 0.4 mM CALCULATIONS
Sodium molybdate 0.1 mM a) CSF
Sodium oxalate 1.2 mM
Sodium benzoate 2.6 mM SA Abs.
x 100 = mg of prot/dL
Surfactants 1% ST Abs.
Standard: Aqueous solution equivalent to 100 mg of proteins /dL (1 g/L). Where:

Protein control: Aqueous protein solution. See the concentration on the vial label. Abs PR: Sample absorbance
Abs ST: Standard absorbance
b) Urine
SA Abs.
x 1000 x L urine/24 h = mg prot./24 h.
ST Abs.
General laboratory equipment. To express the results in μg/min, multiply the mg / 24 h by the factor: 0.6944:
Spectrophotometer or photometer thermostatable at 37ºC. Cuvette: 1 cm light-path. (mg / 24 h) x 0.6944 = μg / min
SAMPLE REFERENCES VALUES

Urine collected for 24 h using the procedure established. Can be stored 8 days when CSF.:
refrigerated at 2-8ºC. Adults: 15-45 mg/dL
Cerebrospinal fluid, (CSF) is stable 4 days at 2-8ºC. Children:10-40 mg/dL
Urine:
CAUTION Adults: <100 mg/24h
The safety statements are on the label. It is advisable to look at the SDS before using The stated values are for guidance. It is advisable that each laboratory determines its own
the reagent. The calibrator must be considered as a human sample, and, thus, potentially reference values.
The disposal of the residues has to be made according to local regulations. PERFORMANCE CHARACTERISTICS
QUALITY CONTROL using a manual method.
Control serum should be included in each test series. Each particular laboratory should
establish its own control program. Sensitivity, as detection limit: 4.0 mg/dL
Linearity: up to 200 mg/dl for the normal procedure. The high sensitivity assay is linear up to
AUTOANALYZERS 400 mg/dL, but higher imprecision can be obtained with normal Abs values.
Adaptations to different autoanalyzers are available on request. Accuracy: 95.5 %
Repetitivity, as Variation Coefficient: 2.68%
INTERFERENCES
No remarkable interferences are known.
Traces of detergent may alter the final result. Thus, it is recommended to work with very
clean material or plasticware to avoid any undiserable contamination. Protect from direct
sunlight.
REFERENCES
Mori, I., Fujita, Y., Fujita, K., Kitano, S., Ogawa, I., Kawabe, H., Koshiyama, Y., Tanaka, T.,
(1986), Bull. Chem. Soc. Jpn., 59, 955-957.
Watanabe, N., Kamei, S., Ohkubo, A., Yamanaka, M., Ohsawa, S., Makin, K., Tokuda, K.
(1986), Clin. Chem., 32, 1551-1554.
ISO 9001 / ISO 13485

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CALCIUM
O-CRESOLPHTHALEIN METHOD
For “in vitro” determination of calcium in serum, plasma or urine
PRINCIPLE PROCEDURE
At alkaline pH values, serum calcium forms a coloured complex with
Technique BL mL ST mL SA mL
o-cresolphthalein, 8-hydroxyquinolein is included in the reagent as a chelating
agent of magnesium ions which can interfere with the reaction. Sample -- -- 0.01
DIAGNOSTIC USE Standard -- 0.01 --
Calcium is the most abundant cation in the human body (99%) and is distributed
Working reagent 1.00 1.00 1.00
mainly in the bones.
High calcium levels are found in the primary and tertiary hyperparathyroidism,
Mix well and incubate at room temperature (20-25ºC) for 5 min.
cancer diseases (metastatic breast carcinoma, kidney or lung, leukemia,
multiple myeloma), vitamin D intoxication, increased renal retention, sarcoidosis, Reading
osteoporosis, thyrotoxicosis. Wavelength: 565 nm
Low calcium levels can be attributed to renal failure, hypoparathyroidism, Blank: BL contents
vitamin D deficiency, malnutrition or malabsorption. Colour stability: a minimum of 1 hour
Single test result could not be used to make a clinical diagnosis. It should CALCULATIONS
SA Abs
x 10 = mg de calcium / dL
REAGENTS ST Abs
SA Abs
A. 1 x 100 mL o-Cresolphthalein Ref. 99 99 36 x 10 x 2 x vol(dL) urine/24h = mg de calcio / 24h (urine)
B. 1 x 100 mL Colour developer Ref. 99 47 42 ST Abs
C. 1 x 5 mL Standard. Ref. 99 19 21 Where:
SA Abs: Sample Absorbance
WORKING REAGENT PREPARATION ST Abs: Standard Absorbance
The components of the kit are ready to use. To prepare working reagent, mix
equal volumes of each reagent (A and B). SI Units
The reagent composition is as follows: REFERENCE VALUES
2-amino-2-methyl-1-propanol 0.35 M Serum
o-cresolphthalein 0.04 mM Newborn: 8.0 – 13.0 mg/dL
8-hydroxyquinolein 12 mM Children: 8.8 – 12.0 mg/dL
HCl 2.5 mM Adults: 8.8 – 10.5 mg/dL
Preservatives and stabilizers Urine: 100 - 300 mg/24 h
Standard: Aqueous solution of calcium equivalent to 10 mg/dL (2.49 mmol/L).
The stated values are for guidance. Each particular laboratory should establish
and test procedures.
The components of the kit, when stored at 2 - 8ºC, will remain stable until the
The working reagent will remain stable 3 days at room temperature (≤ 25ºC) and
15 days at 2-8ºC
Signs of reagent deterioration: obtained using a manual method.
ADDITIONAL EQUIPMENT Sensitivity, as detection limit: 0.9 mg/dL
General laboratory equipment. Linearity:20 mg/dL (4.99 mmol/L).Samples that give higher concentration should
Spectrophotometer or photometer thermostatable at 37ºC. Cuvette: 1 cm light- be diluted in deionised water and the final result has to be multiplied by the
path. dilution factor.
Accuracy: 96.8%
SAMPLE
Serum or plasma with heparin or urine. Serum calcium remains unchanged for
10 days at 2-8ºC.
It is advisable acidifying the samples of 24 hr urine to avoid precipitation of
calcium and dilute with deionized water (1+1) prior to the determination. Multiply
the final result by 2.
CAUTION INTERFERENCES
The safety statements are on the label. Handle the reagent with care. Bilirubin concentrations higher than 20 mg/dL, and phosphates higher than
It is advisable to read the SDS before the reagent manipulation. 40 mg/dL, will interfere with the assay.
The disposal of the residues has to be made according to local regulations. Magnesium ions up to tenfold their normal upper limit in blood will not disturb
QUALITY CONTROL the final results.
Control serum, Seriscann Normal (Ref. 99 41 48) and Seriscann Anormal (Ref. Those anticoagulants which can behave as chelating agents shall never be
99 46 85) should be included in each test series. Each particular laboratory used with this test (EDTA, fluoride, oxalate...).
should establish its own control program. To avoid undesirable contaminations it is expressly recommended the use of
disposal plasticware.
AUTOANALYZERS
Adaptations to different autoanalyzers are available on request. REFERENCES
Kessler, G.,Wolfman, M. (1964) Clin. Chem.,10, 686- 703.
Connerty, H.V., Briggs, A.R. (1965) Am. J. Clin. Pathol., 45, 290 - 296.
Gitelman, H. J., 1967) Anal. Biochem., 18, 521 - 531.
Biggs, H.G., Moorehead, W.R. (1974). Clin. Chem., 20, 1458 - 1460.
Tiezt, NW., Textbook of Clinical Chemistry 5th Edition, W.B. Saunders,
ISO 9001 / ISO 13485

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CALCIUM-ARSENAZO III
COLORIMETRIC METHOD
For “in vitro” determination of calcium in serum, plasma or urine
PRINCIPLE PROCEDURE
At pH 8.5 arsenazo III (2,2’-[1,8-Dihydroxy-3,6-disulpho-2,7-naphthalene-bis-(azo)]
dibenzenearsonic acid) reacts with calcium ion to form a coloured complex. The
BL ST SA
intensity of colour developed is proportional to the calcium concentration in the sample. Procedure
mL mL mL
DIAGNOSTIC USE Sample -- -- 0,01
Calcium is the most abundant cation in the human body (99%) and is distributed
Standard -- 0,01 --
mainly in the bones.
High calcium levels are found in the primary and tertiary hyperparathyroidism, cancer Working reagent 1,00 1,00 1,00
diseases (metastatic breast carcinoma, kidney or lung, leukemia, multiple myeloma),
vitamin D intoxication, high renal retention, sarcoidosis, osteoporosis, thyrotoxicosis. Mix well and incubate at room temperature 20-25ºC for 3min. Read absorbance
Low calcium levels can be attributed to renal failure, hypoparathyroidism, vitamin D (AbsSA o AbsST) of the sample and the standard.
deficiency, bad nutrition and bad calcium absorption.
Single test result could not be used to make a clinical diagnosis. It should integrate Reading
clinical and laboratory data. Wavelength: 650 nm.
Electrolytes
Blank: BL contents.
REAGENTS Colour stability: a minimum of 1 hour.
A. 2 x 100 mL. Arsenazo III reagent Ref. 99 86 10 CALCULATIONS
B. 1 x 5 mL. Standard Ref. 99 87 19 SA Abs
x 10 = mg de calcium / dL
ST Abs
the components of the kit are ready to use. SA Abs
x 10 x 2 x vol(dL) urine/24h = mg de calcio / 24h (urine)
REAGENT COMPOSITION ST Abs
The reagent´s composition is as follows:
Borate buffer pH 8.5 50 mM Where:
Arsenazo III 0.1 mM SA Abs: Sample Absorbance
8-Hydroxyquinolein 12 mM ST Abs: Standard Absorbance
Tensoactives
Preservatives and stabilizers SI Units
Standard: Aqueous solution of calcium equivalent to 10 mg/dL (2.49 mmol/L). (mg/dL) x 0.2495 = mmol/L
STORAGE AND STABILITY REFERENCE VALUES

The components of the kit, when stored at room temperature (≤ 25ºC), will remain Newborn: 8.0 – 13.0 mg/dL
stable until the expiration date stated on the label. Children: 8.8 – 12.0 mg/dL
Adults: 8.8 – 10.5 mg/dL
Signs of reagent deterioration: Urine: 100 - 300 mg/24 h
ADDITIONAL EQUIPMENT The stated values are for guidance. Each particular laboratory should establish its
General laboratory equipment. own normal range, using its own instrumentation, blood collection methods and test
Spectrophotometer or photometer thermostatable at 37ºC. Cuvette: 1 cm light-path. procedures.

Serum or plasma with heparin or urine. Serum calcium remains unchanged for 10 The performance characteristics depend on the method used. It is recommended
days at 2-8ºC. to calculate these data for each particular test protocol. These results have been
It is advisable acidifying the samples of 24 hr urine to avoid precipitation of calcium obtained using a manual method.
and dilute with deionized water (1 +1) prior to the determination.
Linearity: 15 mg/dL (3.75 mmol/L). Samples that give higher concentration should be
CAUTION
diluted in deionised water and the final result has to be multiplied by the dilution factor.
Accuracy: 96.3%
QUALITY CONTROL Trueness: Results obtained with this reagent did not show systematic differences
Control serum, Seriscann Normal (Ref. 99 41 48) and Seriscann Anormal (Ref. 99 46 when compared with reference reagent.
85) should be included in each test series. Each particular laboratory should establish Details of the comparison experiments are available on request.
INTERFERENCES
AUTOANALYZERS Bilirubin concentrations higher than 20 mg/dL and phosphates higher than 40 mg/dL,
Adaptations to different autoanalyzers are available on request. will interfere with the assay.
Those anticoagulants which can behave as chelating agents shall never be used
with this test (EDTA, fluoride, oxalate...).
To avoid undesirable contaminations it is expressly recommended the use of disposal
plasticware.
REFERENCES
Bauer, P.J. (1981). Anal. Biochem., 110, 61 - 72.
Kratochvil, B., Xi-Wen-He. (1990) Can. J. Chem., 68,1932 -1935.
Jansen, J.W,. Helbing, A.R., (1991) Eur. J. Clin. Chem., 29,197-201.
Tiezt, NW., Textbook of Clinical Chemistry 5th Edition, W.B. Saunders, Philadelphia
(2012)
ISO 9001 / ISO 13485

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CHLORIDE
THIOCYANATE METHOD
For “in vitro” determination of chloride in serum, plasma CSF or urine
PRINCIPLE
Chloride ions from the sample react with mercury thiocyanate releasing thiocyanate ions. In the presence of ferric ions, free thiocyanate forms a colored complex which is quantified by
spectrophotometry.
2 Cl- + Hg(SCN)2 → HgCl2 + 2 SCN-

3 SCN- + Fe3+ → Fe(SCN)3

The chloride ion is necessary for proper metabolic functioning. It is also needed to maintain
acid-base balance.
Higher chloride values are associated with dehydration, renal disorders and cystic fibrosis. BL ST SA
Procedure
Low values can be detected in prolonged vomiting, excessive sweating and gastrointestinal mL mL mL
disorders. Sample -- -- 0.01
and laboratory data. Standard -- 0.01 --
REAGENTS Blank reagent 0.01 -- --
Kit 2 x 100 mL. (Ref.99 72 72). Contents:
A. 2 x 100 mL Thiocyanate reagent. Ref. 99 71 07 Reagent A 1.00 1.00 1.00
B. 1 x 5 mL Blank reagent. Ref. 99 72 44
C. 1 x 5 mL Standard. Ref. 99 71 97 Mix and let stand for 5 min. at room temperature (20-25ºC).
the components of the kit are ready to use. Reading
Wavelength: 450 nm.
REAGENTS COMPOSITION Blank: Reagent A. (Thiocyanate)
The reagent composition is as follows: Colour stability: a minimum of 1 hour when protected from direct sunlight.
Reagent A.
Mercuric thiocyanate 0.4 mM CALCULATIONS
Iron (III) nitrate 21.8 mM SA Abs. - BL Abs.
Stabilizers x 100 = mEq Cl- / L
ST Abs. - BL Abs.
Reagent B.
Where:
NaCl 30.7 mM
SA Abs: Sample absorption
Standard: Aqueous solution of Chloride equivalent to 100 mEq/L (100 mmol/L). ST Abs: Standard absorption
BL Abs: BL tub absorption
When stored at room temperature (≤ 25ºC), the components of the kit will remain stable until S.I. Units
the expiration date stated on the label. Protect them from direct sunlight. mEq/L = mmol/L
REFERENCES VALUES
Serum: 95 - 108 mEq/L.
L.C.R.: 119 - 130 mEq/L.
ADDITIONAL EQUIPMENT Urine: 110 - 225 mEq/24 horas.
General laboratory equipment. Each particular laboratory should establish its own reference range.
Serum, plasma with heparin, C.S.F. or urine. Samples can be stored at 2-8ºC. for up to 1 The performance characteristics depend on the method used. It is recommended to
week. calculate these data for each particular test protocol. These results have been obtained
CAUTION
The reagents contain Sodium azide and the reagent A contains mercuric thiocyanate . Sensitivity, as detection limit: 4 mEq/L
Handle with care and use security pipettes. Linearity: 140 mEq/L). Samples that give higher concentration should be diluted in deionised
The safety statements are on the label. It is advisable to look at the SDS before using the water and multiply the result by the dilution factor.
reagent.The disposal of the residues has to be made according to local regulations. Accuracy: 97.7%
QUALITY CONTROL Repetitivity, as CV%: 1.82%
Control serum, Seriscann Normal (Ref. 99 41 48) and Seriscann Anormal (Ref. 99 46 85) Reproducibility, as CV%: 2.52%
should be included in each test series. Each particular laboratory should establish its own Trueness: Results obtained with this reagent did not show systematic differences when
control program. compared with reference reagent.
AUTOANALYZERS
INTERFERENCES
Turbid, hemolyzed or icteric serum samples will give abnormally high values. Bromide
interferes with the reaction.
Chloride traces in the glassware can alter the final result. It is advisable using disposable
plasticware.
REFERENCES
Skeggs, L.T., Hochstrasser, H.C. (1964). Clin. Chem., 10, 918-936.
Young DS. Effects of drugs on Clinical Lab. Tests, 4th ed AACC Press, 1995.
Young DS. Effects of disease on Clinical Lab. Tests, 4th ed AACC 2001.
Burtis A et al. Tietz Textbook of Clinical Chemistry, 3rd ed., AACC, 1999.
Tietz N W et al. Clinical Guide to Laboratory Tests, 3rd ed., AACC, 1995.
ISO 9001 / ISO 13485

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COPPER
COLORIMETRIC METHOD
For “in vitro” determination of copper in serum or plasma
PRINCIPLE PROCEDURE
In acid solution (pH 4.7), copper (II) is released from the ceruloplasmin protein
and reduced to copper (I). The cuprous ion reacts with the 3,5 dibromo-2-
Procedure BL ST SA
pyridylazo-N-ethyl-N (-3-sulfopropyl) -aniline (3,5-diBr-PAESA), and forms a
mL mL mL
stable coloured complex, which is photometrically measured at 582 nm.
The colour intensity is directly proportional to the concentration of copper ions Sample -- -- 0.05
present in the sample. Standard -- 0.05 --
DIAGNOSTIC USE Reagent 1.00 1.00 1.00
Serum copper levels increase in hemochromatosis, biliary cirrhosis, leukemia,
Hodgkin’s disease, anemia, rheumatoid arthritis, hypothyroidism, malignancy, Mix well and let stand 5 min / 37ºC
collagen disease, among others. Read absorbance of the sample and the standard.
The serum copper decreases in Wilson’s disease, Menkes syndrome, nephrotic
syndrome and burns. Reading
Wavelength: 582 nm (570 – 590 nm)
Electrolytes
The determination of levels of copper in the urine is used in the study of Wilson’s
disease (hepatolenticular degeneration) Blank: Contents of BL
Colour stability: 60 min
A single test result could not be used to make a clinical diagnosis. It should
integrate clinical and laboratory data. CALCULATIONS
SA Abs.
REAGENTS x 100 = µg of copper / dL
ST Abs.
Where:
Reagent and standard are ready to use
SI Units
(µg/dL) x 0.157 = µmol/L
REAGENT COMPOSITION
REFERENCES VALUES
Acetate buffer pH 4.7 160 mM
Men: 70 – 140 µg /dL
3,5-di Br-PAESA 0.2 M
Women: 80 – 155 µg /dL
Surfactants 1%
Preservatives and stabilizers
The stated values are for guidance. It is advisable that each laboratory
determines its own reference values.
Standard: Solution of copper in water/HCl equivalent to 100 µg/dL (15.7 µmol/L).
When stored at room temperature (≤ 25ºC), the components of this kit will
remain stable until the expiration date stated on the label.
Presence of particles or turbidity in the reagent. Working reagent blank > 0,300. Sensitivity, as detection limit: 2 µg/dL
Linearity: 500 µg/dL (78.5 µmol/L). Samples that give higher concentration
Note should be diluted in saline solution (1+1) and the final result has to be multiplied
The reagent may precipitate when stored at low temperature. If so, warm gently per 2.
at 37ºC for a few minutes. Accuracy: 95.2%
ADDITIONAL EQUIPMENT Reproducibility, as CV%: 2.96%
General laboratory equipment. Trueness: Results obtained with this reagent did not show systematic differences
Spectrophotometer or photometer thermostatable at 37ºC. Cuvette: 1 cm light-path. when compared with reference reagent.
SAMPLE INTERFERENCES
Serum, non-hemolyzed plasma. Hemolyzed serum samples should be discarded. It is recommended to
use disposable plasticware to run the test to avoid any possible undesirable
CAUTION contamination.
QUALITY CONTROL
AUTOANALYZERS
REFERENCES
Abe, A., Yamashita, S., Noma, A., (1989) Clin. Chem., 35, 552 – 554.
ISO 9001 / ISO 13485

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INORGANIC PHOSPHORUS U.V.
FISKE – SUBBAROW METHOD
For “in vitro” determination of phosphorus in serum, plasma or urine
PRINCIPLE PROCEDURE
The phosphate ion reacts with molybdate to produce phospho-molybdate which is finally
reduced to a molybdenum blue, which is photometrically measured in the UV range.
Procedure BL ST SA
DIAGNOSTIC USE mL mL mL
Serum phosphate concentration, together with serum calcium concentration, is determined Sample -- -- 0.01
by the equilibrium that occurs between the absorption and excretion by the kidneys and the
intestine and formation of bone tissue and cellular metabolism. Approximately 85% of the Standard -- 0.01 --
phosphorus in the body is in bone and teeth
Reagent 1.00 1.00 1.00
High levels of phosphorus are found in cases of renal failure, hypoparathyroidism and
vitamin D intoxication.
Mix well and let stand for 10 min at room temperature (20-25ºC)
Lower than usual values can be found in patients with alcoholism, or subject to parenteral
Read absorbance of the sample and the standard
nutrition
Reading
Wavelength: 340 nm
Blank: BL contents
REAGENTS Colour stability: a minimum of 1 hour
A. 3 x 100 mL Reagent Ref. 99 10 67 CALCULATIONS
B. 1 x 5 mL Standard Ref. 99 00 20 SA Abs.
WORKING REAGENT PREPARATION x 4 = mg of phosphorus/dL
The components of the kit are ready to use ST Abs.
Where:
REAGENT COMPOSITION
ST Abs: Standard absorbance
Ammonium molybdate 12 mM
S.I. Units
Sulfuric acid 2.2 N
REFERENCES VALUES
Standard: Aqueous solution of phosphorus equivalent to 4 mg/dL (1.29 mmol/L).
Sera:
STORAGE AND STABILITY Children up to 15 years: 4.0 – 7.0 mg/dL
When stored at 2 -8ºC, the components of the kit will remain stable until the expiration date Adults: 2.5 – 5.0 mg/dL
Urine: 0.3 – 1.0 g/24 hours
Presence of particles or turbidity in the reagent The stated values are for a guidance. Each particular laboratory should establish its own
Working reagent blank >0.300 reference range.
ADDITIONAL EQUIPMENT PERFORMANCE CHARACTERISTICS
General laboratory equipment. The performance characteristics depend on the method used. It is recommended to
Spectrophotometer or photometer thermostatable at 37ºC. Cuvette: 1 cm light-path. calculate these data for each particular test protocol. These results have been obtained
Serum, heparinised plasma or urine.
Do not use plasma obtained with oxalate, citrate or EDTA, because can interfere the test. Sensitivity, as detection limit: 0.2 mg/dL
When assaying urine samples, they shall be diluted 1/20 in deionized water prior to testing. Linearity: 15 mg/dL (4.84 mmol/L). Samples that give higher concentration should be diluted
Final result will be, therefore, multiplied by 20. in deionised water (1+1) and the final result has to be multiplied per 2.
Accuracy: 96.4%
Serum phosphate remains stable one week when kept at 2-8ºC. In urine and at an acid pH,
phosphate will become stable for up to 3 months if the sample is kept at 2-8ºC. Trueness: Results obtained with this reagent did not show systematic differences when
CAUTION compared with reference reagent.
The reagents contain sodium azide at 0.09%. Handle with care. Details of the performance studies are available on request.
the reagent. The calibrator must be considered as a human sample, and, thus, potentially INTERFERENCES
infectious. Use adequate protection. Lipemic and hemolyzed sera will interfere with the assay.
The disposal of the residues has to be made according to local regulations. Detergents currently used in the laboratory contain phosphates and other substances that
may interfere with the reaction. Therefore it is recommended to rinse the glassware with
diluted nitric acid and finally with deionized water.
It is advisable to use disposable plasticware to run the test to avoid any possible
contamination.
QUALITY CONTROL
control program.
AUTOANALYZERS
REFERENCES
Fiske, C. H., Subbarow, (1925). J. Biol. Chem., 66, 375 - 400.
Goodwin, J.F., (1970) Clin. Chem., 16 (9), 776 -780.
Burtis A. et al. Tietz Textbook of Clinical Chemistry, 3rd ed. AACC 1999.6.
Tietz N W et al. Clinical Guide to Laboratory Tests, 3rd ed. AACC 1995.
ISO 9001 / ISO 13485

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INORGANIC PHOSPHORUS VISIBLE
FISKE – SUBBAROW METHOD
For “in vitro” determination of phosphorus in serum, plasma or urine
PRINCIPLE PROCEDURE
Phosphate ion reacts with molybdate to produce phospho-molybdate, which is
finally reduced to molybdenum blue, which is photometrically measured.
Procedure BL ST SA
mL mL mL
DIAGNOSTIC USE
Serum phosphate concentration, together with serum calcium concentration, is Sample -- -- 0.05
determined by the equilibrium that occurs between the absorption and excretion Standard -- 0.05 --
by the kidneys and the intestine and formation of bone tissue and cellular
metabolism. Approximately 85% of the phosphorus in the body is in bone and Reagent 1.00 1.00 1.00
teeth.
High levels of phosphorus are found in cases of renal failure, hypoparathyroidism Mix well and let stand for 10 min at room temperature (20-25ºC)
and vitamin D intoxication.
Lower than usual values can be found in patients with alcoholism, or subject to Reading
parenteral nutrition Wavelength: 650 nm
Blank: BL contents
Electrolytes
Single test result could not be used to make a clinical diagnosis. It should Colour stability: a minimum of 2 hours
CALCULATIONS
REAGENTS
Kit 3 x 100 mL. (Ref. 99 44 90). Contents: SA Abs
x 4 = mg of phosphorus/dL
A. 3 x 100 mL Fiske-Subbarow reagent Ref. 99 44 16 ST Abs
Where:
WORKING REAGENT PREPARATION SA Abs: Sample absorbance
Reagent and standard are ready to use ST Abs: Standard absorbance
REAGENT COMPOSITION S.I. Units

The reagent composition is as follows: (mg/dL) x 0.3229 = mmol/L
Ammonium molybdate 7 mM
Sulfuric acid 1.7 N REFERENCES VALUES
Iron (II) sulphate 8 mM Sera:
Preservatives and stabilizers Children up to 15 years: 4.0 – 7.0 mg/dL
Adults: 2.5 – 5.0 mg/dL
Standard: Aqueous solution of phosphorus equivalent to 4 mg/dL (1.29 mmol/L)
Urine: 0.3 – 1.0 g/24 h
When stored at 2 -8ºC, the reagent will remain stable until the expiration date The stated values are for a guidance. Each particular laboratory should establish
stated on the label. Likewise, the standard will remain stable until the expiration its own reference range.
date stated on the label, if kept at room temperature (≤ 25ºC).
Signs of reagent deterioration: The performance characteristics depend on the method used. It is recommended
Presence of particles or turbidity in the reagent. Working reagent blank >0.300. to calculate these data for each particular test protocol. These results have been
General laboratory equipment. Sensitivity, as detection limit: 0.2 mg/dL
Spectrophotometer or photometer thermostatable at 37ºC. Cuvette: 1 cm light- Linearity: 15 mg/dL (4.84 mmol/L). Samples that give higher concentration should
path. be diluted in deionised water (1+1) and the final result has to be multiplied per 2.
SAMPLE Accuracy: 96.6%
Serum, heparinised plasma or urine. Repetitivity, as CV%: 2.36%
Do not use plasma obtained with oxalate, citrate or EDTA, because can interfere Reproducibility, as CV%: 2.82%
the test. Trueness: Results obtained with this reagent did not show systematic differences
When assaying urine samples, they shall be diluted 1/20 in deionized water prior when compared with reference reagent.
to testing. Final result will be, therefore, multiplied by 20. Details of the performance studies are available on request.
Serum phosphate remains stable one week when kept at 2-8ºC. In urine and at INTERFERENCES
an acid pH, phosphate will become stable for up to 3 months if the sample is Lipemic and hemolyzed sera will interfere with the assay.
kept at 2-8º C. Detergents currently used in the laboratory contain phosphates and other
substances that may interfere with the reaction. Therefore it is recommended
PRECAUCIONES to rinse the glassware with diluted nitric acid and finally with deionized water.
Los reactivos contienen azida sódica al 0,09%, manipular con precaución. It is advisable to use disposable plasticware to run the test to avoid any possible
Las indicaciones de seguridad se encuentran en la etiqueta de los productos. contamination.
El calibrador debe considerarse como una muestra humana y por lo tanto
potencialmente infeccioso. Utilizar protección adecuada. Se aconseja consultar AUTOANALYZERS
la ficha de datos de seguridad antes de la manipulación del reactivo. La Adaptations to different autoanalyzers are available on request.
eliminación de residuos debe hacerse según la normativa local vigente.
REFERENCES
CONTROL DE CALIDAD Fiske, C. H., Subbarow, Y., (1925) J. Biol. Chem., 66, 375 - 400.
Es recomendable la inclusión de sueros control, Seriscann Normal (Ref. 99 Goodwin, J.F., (1970) Clin. Chem., 16 (9), 776 -780.
41 48) y Seriscann Anormal (Ref. 99 46 85) en cada proceso de medida para
verificar los resultados.
Se aconseja que cada laboratorio establezca su propio programa de control
de calidad y los procedimientos de corrección de las desviaciones detectadas.
ISO 9001 / ISO 13485

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IRON - CAB
CHROMAZUROL B METHOD
For “in vitro” determination of iron in serum or plasma
PRINCIPLE PROCEDURE
Iron reacts with Chromazurol B and cetyltrimethyl-ammonium bromide (CTMA) to form
a coloured ternary complex with an absorbance maximum at 620 nm. The intensity of
Technique BL ST SA
the colour produced, is directly proportional to the concentration of iron in the sample.
mL mL mL
DIAGNOSTIC USE Sample -- -- 0.05
Iron is distributed in the body forming part of hemoglobin and myoglobin.
Standard -- 0.05 --
Low iron concentations are found in infectious diseases and anemia. High levels of
iron are presented in liver disease, hemochromatosis, and at high levels of Transferrin. Reagent 1.00 1.00 1.00
Mix well and incubate 10-15 min at room temperature (20-25ºC).
Single test result could not be used to make a clinical diagnosis. It should integrate
Reading
REAGENTS Wavelength: 620 nm
Kit 2 x 100 mL. (Ref. 99 11 83). Contents: Blank: Contents of BL
A. 2 x 100 mL CAB reagent Ref. 99 12 00 Colour stability: 90 min
CALCULATIONS
Reagent and standard are ready to use. SA Abs
x 200 = μg de hierro / dL
ST Abs
REAGENT COMPOSITION
The reagent´s composition is as follows: Where:
Sodium acetate buffer pH 4.7 50 mM ST Abs: Standard Absorbance
CAB 0.16 mM
CTMA 2.5 mM SI Unit
Guanidinium chloride 2.1 M (μg/dL) x 0,1791 = μmol/L
REFERENCES VALUES
Standard: Aqueous solution of iron equivalent to 200 μg/dL (35.8 μmol/L). Men: 60 - 160 µg/dL
Women: 37 - 145 µg/dL
WARNING
The stated values are for guidance. It is advisable that each laboratory determines its
The standard included in the kit has been adjusted for the
own reference values.
reagent. If desired you can use a serum-based calibrator.
(QCA Ref. 99 62 80). PERFORMANCE CHARACTERISTICS
When stored at room temperature (≤ 25ºC), the components of this kit will remain Sensitivity, as detection limit: 15 µg/dL
stable until the expiration date stated on the label. Once opened the CAB reagent is Linearity: 500 µg/dL. Samples that give higher concentration should be diluted in
stable for 6 weeks at room temperature (≤ 25ºC). deionised water (1+1) and the final result has to be multiplied per 2.
Accuracy: 104.2%
Signs of reagent deterioration: Repetitivity, as CV%: 2.24%
Presence of particles or turbidity in the reagent. Working reagent blank > 0.600. Reproducibility, as CV%: 1.10%
ADDITIONAL EQUIPMENT when compared with reference reagent.
Spectrophotometer or photometer thermostatable at 37ºC. Cuvette: 1 cm light-path. Details of the performance studies are available on request
SAMPLE
INTERFERENCES
Non hemolyzed serum or plasma.
Hemolyzed serum samples should be discarded.
Serum iron is stable for up to 7 days at 2-8ºC.
Concentrations of bilirubin up to 15 mg/dL and copper up to 500 µg/dL do not interfere
with the assay.
CAUTION
It is recommended to use disposable plasticware to run the tests to avoid any possible
undesirable contamination.
Do not introduce pipettes into the reagent bottle, to avoid any possible undiserable
contamination.
QUALITY CONTROL
Control serum, Seriscann Normal (Ref. 99 41 48) and Seriscann Anormal (Ref. 99 46
REFERENCES
85) should be included in each test series. Each particular laboratory should establish
Ihara, K., Hasegawa, S., Naito, K. (2003) Anal. Sci., 19, 265-268.
Tabacco, A., Moda, E., Tarli, P., Veri, P., (1981) Clin. Chim. Acta, 114, 287-290.
Brivio, G., Brega, A., Torelli, G., (1986) La Ricerca Clin. Lab. 16, 523-532.
AUTOANALYZERS
ISO 9001 / ISO 13485

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IRON - FERROZINE®
COLORIMETRIC METHOD
For “in vitro” determination of iron in serum or plasma
PRINCIPLE PROCEDURE
In a slightly acid medium and in the presence of guanidine hydrochloride, iron is released
from transferrin and reduced by hydroxylamine from Fe (III) to Fe(II).
Divalent iron forms a coloured complex with FerroZine®, quantifiable spectrophotometrically. Procedure BR BSA SA ST
mL mL mL mL
(FerroZine®: Hach Chemical Co., Ames, IOWA, U.S.A).
Deionized water 0.20 -- -- --
DIAGNOSTIC USE Standard -- -- -- 0.20
Iron is distributed in the body forming part of hemoglobin and myoglobin.
Sample -- 0.20 0.20 --
Low iron concentations are found in infectious diseases and anemia. High levels of iron are
presented in liver disease, hemochromatosis, and at high levels of Transferrin. Reagent A -- 1.00 -- --
Working reagent 1.00 -- 1.00 1.00
Mix and incubate 10 min at room Temp. (20 - 25ºC) or 5 min at 37º C
REAGENTS
Electrolytes
A. 2 x 100 mL Buffer solution Ref. 99 06 20 Wavelength: 578 nm, 562 nm
B. 1 x 15 mL Colour reagent Ref. 99 04 12 Blank: deionized water
C. 1 x 5 mL Standard Ref. 99 02 90 Colour stability: 30 min

The components of the kit are ready to use. To prepare working reagent, add 1.5 mL
of colour reagent (Reagent B) to 40 mL of buffer solution (Reagent A). SA Abs - (BSA Abs + BR Abs)
x 200 = µg/dL
WORKING REAGENT COMPOSITION ST Abs - BR Abs
Acetate buffer pH 4.9 200 mM BSA Abs: Sample blank absorbance
Guanidine hydrochloride 3.4 M BR Abs: Blank reagent absorbance
Hydroxylamine hydrochloride 58 mM ST Abs: Standard absorbance
FerroZine 1.5 mM
Preservatives and stabilizers Note: A sample blank (BSA) is included to eliminate possible interference from sample
Standard: Aqueous solution of iron equivalent to 200 μg/dL (35.8 μmol/L). turbidity.
When stored at 2-8ºC the components of the kit will remain stable until the expiration date SI Units
stated on the label. (μg/dL) x 0.1791 = μmol/L
The working reagent will remain stable for 3 months at room temperature (≤ 25ºC) and for
REFERENCES VALUES
4 months at 2-8ºC.
Men: 60 - 160 µg/dL
Women: 37 - 145 µg/dL
The stated values are for guidance. It is advisable that each laboratory determines its own
reference values.
SAMPLE The performance characteristics depend on the method used. It is recommended to
Non hemolyzed serum or plasma. calculate these data for each particular test protocol. These results have been obtained
Serum iron is stable for up to 7 days at 2-8ºC. using a manual method.
CAUTION Sensitivity, as detection limit: 18 µg/dL

The safety statements are on the label. Handle the reagent with care. Linearity: 1000 µg/dL (179 µmol/L). Samples that give higher concentration should be
It is advisable to read the SDS before the reagent manipulation. diluted in deionised water (1+1) and the final result has to be multiplied per 2.
The disposal of the residues has to be made according to local regulations. Accuracy: 97.3%
Control serum, Seriscann Normal (Ref. 99 41 48) and Seriscann Anormal (Ref. 99 46 85) Reproducibility, as CV%: 2.39%
should be included in each test series. Each particular laboratory should establish its own Trueness: Results obtained with this reagent did not show systematic differences when
control program. compared with reference reagent.
AUTOANALYZERS
Adaptations to different autoanalyzers are available on request. INTERFERENCES
Do not introduce pipettes into reagents bottles, to avoid any possible undiserable
contamination.
In case of accidental contamination of the standard, it is advisable to use a control serum as
a standard, such as Seriscann Normal (Ref. 99 41 48).
REFERENCES
Stookey L.L., (1970). Anal. Chem., 42, 779 - 781.
Yee,H.Y.and Zin,A. (1971). Clin. Chem. 17, 950 - 953.
Persijn, J.P.and col.,(1971).Clin Chim.Acta.35, 91 - 98.
Williams J.H., Johnson D.J. and Haut, M.J. (1977). Clin. Chem. 23, 237 - 240.
Thompsen J.C. and Mottola H.A.,(1984). Anal. Chem. 56, 755 - 757.
ISO 9001 / ISO 13485

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MAGNESIUM
XYLIDYL BLUE METHOD
For “in vitro” determination of magnesium in serum, plasma C.S.F. and urine
PRINCIPLE PROCEDURE
In an alkaline medium, the magnesium ions of the sample will produce a coloured
complex with Xylidyl blue. Colour intensity is directly proportional to the magnesium ions’ Technique BL ST SA
concentration present in the sample. Ehylene glycol-bis(2-aminoethylether)-N,N,N′,N mL mL mL
tetraacetic acid (EGTA) performs as a chelating agent.
Sample --- --- 0.01
DIAGNOSTIC USE Standard --- 0.01 ---
Magnesium is found in the bone and in the cells of tissues and organs. Magnesium is
necessary for nearly all biochemical processes in the body. It helps to maintain normal Reagent 1.00 1.00 1.00
muscle and nerve function, it keeps the bone strength, it controls heartbeat and it helps to
regulate blood pressure. Magnesium also controls the levels of sugar in the blood and it Mix well and let stand 10 min at room temperature (20-25ºC).
helps strengthen the body’s immune system.
Main causes of magnesium deficiency are intestinal malabsorption, pancreatitis and Reading
hypoparathyroidism. High concentrations are found in dehydration, renal insufficiency, Wavelength: 520 nm
Addison disease, hypothyroidism and ingestion of anti-acids. Blank: Contents of BL
Colour stability: 1 hour
and laboratory data. CALCULATIONS
REAGENTS SA Abs.
x 4 = mg of magnesium / dL
Kit 2 x 100 mL. (Ref. 99 29 85). Contents: ST Abs
A. 2 x 100 mL Xylidyl blue reagent Ref. 99 35 10
B. 1 x 5 mL Standard Ref. 99 06 40 SA Abs
x 4 x 5 = mg of magnesium / dL (urine)
WORKING REAGENT PREPARATION ST Abs
SA Abs
x 4 x 5 x vol(dL) urine/24h = mg de magnesio/ 24h (urine 24h)
WORKING REAGENT COMPOSITION ST Abs
Where:
Tris buffer, pH 11 0.3 mM SA Abs: Sample absorbance
Xylidyl blue 0.1 mM ST Abs: Standard absorbance
EGTA 50 μM
K2CO3 75 mM SI Units
Surfactants 2% (mg/dL) x 0.4113 = mmol/L
Preservatives and stabilizers REFERENCES VALUES
Standard: Aqueous solution of magnesium equivalent to 4 mg/dL (1.645 mmol/L). Serum, palsma: 1.9 – 2.5 mg/dL
C.S.F.: 2.5 – 3.5 mg/dL
STORAGE AND STABILITY Urine: 1.0 - 10 mg/dL
When stored at room temperature (≤ 25ºC), the components of the kit will remain stable until 24 hours Urine: 50 - 150 mg/24 h
the expiration date stated on the label. The stated values are for guidance. It is advisable that each laboratory determines its own
reference values.
Signs of reagent deterioration: PERFORMANCE CHARACTERISTICS
Presence of particles or turbidity in the reagent. Working reagent blank > 1.000 The performance characteristics depend on the method used. It is recommended to
ADDITIONAL EQUIPMENT calculate these data for each particular test protocol. These results have been obtained
General laboratory equipment. using a manual method.
SAMPLE
Linearity: 5 mg/dL. Samples that give higher concentration should be diluted in deionised
Non-hemolyzed serum, heparinized plasma, C.S.F., and urine. Serum magnesium is
water (1+1) and the final result has to be multiplied per 2.
stable for up to 7 days at 2-8º C.
Accuracy: 104.3%
To run a urine sample, this should be previously taken to an acid pH value (3-4) by adding
some drops of HCl. Then, dilute with deionized water (1+4). Multiply the final result by 5.
The reagents contain sodium azide at 0.09%. Handle with care. compared with reference reagent.
The safety statements are on the label. It is advisable to look at the SDS before using Details of the performance studies are available on request.
infectious. Use adequate protection. INTERFERENCES
The disposal of the residues has to be made according to local regulations. Hemolyzed serum samples should be discarded.
Lipaemic sera as well as bilirubin concentration up to 20 mg/dL do not interfere with the
assay.
QUALITY CONTROL
control program.
AUTOANALYZERS
REFERENCES
Mann, C. K., Joe, J. H., (1956) Anal. Chem., 28, 202-205.
Mann, C. K., Joe, J. H., (1957) Anal. Chim. Acta, 16, 155-160.
Bohuon, C., (1962) Clin. Chim. Acta, 7, 811-817
Tiezt, NW., Textbook of Clinical Chemistry 5th Edition, W.B. Saunders, Philadelphia (2012)
ISO 9001 / ISO 13485

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MAGNESIUM - CALMAGITE
COLORIMETRIC METHOD
For “in vitro” determination of magnesium in serum, plasma, C.S.F. and urine
PRINCIPLE PROCEDURE
In an alkaline medium, the magnesium ions of the sample will produce a coloured complex
with Calmagite. Colour intensity is directly proportional to the magnesium ions’ concentration Technique BL ST SA
present in the sample. EGTA eliminates the interference due to calcium. mL mL mL
DIAGNOSTIC USE Sample --- --- 0.01

Magnesium is found in the bone and in the cells of tissues and organs. Magnesium is Standard --- 0.01 ---
necessary for nearly all biochemical processes in the body. It helps to maintain normal
muscle and nerve function, it keeps the bone strength, it controls heartbeat and it helps to Working reagent 1.00 1.00 1.00
regulate blood pressure. Magnesium also controls the levels of sugar in the blood and it
helps strengthen the body’s defense system (immune system). Mix well and incubate 1 or 2 min at room temperature (20-25ºC).
Main causes of magnesium deficiency are intestinal malabsorption, pancreatitis and
hypoparathyroidism. High concentrations are found in dehydration, renal insufficiency, Reading
Addison disease, hypothyroidism and ingestion of anti-acids. Wavelength: 546 nm; 520 nm
Blank: Contents of BL
A single test result could not be used to make a clinical diagnosis. It should integrate clinical Colour stability: 1 hour
Electrolytes
CALCULATIONS
REAGENTS SA Abs
x 2 = mg of magnesium / dL
Kit 2 x 100 mL. (Ref. 99 33 10). Contents: ST Abs
A. 1 x 100 mL Colour reagent Ref. 99 33 15
B. 1 x 100 mL Buffer reagent Ref. 99 33 20 SA Abs
C. 1 x 5 mL Standard Ref. 99 33 25 x 2 x 5 = mg of magnesium / dL (urine)
ST Abs
Mix equal volumes of each reagent, (A+B). Standard is ready to use. SA Abs
x 2 x 5 x vol(dL) urine/24h = mg de magnesio/ 24h (urine 24h)
ST Abs
The reagent composition is as follows: Where:
SA Abs: sample absorbance
Triethanolamine buffer pH 11.5 65 mM ST Abs: standard absorbance
Calmagite 0.2 mM
EGTA 78 μM S.I. Units
Surfactants 2% (mg/dL) x 0.4113 = mmol/L
REFERENCES VALUES
Standard: Aqueous solution equivalent to 2 mg/dL (0.823 mmol/L). Serum, palsma: 1.9 – 2.5 mg/dL
C.S:F.: 2.5 – 3.5 mg/dL
STORAGE AND STABILITY Urine: 1.0 - 10 mg/dL
When stored at 2-8ºC, the components of the kit will remain stable until the expiration date 24 hours Urine: 50 - 150 mg/24 h
stated on the label. The stated values are for guidance. It is advisable that each laboratory determines its own
reference values.
Working reagent stability: 15 days at room temperature (≤ 25ºC) or 30 days at 2 -8ºC.
Signs of reagent deterioration: The performance characteristics depend on the method used. It is recommended to
Presence of particles or turbidity in the reagent. Working reagent blank > 1.500. calculate these data for each particular test protocol. These results have been obtained
General laboratory equipment. Sensitivity, as detection limit: 0.2 mg/dL
Spectrophotometer or photometer. Linearity: 4.5 mg/dL. Samples that give higher concentration should be diluted in deionised
water (1+1) and the final result has to be multiplied per 2.
SAMPLE Accuracy: 97.1%
Non-hemolyzed serum, heparinized plasma, C.S.F., and urine. Repetitivity, as CV%: 2.42%
The magnesium in serum is stable for up to one week at 2-8ºC. Reproducibility, as CV%: 2.75%
To run a urine sample, this should be previously taken to an acid pH value (3-4) by adding compared with reference reagent.
some drops of HCl. Then, dilute with deionized water (1+4). Multiply the final result by 5. Details of the performance studies are available on request.
INTERFERENCES
CAUTION Hemolyzed serum samples should be discarded.
Handle with care. The reagent contains sodium azide at 0.09%. The safety statements are It is recommended to use disposable plasticware to run the tests to avoid any possible
on the label. It is recommended to read the MSDS before reagent handling. undesirable contamination.
Waste products must be handled as per local regulations. Lipaemic sera as well as bilirubin concentration up to 20 mg/dL do not interfere with the
assay.
QUALITY CONTROL
control program.
AUTOANALYZERS
REFERENCES
Ingman, F., Ringbom, A., (1966) Microchem. J.,10, 543-553.
Chauhan, U.P.S., Sark, B. C. R., (1969). Anal. Biochem., 32, 70-80.
Abernethy, M.H., Fowler, R. T. (1982) Clin. Chem., 28, 520-522.
Tiezt, NW., Textbook of Clinical Chemistry 5th Edition, W.B. Saunders, Philadelphia (2012)
ISO 9001 / ISO 13485

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POTASSIUM
TPB-NA METHOD
For “in vitro” determination of potassium in serum or plasma
PRINCIPLE PROCEDURE
After deproteinization of serum with trichloracetic acid (TCA), potassium ions precipitate
with sodium tetraphenylboron (Na-TPB) in an alkaline medium, resulting in a stable
Technique BR SA ST
suspension of potassium tetraphenylborate.
The turbidity produced is proportional to the potassium concentration of the sample. mL mL mL
DIAGNOSTIC USE Sample --- 0.1 ---

Potassium determination is used for electrolytic balance evaluation. Potassium levels Reagent A --- 1.0 ---
are controlled by aldosterone hormone. This hormone, released by suprarenal glands,
increases the sodium absorption and potassium excretion in the kidneys. Mix well and centrifuge at 2000 x g / 5 minutes
High levels of potassium in serum may be caused by the intake of potassium supplements Keep the supernatant.
or acute renal failure. Low potassium levels are found in cases of decreased potassium
intake or potassium loss from the body through intestinal secretions or urine. Working reagent 2.0 2.0 2.0
Supernatant --- 0.2 ---
and laboratory data. Standard --- --- 0.2
REAGENTS Mix thoroughly to obtain a homogeneous turbidity

Kit 3 x 100 mL. (Ref. 99 11 11). Contents: Read after 5-10 minutes at room temperature (20-25ºC). Mix once again before reading.
A. 1 x 100 mL Trichloracetic acid 0.3 M Ref. 99 11 66
B. 1 x 100 mL Na-TPB 0.1 M Ref. 99 12 02 Note: It is necessary to homogenize thoroughly before reading, because sample
C. 1 x 100 mL Sodium hydroxide 1.5 M Ref. 99 08 10 turbidity is measured.
Reading
WORKING REAGENT PREPARATION Wavelength: 580 nm
Mix equal parts of reagent B and reagent C. Let stand for 15 min. prior to use. Blank: Contents of BL
Signal stability: 30 min
Reagent A as well as the standard are ready-to-use.
CALCULATIONS
WORKING REAGENT COMPOSITION SA Abs
The reagent composition is as follows: x 5 = mmol of potassium / L
ST Abs
Na TPB 0.05 M
Sodium hydroxide 0.75 M
Where:
Standard: Aqueous solution of potassium equivalent to 5 mmol/L.
S.I. Units
mmol/L
When stored at room temperature (≤ 25ºC), the components of the kit will remain stable until
the expiration date stated on the label. Semimicro method
The working reagent prepared this way is stable 60 days at room temperature (≤ 25ºC) It can be carried out with half of the volumes as per the macro method.
and at 2-8ºC.
REFERENCES VALUES
Signs of reagent deterioration: Serum / plasma: 3.6 – 5.5 mmol/L.
Presence of particles or turbidity in the reagent. Working reagent blank > 0.700. The stated values are for guidance. It is advisable that each laboratory determines its own
reference values.
General laboratory equipment. PERFORMANCE CHARACTERISTICS
Spectrophotometer or photometer thermostatable at 37ºC. Cuvette: 1 cm light-path. The performance characteristics depend on the method used. It is recommended to
Serum and heparinized plasma. Discard hemolyzed samples.
Potassium in serum is stable for 10 days at 2-8ºC. Sensitivity, as detection limit: 0.5 mmol/L
Linearity: 10 mmol/L. Samples that give higher concentration should be diluted in saline
CAUTION solution (1+1) and the final result has to be multiplied per 2.
The safety statements are on the label. Handle the reagent with care. Accuracy: 94.8%
It is advisable to read the SDS before the reagent manipulation. Repetitivity, as CV%: 2.95%
The disposal of the residues has to be made according to local regulations. Reproducibility, as CV%: 3.41%
QUALITY CONTROL compared with reference reagent.
Control serum, Seriscann Normal (Ref. 99 41 48) and Seriscann Anormal (Ref. 99 46 85) Details of the performance studies are available on request.
control program. INTERFERENCES
Hemolyzed samples shall be discarded since red blood cells contain high concentration of
potassium ions. Serum should be immediately recovered from the clot.
Material intended to be used for this test must be perfectly clean since traces of detergent
can interfere with the assay. It is recommended to use disposable plasticware.
REFERENCES
Kim, E., Waddell, L., Logan, J., (1972) Clin. Chem, 18, 124-128.
Henry, R.J., (1974) Clin. Chem. Harper & Row, New York, Sec. Edit, 644-646.
ISO 9001 / ISO 13485

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TIBC
FeCl3 + MgCO3 method
For “in vitro” determination of total iron binding capacity in serum
PRINCIPLE PROCEDURE
Excess of ferric iron (FeCl3) is added to the specimen to saturate the transferrin.
Remaining ferric iron is absorbed on MgCO3 and discarded after centrifugation.
Technique mL
The fixed iron which remains in the supernatant, is the total iron binding capacity
(TIBC) , which is determined using the same technique as for serum iron. Sample 1.0
FeCl3 solution 2.0
DIAGNOSTIC USE
Iron is distributed in the body as part of hemoglobin and myoglobin. Mix and let stand 10 min at room temp (20-25ºC).
The total binding capacity of iron (TIBC) reflects the concentration in transferrin. MgCO3 1 dose
His determination is performed in conjunction with serum iron and indicates the
ability to transport iron in the blood. Cover with common plastic film and shake vigorously for 15-20 sec
Increased TIBC may be due to iron deficiency anaemia, acute hepatitis or Let stand for 30 min with occasional shakings (4-5) during the period.
advanced pregnancy. Centrifuge and decant the supernatant.
The decrease in TIBC occurs in iron deficiency anaemia, hemochromatosis, Determine the supernatant iron concentration with the corresponding iron test.
Electrolytes
malignant tumours and chronic infections.
CALCULATIONS
Single test result could not be used to make a clinical diagnosis. It should 1. Total iron binding capacity (TIBC):
integrate clinical and laboratory data. Iron concentration in the supernatant (μg/dL) x 3 (dilution factor) = Serum TIBC
(μg/dL)
REAGENTS
Kit (Ref. 99 14 62) for 100 tests. Contents:
2. Unsaturated iron binding capacity (UIBC):
A. 2 x 100 mL FeCl3 solution Ref. 99 19 55
TIBC (μg/dL) - serum iron (μg/dL) = UIBC (μg/dll)
B. 1 x 20 g MgCO3 Ref. 99 24 15
C. 1 Dispensing spoon
3. Siderophilin or transferrin:
WORKING REAGENT PREPARATION Assuming that 1 mg of transferrin may bind 1.2 μg of iron as a maximum, a very
Reagents are ready to use approximate value can be obtained by using the following expression:
REAGENTS COMPOSITION TIBC (μg/dL)

= transferrin (mg/dL).
The reagent composition is as follows: 1.2
Reagent A
HCl 1N 5.0 mL/L S.I. Units
p-Hydroxybenzoic acid 14.5 mM (μg/dL) x 0.1791 = μmol/L
FeCl3 0.11 mM
REFERENCES VALUES
Reagent B TIBC: 258 - 388 μg/dL
MgCO3 100 % UIBC: 160 - 280 μg/dL
Transferrin: 210 – 360 mg/dL
When stored at (≤ 25ºC) the components of the kit will remain stable until the The stated values are for guidance. It is advisable that each laboratory
expiration date stated on the label. determines its own reference values.
Signs of reagent deterioration: PERFORMANCE CHARACTERISTICS

Presence of particles or turbidity in the reagent. Reagent blank > 0.300. The performance characteristics depend on the method used. It is recommended
ADDITIONAL EQUIPMENT obtained using a manual method.
Spectrophotometer or photometer thermostatable at 37ºC. Cuvette: 1 cm light-path. Sensitivity, as detection limit: 15 µg/dL
Accuracy: 96.6%
SAMPLE Reproducibility, as CV%: 2.98%
Non-hemolyzed serum. If sample is to be assayed after 8h, store at 2-8ºC Trueness: Results obtained with this reagent did not show systematic differences
Should the analysis be carried out after 24 h., freeze the sample. Specimen when compared with reference reagent.
should not contain any iron-chelating agent. Details of the performance studies are available on request.
CAUTION QUALITY CONTROL

The safety statements are on the label. Handle the reagent with care. Control serum, Seriscann Normal (Ref. 99 41 48) and Seriscann Anormal (Ref. 99 46 85)
It is advisable to read the SDS before the reagent manipulation. should be included in each test series. Each particular laboratory should establish its own
The disposal of the residues has to be made according to local regulations. control program.
AUTOANALYZERS
REFERENCES
Kunesh, J.P., Small, L.L., (1970) Clin. Chem., 16, 148– 149.
ISO 9001 / ISO 13485

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ZINC
COLORIMETRIC METHOD
For “in vitro” determination of zinc in serum, plasma or urine
PRINCIPLE PROCEDURE
In an alkaline solution, the zinc ions of the sample will produce a red coloured
complex with 2- (5 - bromo - 2 - pyridylazo) - 5 - [- N - propyl -N-(3-sulfopropyl) Technique BL ST SA
amino] - phenol, sodium salt (5 - Br - PAPS).
The colour intensity at 560 nm is directly proportional to the zinc ions’ mL mL mL
concentration present in the sample. Sample --- --- 0.05
DIAGNOSTIC USE Standard --- 0.05 ---

Zinc deficiency causes growth retardation and delayed sexual maturation. Reagent 1.00 1.00 1.00
High levels of zinc in urine can be found when there is an alteration in the zinc -
protein transport binding (nephrosis, alcoholic cirrhosis or penicillin treatment). Mix well and let stand 10 min at room temperature (20-25ºC), or 5 min./37ºC.
Read absorbance of the sample and the standard.
integrate clinical and laboratory data. Reading
REAGENTS Wavelength: 560 nm
Kit 2 x 50 mL. (Ref. 99 28 14). Contents: Blank: Contents of BL
A. 2 x 50 mL Reagent Ref. 99 28 18 Colour stability: 30 min.
The components of the kit are ready to use. SA Abs
x 200 = µg of zinc / dL
ST Abs
REAGENT COMPOSITION
Bicarbonate buffer pH 9.8 185 mM ST Abs: Standard Absorbance
5 - Br - PAPS 0.05 mM
Sodium citrate 125 mM S.I. Units
Dimethylglyoxime 5 mM (µg/dL) x 0.153 = µmol/L
Surfactants 1%
REFERENCES VALUES
Serum / Plasma: 60 – 110 µg /dL
Urine: 150 - 1200 µg / 24h
Standard: Solution of Zinc in water / HCl equivalent to 200 µg/dL (30.6 µmol/L).
The stated values are for guidance. It is advisable that each laboratory
determines its own reference values.
The components of the kit, when stored at room temperature (≤ 25ºC), will
remain stable until the expiration date stated on the label. PERFORMANCE CHARACTERISTICS
Signs of reagent deterioration: to calculate these data for each particular test protocol. These results have been
Presence of particles or turbidity in the reagent. Reagent blank >0.300. obtained using a manual method.
Sensitivity, as detection limit: 4 µg/dL
Linearity: 400 µg/dL (61.2µmol/L). Samples that give higher concentration should
be diluted in deionized water (1+2) and the final result has to be multiplied per 3.
path.
Accuracy: 95.5%
SAMPLE Repetitivity, as CV%: 2.62%
Serum, non-hemolyzed plasma or urine. Reproducibility, as CV%: 2.94%
The safety statements are on the label. Handle the reagent with care. when compared with reference reagent.
The disposal of the residues has to be made according to local regulations. Details of the performance studies are available on request
QUALITY CONTROL
INTERFERENCES
It is recommended to use disposable plasticware to run the test to avoid any
possible undesirable contamination.
AUTOANALYZERS
REFERENCES
Makino, T., Saito, M., Horiguchi, D., Kina, K., (1982) Clin. Chim. Acta, 120, 127
– 135.
Homsher, R., Zak, B., (1985) Clin. Chem., 31 (8), 1310 – 1313.
Johsen, G., Eliasson, R., (1987) Intern. J. Andrology, 10, 435 – 440.
ISO 9001 / ISO 13485

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Quality Control
ISO 9001 / ISO 13485

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CALIBRATOR FOR AUTOANALYZERS
PRODUCT DESCRIPTION
The QCA Calibrator for Autoanalyzers is a lyophilized calibrator serum based
on human serum.
This Calibrator is designed to provide suitable calibration levels for most of the
common autoanalyzers.
The values for each constituent are derived from interlaboratory data.
REAGENTS
Calibrator for Autoanalyzers (Ref. 99 62 80)
Contents:
A. 1 x 7 mL Lyoph. calibration serum. Ref.99 68 20
B. 1 x 10 mL Diluent. Ref.99 68 80
CAUTION
Human serum was used in the manufacture of this product. Each donor unit was
tested with licensed reagents and found negative for HBsAg and nonreactive for
the HIV antibody.
Because of no test method can offer complete assurance that products derived
from human blood will not transmit infection agents, it is recommended that this
product be handled with the same precautions used for patient specimens.
PREPARATION
Open a lyophilized vial (1) very carefully, and add exactly 7 mL of diluent from
vial (2). Close the vial carefully and dissolve the contents completely.
Avoid the formation of foam.

The Calibrator for autoanalyzers when stored at 2 - 8ºC, will remain stable until
the expiration date stated on the label. Once rehydrated, the constituents are
stable for:
12 hours at 25ºC.
5 days at 2-8ºC.
1 month at -20ºC.
Bilirubin and alkaline phosphatase are stable for only:
8 hours at 25ºC.
2 days at 2-8ºC.
1 month at -20ºC.
ASSIGNED VALUES
The assigned concentrations for each parameter are lot specific.
The following table shows the concentration values and the traceability for each
parameter.
REFERENCES
Hyltoft Petersen, P.; Ricos, C.; Stölckl, D. Proposed guidelines for the internal
quality control of analytical results in the medical laboratory. Eur J Clin Chem
Biochem, 1996, 34, 983:999.
Lawson, NS.; Haven GT.; Williams, GW. Analyte stability in clinical chemistry
quality control materials. CRC Crit Rev Clin Lab Sci, 1982, 17, 1:50.
ISO 9001 / ISO 13485

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CALIBRATOR FOR AUTOANALYZERS
Constituent Method Calibration value SI Units Uncertainly Traceability

α-Amylase BPS (Benzylidene-G7PNP) 37 ºC 233 U/L 233 U/L
± 7.70 Internal master
IFCC liquid 37 ºC 213 U/L 213 U/L
CK NAC IFCC
37 ºC 429 U/L 429 U/L ± 7.50 Internal master
Alkaline Phosphatase DGKC 37 ºC 403 U/L 403 U/L ± 4.63

Internal master
IFCC 37 ºC 260 U/L 260 U/L ± 9.30
Gamma GT Szasz 405 nm
GOT/AST IFCC
GPT/ALT IFCC
LDH SFBC (Liquid) 37 ºC

552 U/L 552 U/L ± 14.0 Internal master
Kinetic, optimized 37 ºC
Lipase Enzymatic colorimetric
Quality Control
Cholesterol CHOD-POD
202 mg/dl 5.23 mmol/L ± 8.30 NIST 909b
Cholesterol HDL Enzymatic-Direct

86 mg/dl 2.27 mmol/L ± 1.80 Internal master
Triglycerides GPO
178 mg/dl 2.01 mmol/L ± 7.20 NIST 909b
Calcium Arsenazo III 9.40 mg/dl 2.35 mmol/l ± 0.25

NIST 909b
o-Cresolphthalein 8.70 mg/dl 2.17 mmol/l ± 0.32
Chloride Thiocyanate
92 mEq/L 92 mmol/L ± 5.00 NIST 909b
Inorganic Phosphorus Phosphomolybdate

5.20 mg/dl 1.68 mmol/l ± 0.5 QCA Standard*
Iron Ferrozine
152 μg/dl 27.2 μmol/l ± 8.70 QCA Standard*
Magnesium Xylidyl blue

3.00 mg/dl 1.23 mmol/L ± 0.10 NIST 909b
Albumine BCG
2.35 g/dl 23.5 g/L ± 0.38 NIST 927d
Total protein Biuret

3.50 g/dl 35.0 g/L ± 0.99 NIST 927d
Uric Acid Uricasa(e) – POD

6.73 mg/dl 400 μmol/L ± 0.20 NIST 909b
Bilirubin Jendrassik – Gróf Total 3.70 mg/dl 63.3 μmol/L ± 0.64 NIST 909b
Jendrassik – Gróf Direct 1.10 mg/dl 18.8 μmol/L ± 0.05 NIST 909b
Creatinine Kinetic Jaffé
3.06 mg/dl 270.5 μmol/L ± 0.20 NIST 909b
Glucose GOD-POD
205 mg/dl 11.4 mmol/l ± 4.90 NIST 965a
Urée Ureasa(e) GLDH

78 mg/dl 13.0 mmol/L ± 4.60 NIST 909b
Lot:133120
Exp.: 2017.09.30
*QCA standard is prepared according to:

American Chemical Society Specification Oficial (1/4/1993). Reagent Chemicals, 8 Ed., ISBN 0-8412-2502-8 (1993).
Remark:
These values correspond to a production lot for instance.
The correct values are those given in the prospectus inside the kit.
ISO 9001 / ISO 13485

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SERISCANN® ABNORMAL
ABNORMAL CONTROL SERUM Remark:
LOT 133100 / EXP 2017.09 The correct values are those given in the prospectus inside the kit.
PRODUCT DESCRIPTION ADDITIONAL EQUIPMENT ASIGNED VALUES

The control serum is prepared from human Volumetric pipettes. The assigned concentrations for each parameter
serum with human and nonhuman enzymes and are lot specific.
nonprotein constituents added. Bacteriostatic PREPARATION
The value and expected range for each constituent
agents have been added. Add exactly 5 ml. of deionised water to each vial.
Allow to sit 5 to 10 minutes. Swirl the contents until are derived from interlaboratory data and they are
REAGENTS homogeneous. given for orientation only; each laboratory should
Kit 10 x 5 ml. (Ref. 99 93 29). Contents: This control serum should be handled as if a sample establish its own acceptation range.The mean
10 Freeze-dried vials of abnormal control serum. from human origin. of several determinations may not duplicate the
Analytical values should fall in between the assay value printed on the insert but should fall within the
1 x 5 ml. (Ref.99 46 85). Contents: range. expected range.
1 Freeze-dried vial of abnormal control serum. To determine the accuracy and precision of a certain
STORAGE AND STABILITY analytical method, it is advisable to run a Normal as
CAUTION Before reconstitution, the product remains stable, well an Abnormal control serum samples.
Human serum was used in the manufacture of this when stored at 2 - 8º C., until the expiration
product. Each donor unit was tested with licensed date stated on the label. Once rehydrated, the REFERENCES
reagents and found negative for HBsAg and constituents are stable for 7 days, when stored at Hyltoft Petersen, P.; Ricos, C.; Stölckl, D. Proposed
nonreactive for the HIV antibody. 2 - 8º C, 8 hours at 25ºC and 1 month at -20ºC. guidelines for the internal quality control of analytical
Because of no test method can offer complete Except for Acid phosphatase, only stable for 48 h. results in the medical laboratory. Eur J Clin Chem
assurance that products derived from human blood at 2 - 8º C and 1 month at -20ºC. Biochem, 1996, 34, 983:999.
will not transmit infection agents, it is recommended For Alkaline phosphatase, rehydrate the serum and Lawson, NS.; Haven GT.; Williams, GW. Analyte
that this product be handled with the same let stand for one hour at room temperature. stability in clinical chemistry quality control
precautions used for patient specimens. Bilirubin is stable for 4 days at 2-8ºC. It is materials. CRC Crit Rev Clin Lab Sci, 1982, 17,
The disposal of the residues has to be made recommended not to store at room temperature or 1:50.
according to local regulations. freeze. Since Bilirubin is a light-sensitive metabolite
it is advisable, for the sake of better storage
conditions, to keep the control in the dark.
ENZYMES
Component Method T (ºC) Value Range Units
IFCC method 37º 283 246 - 320 U/ L
α-Amylase Substrate BPS-Blocked maltoheptaoside 37º 297 247 - 347 U/ L
CK NAC activated IFCC 37º 552 460 - 645 U/ L
CK-MB Inmunological 37º 40.6 26 - 56 U/ L
Cholinesterase Butyrylthiocholine iodide 37º 4540 3634-5452 U/ L
Acid Phosphatase α-Naphthyl phosphate 37º 43.7 32.2 - 56.2 U/ L
Prostatic acid phos. α-Naphthyl phosphate 37º 25.0 13.5 - 36.5 U/ L
DGKC (p-NPP) 37º 540 477 - 603 U/ L

Alkaline Phosphatase IFCC liquid 37º 380 303 - 457 U/ L
Phenolphthalein monophosphate 37º 193 136 - 250 U/ L
Gamma GT Szasz 405 nm 37º 159 134 - 184 U/ L
GLDH DGKC 37º 25 19 - 31 U/ L

IFCC 37º 125 91.0 - 159 U/ L
GOT/AST
Reitman-Frankel 37º 131 91 - 171 U/ ml
IFCC 37º 129 102 - 156 U/ L
GPT/ALT
Reitman-Frankel 37º 89.6 71 - 108 U/ ml
LDH SFBC (Pir. → Lact.) 37º 707 593 - 821 U/ L
Lipase Coloration 37º 66.0 37.0 - 95.0 U/ L
LIPIDS
265 220 - 308 mg/dl
Cholesterol CHOD-POD
6.86 5.70 - 7.98 mmol/ L
120 94.0 - 148 mg/dl
HDL - Cholesterol Enzymatic
3.13 2.43 - 3.83 mmol/ L
168 123 - 209 mg/dl
LDL - Cholesterol Enzymatic
4.30 3.20 - 5.41 mmol/ L
235 208 - 262 mg/dl
Triglycerides GPO
2.66 2.35 - 3.96 mmol/ L
ISO 9001 / ISO 13485

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SERISCANN® ABNORMAL
ABNORMAL CONTROL SERUM
LOT 133100 / EXP 2017.09
IONS
Component Method Value Range Units
11.0 8.95 - 13.1 mg/dl
Arsenazo III 2.74 2.23 - 3.27 mmol/ L
Calcium
o-Cresolphthalein 11.6 9.30 - 13.9 mg/dl
2.90 2.32 - 3.47 mmol/ L
mEg/L
Ion-selective electrode 111 87.0 - 135 mmol/ L
Chloride
Thiocyanate method 104 85.7 - 122 mEg/L
mmol/ L
76.0 59.0 - 93.0 µg/dl

Copper Direct Colorimetric method
11.9 9.26 - 14.6 µmol/L
6.74 5.70 - 7.80 mg/dl
Inorganic phosphorus Phosphomolybdate method
2.18 1.84 - 2.52 mmol/L
187 176 - 198 μg/dl
FerroZine 33.5 31.5 - 35.5 μmol/L
Iron
179 141 - 218 μg/dl
Quality Control
CAB Method
32.0 25.2 - 39.0 μmol/L
3.96 3.21 - 4.68 mg/dl

Calmagite dye 1.63 1.32 - 1.92 mmol/L
Magnesium
Xylidyl blue 3.90 3.21 - 4.60 mg/dl
1.60 1.32 - 1.90 mmol/L
mEg/L
Ion selective electrode 6.02 5.40 - 6.63 mmol/L
Potassium
Turbidimetric method / TPB-Na 8.50 7.64 - 9.36 mEg/L
mmol/L
mEg/L
Sodium Ion-selective electrode 159 135 - 184
mmol/L
249 180 - 320 μg/dl
TIBC FeCI3; MgCO3 44.6 32.2 - 57.3 μmol/L
144 89 - 139 μg/dl
Zn Br-PAPS method
17.4 13.6 - 21.3 μmol/L
PROTEINS
3.00 2.20 – 3.80 g/dl
Albumine BCG method
30.0 22.0 – 38.0 g/l
4.63 4.12 – 5.13 g/dl
Protéines Totales Biuret 46.3 41.2 – 51.3 g/l
OTHER METABOLITES
Uric Acid Uricase POD 9.25 7.70 – 10.80 mg/dl

548 458 – 643 μmol/L
Jendrassik – Gróf Manual 4.85 4.00 – 5.70 mg/dl

83.0 68.4 – 97.5 μmol/L
Total Bilirubin
Jendrassik – Gróf Automatic 5.00 4.20 – 5.80 mg/dl
85.5 72.0 – 99.2 μmol/L

34.2 21.2 – 48.0 μmol/L
Direct Bilirubin
Jendrassik – Gróf Automatic 2.35 1.65 – 3.05 mg/dl
41.6 28.2 – 52.2 μmol/L
Creatinine Kinetic Jaffé 3.50 2.78 – 4.22 mg/dl

309 246 – 373 μmol/L
Glucose GOD-POD 272 242 – 302 mg/dl

15.1 13.4 – 16.7 μmol/L
Urease GLDH 110 71 – 129 mg/dl

16.7 11.8 – 21.5 μmol/L
Uree
114 87 – 141 mg/dl
Urease Berthelot 19.0 14.5 – 23.5 μmol/L
ISO 9001 / ISO 13485

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SERISCANN® NORMAL
NORMAL CONTROL SERUM Remark:
LOT 142830 / EXP. 2018.09 The correct values are those given in the prospectus inside the kit.
PRODUCT DESCRIPTION ADDITIONAL EQUIPMENT ASIGNED VALUES

The control serum is prepared from human Volumetric pipettes. The assigned concentrations for each parameter
serum with human and nonhuman enzymes and are lot specific.
nonprotein constituents added. Bacteriostatic PREPARATION The value and expected range for each constituent
agents have been added. Add exactly 5 ml. of deionized water to each vial.
Allow to sit 5 to 10 minutes. Swirl the contents until are derived from interlaboratory data and they are
REAGENTS homogeneous. given for orientation only; each laboratory should
Kit 10 x 5 ml. (Ref. 99 65 71). Contents: This control serum should be handled as if a sample establish its own acceptation range.The mean
10 Freeze-dried vials of normal control serum. from human origin. of several determinations may not duplicate the
Analytical values should fall in between the assay value printed on the insert but should fall within the
1 x 5 ml. (Ref.99 41 48). Contents: range. expected range.
1 Freeze-dried vial of normal control serum To determine the accuracy and precision of a certain
STORAGE AND STABILITY analytical method, it is advisable to run a Normal as
CAUTION Before reconstitution, the product remains stable, well an Abnormal control serum samples.
Human serum was used in the manufacture of this when stored at 2 - 8º C., until the expiration
product. Each donor unit was tested with licensed date stated on the label. Once rehydrated, the REFERENCES
reagents and found negative for HBsAg and constituents are stable for 7 days, when stored at Hyltoft Petersen, P.; Ricos, C.; Stölckl, D. Proposed
nonreactive for the HIV antibody. 2 - 8º C, 8 hours at 25ºC and 1 month at -20ºC. guidelines for the internal quality control of analytical
Because of no test method can offer complete Except for Acid phosphatase, only stable for 48 h. results in the medical laboratory. Eur J Clin Chem
assurance that products derived from human blood at 2 - 8º C and 1 month at -20ºC. Biochem, 1996, 34, 983:999.
will not transmit infection agents, it is recommended For Alkaline phosphatase, rehydrate the serum and Lawson, NS.; Haven GT.; Williams, GW. Analyte
that this product be handled with the same leave for one hour at room temperature. stability in clinical chemistry quality control
precautions used for patient specimens. Bilirubin is stable for 4 days at 2-8ºC. It is materials. CRC Crit Rev Clin Lab Sci, 1982, 17,
The disposal of the residues has to be made recommended not to store at room temperature or 1:50.
according to local regulations. freeze. Since Bilirubin is a light-sensitive metabolite
it is advisable, for the sake of better storage
conditions, to keep the control in the dark.
ENZYMES
Component Method T (ºC) Value Interval Units
IFCC method 37º 91 81 - 101 U/ L
α-Amylase Substrate BPS-Blocked maltoheptaoside 37º 93 79.3 - 106.7 U/ L
CK NAC activated IFCC 37º 206 229 - 292 U/ L
CK-MB Inmunological 37º 18 14 - 22 U/ L
Cholinesterase Butyrylthiocholine iodide 37º 5021 3621 - 6421 U/ L
Acid Phosphatase α-Naphthyl phosphate 37º 15.4 8.2 - 22.6 U/ L
Phosphatase acide prost. α-Naphthyl phosphate 37º 7.9 4.9 - 10.9 U/ L
DGKC (p-NPP) 37º 246 210 - 282 U/ L

Alkaline Phosphatase IFCC liquid 37º 165 132 - 198 U/ L
Phenolphthalein monophosphate 37º 62 53 - 72 U/ L
Gamma GT Szasz 405 nm 37º 52 42 - 62 U/ L

GLDH DGKC 37º 12.6 9.4 - 15.8 U/ L
IFCC 37º 43.5 32.0 - 55.0 U/ L

GOT/AST
Reitman-Frankel 37º 26.7 14.7 - 38.7 U/ ml
IFCC 37º 32.5 22.5 - 42.5 U/ L

GPT/ALT
Reitman-Frankel 37º 12.3 5.6 - 19.0 U/ ml
LDH SFBC (Pir. → Lact.) 37º 450 372 - 528 U/ L
Lipase Coloration 37º 49 30 - 68 U/ L
LIPIDS
157 134 - 180 mg/dl
Cholesterol CHOD-POD 4.07 3.47 - 4.66 mmol/ L
57 48 - 66 mg/dl
HDL - Cholesterol Enzymatic
1.48 1.24 - 1.71 mmol/ L
86 71 - 101 mg/dl
LDL - Cholesterol Enzymatic
2.22 1.83 - 2.62 mmol/ L
Triglycerides GPO 91 78 - 104 mg/dl

1.04 0.89 - 1.19 mmol/ L
ISO 9001 / ISO 13485

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SERISCANN® NORMAL
NORMAL CONTROL SERUM
LOT 142830 / EXP. 2018.09
IONS
Component Method Value Interval Units
8.80 6.60 - 11.00 mg/dl
Arsenazo III 2.20 1.65 - 2.74 mmol/ L
Calcium
o-Cresolphthalein 8.60 6.90 - 10.30 mg/dl
2.15 1.72 - 2.57 mmol/ L
mEg/L
Ion-selective electrode 101 90 - 112 mmol/ L
Chloride
Thiocyanate method 99.5 79.5 - 119.5 mEg/L
mmol/ L
84.7 65.7 - 103.7 µg/dl
Copper Direct Colorimetric method
13.3 10.3 - 16.3 µmol/L
4.60 3.50 - 5.70 mg/dl
Phosphore inorganique Phosphomolybdate method
1.48 1.13 - 1.84 mmol/L
119 97 - 141 μg/dl
FerroZine 21.3 17.4 - 25.3 μmol/L
Iron
CAB Methode 131 101 - 161 μg/dl
Quality Control
23.5 18.1 - 28.8 μmol/L
2.10 1.36 - 2.84 mg/dl

Calmagite dye 0.86 0.56 - 1.17 mmol/L
Magnésium
Xylidyl Blue 2.37 1.57 - 3.17 mg/dl
0.97 0.65 - 1.3 mmol/L
mEg/L
Ion selective electrode 3.95 3.71 - 4.19 mmol/L
Potassium
Turbidimetric method / TPB-Na 3.60 2.35 - 4.85 mEg/L
mmol/L
mEg/L
Sodium Ion-selective electrode 140 101 - 179
mmol/L
274 213 - 335 μg/dl
TIBC FeCI3; MgCO3 49.0 38.1 - 60.0 μmol/L
129 91 - 167 μg/dl
Zn Br-PAPS method
19.7 13.9 - 25.6 μmol/L
PROTEINS
4.13 3.73 – 4.53 g/dl
Albumine BCG Method
41.3 37.3 – 45.3 g/L
5.76 5.05 – 6.47 g/dl
Totales Proteins Biuret
57.6 50.5 – 64.7 g/L
OTHER METABOLITES
6.90 4.60 – 7.20 mg/dl
Uric Acid Uricase POD
351 274 – 428 μmol/L

26.8 22.2 – 31.5 μmol/L
Total Bilirubin
1.63 1.35 – 1.91 mg/dl
Jendrassik – Gróf Automatic 27.8 23.1 – 32.7 μmol/L

21.4 18.3 – 24.5 μmol/L
Direct Bilirubin
1.02 0.62 – 1.42 mg/dl
Jendrassik – Gróf Automatic 17.4 10.6 – 24.3 μmol/L
1.50 1.00 – 2.00 mg/dl
Creatinine Kinetic Jaffé 132.6 88.4 – 176.8 mmol/L
108 83.6 – 132.4 mg/dl
Glucose GOD-POD 6.00 4.64 – 7.35 mmol/L
44 38.4 – 48.6 mg/dl
Urease GLDH 7.33 6.40 – 8.26 μmol/L
Uree 45 38.6 – 51.4 mg/dl
Urease Berthelot 7.50 6.42 – 8.56 μmol/L
ISO 9001 / ISO 13485

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ANTI-n-DNA LATEX
SLIDE TEST
For “in vitro” diagnostic
PRINCIPLE PROCEDURE
Lupus Erythematosus (LE) is an autoimmune disease in which antibodies
against deoxyribonucleic acid (DNA), as well as other nuclear components, are Technique
produced.
In 75-80% of those patients suffering from LE, it can be detected what has 1. Bring reagents and samples to room temperature prior to assay.
been called the LE cell, the formation of which is believed to be due to those 2. Place one drop (40 μL) of the sample onto a slide circle.
antibodies. 3. Shake well the latex reagent and add one drop over the serum
When the latex reagent, which has been coated with native deoxyribonucleic sample. Mix with the stirrer.
acid (n-DNA), is brought into contact with the serum sample, a clear agglutination 4. Tilt the slide and observe the presence or absence of agglutination
will be observed if anti-n-DNA antibodies are present. (Positive result). after 3 minutes
DIAGNOSTIC USE Semiquantitative technique

High levels of ANA are found in patients with lupus erythematosus. They also Using physiological saline, dilute the specimens 1/2, 1/4, 1/8, 1/16 and proceed
appear in patients suffering from youth rheumatic arthritis, sclerosis, connective as stated in procedure.
tissue diseases and autoimmune diseases. Titter: last dilution that produces latex agglutination.
Latex anti-n-DNA reagent does not give a diagnosis of the disease. Results have
to be evaluated within a total clinical study.
RESULTS
REAGENTS POSITIVE: Agglutination after 3 minutes.
Kit (Ref. 99 86 33)* for 20 tests. Contents: NEGATIVE: No agglutination after 3 minutes.
A. 1 x 1.0 mL Latex reagent Ref. 99 91 04
B. 1 x 0.5 mL Positive control Ref. 99 38 23
C. 1 x 0.5 mL Negative control Ref. 99 50 11 PERFORMANCE CHARACTERISTICS
D. Reaction slide and disposable stirrers. The performance characteristics depend on the method used. It is recommended
to calculate these data for each particular test protocol.
A. 1 x 2.0 mL Latex reagent Ref. 99 05 00 The reagent is designed to agglutinate in the presence of levels of anti-nuclear
B. 1 x 0.5 mL Positive control Ref. 99 38 23 antibodies (ANA) commonly found in systemic lupus erythematosus.
C. 1 x 0.5 mL Negative control Ref. 99 50 11
D. Reaction slide and disposable stirrers. Test gave positive reactions with 92% of the samples having titters of ANA ≥1/20
by immunofluorescence method.
REAGENT PREPARATION No false positive reactions were observed with latex reagent in testing
Reagent and controls are ready to use. performance of samples from healthy individuals.
Sensitivity, as dilution limit of the internal control (pool of sera from subjects
REAGENT COMPOSITION affected by LE): 1/4 ± 1 dil.
Latex reagent: Suspension of polystyrene latex particles coated with
deoxiribonucloeproteins in a stabilization buffer. Details of the performance studies are available on request.
Positive control: Pool of human sera known to have a positive reaction with anti-
n-DNA latex reagent. INTERFERENCES
Negative control: Pool of human sera known to have a negative reaction. Highly lipemic or haemolyzed samples may interfere in the agglutination
reaction.
STORAGE AND STABILITY Those patients suffering from a variety of connective tissue diseases may
When stored at 2-8ºC, the components of this kit will remain stable until the agglutinate the reagent.
expiration date stated on the label. Do not freeze.
Signs of reagent deterioration: QUALITY CONTROL

Presence of aggregates and difficulty of homogenization of the latex reagent .. Control serum should be included in each test series. Each particular laboratory should establish
Discard those controls in which, in spite of containing sodium azide, a microbial
growth is apparent.
REFERENCES
Christian, C.L., Hatfield, W.B., Chase, P.H., (1963) J. Clin. Invst., 42, 823-829.
Friou, G. J., FInch, S. C., Detree, K. D. (1958). J. Immunol., 80, 324 - 329.
Chaomao, J. C., (1976). Ara. J. Med. Tech., 42, 154 - 157.
SAMPLE
Fresh serum or stored at 2-8ºC for no longer than 48h. It is necessary to freeze
the sample when the assay is to be carried out after that period of time. Discard
contaminated or hemolyzed sera.
CAUTION
The safety statements are on the label. It is recomended to read SDS before reagent manipulation
Human serum used in the control preparation have been found negative in the reaction to HBsAg
and HIV I/II. However they should always be handled with care. All reagents contain sodium azide
0.09% as preservative. It is recommended to read SDS before reagent manipulation.
Waste products must be handled as per local regulations.
ISO 9001 / ISO 13485

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AS DIRECT LATEX
SLIDE TEST
For “in vitro” determination of anti-SLO
PRINCIPLE PROCEDURE
The AS Direct Latex reagent is a suspension of polystyrene particles sensitized 1. Bring reagents and serum samples to room temperature.
with streptolysin O. When the reagent is faced against the serum with anti-SLO 2. Place 40 µL of undiluted serum onto a slide’s black area.
antibodies, an antigen-antibody reaction takes place being easily visualized 3. Mix well the Latex reagent and add one drop (40 µL) over the serum drop.
because of the latex agglutination. 4. Mix with the aid of a stirrer both drops and title the slide.
5. Observe the presence or absence of agglutination within a period no
DIAGNOSTIC USE longer than 3 minutes.
Anti-streptolysin O antibodies are produced in those infections promoted by
beta-hemolytic streptococci, due to the presence of the streptolysin O (SLO) Semiquantitative technique
liberated from the bacteria. Information on the extent and degree of the infection Prepare serial two-fold dilutions in physiological saline (NaCl 0.9%) and then
can be obtained from the measurement of the antibodies level of serum. proceed with each diluted sample as described for undiluted serum.
Single test result could not be used to make a clinical diagnosis. It should The approximate anti-SLO level in a serum sample can be calculated by the
integrate clinical and laboratory data. following formula:
REAGENTS AS Titter IU/mL = Highest dilution with positive reaction x reagent sensitivity
Kit (Ref. 99 27 15) for 50 tests. Contents: (200 IU/mL)
A. 1 x 2.0 mL Latex reagent Ref. 99 10 55
B. 1 x 0.5 mL Positive control Ref. 99 00 03 INTERPRETATION OF RESULTS
C. 1 x 0.5 mL Negative control Ref. 99 82 74 Latex agglutination will mean that the anti-SLO level is equal or higher than 200
D. Slide and disposable stirrers. IU/mL.
Kit (Ref. 99 27 17) for 100 tests. Contents: REFERENCES VALUES

A. 1 x 4.0 mL Latex reagent Ref. 99 27 10 Concentrations up to 200 UI/mL are considered normal values.
C. 1 x 0.5 mL Negative control Ref. 99 82 74 Each particular laboratory should establish its own reference range.
D. Slide and disposable stirrers.
REAGENT PREPARATION
Reagent and controls are ready to use. Sensitivity: the reagent is designed to agglutinate in the presence of levels of
anti-SLO higher than 200 ± 50 UI/mL IU/mL. It is standardized against WHO
REAGENT COMPOSITION international standard for ASO, Ref. 1st IRP AST91.
A. Latex reagent: Suspension of polystyrene latex particles coated with SLO in
a stabilization buffer. Specificity: the correlation of results with the titration method of Rantz and
B. Positive control: Pool of human sera containing an anti-SLO titter higher than Randall is 100% for titters lower than 166 IU/mL or higher than 250 IU/mL. Titters
200 IU/mL. between these levels can give doubtful results.
C. Negative control: Pool of human sera containing an anti-SLO titter lower than
200 UI/mL.
Immunology
There is no prozone phenomenon for titers ≤ 1100 IU/mL.
STORAGE AND STABILITY Details of the performance studies are available on request.
The components of the kit, stored at 2-8ºC, will remain stable until the expiration
date stated on the label. Do not freeze. INTERFERENCES
Signs of reagent deterioration: There is no interference by RF until 540 IU/mL or by CRP until 400 mg/L.
Presence of particles in the reagent after homogenization. Lipemic and highly hemolyzed sera, as well as plasma, interfere with the assay.
Agglutination with negative control.
Discard those controls in which, in spite of containing sodium azide, a microbial LIMITATIONS OF THE PROCEDURE
growth is apparent. Occasional agglutinations produced after 3 min. have no diagnostic significance.
The AS latex is a screening reagent, any positive or negative result must be
ADDITIONAL EQUIPMENT evaluated together with all the clinical and diagnostic tests.
QUALITY CONTROL
SAMPLE Control serum should be included in each test series. Each particular laboratory should establish
Fresh sera or stored at 2-8ºC for no longer than 48h. It is necessary to freeze its own control program.
contaminated or hemolyzed sera.
REFERENCES
PRECAUCIONES Klein, G.C., Baker, C.N., Moody, M.D. (1970). Appl. Microbiol., 19, 60-61.
Las indicaciones de seguridad se encuentran en la etiqueta de los productos. Se aconseja
consultar la ficha de datos de seguridad antes de la manipulación del reactivo. Oberley, T.D., Duncan, J.L. (1971).Infect. Immun., 4, 683-687.
Los sueros humanos utilizados en la preparación del calibrador y el control han resultado negativos Ginsburg, I. (1972). J. Infect. Dis., 126, 294-340.
en la reacción con el HBsAg y el HIV I/II. A pesar de ello, deberán manejarse con precaución. Por Bernheimer, A.W., Linder, R., Avigad, L.S., (1979), Infect. Immun., 23, 838-844.
otra parte, todos los reactivos contienen azida sódica al 0,09% como conservante.
Se aconseja consultar la ficha de datos de seguridad antes de la manipulación del reactivo.
La eliminación de los residuos debe hacerse según la normativa local vigente.
ISO 9001 / ISO 13485

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CRP DIRECT LATEX
SLIDE TEST
For “in vitro” determination of C reactive protein
PRINCIPLE PROCEDURE
The CRP Direct latex reagent is a suspension of polystyrene particles sensitized 1. Bring reagents and serum samples to room temperature.
with anti-human C-reactive protein. When the reagent is faced against the 2. Place 40 µL of undiluted serum onto a slide’s black area.
serum, an antigen-antibody reaction takes place being easily visualized because 3. Mix well the Latex reagent and add one drop (40 µL) over the serum drop.
of the latex agglutination. 4. Mix with the aid of a stirrer both drops and title the slide.
C-reactive protein is a γ-globulin, the level of which increases in inflammatory
processes, in the acute phase of different diseases and after surgical operations. Semiquantitative technique
The main diagnostic value of its determination is in the detection of inflammatory Prepare serial two-fold dilutions in physiological saline (NaCl 0.9%) and then
processes having a rheumatic origin: acute articular rheumatism or chronic proceed with each diluted sample as described for undiluted serum.
polyarthritis in its acute phase, and the control of the evolution of inflammatory The approximate C-reactive protein level in a serum sample can be calculated
or infectious processes after therapy. by the following formula:
integrate clinical and laboratory data. CRP Titter mg/L = Highest dilution with positive reaction x reagent sensitivity
(7.5 mg/L)
REAGENTS
Kit (Ref. 99 00 92) for 50 tests. Contents: INTERPRETATION OF RESULTS
A. 1 x 2.0 mL Latex reagent Ref. 99 00 95 Latex agglutination will mean that the C-reactive protein level is higher than 7.5
B. 1 x 0.5 mL Positive control Ref. 99 77 20 mg/L.
D. Slide and disposable stirrers. REFERENCES VALUES
Concentrations up to 7.5 mg/L are considered normal values.
A. 1 x 4.0 mL Latex reagent Ref. 99 02 09 Each particular laboratory should establish its own reference range.
Sensitivity: The reagent is designed to agglutinate in the presence of CRP
REAGENT PREPARATION concentrations higher than 7.5 mg/dL, standardized against WHO standard Ref.
Reagent and controls are ready to use. CRM 470.
REAGENTS COMPOSITION Specificity: The reagent agglutinates in the presence of human CRP.
A. Latex reagent: Suspension of polystyrene latex particles sensitized with anti-
human CRP in a stabilization buffer. Prozone phenomena start appearing from 100 mg/L.
B. Positive control: Pool of human sera containing a CRP titter higher than 8
mg/L. Details of the performance studies are available on request.
C. Negative control: Pool of human sera containing a CRP titter lower than 7
mg/L. INTERFERENCES
There is no interference by ASO until 1100 IU/mL.
STORAGE AND STABILITY Rheumatoid factors in the sample may agglutinate the reagent.
The components of the kit, when stored at 2-8ºC, will remain stable until the Lipemic and highly hemolyzed sera, as well as plasma interfere with assay.
expiration date stated on the label. Do not freeze.
LIMITATIONS OF THE PROCEDURE
Signs of reagent deterioration: Occasional agglutinations produced after 3 min have no diagnostic significance.
Presence of particles in the reagent after homogenization. The CRP latex is a screening reagent, any positive or negative result must be
Agglutination with negative control. evaluated together with all the clinical and diagnostic tests.
Discard those controls in which, in spite of containing sodium azide, a microbial
growth is apparent. QUALITY CONTROL
Control serum should be included in each test series. Each particular laboratory should establish
SAMPLE REFERENCES
Fresh sera or stored at 2-8ºC for no longer than 48h. It is necessary to freeze Bienvenu, J. (1984); Ann. Biol. Clin., 42, 47-52.
the sample when the assay is to be carried out after that period of time. Discard Deyo, R. A., Pope, R. M., Perselin, R.H. (1980); J. Rheumatol., 7, 279-287
contaminated or hemolyzed sera. Engler, R. (1988); Ann. Biol. Clin., 46, 336-342.
Kindmark, C. O. (1972); Scand. J. Lab. Invest., 29, 401-411.
CAUTION Laurent, P. (1984); Ann. Biol. Clin., 42, 53-59.
The safety statements are on the label. It is recomended to read SDS before reagent manipulation Pepys, M. B. Immunoassays for acute phase proteins. In Voller, A., Bartlett, A.,
eds. Immunoassays for the 80’s. Lancaster, U.K., MTT Press, (1981); 341-352.
ISO 9001 / ISO 13485

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RF DIRECT LATEX
RAPID SLIDE TEST
For “in vitro” determination of rheumatoid factors
PRINCIPLE PROCEDURE
The RF Direct Latex reagent is a suspension of polystyrene particles sensitized 1. Bring reagents and serum samples to room temperature.
with human gamma-globulin. When the reagent is faced against the rheumatoid 2. Place 40 µL of undiluted serum onto a slide’s black area.
factors of serum, an antigen-antibody reaction takes place being easily visualized 3. Mix well the Latex reagent and add one drop (40 µL) over the serum drop.
because of the latex agglutination. 4. Mix with the aid of a stirrer both drops and title the slide.
In most patients suffering from evolutive chronic polyarthritis, the presences
of macroglobulins (rheumatoid factors), which are able to agglutinate inert Semiquantitative technique
particles sensitized with human gamma-globulin, are detected. The test of the Prepare serial two-fold dilutions in physiological saline (NaCl 0.9%) and then
latex agglutination allows distinguishing between this disease and the other one proceed with each diluted sample as described for undiluted serum.
known as articular rheumatism or rheumatic fever, in which rheumatoid factors The approximate rheumatoid factor level in a serum sample can be calculated
are not present. by the following formula:
integrate clinical and laboratory data. RF Titter IU/mL = Highest dilution with positive reaction x reagent sensitivity (20
IU/mL)
REAGENTS
Kit (Ref. 99 45 21) for 50 tests. Contents: INTERPRETATION OF RESULTS
A. 1 x 2.0 mL Latex reagent Ref. 99 10 75 Latex agglutination will mean that the rheumatoid factor level is equal or higher
B. 1 x 0.5 mL Positive control Ref. 99 61 81 than 20 IU/mL.
D. Slide and disposable stirrers. REFERENCES VALUES
Concentrations up to 30 IU/mL are considered normal values.
A. 1 x 4.0 mL Latex reagent Ref. 99 45 05 Each particular laboratory should establish its own reference range.
Sensitivity: the reagent is designed to agglutinate in the presence of levels of
REAGENT PREPARATION RF higher than 20 IU/mL. Standardized against WHO international standard for
Reagent and controls are ready to use. RF, Ref. W1066.
REAGENT COMPOSITION Specificity: the reagent agglutinates only in the presence of rheumatoid factors.
A. Latex reagent: Suspension of polystyrene latex particles coated with human
gamma-globulin in a stabilization buffer. There is no prozone phenomenon for titters ≤ 1129 IU/mL.
B. Positive control: Pool of human sera containing a RF titter higher than 20 IU/
mL.
Immunology
C. Negative control: Pool of human sera without RF concentration.
INTERFERENCES
STORAGE AND STABILITY There is no interference by ASO until 1000 IU/mL or by CRP until 448 mg/L
The components of the kit, stored at 2-8ºC, will remain stable until the expiration Lipemic and highly hemolyzed sera as well as plasma interfere with the assay.
date stated on the label. Do not freeze.
Signs of reagent deterioration: LIMITATIONS OF THE PROCEDURE
Presence of particles in the reagent after homogenization. Occasional agglutinations produced after 3 min have no diagnostic significance.
Agglutination with negative control. The latex FR is a screening reagent, any positive or negative result must be
Discard those controls in which, in spite of containing sodium azide, a microbial evaluated together with all the clinical and diagnostic tests.
growth is apparent. In some cases (20-30%) samples from patients suffering from rheumatoid
arthritis are negative.
General laboratory equipment. QUALITY CONTROL
SAMPLE its own control program.
Fresh serum or stored at 2-8ºC for no longer than 48h. It is necessary to freeze
contaminated or hemolyzed sera. REFERENCES
Waaler, E. (1940), Acad. Path. Microbiol. Scan. 17, 172–187.
CAUTION Singer, J.M, Plotz, C.M., (1965), Am. J. Mad., 21, 888-892
Edelman, G.M., Kunkel, H.G., Franklin, E.C., (1958), J. Exp. Med., 108 , 105-
and HIV I/II. However they should always be handled with care. All reagents contain sodium azide 120.
0.09% as preservative. It is recommended to read SDS before reagent manipulation. Singer, J.M. (1961), Am. J. Med. , 31, 766-779.
Waste products must be handled as per local regulations. Jones, W.L., Wiggins, G.L. (1973), Am. J. Clin. Pathol., 60, 703-706.
ISO 9001 / ISO 13485

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AS TURBIDIMETRIC LATEX 1:5
QUANTITATIVE DETERMINATION
For “in vitro” diagnostic of anti-SLO antibodies
PRINCIPLE PROCEDURE
The turbidimetric AS LATEX reagent allows quantifying the level of anti-SLO antibodies 1. Mix the working reagent. Bring the working reagent and the instrument to 37ºC.
present in serum samples by comparing the turbidimetric response produced with that 2. Pipette into the cuvette:
obtained with known concentrations of anti-SLO.
The reagent consists of a suspension of latex particles of homogeneous size sensitized
with SLO, capable of aggregation in the presence of anti-SLO. This aggregation process Monoreagent Method
produces an increase in the size of the particles which in turn produces an increase in the Working reagent 1.0 mL
absorbance of the system.
sample, STD or control 10 µL
DIAGNOSTIC USE Bireagent Method
Anti-Streptolysin O antibodies are produced in those infections promoted by beta-hemolytic
streptococci, due to the presence of the streptolysin O (SLO) liberated from the bacteria. Latex 200 µL
Information on the extent and degree of the infection can be obtained from the measurement Buffer 800 µL
of the antibodies level of serum.
Single test result could not be used to make a clinical diagnosis. It should integrate clinical Mix well both reagents.
REAGENTS
Kit 50 mL Ref. 99 22 40 3. Mix gently and start stopwatch. Record the absorbance at 540nm immediately (Absi).
Contents: 4. Record a new absorbance after 1 minute (Absf).
A. 1 x 10 mL Turbidimetric AS latex Ref. 99 03 80
B. 1 x 40 mL Buffer solution Ref. 99 63 10 CALCULATIONS
C. 1 x 1 mL Standard. Ref. 99 03 82 ∆ Abs = Absf – Absi
The standard concentration is stated in the label on the vial. For each sample, STD or control.
Kit 100 mL Ref. 99 22 60 ASO concentration in the sample is:

Contents:
A. 1 x 20 mL Turbidimetric AS latex Ref. 99 03 83 ∆ Abs sample
B. 1 x 80 mL Buffer solution Ref. 99 63 16 × Conc.standard= UI/mL ASO
C. 1 x 1 mL Standard. Ref. 99 03 82 ∆ Abs Standard
The standard concentration is stated in the label on the vial.
The method described above is only indicative, as it should be specifically adapted to each
Kit 300 mL Ref. 99 22 70 autoanalyzer.
Contents: REFERENCE VALUES
A. 1 x 60 mL Turbidimetric AS latex Ref. 99 03 86 Titers higher than 200-250 IU/mL are considered to indicate a sickness in progress.
B. 3 x 80 mL Buffer solution Ref. 99 63 16 Each particular laboratory should establish its own reference range.
C. 1 x 1 mL Standard. Ref. 99 03 82 PERFORMANCE CHARACTERISTICS
The standard concentration is stated in the label on the vial. The performance characteristics depend on the method used and on the instrument used.
It is recommended to calculate these data for each particular test protocol. These results
1 x 1mL Rheuma Line Control High (Polyvalent) Ref.99 21 50 have been obtained using an autoanalyzer using bireagent method.
1 x 1mL Rheuma Line Control Low (Polyvalent) Ref.99 21 55
The concentration of each component is stated in the label on the vial. Sensitivity, as detection limit: 11 UI/mL
WORKING REAGENT PREPARATION Linearity: 800 UI/mL. Samples with higher concentration should be diluted in saline NaCl
Monoreagent method: Mix thoroughly the latex reagent. Prepare the volume necessary with 0.9% (1+4) and the final result have to be multiplied per 5. Linearity could change depending
the ratio 1 mL A latex reagent + 4 mL B buffer solution. Working reagent is stable 7 days at on the instrument used.
2-8ºC. Homogenize before use. Accuracy: 98.4%
Bireagent method: Use reagents directly. Withinrun precision as CV%: 5.2%
Standard and controls are ready to use. Total precision, as CV%: 9.2%
No prozone effect was observed up to 5500 UI/mL
REAGENTS COMPOSITION Trueness: Results obtained with this reagent did not show systematic differences when
A. Latex reagent: Suspension of latex particles sensitized with streptolysin O. Sodium azide compared with reference reagent.
0.9 g/L.
B. Buffer: phosphate buffer 10mM, pH 7.2 Sodium azide 0.9 g/L. Details of the performance studies are available on request.
C. Standard: pool of human sera with known titter. Concentration is traceable to a
International Standard ASO NIBSC 94/662. INTERFERENCES
Rheuma Line Control: pool of human sera with anti-SLO, C reactive protein and rheumatoid No interference was observed by bilirubin (171 µmol/L), haemoglobin (5 g/L), triglycerides
factor. Sodium azide 0.9 g/L. (2.28 mmol/L), RF (210UI/mL). Other drugs and substances may interfere in the test (See
references)
STORAGE AND STABILITY QUALITY CONTROL
The components of the kit, when stored at 2 - 8ºC, will remain stable until the expiration date Rheuma Line Control High Polyvalent (99 21 50) and Rheuma Line Control Low Polyvalent
stated on the label. Do not freeze. (99 21 55) should be included in each test series. Each particular laboratory should establish
The standards and controls are stable until the expiration date stated on the label, when its own control program and correctives actions of measure deviations.
kept at 2-8ºC. When opened they are stable for 30 days at 2-8ºC.
Signs of reagent deterioration: AUTOANALYZERS
Presence of particles or turbidity in buffer or standard. Working reagent blank > 1500 at Adaptations to different autoanalyzers are available on request.
540nm. REFERENCES
ADDITIONAL EQUIPMENT Manual of clinical lab. Inmuno., ED by Rose, N.R., Friedman, H., Fahey, J.L., 3a ed. 336-
General laboratory equipment. 339.
Spectrophotometer or photometer thermostatable at 37ºC. Cuvette: 1 cm light-path. Wannamaker, L.W., Ayoub, E.M.(1960). Circulation, 21, 598 - 614.
Klein, G.C., Baker, C.N., Jones, W.C. (1971). Appl. Microbiol. V21 (6), 999 -1001.
Winkles, J., Lunec, J., Deverill, I. (1987). Clin. Chem. 33 (5), 685 - 689.
SAMPLE Winkles, J.W., Lunec, J., Gray, L. (1989). Clin. Chem.35 (2), 303 – 307.
Fresh sera or stored at 2 - 8ºC for no longer than 48 h. It is necessary to freeze the sample Young DS. Effects of drugs on clinical laboratory tests, 5th ed. AACC Press, 2000.
when the assay is to be carried out after that period of time. Discard contaminated or
hemolyzed sera.
CAUTION
The safety statements are on the label. It is recomended to read SDS before reagent
manipulation Human serum used in the control preparation have been found negative in
the reaction to HBsAg and HIV I/II. However they should always be handled with care. All
reagents contain sodium azide 0.09% as preservative. It is recommended to read SDS
before reagent manipulation.
ISO 9001 / ISO 13485

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CRP TURBIDIMETRIC LATEX 1:5
For “in vitro” determination of C Reactive Protein
PRINCIPLE PROCEDURE
The turbidimetric CRP LATEX reagent allows quantifying the level of CRP present in 1. Mix the working reagent. Bring the working reagent and the instrument to 37ºC.
the sera samples by comparing the turbidimetric response produced by these with those 2. Pipette into the cuvette:
obtained from sample with known concentrations of CRP.
The reagent consists of a suspension of latex particles of homogeneous size sensitized
with anti-CRP, capable of aggregation in the presence of CRP. This aggregation process Monoreagent Method
produces an increase in the size of the latex particles which in turn produces an increase in Working reagent 1.0 mL
the absorbance of the system.
DIAGNOSTIC USE Bireagent Method
C-Reactive protein is a γ-globulin, the level of which increases in inflammatory processes,
in the acute phase of different diseases and after surgical operations. The main diagnostic Latex 200 µL
value of its determination is in the detection of inflammatory processes having a rheumatic Buffer 800 µL
origin.
Single test result could not be used to make a clinical diagnosis. It should integrate clinical Mix well both reagents. Incubate for 3 minutes.
REAGENTS
Contents: 4. Record a new absorbance after 40 seconds (Absf).
A. 1 x 10 mL Turbidimetric CRP latex Ref. 99 04 00
B. 1 x 40 mL Buffer solution Ref. 99 04 15 CALCULATIONS
C. 1 x 1 mL Standard Ref. 99 03 28 ∆ Abs = Absf – Absi
The standard concentration is stated in the label on the vial. For each sample, STD or control.
Kit 100 mL Ref. 99 74 95 CRP concentration in the sample is:

Contents:
A. 1 x 20 mL Turbidimetric CRP latex Ref. 99 04 13 ∆ Abs sample
B. 1 x 80 mL Buffer solution Ref. 99 04 23 × Conc. standard=mg/L CRP
C. 1 x 1 mL Standard Ref. 99 03 28 ∆ Abs Standard
The standard concentration is stated in the label on the vial.
Kit 300 mL Ref. 99 75 00 autoanalyzer.
Contents:
REFERENCE VALUES
A. 1 x 60 mL Turbidimetric CRP latex Ref. 99 04 14
Concentrations up to 7,5 mg/L are considered normal values.
Each particular laboratory should establish its own reference range.
The standard concentration is stated in the label on the vial. PERFORMANCE CHARACTERISTICS
1 x 1mL Rheuma Line Control High (Polyvalent) Ref. 99 21 50 calculate these data for each particular test protocol. These results have been obtained
1 x 1mL Rheuma Line Control Low (Polyvalent) Ref. 99 21 55 using an autoanalyzer with bireagent method.
The concentration of each component is stated in the label on the vial.
Sensitivity, as detection limit: 2 mg/L
Immunology
Linearity: up to 150 mg/L. Samples with higher concentration should be diluted in saline
Monoreagent method: mix thoroughly the latex reagent. Prepare the volume necessary with
NaCl 0.9% (1+4) and the final result have to be multiplied per 5.
the ratio 1 mL A. latex reagent + 4 mL B. buffer solution. Incubate for 3 minutes. Working
Linearity could change depending on the instrument used.
reagent is stable 4 days at 2-8ºC. Homogenize before use.
Accuracy: 96.1%
Bireagent method.: Use reagents directly.
Withinrun precision, as CV%: 5.3%
Standard and controls are ready to use.
Total precision, as CV%: 7.5%
No prozone effect was observed up to 350 mg/L
Trueness: results obtained with this reagent did not show systematic differences when
A. Latex reagent: suspension of latex particles sensitized with anti-human CRP. Sodium
azide 0.9g/L
B. Buffer solution: phosphate buffer 100mM, pH 6.5; sodium azide 0.9g/L.
C. Standard: pool of human sera with known titter. Concentration is traceable to international
reference material DA472k/IFCC.
INTERFERENCES
Rheuma Line Control: pool of human sera with anti-SLO, C reactive protein and rheumatoid
No interference was observed by bilirubin (171 µmol/L), haemoglobin (5 g/L), triglycerides
factor. Sodium azide 0.9g/L.
(2.28 mmol/L), RF (210UI/mL). Other drugs and substances may interfere in the test (see
references)
The components of the kit, when stored at 2 - 8ºC, will remain stable until the expiration date QUALITY CONTROL
stated on the label. Do not freeze. Rheuma Line Control High Polyvalent (99 21 50) and Rheuma Line Control Low Polyvalent
The standards and controls are stable until the expiration date stated on the label, when (99 21 55) should be included in each test series. Each particular laboratory should establish
kept at 2-8ºC. When opened they are stable for 30 days at 2-8ºC. its own control program and correctives actions of measure deviations.
Signs of reagent deterioration: AUTOANALYZERS
Presence of particles or turbidity in buffer or standard. Working reagent blank > 1300 at Adaptations to different autoanalyzers are available on request.
540nm. REFERENCES
ADDITIONAL EQUIPMENT Pepys,M.B., Immunoassays for acute phase proteins, in Voller, A., Bartlett, A. eds.
General laboratory equipment. Immunoassays for the 80’s. Lancaster U.K., MTT Press, (1981). 341-352.
Spectrophotometer or photometer thermostatable at 37ºC. Cuvette: 1 cm light-path. Engler, R. (1988). Ann. Biol. Clin. 46, 336-342.
Kindmark, C.O. (1972). Scand. J. Lab. Invest., 29, 407-411.
SAMPLE Winkles, J., Lunec, J., Deverill, I. (1987). 685-689.
Fresh sera or stored at 2-8ºC for no longer than 48 h. It is necessary to freeze the sample Young DS. Effects of drugs on clinical laboratory tests, 5th ed. AACC Press, 2000.
when the assay is to be carried out after that period of time. Discard contaminated or
hemolyzed sera.
CAUTION
the reaction to HBsAg and HIV I/II. However they should always be handled with care. All
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RF TURBIDIMETRIC LATEX 1:5
For “in vitro” determination of Rheumatoid Factors
PRINCIPLE PROCEDURE
The turbidimetric RF LATEX reagent allows quantifying the rheumatoid factors present in the 1. Mix the working reagent. Temper the working reagent and the instrument to 37ºC.
sera samples by comparing the turbidimetric response produced by these with those obtained from 2. Pipette into the cuvette:
sample with known concentrations of rheumatoid factor.
The reagent consists of a latex particle suspension sensitised with human gamma globulin,
capable of aggregation in the presence of rheumatoid factors. This aggregation process produces Monoreagent Method
an increase in the size of the particles which in turn produces an increase in the absorbance of Working reagent 1.0 mL
the system.
DIAGNOSTIC USE
Bireagent Method
In most patients suffering from evolutive chronic polyarthritis, the presences of macroglobulins,
rheumatoid factors (RF), which are able to agglutinate inert particles sensitized with human Latex 200 µL
gamma-globulin, are detected. The test of the latex agglutination allows distinguishing between
this disease and the other one known as articular rheumatism or rheumatic fever, in which Buffer 800 µL
rheumatoid factors are not present.
Mix well both reagents.
Single test result could not be used to make a clinical diagnosis. It should integrate clinical and
laboratory data. sample, STD or control 33 µL
REAGENTS
Contents: 4. Record a new Absorbance after 150 seconds (Absf).
A. 1 x 10 mL Turbidimetric RF latex Ref. 99 04 30
B. 1 x 40 mL Buffer solution Ref. 99 04 35 CALIBRATION
C. 1 x 1 mL Standard Ref. 99 04 32 Prepare the following RF standard dilution using saline solution (NaCl 0.9%). To obtain the RF
The standard concentration is stated on the label. concentration of each dilution, multiply the standard concentration by the corresponding factor
indicated in the table.
Kit 100 mL Ref. 99 54 90
0 1 2 3 4 5
Contents:
A. 1 x 20 mL Turbidimetric RF latex Ref. 99 04 34 Std. FR (µL) -- 25 50 100 200 400
C. 1 x 1 mL Standard Ref. 99 04 32 Saline sol. (µL) 400 375 350 300 200 --
The standard concentration is stated on the label. Factor 0 0,0625 0,125 0,25 0,5 1,0
Kit 300 mL Ref. 99 55 00
CALCULATIONS
Contents:
∆ Abs = Absf – Absi
A. 1 x 60 mL Turbidimetric RF latex Ref. 99 04 36
For each sample, STD or control.
Calibration curve: plot the values of ∆Abs of each dilution against the corresponding RF
The standard concentration is stated on the label.
concentration.
It is recomended to recalibrate when changing lot of reagent, when instrument is adjusted or when
1 x 1mL Rheuma Line Control High (Polyvalent) Ref.99 21 50
control results are out of acceptance range.
1 x 1mL Rheuma Line Control Low (Polyvalent) Ref.99 21 55
The concentration of each component is stated on the label.
RF concentration of the sample is obtained by the interpolation of ∆Abs of each sample in the
WORKING REAGENT PREPARATION calibration curve.
Monoreagent method: mix thoroughly the latex reagent. Prepare the volume necessary with the In some instruments, calibration curve is linear in the range 25-75 UI/mL, in that case, it is possible
ratio 1 mL A latex reagent + 4 mL B buffer solution. Working reagent is stable 10 days at 2-8ºC. to perform the calibration with only one point (the standard of 50 UI/mL). For more accuracy,
Homogenize before use. we recommend the use of standard curve for calibration. The method described above is only
Bireagent method: use reagents directly. indicative, as it should be specifically adapted to each autoanalyzer.
Standard and controls are ready to use.
REFERENCE VALUES
Concentrations up to 30 IU/mL are considered normal values.
REAGENT COMPOSITION
Each particular laboratory should establish its own reference range.
A. Latex reagent: suspension of latex particles sensitised with human gamma-Globulins in a
stabilization buffer. Sodium azide 0.9 g/L PERFORMANCE CHARACTERISTICS
B. Buffer: glicine buffer, pH 8.1; Sodium azide 0.9 g/L The performance characteristics depend on the method used. It is recommended to calculate these
C.Standard: pool of human sera with known concentration of RF. Concentration is traceable to a data for each particular test protocol. These results have been obtained using an autoanalyzer with
WHO International Standard RF, Ref. W1066. bireagent method.
Rheuma Line Control: pool of human sera with anti-SLO, C reactive protein and rheumatoid factor
antibodies. Sodium azide 0.9 g/L Sensitivity, as detection limit: 2.4 UI/mL
Reaction range: 2.4-100 UI/mL. Samples with higher concentration should be diluted in saline
STORAGE AND STABILITY NaCl 0.9% (1+4) and the final result have to be multiplied per 5. Reaction range could change
The components of the kit, when stored at 2-8ºC, will remain stable until the expiration date stated depending on the analyzer used.
on the label. Do not freeze. Accuracy: 102.8%
The standards and controls are stable until the expiration date stated on the label, when kept at Repetitivity, as CV%: 1.4%
2-8ºC. When opened they are stable for 30 days at 2-8ºC. Reproducibility, as CV%: 3.95%
Signs of reagent deterioration: No prozone effect was observed up to 140 UI/mL.
Presence of particles or turbidity in buffer or standard. Working reagent blank > 1400 at 630nm. Trueness: Results obtained with this reagent did not show systematic differences when compared
INTERFERENCES
SAMPLE No interference was observed by bilirubin (333 µmol/L), haemoglobin (10 g/L), triglycerides (50
Fresh sera or stored at 2-8ºC for no longer than 48 h It is necessary to freeze the sample when g/L), ASO (1000 UI/mL), CRP (165 mg/L). Other drugs and substances may interfere in the test
the assay is to be carried out after that period of time. Discard contaminated or hemolyzed sera. (see references).
AUTOANALYZERS
CAUTION Adaptations to different autoanalyzers are available on request.
REFERENCES
Anderson, B., Antigens associated with Rheumatoid Artritis, in Natelson, S., Pesce, A.J., Dietz,
A.A. eds. Current Topics in Clinical Chemistry, V3: Clinical Immunochemistry. (1979). AA.CC.
176-190.
Johnson, P.M., Faulk, W.P., (1976). Clin. Immunol. Immunopathol., 6, 414-440
QUALITY CONTROL Taborn, J. D., Walker, S. E., (1979). Lab. Med., 10, 392 - 395.
Rheuma Line Control High Polyvalent (99 21 50) and Rheuma Line Control Low Polyvalent (99 Witherigton, R.H., Teitsson, I., Valdimarsson, H., Seifert, M.H. (1984).Ann. Rheum. Dis., 42, 679-
21 55) should be included in each test series. Each particular laboratory should establish its own 685.
control program and correctives actions of measure deviations. Winkles, J. W., Lunec, J., Gray, L. (1989). Clin. Chem. 35 (2), 303-307.
ISO 9001 / ISO 13485

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RHEUMA LINE CONTROL HIGH POLYVALENT
FOR ASO, RF, CRP
PRODUCT DESCRIPTION
The Rheuma Line is a liquid control serum which is prepared from human serum.
The Rheuma Line control is intended to be used for internal quality control in
determinations of levels of ASO, RF and CRP by immunoturbidimetry.
REAGENTS
1 x 1mL Rheuma Line Control High Polyvalent Ref. 99 21 50
Contents:
Serum calibrated in ASO, RF and CRP.
CAUTION
The safety statements are on the label. It is recomended to read SDS before
reagent manipulation Human serum used in the control preparation have been
found negative in the reaction to HBsAg and HIV I/II. However they should al-
ways be handled with care. All reagents contain sodium azide 0.09% as pre-
servative. It is recommended to read SDS before reagent manipulation.
REAGENT PREPARATION
COMPOSITION
Pool of human plasma samples with stabilizers.

The reagent, when stored at 2-8ºC, will remain stable until the expiration date
Signs of reagent deterioration: Presence of particles in the reagent.
Immunology
ASSIGNED VALUES
The concentration values are traceable to reference material: ASO: WHO 1st
IRP AST91; RF: WHO W1066; CRP: WHO Standard CRM-470.
The values and expected range for each constituent are derived from interlabo-
ratory data and they are only given for orientation; each laboratory should
establish its own acceptation range.
REFERENCES
Biochem, 1996, 34, 983:999.
ISO 9001 / ISO 13485

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RHEUMA LINE CONTROL LOW POLYVALENT
FOR ASO, RF, CRP
PRODUCT DESCRIPTION
The Rheuma Line is a liquid control serum which is prepared from human serum.
The Rheuma Line control is intended to be used for internal quality control in
determinations of levels of ASO, RF and CRP by immunoturbidimetry.
REAGENTS
1 x 1mL Rheuma Line Control Low Polyvalent Ref. 99 21 55
Contents:
Serum calibrated in ASO, RF and CRP.
CAUTION
found negative in the reaction to HBsAg and HIV I/II. However they should al-
ways be handled with care. All reagents contain sodium azide 0.09% as preserv-
ative. It is recommended to read SDS before reagent manipulation.
REAGENT PREPARATION
COMPOSITION
Pool of human plasma with stabilizers.

ASSIGNED VALUES
The concentration values are traceable to reference material: ASO: WHO 1st
IRP AST91; RF: WHO W1066; CRP: WHO Standard CRM-470.
The values and expected range for each constituent are derived from interla-
boratory data and they are given for orientation only; each laboratory should
establish its own acceptation range.
REFERENCES
Biochem, 1996, 34, 983:999.
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BENGAL ROSE
DETECTION OF BRUCELLOSIS BY SLIDE AGGLUTINATION
For “ in vitro” diagnostic
PRINCIPLE PROCEDURE
The Bengal Rose is a fast diagnostic test for the detection of anti-Brucella
antibodies. The reagent is standardized microorganism suspension at acidic pH 1. Bring reagent, samples and controls to room temperature.
which agglutinates in the presence of antibodies to Brucella sp. Softly homogenize the reactive suspension including the volume inside
the dropper.
DIAGNOSTIC USE 2. Dispense one drop each (40 μL) of serum and controls on different
Brucella sp. induces a humoral immune response with production of anti-Brucella slide reaction circles.
antibodies. The Bengal Rose test reagent allows detection of these antibodies 3. On each, add one drop (40 μL) of the reagent and mix with the aid of
generated as result of infection. Serodiagnostic test should be performed in a disposable stirrer.
parallel with cultures suitable for the isolation and identification of the causative 4. Tilt the slide for 4 min and observe for the presence or absence of
organism. agglutination.
integrate clinical and laboratory data. RESULTS INTERPRETATION
POSITIVE: Clear agglutination.
REAGENTS NEGATIVE: The suspension remains homogeneous.
Kit for 100 tests Ref. 99 77 09
Contents: REFERENCES VALUES
1 x 5 mL Reagent Ref. 99 78 18 The titers considered as reference values for anti-Brucella antibodies are ≤ 25
1 x 1 mL Positive control Ref. 99 46 12 IU/mL.
1 x 1 mL Negative control Ref. 99 32 15 Each particular laboratory should establish its own reference range.
Slide and disposable stirrers.
REAGENT PREPARATION Sensitivity: Positive reactions are obtained at levels higher than 25 IU/mL (anti-
Reagent and controls are ready to use. Brucella serum, 2nd International Standard, NIBS/WHO)
There is no prozone effect for titers ≤ 1000 IU/mL.
REAGENT COMPOSITION The results obtained showed no significant differences when comparing them
Reagent: bacterial suspension of inactivated B. abortus at an acid pH, stained with those obtained with the reference reagent.
with Begal Rose and an added 0.5% of phenol.
Positive control: Pooled sera of animal origin with anti -Brucella antibodies. Details of the performance studies are available on request.
Negative control: Pooled sera of animal origin. Nonreactive
INTERFERENCES
STORAGE AND STABILITY Hemoglobin >10 mg/mL, highly lipemic sera (Lipids >10 mg/mL) or bilirubin ≥ 2.5
When stored at 2-8ºC, the components of the kit will remain stable until the mg/dl can interfere with the assay.
expiration date stated on the label. Do not freeze. Rheumatoid factors ≤ 300 IU/mL do not interfere.
Signs of reagent deterioration: METHOD LIMITATIONS

Presence of particles in the reagent after the homogenization. The Bengal Rose is a screening reagent. Any positive or negative result must be
Immunology
Discard those controls in which, in spite of containing sodium azide, a microbial evaluated together with all the clinical and diagnostic tests.
growth is apparent. False negative results can appear in the early infection or in the last stages of
the sickness.
ADDITIONAL EQUIPMENT False positive results can be obtained in samples with bacterial contamination as
General laboratory equipment. well as ín sera from individuals vaccinated with the strain 19.
SAMPLE Note: In veterinary clinics, the reagent can be used as a screening technique.
Non hemolyzed serum stored at 2-8ºC for no longer than one week. It is The specifications for doing the test are fixed by the particular regulations in
recommended to freeze the sample if desired to store it for a longer time. each country.
Discard contaminated sera. The samples must be free from precipitates; All positive results must be confirmed with a specific test (complement fixation).
otherwise anomalous results can be obtained. Manipulate all samples as if they
were potentially infectious. REFERENCES
Ruiz-Mesa, JD., Sánchez-Gonzalez, J. , Reguera, JM.; Martín, L., Lopez-
CAUTION Palmero, S., Colmenero, J. D. (2005) Clin. Microbiol. Infect., 11, 211-225.
The safety statements are on the label. It is recomended to read SDS before reagent manipulation Abdou, AE., (2000) Eastern Mediterranean Health J., 6, 796-807.
Lucero, NE., Bolpe, JE., (1998) J. Clin. Microbiol., 36, 1425-1427.
0.09% as preservative. It is recommended to read SDS before reagent manipulation. Vogel, H., Cherubim, CE., Millan, SJ., (1970) Am. J. Clin. Path., 53, 932-938.
Waste products must be handled as per local regulations. Currier, RW., DVM, MPH, (1995) J. Am. Vet. Med. Assoc. (AVMA): Brucellosis.
QUALITY CONTROL
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FEBRILE ANTIGENS
QUALITATIVE AND QUANTITATIVE DETERMINATION
PRINCIPLE
B. Slide agglutination. Semiquantitative determination.
Febrile antigens are bacterial suspensions specially prepared for use in slide and tube agglutination
procedure for detecting specific agglutinins implicated in certain febrile diseases such as Brucellosis and
Technique
Salmonellosis. When the reagent is faced against the serum, an antigen-antibody reaction takes place
1. Bring sample, reagent and controls to room temperature.
being easily visualized because of the agglutination.
2. Softly homogenize the bacterial suspension, including the volume in the dropper
3. Dispense 80, 40, 20, 10 and 5 µl of serum and 20 µL of each control on different zones of the
DIAGNOSTIC USE
slide. Add one drop (40 µL) of the bacterial suspension on each zone.
Febrile antigens are used to detect antibodies against Salmonella, Brucella and Proteus OX-19 in patient
4. Mix with the aid of a disposable stirrer, tilts the slide and observe the presence or absence of
sera. They are used for diagnostic of diseases that cause fever as for example typhoid fever (Salmonella),
agglutination within a period no longer than1 minute.
Malta fever (Brucellosis).
Infection by these microorganisms induces humoral immunity response which produces antibodies that
RESULTS
can be detected with the specific antigen.
The serum volumes dispensed above correspond approximately to titres of 1:20, 1:40, 1:80, 1:160 and
Febrile serodiagnostics tests should be used in parallel with appropriate cultural techniques for the isolation
1:320 by the Tube technique.
and identification of the causative organism.
POSITIVE: Agglutination
NEGATIVE: No agglutination
data.
RESULTS INTERPRETATION
REAGENTS
Titres equal or higher than 1:80 have probable diagnostic significance. However, the precise diagnosis
S. Typhi H (FlagellarAg., d-H) 5 mL Ref. 99 01 88 Contents: Ref. 99 02 39
of the disease depends not only on the analytical results but also on a whole clinical evaluation.
S. Typhi O (Somatic Ag., 9, 12-O) 5 mL Ref. 99 81 01 Contents: Ref. 99 03 33
S. Paratyphi A H (Flagellar Ag, a-H) 5 mL Ref. 99 99 17 Contents: Ref. 99 03 49
S. Paratyphi B H (Flagellar Ag, b-H) 5 mL Ref. 99 08 70 Contents: Ref. 99 02 47 C. Tube agglutination. Quantitative determination
S. Paratyphi C H (Flagellar Ag, c-H) 5 mL Ref. 99 76 25 Contents: Ref. 99 03 29
S. Paratyphi A O (Ag. Somatic, 1,2,12-O) 5 mL Ref. 99 72 12 Contents: Ref. 99 03 23 It is recommended to confirm each positive reaction by the Tube technique.
S. Paratyphi B O (Ag. Somatic, 1,4,5,12-O) 5 mL Ref. 99 75 07 Contents: Ref. 99 03 25 The test can be done with the same regents used for slide test according to the following method.
S. Paratyphi C O (Ag. Somatic, 6,7-O) 5 mL Ref. 99 77 33 Contents: Ref. 99 03 30
Brucella Abortus 5 mL Ref. 99 90 01 Contents: Ref. 99 03 39 Technique
Brucella Melitensis 5 mL Ref. 99 82 79 Contents: Ref. 99 03 35 Prepare the dilutions (7 Kahn tubes ) for each antigen, as indicated in the following table.
Proteus OX 19 5 mL Ref. 99 61 15 Contents: Ref. 99 03 13
Salmonella Positive Control 1 mL Ref. 99 91 29 Contents: Ref. 99 77 06
Brucella Positive Control 1 mL Ref. 99 46 11 Contents: Ref. 99 77 08 Tube Nº 1 2 3 4 5 6 7
Proteus Positive Control 1 mL Ref. 99 91 31 Contents: Ref. 99 77 07
Negative Control 1 mL Ref. 99 39 10 Contents: Ref. 99 02 89 Saline (mL) 1.9 1.0 1.0 1.0 1.0 1.0 1.0
Serum (mL) 0.1 1.0 1.0 1.0 1.0 1.0 1.0

Kit Febrile Antigens (Widal) 8 x 5mL Ref. 99 51 80 Contents:
Ref. 99 02 39 / Ref. 99 03 33/ Ref. 99 03 49 / Ref. 99 02 47 / Ref. 99 03 29 / Ref. 99 03 23 / Ref. 99 03 25/
→ → → → → →
Ref. 99 03 30 / Ref. 99 02 89 / Ref. 99 77 06 Antigen (µL) 50 50 50 50 50 50 50
WORKING REAGENT PREPARATION Titre 1:20 1:40 1:80 1:160 1:320 1:640 1:1280
Reagents and controls are ready to use.
REAGENTS COMPOSITION 1. Pipette 1.9mL of saline into the tube Nº 1 of each row and 1.0mL into the remaining tubes.
Antigens: stabilized and colored suspension of non viable bacteria in a buffered solution. 2. Add 0.1mL of the serum to be assayed into the first tube, mix well, and transfer 1.0mL to the
Positive controls: serum from animal origin containing antibodies of anti-Salmonella, anti-Brucella or anti- tube Nº 2.
Proteus. 3. Mix well and transfer again 1.0mL from tube Nº 2 to tube Nº 3 and so on. Discard 1.0mL from
Negative Control: non-reactive serum from animal origin. tube Nº 7 after mixing thoroughly.
4. Add 50 μL of the desired antigen to each of the 7 tubes shake the rack and incubate at 37ºC
for 24/48h.
When stored at 2-8ºC, the reagents will remain stable until the expiration date stated on the label. Do not
5. After incubation time, read the results.
freeze.
Reading
Presence of particles in the reagent after the homogeneization.
Using a proper lamp and reading against a black background, record the degree of agglutination as
ADDITIONAL EQUIPMENT indicated.
For all methods ++++ 100% of the microorganisms are agglutinated. Supernatant is clear
Pipettes, saline solution (NaCl 0.9%). +++ Approximately 75% of the microorganisms are agglutinated. Supernatant is clear.
For A and B methods ++ Approximately 50% of the microorganisms are agglutinated. Supernatant is moderately
Glass or plastic slides, disposable stirrers. cloudy.
For C method: + Approximately 25% of the microorganisms are agglutinated. Supernatant is cloudy
Khan tubes, thermostated water bath or drying oven at 37ºC. - No agglutination. Microorganisms remain as a cloudy suspension.
SAMPLE
Serum. Can be stored up to 1 week at 2-8ºC.
CAUTION NOTE
Somatic antigens contain 0,5% phenol and flagellar antigens contain 0,5% formalin as preservatives.; they The tone and colour intensity of the antigen suspension can be modified from lot to lot. This variation will
should be handled with care. The safety statements are on the label. It is recommended to read MSDS not affect the final results.
before reagent handling.
Waste products must be handled as per local regulations REFERENCE VALUES
In early infections: Salmonela and Brucela have titers higher than 1/80 ( Somatic antigens and brucela) and
titer higher than 1/160 (flagellar antigens).
Proteus: Titers lower than 1/160 are not significatives.
Titre rises considerably between the acute phase and the convalescence.
QUALITY CONTROL
Control serum, Seriscann Normal (Ref. 99 41 48) and Seriscann Anormal (Ref. 99 46 85) The above values are for guidance. Each particular laboratory should establish its own reference range.
control program.
The antigenicity of all febrile antigens has been standardized with control antiserum of established titter, as
there is not any reference material which allows to determine reagent sensitivity.
PROCEDURE
A. Slide agglutination. Qualitative determiantion. Sensitivity, as the dilution limit able to agglutinate with the reagent is:
1/32 ± 1 step dilution (Salmonella), 1/64 ± 1 step dilution (Brucella), 1/16 ± 1 step dilution (Proteus).
Technique In some cases, prozone phenomena can be found; thus, when suspecting a high titre, it is advisable to
1. Bring sample, reagent and controls to room temperature. dilute the sample to 1:20 with physiological saline (NaCl 0.9 %) and perform the test once again.
2. Softly homogenize the bacterial suspension, including the volume in the dropper
3. Dispense 20 µL of sera and each control on different zones of the slide. Add one drop (40 µL) The performance characteristics of each bacterial antigen may be found in the corresponding Technical
of the bacterial suspension on each zone. Mix with the aid of a disposable stirrer, tilts the slide. Annex and they are available on request.
4. Observe the presence or absence of agglutination within a period no longer than1 minute.
INTERFERENCES
RESULTS There is no interference by RF at titters lower than 300 IU/mL. hemoglobin (≤10 gL/L); lipids (≤10 g/L) or
POSITIVE: Agglutination. bilirubin (≤20 mg/dL) do not interfere the test.
NEGATIVE: No agglutination. Traces of detergent in the labware may give falsely positive results, as well as the use of an old or
contaminated saline solution.
RESULTS INTERPRETATION Negative results can be obtained with patients at the early stages of the disease or with those under
Positive results are equivalent to titer equal or higher than 1:80. antibiotic treatment.
REFERENCES
Vogel,H., Cherubin, C.E., Milian, S.J. (1970). Amer. J. Clin. Pathol., 53, 932 – 938.
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INFECTIOUS MONONUCLEOSIS
HEMAGGLUTINATION SLIDE TEST
PRINCIPLE PROCEDURE
Specially processed horse erythrocytes are used in this test. They are able to
agglutinate the mononucleosis associated heterophilic antibodies. Forsmann 1. Bring reagent and serum samples to room temperature.
antibodies are not agglutinated in the reaction. 2. Mix thoroughly the erythrocyte suspension for total homogenisation.
3. Place 40 μL of serum and one drop (40 μL) of reagent and controls on
DIAGNOSTIC USE different reaction zones of the slide.
Mononucleosis is an infectious disease produced by the Epstein-Barr virus 4. On each, add one drop (40 μL) of reagent and mix with the aid of a
(EBV). While the infection is in course, in the patient serum, a certain type of disposable stirrer.
heterophilic antibodies appear, that are able to agglutinate horse as well as 5. Tilt the slide during 2 min and observe for the presence or absence of
sheep erythrocytes. agglutination. Interpretation of results
Single test result could not be used to make a clinical diagnosis. It should Semiquantitative technique
integrate clinical and laboratory data. Prepare serial two-fold dilutions of the sample, with physiological saline (NaCl
0.9%), and proceed with each and every dilution with the test as described
REAGENTS before.
A. 1 x 1.0 mL Reagent Ref. 99 15 26 RESULTS
B. 1 x 0.5 mL Positive control Ref. 99 18 41 POSITIVE: A visible agglutination within 2min.
C. 1 x 0.5 mL Negative control Ref. 99 24 90 NEGATIVE: No agglutination.
D. Slide and disposable stirrers
RESULTS INTERPRETATION
REAGENT PREPARATION The hemagglutination test is a screening method. To have a diagnostic
Reagent and controls are ready to use. confirmation of mononucleosis, the positive results should be considered within
a complete clinical evaluation.
REAGENT COMPOSITION
Reagent: Suspension of horse erythrocytes stabilized in a buffered saline PERFORMANCE CHARACTERISTICS
solution.
Positive control: Pool of human sera. Sensitivity: as the dilution limit able to agglutinate with the reagent: dil. 1/8 ± 1
Negative control: Pool of non-reactive human sera. step dilution of positive control. There is no international standard for Infectious
Mononucleosis.
ADDITIONAL EQUIPMENT Specificity: The reagent shows a 98% correlation with the differential method of
General laboratory equipment. Davidshon.
The results are positive in 80% of patients suffering from IM during the first week
STORAGE AND STABILITY of the onset of symptoms and rise to 90% within the next 3 weeks.
When stored at 2-8ºC, the components of the kit will remain stable until the
expiration date stated on the label. Do not freeze. INTERFERENCES
There is no interference by Forsmann antibodies. There is no interference by
Immunology
Signs of reagent deterioration: bilirrubin until 40 mg/dL, by ascorbic acid until 20 mg/dL.
Difficulty for erythrocytes dispersion. Discard those controls in which, in spite of Lipemic or hemolyzed samples may interfere the agglutination reaction
containing sodium azide, a microbial growth is apparent.
METHOD LIMITATIONS
SAMPLE To have satisfactory results it is essential that the reagent and serum samples
Fresh serum or stored at 2-8ºC for no longer than 48 hours. Discard contaminated be approximately of the same size.
or hemolyzed sera. Occasional agglutinations produced after 2 min have no diagnostic significance.
Sera from some patients with hematological and clinical evidence of Infectious
CAUTION Mononucleosis remain persistently negative.
REFERENCES
0.09% as preservative. It is recommended to read SDS before reagent manipulation. Paul, J. R. & Bunell, W. (1932). Am. J. Med. Sci., 183,90-92.
Waste products must be handled as per local regulations. Davidshon, I. (1937).J.A.M.A.,108, 289-295.
QUALITY CONTROL Lee, C.L., Davidshon, I. & Slaby, R. (1968).Am. J. Clin. Path., 49, 3-10.
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RPR CARBON
SERUM / PLASMA SYPHILIS SLIDE TEST
PRINCIPLE PROCEDURE
In patients suffering from infection of Treponema pallidum are detected plasma
reagins, antibodies capable of reacting with the nontreponemal antigen VDRL A. Qualitative technique.
(Venereal Disease Research Laboratory). Bring reagents and samples to room temperature prior to assay.
RPR Carbon antigen is a modification of the VDRL antigen in which are included Mix the RPR Antigen suspension and open the vial. With the aid of the plastic
carbon particles, specially treated in order to facilitate the visualization of agglutination dispenser coupled to the needle, suck the suspension. Mix thoroughly the suspension.
Onto a card reaction circle add one drop of the sample (40 μL). Then add a drop of
DIAGNOSTIC USE the RPR Antigen (20 μL) from the antigen dispenser.
The RPR Carbon test for serum or plasma allows the evaluation of the presence Mix well, and tilt the reaction card with a rotative agitator for 8 min at 100 rpm. Read
of plasma reagins, providing information about the possible presence and extent of the results.
T. pallidum infection. As a screening test, the result should always be confirmed by
specific tests. B. Semiquantitative technique
Single test result could not be used to make a clinical diagnosis. It should integrate In those cases in which it is desired to find out the titer of a positive sample, it is
clinical and laboratory data. feasible, by the serial dilution methodology, to carry out a semiquantitative titration.
From this point, proceed as in the case of the qualitative technique for each dilution.
REAGENTS
Kit (Ref. 99 14 00) 1 x 5 mL, RPR antigen for 250 tests. INTERPRETATION OF RESULTS
Positive reaction: Gross clumps around the periphery
Kit (Ref. 99 17 97) for 100 tests. Contents: Weak positive reaction: Fine clumps forming a ring shape around the center of the
A. 1 x 2 mL. RPR Carbon antigen Ref. 99 74 56 circle.
B. 1 x 0.5 mL. Positive control. Ref. 99 20 43 Negative reaction: The suspension remains homogeneous.
C. 1 x 0.5 mL. Negative control. Ref. 99 95 21 In the semiquantitative technique the titer will be the highest dilution at which a clear
D. 1 Plastic dispenser. agglutinationis observed.
E. 1 Dispensing needle.
F. 13 Reaction cards. C. Microtiter plate agglutination.
G. 50 Disposable stirrers Use microtiter plate with flat bottom. Place in a well of the microtiter plate one sample
drop (40 μL).
Kit (Ref. 99 24 05) for 500 tests. Contents: Then add a drop of the RPR Antigen (20 μL) from the antigen dispenser.
A. 2 x 5 mL. RPR Carbon Antigen Ref. 99 02 64 Mix well the microtiter plate with a rotative agitator for 20 min. at 200 rpm.
B. 1 x 1 mL. Positive control. Ref. 99 20 40 Read macroscopically, over a white surface.
C. 1 x 1 mL. Negative control. Ref. 99 95 19
D. 1 Plastic dispenser. INTERPRETATION OF RESULTS
E. 1 Dispensing needles. Positive reaction: Gross clumps in the well.
F. 63 Reaction cards. Weak positive reaction: Fine clumps.
G. 250 Disposable stirrers. Negative reaction: The suspension remains homogeneous.
WORKING REAGENT PREPARATION PERFORMANCE CHARACTERISTICS

Reagent and controls are ready to use. The performance characteristics of the product depend on the reagent and methodical
reading of results.
REAGENT COMPOSITION Specificity: the reagent agglutinates only in presence of plasmatic reagines present
The RPR Carbon suspension components are: in the sample.
Buffer sodium/ potassium phosphate 10 mM The sensitivity of the reagent is adjusted batch to batch as it is indicated in the
Choline chloride 10.0 % specifications of the CDC (Center for Disease Control, USA
Lipids 0.12 % RPR Carbon test is a screening determination. With positive samples, must be
Charcoal 0.02 % obtained a confirmation with the aid of specific treponemal methodologies.
EDTA 12.5 mM
Preservatives and stabilizers INTERFERENCES
False positive results can be obtained in those samples with infectious
Positive control: Serum of human origin, reactive against the RPR Carbon antigen. mononucleosis, leprosy, lupus erithematosus, toxoplasmosis, etc…, as well as in
Negative control: Pool of non-reactive animal sera. some drug addictions.
QUALITY CONTROL
The components of the kit, when stored at 2-8ºC, will remain stable until the expiration Control serum should be included in each test series. Each particular laboratory should establish
date stated on the label. Do not freeze. Once the antigen is in the plastic dispenser, it its own control program.
will remain stable for up to 6 months if stored at 2-8ºC.
Signs of product deterioration: REFERENCES

Reagent: Presence of agglutinated charcoal or difficulty in reagent homogenization Portnoy,J., Brewer, J.H., Harris,A.D. (1962). Pub Health Repo., 77, 645-652.
Controls: Discard those controls in which, in spite of containing preservative, a Portnoy, J., (1963). Amer. J. Clin. Path., 40, 473-479.
microbial growth is apparent. Falcone, V.H., Stout, G.V., Moore, M.B. (1964). Pub. Health Repo., 79, 491-495.
Scotti, A.G., McKey, D.M., Trautman, J.R. (1970). Arch. Derm., 101, 328-330.
ADDITIONAL EQUIPMENT Dorwart, B.B.,Myers, A.R.(1974).Brit.J.Vener. Dis. 50, 435-436.
General laboratory equipment. Kaufman, R.E., Weiss, S., Moore, J.D., Falcone, V., Wiesner, P.J. (1974). Brit. J.
Vener. Dis., 50, 350-353.
SAMPLE
Serum or plasma. Inactivation is not necessary. At 2-8ºC, the serum is stable up to
48h. Freezing is required if the assay will be delayed. If assaying a plasma sample,
the tests should be done within 20h. after the blood collection.
Discard the hemolyzed or contaminated samples.
CAUTION
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α-1 ACID GLYCOPROTEIN
QUANTITATIVE DETERMINATION BY IMMUNOTURBIDIMETRY
PRINCIPLE PROCEDURE
The reagent allows quantifying the level of human α-1 acid glycoprotein present in the Technique
sample by comparing the turbidimetric response produced with that obtained from standard To be used in clinical chemistry analyzers.
curve of known concentrations of human α-1 acid glycoprotein.
The reagent is an antiserum anti-human α-1 acid glycoprotein which reacts with the α-1 acid 1. Shake gently the reagents before using it. Use the sera sample without dilution.
glycoprotein of the serum giving protein aggregates. This aggregation process produces an
increase in the Abs. of the system.
Method
DIAGNOSTIC USE Reagent A 240 µL
The α-1 acid glycoprotein (AGG), also known as orosomucoid, is a major constituent of
the seromucoid fraction of plasma. AGG is synthesized mainly by hepatocytes. AGG is sample, calibrator or control 3µL
a lipocalin, a family of protein which bind hydrophobic compounds as progesterone and Mix, incubate 20 seconds at 37ºC and read absorbance (Absi) at 340nm.
related hormones. Then add Reagent B
Plasma AGG concentration increase up to fourfold in response acute-phase(trauma,
inflammation or tissue necrosis) and serves as an indicator of the clinical activity of ulcerative Reagent B 60 µL
colitis. AGG concentration are also increased by glucocorticoid hormones.
Synthesis and plasma AGG concentration are decreased by estrogens. 2. Mix well, incubate during 4 minutes then read the absorbance (Absf) at 340 nm.
Single test result could not be used to make a clinical diagnosis. It should integrate clinical CALIBRATION
and laboratory data. Prepare the standard curve by serial dilutions of calibrator with saline solution. (Protein
REAGENTS Calibrator Ref. 99 99 39). Use saline solution for concentration 0 mg/L.
Kit (Ref. 99 40 05) Contents:
A. 1 x 16.0 mL buffer solution Ref. 99 05 44 Calibration curve: plot the values ∆Abs of each dilution against α-1 acid glycoprotein
B. 1 x 4.0 mL anti-human AGG serum Ref. 99 04 58 concentration of each dilution. To obtain the α-1 acid glycoprotein concentration of each
dilution, use the corresponding dilution factor.
Protein Calibrator. Ref. 99 99 39
1 x 1 mL Pool of sera samples calibrated in Antithrombin III, IgA, IgG, IgM,Transferrin, C3, CALCULATIONS
C4, antitripsin and α-1 acid glycoprotein. ∆ Abs = Absf – Absi
The concentration is stated on the insert. For each sample, calibrator or control.
Control of Proteins Ref. 99 66 86 α-1 acid glycoprotein concentration in the sample is obtained by the interpolation of ∆Abs of
1 x 1 mL Sera sample positive for Antithrombin III, IgA, IgG, IgM, Transferrin, C3, C4, each sample in the calibration curve.
antitripsin and α-1 acid glycoprotein.
The concentration is stated on the insert. The method described above is only indicative, as it should be specifically adapted to each
autoanalyzer.
REAGENT PREPARATION
Reagents are ready to use. REFERENCES VALUES
50-120 mg/dL
REAGENT COMPOSITION Each particular laboratory should establish its own normal range, obtained from samples
A. Sodium phosphate/saline buffer pH 7.4 with stabilizers and preservatives. of a representative population, using its own instrumentation, blood collection methods and
B. Goat anti serum anti-human AGG in buffered saline pH 7.4 with stabilizers and preservatives. assaying procedures.
Immunology
STORAGE AND STABILITY The performance characteristics depend on the method used. It is recommended to
The components of the kit are stable up to the expiry date indicated on the label when kept calculate these data for each particular test protocol. These results have been obtained
at 2-8ºC. using an autoanalyzer.
Do not freeze antisera or buffer solution.
Presence of particles in the reagent. Quality control values outside acceptation range. Reaction range: 250 mg/dL. Samples that give higher concentration should be diluted in
ADDITIONAL EQUIPMENT saline NaCl 0.9% (1+1) and the final result have to be multiplied per 2.
General laboratory equipment. Accuracy: 91.8%
Spectrophotometer or photometer thermostatable at 37ºC. Cuvette: 1 cm light-path. Repetitivity, as CV%: 3.01%
No prozone effect was observed up to 600 mg/dL
The safety statements are on the label. It is recomended to read SDS before reagent compared with reference reagent.
the reaction to HBsAg and HIV I/II. However they should always be handled with care. All Details of the comparison experiments are available on request.
before reagent manipulation. INTERFERENCES
Waste products must be handled as per local regulations. No interference was observed by hemoglobin (62.5 mg/dL), bilirubin (30 mg/dL), triglycerides
SAMPLE (1250 mg/dL), FR (300 UI/mL). Hemolyzed sera and other substances could interfere (See
Recent serum or plasma (Heparin or EDTA plasma) kept at 2-8ºC for not more than 48h. If references).
the test should be performed later, it is recommended to freeze the sample. Avoid successive Samples that give an Abs. higher than that obtained for the last point in the standard curve
freezing and thawing. Discard hemolyzed or contaminated samples. should be diluted in saline (NaCl 0.9%) and the determination repeated.
QUALITY CONTROL General laboratory equipment.
Control serum should be included in each test series. Each particular laboratory should Spectrophotometer or photometer thermostatable at 37ºC. Cuvette: 1 cm light-path.
establish its own control program.
REFERENCES
Dati et al., Eur J Clin Chem Clin Biochem, 34, 517 - 520 (1996).
Young, D.S.; Effects of Preanalytical Variables on Clinical Laboratory Tests. 3rd Ed.
AACCPress 2000.
Young, D.S.; Effects of drugs on Clinical Laboratory Tests. 4th Ed. AACC Press. 2000.
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α1-ANTITRYPSIN
PRINCIPLE PROCEDURE
The reagent allows quantifying the level of human α1-antitrypsin present in the sample by Technique
comparing the turbidimetric response produced with that obtained from the standard curve To be used in clinical chemistry analyzers.
of known concentrations of human α1-antitrypsin.
The reagent is an antiserum anti-human α1-antitrypsin which reacts with the α1-antitrypsin 1. Shake gently the reagents before use. The sample shall be processed without dilution.
of the serum giving protein aggregates. This aggregation process produces an increase in Conditions:
the Abs. of the system. Volume of buffer solution (A): 240 μL
Volume of antibody solution (B): 60 μL
DIAGNOSTIC USE Volume of sample: 2 μL
α1-antitrypsin (AAT), is a protein synthesized in the liver by the hepatocytes. AAT is the most Reaction temperature: 37ºC
abundant protease inhibitor in plasma. AAT covalently binds to the active sites of serine
proteases, thus blocking their enzymatic activity. AAT inhibits many proteases, however one 2. Mix well; start the stopwatch and read the Abs (Absi). Repeat the reading after 5min. of
of the most critical is the elastase, which is released from neutrophils. reaction (Absf) at 340 nm
AAT deficit is a genetic defect. It is associated with the risk to suffer pulmonary emphysema.
It could also be associated with liver diseases as hepatitis, cirrhosis or hepatocelular CALCULATIONS
carcinoma. Determine the ΔAbs (Absorbance increase) as (Absf – Absi) for each sample, standard or
Plasma AAT concentrations are increased in acute inflammatory or necrotic processes. It control. α1-antitripsin concentrations are obtained by the interpolation of the ∆Abs value in
is also increased by estrogens stimulation, particularly during late pregnancy and during the calibration curve.
estrogen therapy.
Calibration curve
Single test result could not be used to make a clinical diagnosis. It should integrate clinical Prepare the curve by serially diluting the calibrator (Protein Calibrator Ref. 99 99 39).
and laboratory data. To obtain the concentration of each dilution, multiply the calibrator concentration by the
REAGENTS corresponding factor indicated in the table:
A. 1 x 16.0 mL Buffer solution. Ref. 99 04 71
B. 1 x 4.0 mL anti-human AAT serum Ref. 99 04 77 0 1 2 3 4 5
CAL (μL) -- 25 50 100 200 400
Protein Calibrator. Ref. 99 99 39
1 x 1 mL Pool of human plasma samples calibrated in antithrombin III, IgA, IgG, IgM, Saline sol. (μL) 400 375 350 300 200 --
transferrin, C3, C4, α1-antitrypsin and α-1 acid glycoprotein. Factor 0 0.0625 0.125 0.25 0.5 1.0
The concentration is stated on the insert.
Protein Control Ref. 99 66 86 The method described above is only indicative, as it should be specifically adapted to each
1 x 1 mL Samples positive for antithrombin III, IgA, IgG, IgM, transferrin, C3, C4, α1- autoanalyzer.
antitrypsin and α-1 acid glycoprotein.
REFERENCES VALUES
90 - 200 mg/dL
REAGENT PREPARATION Each particular laboratory should establish its own normal range, obtained from samples
Reagents are ready to use. of a representative population, using its own instrumentation, blood collection methods and
assaying procedures.
REAGENT COMPOSITION
A. Sodium phosphate/saline buffer pH 7.4 with stabilizers and preservatives.
B. Goat antiserum anti-human AAT in buffered saline pH 7.4 with stabilizers and
preservatives.
using an autoanalyzer.
The components of the kit are stable up to the expiry date indicated on the label when kept
Reaction range: 400 mg/dL. Samples that give higher concentration should be diluted in
at 2-8ºC.
saline NaCl 0.9% (1+1) and the final result has to be multiplied by 2.
Accuracy: 101.1%
Presence of particles in the reagent. Quality control values outside acceptation range.
ADDITIONAL EQUIPMENT Trueness: Results obtained with this reagent did not show systematic differences when
General laboratory equipment. compared with the reference reagent.
CAUTION
The safety statements are on the label. It is recomended to read SDS before reagent INTERFERENCES
manipulation Human serum used in the control preparation have been found negative in No interference was observed by hemoglobin (500 mg/dL), bilirubin (20 mg/dL) and RF (600
the reaction to HBsAg and HIV I/II. However they should always be handled with care. All UI/mL). Lipemic sera and other substances could interfere (See references).
reagents contain sodium azide 0.09% as preservative. It is recommended to read SDS Samples that give an Abs. higher than that obtained for the last point in the standard curve
before reagent manipulation. should be diluted in saline (NaCl 0.9%) and the determination repeated.
Waste products must be handled as per local regulations. AUTOANALYZERS
SAMPLE Adaptations to different autoanalyzers are available on request.
Recent serum or plasma (Heparin or EDTA plasma) kept at 2-8ºC for no longer than 48h. If REFERENCES
the test is to be performed later, it is recommended to freeze the sample. Avoid successive Tiezt, NW., Textbook of Clinical Chemistry 5th Edition, W.B. Saunders, Philadelphia (2012).
freezing and thawing. Discard hemolyzed or contaminated samples. Dati et al., Eur J Clin Chem Clin Biochem, 34, 517 - 520 (1996).
QUALITY CONTROL AACCPress 2000.
Control serum should be included in each test series. Each particular laboratory should Young, D.S.; Effects of drugs on Clinical Laboratory Tests. 4th Ed. AACC Press. 2000.
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APOLIPOPROTEIN A1
PRINCIPLE PROCEDURE
The reagent allows quantifying the level of human apolipoprotein A1 present in the sample Technique
by comparing the turbidimetric response produced with that obtained from the standard To be used in clinical chemistry analyzers.
curve of known concentrations of human apolipoprotein A1.
The reagent is an antiserum anti-human apolipoprotein A1 which reacts with the 1. Shake gently the reagents before use. The sample shall be processed without dilution.
apolipoprotein A1 of the serum giving protein aggregates. This aggregation process Conditions:
produces an increase in the Abs. of the system. Volume of buffer solution (A): 240 μL
The apolipoprotein A1 (APO A1) is the most important structural protein of HDL lipoprotein, Reaction temperature: 37ºC
it is about 70% of total HDL lipoprotein. It is synthesized in the small intestine and in the
liver. APO A1 acts as an specific activator of LCAT (lecithin cholesterol acyl transferase), the 2. Mix well; start the stopwatch and read the Abs (Absi). Repeat the reading after 3min. of
enzyme responsible for forming cholesteryl esters in plasma and to transport the cholesterol reaction (Absf) at 340nm
cells to the liver to be excreted.
Low APO A1 concentrations can be associated with coronary heart diseases risk. Genetic CALCULATIONS
disorders could also decrease APO A1 in serum or plasma, for example, Tangier disease, Determine the ΔAbs (Absorbance increase) as (Absf – Absi) for each sample, standard
in which the decrease of APO A1 concentration are due to increase on cholesteryl esters or control. Apolipoprotein A1 concentrations are obtained by the interpolation of the ∆Abs
in body tissues. value in the calibration curve.
High levels of APO A1 can be detected on patients with diet, physic exercise or ingest some
drugs as oestrogens or niacin. Calibration curve
Prepare the curve by serially diluting the calibrator with saline (apolipoprotein calibrator Ref.
Single test result could not be used to make a clinical diagnosis. It should integrate clinical 99 36 90). To obtain the concentration of each dilution, multiply the calibrator concentration
and laboratory data. by the corresponding factor indicated in the table:
REAGENTS
Kit (Ref. 99 34 02). Contents: 0 1 2 3 4 5
A. 1 x 16.0 mL Buffer solution. Ref. 99 04 57
CAL (µL) -- 25 50 100 200 400
B. 1 x 4.0 mL Anti-human APO A1 serum Ref. 99 04 78
Saline sol. (µL) 400 375 350 300 200 --
Apolipoprotein Calibrator Ref. 99 36 90
1 x 1 mL Pool of human plasma samples calibrated in apolipoprotein A1 and apolipoprotein Factor 0 0.0625 0.125 0.25 0.5 1.0
B.
The concentration of each component is stated on the vial label The method described above is only indicative, as it should be specifically adapted to each
autoanalyzer.
Apolipoprotein Control Ref. 99 36 92
1 x 1 mL Samples positive for apolipoprotein A1 and apolipoprotein B. REFERENCES VALUES
The concentration of each component is stated on the vial label 103-209 mg/dL
Each particular laboratory should establish its own normal range, obtained from samples
REAGENT PREPARATION of a representative population, using its own instrumentation, blood collection methods and
Reagents are ready to use. assaying procedures.
Immunology
A. Sodium phosphate/saline buffer pH 7.4 with stabilizers and preservatives. The performance characteristics depend on the method used. It is recommended to
B. Goat antiserum anti-human APO A1 in buffered saline pH 7.4 with stabilizers and calculate these data for each particular test protocol. These results have been obtained
preservatives. using an autoanalyzer.
STORAGE AND STABILITY Sensitivity, as detection limit: 4.0 mg/dL

The antiserum reagent, the buffer solution and the protein calibrator and control are stable Reaction range: 300 mg/dL. Samples that give higher concentration should be diluted in
up to the expiry date indicated on the label when kept at 2-8ºC. saline NaCl 0.9% (1+1) and the final result has to be multiplied by 2.
Do not freeze antisera or buffer solution. Accuracy: 99.5%
Presence of particles in the reagent. Quality control values outside acceptation range. No prozone effect was observed up to 1650 mg/dL
ADDITIONAL EQUIPMENT compared with the reference reagent.
Spectrophotometer or photometer thermostatable at 37ºC. Cuvette: 1 cm light-path. Details of the comparison experiments are available on request.
The safety statements are on the label. It is recomended to read SDS before reagent No interference was observed by hemoglobin (1000 mg/dL), bilirubin (30 mg/dL) and RF
manipulation Human serum used in the control preparation have been found negative in (600 UI/mL). Lipemic sera and other substances could interfere (See references).
the reaction to HBsAg and HIV I/II. However they should always be handled with care. All Samples that give an Abs. higher than that obtained for the last point in the standard curve
reagents contain sodium azide 0.09% as preservative. It is recommended to read SDS should be diluted in saline (NaCl 0.9%) and the determination repeated.
Waste products must be handled as per local regulations. AUTOANALYZERS
SAMPLE Adaptations to different autoanalyzers are available on request.
Recent serum or plasma (heparin or EDTA plasma) kept at 2-8ºC for no longer than 48h. If
REFERENCES
the test is to be performed later, it is recommended to freeze the sample. Avoid successive
freezing and thawing. Discard hemolyzed or contaminated samples.
Richitie, RF. et. al., Clin. Chem., 1991, 37, 1676.
AACCPress 2000.
Young, D.S.; Effects of drugs on Clinical Laboratory Tests. 4th Ed. AACCPress. 2000.
QUALITY CONTROL
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APOLIPOPROTEIN B
PRINCIPLE PROCEDURE
The reagent allows quantifying the level of human apolipoprotein B present in the sample by Technique
of known concentrations of human apolipoprotein B.
The reagent is an antiserum anti-human apolipoprotein B which reacts with the apolipoprotein 1. Shake gently the reagents before use. The sample shall be processed without dilution.
B of the serum giving protein aggregates. This aggregation process produces an increase Conditions:
in the Abs. of the system. Volume of buffer solution (A): 240 μL
The apolipoprotein B (APO B) is the most abundant structural protein in LDL, VDLD Reaction temperature: 37ºC
lipoprotein and chylomicrons. The main function is to transport lipoproteins rich in
triglycerides from the liver to body tissues. 2. Mix well; start the stopwatch and read the Abs (Absi). Repeat the reading after 2min of
Since changes on APO A1 and APO B concentrations are similar to the ones in HDL and reaction (Absf) at 340nm
LDL on patients with coronary heart diseases, the measurement of apolipoproteins can be
applied to the estimation of coronary risk. CALCULATIONS
Increase in APO B concentration could be because a disorder on lipoprotein metabolism Determine the ΔAbs (Absorbance increase) as (Absf – Absi) for each sample, standard or
as hyperapobetalipoproteinemia, in which the concentration of APO B increase and LDL- control. Apolipoprotein B concentrations are obtained by the interpolation of the ∆Abs value
cholesterol levels are normal. in the calibration curve.
APO B concentration decreases in abetalipoproteinemia, in which the secretion of intestinal
and hepatic lipoproteins rich in triglycerides is blocked. Calibration curve
Prepare the curve by serially diluting the calibrator with saline (apolipoprotein calibrator Ref.
Single test result could not be used to make a clinical diagnosis. It should integrate clinical 99 36 90). To obtain the concentration of each dilution, multiply the calibrator concentration
and laboratory data. by the corresponding factor indicated in the table:
REAGENTS
Kit (Ref. 99 34 06). Contents: 0 1 2 3 4 5
A. 1 x 16.0 mL Buffer solution. Ref. 99 04 59 CAL (µL) -- 25 50 100 200 400
B. 1 x 4.0 mL anti-human APO B serum Ref. 99 04 83
Saline sol. (µL) 400 375 350 300 200 --
Apolipoprotein Calibrator. Ref. 99 36 90
Factor 0 0.0625 0.125 0.25 0.5 1.0
1 x 1 mL Pool of human plasma samples calibrated in apolipoprotein A1 and apolipoprotein
B.
The concentration of each component is stated on the vial label.
autoanalyzer.
REFERENCES VALUES
1 x 1 mL Samples positive for apolipoprotein A1 and apolipoprotein B.
59-168 mg/dL
REAGENT PREPARATION of a representative population, using its own instrumentation, blood collection methods and
Reagents are ready to use. assaying procedures.

A. Sodium phosphate/saline buffer pH 7.4 with stabilizers and preservatives. The performance characteristics depend on the method used. It is recommended to
B. Goat antiserum anti-human APO B in buffered saline pH 7.4 with stabilizers and calculate these data for each particular test protocol. These results have been obtained
preservatives. using an autoanalyzer.
STORAGE AND STABILITY Sensitivity, as detection limit: 7.8 mg/dL

The antiserum reagent, the buffer solution and the Calibrator and Control are stable up to Reaction range: 300 mg/dL. Samples that give higher concentration should be diluted in
the expiry date indicated on the label when kept at 2-8ºC. saline NaCl 0.9% (1+1) and the final result has to be multiplied by 2.
Do not freeze antisera or buffer solution. Accuracy: 95.1%
Presence of particles in the reagent. Quality control values outside acceptation range. No prozone effect was observed up to 3000 mg/dL
ADDITIONAL EQUIPMENT compared with the reference reagent.
Spectrophotometer or photometer thermostatable at 37ºC. Cuvette: 1 cm light- Details of the comparison experiments are available on request.
path.
No interference was observed by hemoglobin (125 mg/dL), bilirubin (5 mg/dL) and RF (600
UI/mL). Lipemic sera and other substances could interfere (See references).
reagent manipulation Human serum used in the control preparation have been Samples that give an Abs. higher than that obtained for the last point in the standard curve
found negative in the reaction to HBsAg and HIV I/II. However they should al- should be diluted in saline (NaCl 0.9%) and the determination repeated.
ways be handled with care. All reagents contain sodium azide 0.09% as pre-
servative. It is recommended to read SDS before reagent manipulation. AUTOANALYZERS
Waste products must be handled as per local regulations. Adaptations to different autoanalyzers are available on request.
SAMPLE REFERENCES
Recent serum or plasma (heparin or EDTA plasma) kept at 2-8ºC for no longer than 48h. If Tiezt, NW., Textbook of Clinical Chemistry 5th Edition, W.B. Saunders, Philadelphia (2012).
the test is to be performed later, it is recommended to freeze the sample. Avoid successive Richitie, RF. et. al., Clin. Chem., 1991, 37, 1676.
freezing and thawing. Discard hemolyzed or contaminated samples. Young, D.S.; Effects of Preanalytical Variables on Clinical Laboratory Tests. 3rd Ed.
QUALITY CONTROL AACCPress 2000.
Young, D.S.; Effects of drugs on Clinical Laboratory Tests. 4th Ed. AACCPress. 2000.
ISO 9001 / ISO 13485

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APOLIPOPROTEIN CALIBRATOR
PRODUCT DESCRIPTION
Pool of human plasma for the preparation of the standard curve for protein
determinations in sera or plasma by immunoturbidimetry.
REAGENTS
Apolipoprotein Calibrator Ref. 99 36 90
Contents:
1 x 1 mL Pool of plasma samples titrated in the parameters below.
REAGENT PREPARATION
REAGENT COMPOSITION
Pool of human plasma with stabilizers and preservatives. The pool of plasma is
calibrated in apolipoprotein A1 and apolipoprotein B.

ASSIGNED VALUES
The concentration value for each parameter is indicated on the vial label and
Immunology
they are all traceable to the reference material WHO/SP1-01 (ApoA1) y WHO/
SP3-07 (Apo B).
CAUTION
found negative in the reaction to HBsAg and HIV I/II. However they should always
be handled with care. All reagents contain sodium azide 0.09% as preservative.
It is recommended to read SDS before reagent manipulation.
ISO 9001 / ISO 13485

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APOLIPOPROTEIN CONTROLS
PRODUCT DESCRIPTION
Pool of human plasma for quality control for protein determinations in sera or
plasma by immunoturbidimetry.
REAGENTS
Contents:
REAGENT PREPARATION
Control is ready to use.
REAGENT COMPOSITION
Pool of human plasma with stabilizers and preservatives. The pool of plasma is
titrated in apolipoprotein A1 and apolipoprotein B.

ASSIGNED VALUES
The concentration value for each parameter is indicated on the vial label and
they are all traceable to the reference material WHO/SP1-01 (ApoA1) and WHO/
SP3-07 (Apo B).
The value and expected range for each parameter are derived from interlaboratory
data and they are given for orientation only; each laboratory should establish its
own acceptation range.
CAUTION
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C3 COMPLEMENT
PRINCIPLE PROCEDURE
The reagent allows quantifying the level of human C3 Complement present in the sample by Technique
of known concentrations of human C3 Complement.
The reagent is an antiserum anti-human C3 Complement which reacts with the C3 1. Shake gently the reagents before use. The sample shall be processed without dilution.
Complement of the serum giving protein aggregates. This aggregation process produces Conditions:
an increase in the Abs. of the system. Volume of buffer solution (A): 240 μL
The C3 Complement (C3) is the most abundant component and is cleaved by all pathways Reaction temperature: 37ºC
of complement activation. It is a protein synthesized by the liver in response to bacterial
endotoxin which induces their synthesis by monocytes and fibroblasts. 2. Mix well; start the stopwatch and read the Abs (Absi). Repeat the reading after 2min of
High levels of C3 are found in acute inflammatory reactions, amyloidosis and biliary reaction (Absf) at 340nm
obstruction.
Decreased levels occur with genetic deficiency, recurrent infection by pyogenic bacteria CALCULATIONS
and in neonates. Determine the ΔAbs (Absorbance increase) as (Absf – Absi) for each sample, standard or
control. C3 Complement concentrations are obtained by the interpolation of the ∆Abs value
Single test result could not be used to make a clinical diagnosis. It should integrate clinical in the calibration curve.
Calibration curve
REAGENTS Prepare the curve by serially diluting the calibrator (Protein Calibrator Ref. 99 99 39).
Kit (Ref. 99 32 50). Contents: To obtain the concentration of each dilution, multiply the calibrator concentration by the
A. 1 x 16.0 mL Buffer solution Ref. 99 05 41 corresponding factor indicated in the table:
B. 1 x 4.0 mL anti-human C3 serum Ref. 99 04 52
Protein Calibrator. Ref. 99 99 39 0 1 2 3 4 5

1 x 1 mL Pool of human plasma samples calibrated in antithrombin III, IgA, IgG,
CAL (μL) -- 25 50 100 200 400
IgM,transferrin, C3, C4, α1-antitrypsin and α-1 acid glycoprotein.
The concentration is stated on the insert. Saline sol. (μL) 400 375 350 300 200 --
Factor 0 0.0625 0.125 0.25 0.5 1.0
Protein Control Ref. 99 66 86
1 x 1 mL Samples positive for antithrombin III, IgA, IgG, IgM, transferrin, C3, C4, α1-
antitrypsin and α-1 acid glycoprotein.
autoanalyzer.
REAGENT PREPARATION
REFERENCES VALUES
Reagents are ready to use.
90 - 180 mg/dL
B. Goat antiserum anti-human C3 in buffered saline pH 7.4 with stabilizers and preservatives. assaying procedures.
Immunology
STORAGE AND STABILITY PERFORMANCE CHARACTERISTICS
The antiserum reagent, the buffer solution and the Protein Calibrator and Control are stable The performance characteristics depend on the method used. It is recommended to
up to the expiry date indicated on the label when kept at 2-8ºC. calculate these data for each particular test protocol. These results have been obtained
Do not freeze antisera or buffer solution. using an autoanalyzer.

Presence of particles in the reagent. Quality control values outside acceptation range. Reaction range: 350 mg/dL. Samples that give higher concentration should be diluted in
Accuracy: 96.1%
The safety statements are on the label. It is recomended to read SDS before reagent compared with the reference reagent.
INTERFERENCES
No interference was observed by hemoglobin (500 mg/dL), bilirubin (20 mg/dL) and RF (300
UI/mL). Lipemic sera and other substances could interfere (See references).
SAMPLE Samples that give an Abs. higher than that obtained for the last point in the standard curve
Recent serum or plasma (heparin or EDTA plasma) kept at 2-8ºC for no longer than 48h. If should be diluted in saline (NaCl 0.9%) and the determination repeated.
freezing and thawing. Discard hemolyzed or contaminated samples. AUTOANALYZERS

Control serum should be included in each test series. Each particular laboratory should Tiezt, NW., Textbook of Clinical Chemistry 5th Edition, W.B. Saunders, Philadelphia (2012).
establish its own control program. Dati et al., Eur J Clin Chem Clin Biochem, 34, 517 - 520 (1996).
AACCPress 2000.
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C4 COMPLEMENT
PRINCIPLE PROCEDURE
The reagent allows quantifying the level of human C4 Complement present in the sample Technique
by comparing the turbidimetric response produced with that obtained from standard curve To be used in clinical chemistry analyzers.
of known concentrations of human C4 Complement.
The reagent is an antiserum anti-human C4 Complement which reacts with the C4 1. Shake gently the reagents before use. The sample shall be processed without dilution.
Complement of the serum giving protein aggregates. This aggregation process produces Conditions:
an increase in the Abs. of the system. Volume of buffer solution (A): 240 μL
The C4 Complement (C4) is an essential component for the complement activation Reaction temperature: 37ºC
pathways.
Concentration of C4 are slightly increased by the acute inflammatory reactions (trauma, 2. Mix well; start the stopwatch and read the Abs (Absi). Repeat the reading after 2min of
inflammations or tissue necrosis). reaction (Absf) at 340 nm
Concentration of C4 decreased in genetic deficiencies associated with a high prevalence
of autoimmune or collagen vascular diseases, particularly Systemic Lupus Erythematosus CALCULATIONS
(SLE). C4 plasma concentration also may decrease due to its consumption in the formation Determine the ΔAbs (Absorbance increase) as (Absf – Absi) for each sample, standard or
of immunocomplexes or in neonates. control. C4 Complement concentrations are obtained by the interpolation of the ∆Abs value
in the calibration curve.
and laboratory data. Calibration curve
Prepare the curve by serially diluting the calibrator (Protein Calibrator Ref. 99 99 39).
REAGENTS To obtain the concentration of each dilution, multiply the calibrator concentration by the
Kit (Ref. 99 36 69). Contents: corresponding factor indicated in the table:
A. 1 x 16.0 mL Buffer solution Ref. 99 05 42
B. 1 x 4.0 mL anti-human C4 serum Ref. 99 04 55
0 1 2 3 4 5
Protein Calibrator Ref. 99 99 39
CAL (μL) -- 25 50 100 200 400
1 x 1 mL Pool of human plasma samples calibrated in antithrombin III, IgA, IgG,
IgM,transferrin, C3, C4, α1-antitrypsin and α-1 acid glycoprotein. Saline sol. (μL) 400 375 350 300 200 --
Factor 0 0.0625 0.125 0.25 0.5 1.0
1 x 1 mL Samples positive for antithrombin III, IgA, IgG, IgM, transferrin, C3, C4, α1-
antitrypsin and α-1 acid glycoprotein. The method described above is only indicative, as it should be specifically adapted to each
The concentration is stated on the insert. autoanalyzer.
REAGENT PREPARATION
10 - 40 mg/dL
B. Goat anti serum anti-human C4 in buffered saline pH 7.4 with stabilizers and preservatives. assaying procedures.
The antiserum reagent, the buffer solution and the Protein Calibrator and Control are stable
up to the expiry date indicated on the label when kept at 2-8ºC.
using an autoanalyzer.
Presence of particles in the reagent. Quality control values outside acceptation range.
Accuracy: 98%
Waste products must be handled as per local regulations. No interference was observed by hemoglobin (62.5 mg/dL), bilirubin (20 mg/dL) and RF
(300 UI/mL). Lipemic sera and other substances could interfere (See references).
SAMPLE Samples that give an Abs. higher than that obtained for the last point in the standard curve
Recent serum or plasma (Heparin or EDTA plasma) kept at 2-8ºC for no longer than 48h. If should be diluted in saline (NaCl 0.9%) and the determination repeated.
freezing and thawing. Discard hemolyzed or contaminated samples. AUTOANALYZERS

Control serum should be included in each test series. Each particular laboratory should Tiezt, NW., Textbook of Clinical Chemistry 5th Edition, W.B. Saunders, Philadelphia (2012).
establish its own control program. Dati et al., Eur J Clin Chem Clin Biochem, 34, 517 - 520 (1996).
AACCPress 2000.
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FERRITIN
PRINCIPLE PROCEDURE
Ferritin reagent consists of a suspension of polystyrene latex particles coated with Technique
human anti-ferritin. The agglutination produced by serum ferritin can be detected as To be used in clinical chemistry analyzers.
increase of absorbance due to the aggregation of the sensitized particles. 1. Bring reagents and samples at room temp. Shake gently the reagents before using
it.
DIAGNOSTIC USE 2. Add to a reaction cuvette.
Ferritin is an iron storage protein; the serum ferritin concentration has a good Volume of buffer solution: 0.6 mL
correlation against the total body iron stores. Volume of latex reagent: 0.3 mL
In healthy subjects and in patients with either deficiency or excess of iron, serum Reaction temperature: 37ºC
ferritin is a reliable indicator of the iron pool that can be mobilized. 3. Mix thoroughly for total homogenisation. Add
On the other hand, the serum levels of ferritin are found to be elevated in patients with Volume of sample: 0.1 mL
hepatic diseases, chronical inflammations, several types of cancer, and some types 4. Mix well; start the stopwatch and read the Abs (Absi) and after 7 min of reaction
of rheumatoid arthritis. (Absf) at 630 or 700 nm.
Besides the use of ferritin determination as indicator of metabolism of iron it is used CALCULATIONS
as tumor marker for therapeutic drug monitoring and indicator of evolution of certain Determine the ΔAbs (Absorbance increase) as Absf – Absi for each sample, std or
tumors. control.
REAGENTS Ferritin concentration is calculated as:
Kit 30 mL (Ref. 99 21 05). Contents: ΔAbs sample
A. 1 x 20 mL Buffer Reagent Ref. 99 21 09 x conc. STD = ng Ferritin / mL
B. 1 x 10 mL Latex Reagent Ref. 99 21 07 ΔAbs standard
The concentration is stated on the label. Where:
ΔAbs sample: Absorbance increase of sample
Kit 90 mL (Ref. 99 77 30). Contents: ΔAbs standard: Absorbance increase of standard
A. 1 x 60 mL Buffer Reagent Ref. 99 85 22 Conc. STD: Standard concentration
B. 1 x 30 mL Latex Reagent Ref. 99 80 55 Calibration: Prepare the standard curve using the ferritin calibrator and tris buffer.
C. 1 x 2 mL Standard Ref. 99 21 12 Use tris buffer for concentration 0 ng/mL. (Ref. 992130)
The concentration is stated on the label.
REFERENCES VALUES
Ferritin calibrator (Ref. 99 21 30). Contents: Children/Adolescents: 15 – 120 ng/mL
A. 1 x 2.5 mL Calibrator Ref. 99 21 31 Men: 30 – 300 ng/mL
B. 1 x 5 mL Buffer Ref. 99 21 32 Women: 10 – 160 ng/mL
Ferritin calibrator and tris buffer to prepare calibration curve. The concentration is Women (post-menopausal) 30 – 300 ng/mL
stated on the label. Each particular laboratory should establish its own normal range, obtained from
samples of a representative population, using its own instrumentation, blood collection
Ferritin control (Ref. 99 21 60). Contents: methods and assaying procedures.
1 x 1.0 mL Ferritin valuated serum. PERFORMANCE CHARACTERISTICS
The concentration is stated on the label. The performance characteristics depend on the method used. It is recommended
Immunology
REAGENT PREPARATION to calculate these data for each particular test protocol. These results have been
Reagents are ready to use. obtained using an autoanalyzer.
Linearity: up to 300 ng/mL. Linearity limit may vary depending on the analyzer used.
REAGENT COMPOSITION For concentrations higher than 300 ng/mL calibrate the reagent using the ferritin
Latex reagent: suspension of latex particles sensitized with anti-human ferritin in a calibrator for more accurate results.
stabilisation buffer Sensitivity, as detection limit: 5.2 ng/mL
Buffer solution: sodium phosphate buffer, pH 6.5 Accuracy: 99.8%
Standards and Controls: pool of human sera with stabilizers. Repetitivity, as CV%: 3.56%
STORAGE AND STABILITY No prozone effect was observed up to 10000 ng/mL
The components of the kit, when stored at 2–8ºC, will remain stable until the expiration Trueness: Results obtained with this reagent did not show systematic differences
date stated on the label. Do not freeze. when compared with reference reagent.
Signs of reagent deterioration: Details of the comparison experiments are available on request.
Blank reagent >1500. Quality control values outside acceptation range.
ADDITIONAL EQUIPMENT INTERFERENCES
General laboratory equipment. Bilirrubin does not interfere up to concentrations of 427 μmol/L and Hemoglobin up
Spectrophotometer or photometer thermostatable at 37ºC. Cuvette: 1 cm light-path. to 15 g/L.
Lipaemic or turbid samples must be clarified before the assay by centrifugation.
CAUTION Mix thoroughly the latex reagent before using.
The safety statements are on the label. It is recomended to read SDS before reagent Dilute the samples with Abs values higher than that obtained for the last point in the
manipulation Human serum used in the control preparation have been found negative standard curve and repeat the assay.
in the reaction to HBsAg and HIV I/II. However they should always be handled with AUTOANALYZERS
care. All reagents contain sodium azide 0.09% as preservative. It is recommended to Adaptations to different autoanalyzers are available on request.
read SDS before reagent manipulation. REFERENCES
Waste products must be handled as per local regulations. Bernard, A. Lauwerys, R.; 1984, J. Immunol. Methods. 71: 141 – 147.
SAMPLE Cook, J. D.; et al. 1974, Am. J. Clin. Nutr. 27: 681–687.
Fresh serum; do not use lipaemic or turbid samples. Ferritin in the sample remains Milman, N. et al. 1994, Eur. J. Haematol 53: 16 – 20
stable for 5 days at 2 - 8ºC. Deep-frozen sera can be stored for up to 3 months. Worwood, M. 1990, Blood Reviews, 4: 259 – 269
Repeated freeze-thaw cycles must be avoided.
QUALITY CONTROL
Control serum should be included in each test series. Each particular laboratory
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FERRITIN CALIBRATOR
PRODUCT DESCRIPTION
Pool of human sera with known ferritin concentration for the preparation of the
standard curve for ferritin determination by immunoturbidimetry.
REAGENTS
Kit Ferritin Calibrator Ref. 99 21 30
Contents:
A. 1 x 2.5 mL Ferritin calibrator Ref. 99 21 31
B. 1 x 5 mL Buffer Ref. 99 21 32
The concentration is stated on the label.
REAGENT PREPARATION
REAGENT COMPOSITION
Calibrator: Pool of human sera containing known quantities of ferritin and added
with stabilizers.
Buffer: Tris/HCl 50mM added with stabilizers and preservatives.

Reagents, when stored at 2 - 8ºC, will remain stable until the expiration date
ASSIGNED VALUES
The concentration is stated on the vial label as well as in the certificate of
analysis of the corresponding lot (www.qca.es).
The concentration value is traceable to the reference material: NIST 94/572.
CAUTION
PROCEDURE
Prepare the standard curve by serial dilutions of calibrator with the buffer.
The calibrator dilutions shall be processed as any other serum sample, according
to the test instructions (Ferritin).
It is recommended to use control serum (Ferritin Control, Ref. 99 21 60) to check
measure deviations.
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FERRITIN CONTROL
PRODUCT DESCRIPTION
The Ferritin control is a liquid control serum which is prepared from human se-
rum. The control is intended to be used for internal quality control in determina-
tions of levels of Ferritin by immunoturbidimetry.
REAGENTS
1 x 1mL Ferritin control Ref. 99 21 60
Contents:
Serum calibrated in Ferritin.
The concentration is stated on the vial label as well as in the certificate of analy-
sis of the corresponding lot (www.qca.es).
CAUTION
Human serum was used in the manufacture of this product. Each donor unit was
tested with licensed reagents and found negative for HBsAg and nonreactive for
the HIV antibody.
However, it is recommended that this product be handled with the same precau-
tions used for patient specimens.
MATERIAL NECESARIO NO SUMINISTRADO
Pipetas volumétricas.
REAGENT PREPARATION
COMPOSITION
Pool of human sera samples with stabilizers.
ASSIGNED VALUES
Immunology
The assigned concentration is lot specific.
The concentration value is traceable to the reference material: NIST 94/572.
The value and expected range are derived from interlaboratory data and they
are only given for orientation; each laboratory should establish its own accepta-
tion range.
REFERENCES
Biochem, 1996, 34, 983:999.
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IMMUNOGLOBULIN A (IgA)
PRINCIPLE PROCEDURE
The reagent allows quantifying the level of human IgA present in the sample by comparing Technique
the turbidimetric response produced with that obtained from the standard curve of known To be used in automatic analyzers.
human IgA concentrations.
The reagent is an antiserum anti-human IgA which reacts with the IgA of the serum giving 1. Shake gently the reagents before using it. Use the sera samples without dilution.
protein aggregates. This aggregation process produces an increase in the Abs. of the Conditions:
system. Volume of buffer solution: 250 μL
Volume of antibody solution: 50 μL
The IgA is a dimmer form in secretions. It acts as a protector agent in front of the respiratory Reaction temperature: 37º C
system, urinary tract and intestinal tract infections.
The monomer form is found in serum and its specific role is still unclear; it may be important 2. Mix well; start the stopwatch and read the Abs (Absi) and after 3 min. of reaction (Absf)
in virus neutralisation. at 340nm.
High levels of serum IgA are found in hepatic diseases, tuberculosis, and rheumatoid CALCULATIONS
arthritis. Determine the ΔAbs (Absorbance increase) as (Absf – Absi) for each sample, standard
Low levels are associated with immunodeficiencies congenital or acquired. or control. Immunoglobulin A concentrations are obtained by the interpolation of the ∆Abs
value in the calibration curve.
Single test result can not be used to make a clinical diagnosis. It should integrate clinical
and laboratory data. Calibration curve
REAGENTS Prepare the curve by serially diluting the calibrator (Protein Calibrator Ref. 99 99 39).
Kit (Ref. 99 54 04). Contents: To obtain the concentration of each dilution, multiply the calibrator concentration by the
A. 1 x 20.0 mL Buffer solution Ref. 99 54 74 corresponding factor indicated in the table:
B. 1 x 4.0 mL anti-human IgA serum Ref. 99 54 34
Protein Calibrator Ref. 99 99 39 0 1 2 3 4 5

1 x 1 mL Pool of sera samples calibrated in antithrombin III, IgA, IgG, IgM,transferrin, C3, CAL (μL) -- 25 50 100 200 400
C4, α1-antitrypsin and α-1 acid Glycoprotein.
The concentration is stated on the insert. Saline sol. (μL) 400 375 350 300 200 --
Factor 0 0.0625 0.125 0.25 0.5 1.0
Control of Proteins Ref. 99 66 86
1 x 1 mL Sera sample positive for antithrombin III, IgA, IgG, IgM,transferrin, C3, C4, α1- The method described above is only indicative, as it should be specifically adapted to each
antitrypsin and α-1 acid Glycoprotein. autoanalyzer.
REFERENCES VALUES
REAGENT PREPARATION 70 - 400 mg/dL
Reagents are ready to use. Each particular laboratory should establish its own normal range, obtained from samples
of a representative population, using its own instrumentation, blood collection methods and
REAGENT COMPOSITION assaying procedures.
A. Sodium phosphate/saline buffer pH 7.4 with stabilizers and preservatives.
B. Goat antiserum anti-human IgA in buffered saline pH 7.4 with stabilizers and preservatives. PERFORMANCE CHARACTERISTICS
STORAGE AND STABILITY calculate these data for each particular test protocol. These results have been obtained
The antiserum reagent, the buffer solution and the Protein Controls are stable up to the using an autoanalyzer.
expiry date indicated on the label when kept at 2-8ºC.
Do not freeze antisera or buffer solution. Sensitivity, as detection limit: 6 mg/dL
Signs of reagent deterioration: saline NaCl 0.9% (1+1) and the final result has to be multiplied by 2.
Presence of particles in the reagent. Quality control values out of the acceptation range. Accuracy: 108%
ADDITIONAL EQUIPMENT No prozone effect was observed up to 2000 mg/dL
General laboratory equipment. Trueness: Results obtained with this reagent did not show systematic differences when
Spectrophotometer or photometer thermostatable at 37ºC. Cuvette: 1 cm light-path. compared with the reference reagent.
CAUTION Details of the comparison experiments are available on request.

manipulation Human serum used in the control preparation have been found negative in INTERFERENCES
the reaction to HBsAg and HIV I/II. However they should always be handled with care. All No interference was observed by hemoglobin (500mg/dL), bilirubin (20 mg/dL), triglycerides
reagents contain sodium azide 0.09% as preservative. It is recommended to read SDS (5000 mg/dL), heparine (50 mg/dL), RF (150UI/mL). Hemolyzed sera and other substances
before reagent manipulation. could interfere (see references). No cross reaction under test conditions with IgG or IgM
Waste products must be handled as per local regulations. have been observed. Samples that give an Abs. higher than that obtained for the last point in
SAMPLE the standard curve should be diluted in saline (NaCl 0.9%) and the determination repeated.
Recent serum or plasma (Heparin or EDTA plasma) kept at 2-8ºC no longer than 48h. If AUTOANALYZERS
the test is to be performed later, it is recommended to freeze the sample. Avoid repeated Adaptations to different autoanalyzers are available on request.
freezing and thawing. Discard hemolyzed or contaminated samples. REFERENCES
QUALITY CONTROL Young, D.S.; Effects of Preanalytical Variables on Clinical Laboratory Tests. 3rd Ed.
Control serum should be included in each test series. Each particular laboratory should AACCPress.
establish its own control program. Young, D.S.; Effects of drugs on Clinical Laboratory Tests. 4th Ed. AACCPress.
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IMMUNOGLOBULIN G (IgG)
PRINCIPLE PROCEDURE
The reagent allows to quantify the level of human IgG present in the sample by comparing Technique
human IgG concentrations.
The reagent is an antiserum anti-human IgG that reacts with the IgG of the serum giving 1. Shake gently the reagents before use. Use the sera samples without dilution.
Immunoglobulin G (75% of total Ig’s of serum) is the predominant serum immunoglobulin. Reaction temperature: 37º C
It is of particular importance in the body’s long-term defence against infection.
2. Mix well; start the stopwatch and read the Abs (Absi) and after 3 min. of reaction (Absf)
High levels of serum IgG are found in response to chronic or recurrent infections, at 340nm.
autoimmune diseases and hepatic diseases.
IgG deficiency is associated with recurrent and occasionally severe piogenic infections and CALCULATIONS
with immunodeficiencies acquired or congenital. Determine the ΔAbs (Absorbance increase) as (Absf – Absi) for each sample, standard or
Single test result can not be used to make a clinical diagnosis. It should integrate clinical control. Immunoglobulin G concentrations are obtained by the interpolation of the ∆Abs
and laboratory data. value in the calibration curve.
REAGENTS
Kit (Ref. 99 37 03). Contents: Calibration curve
A. 1 x 20.0 mL Buffer solution Ref. 99 37 73 Prepare the curve by serially diluting the calibrator (Protein Calibrator Ref. 99 99 39).
B. 1 x 4.0 mL anti-human IgG serum Ref. 99 37 33 To obtain the concentration of each dilution, multiply the calibrator concentration by the
corresponding factor indicated in the table:
1 x 1 mL Pool of sera samples calibrated in antithrombin III, IgA, IgG, IgM,transferrin, C3, 0 1 2 3 4 5
C4, α1-antitrypsin and α-1 acid glycoprotein.
The concentration is stated on the insert. CAL (μL) -- 25 50 100 200 400
Saline sol. (μL) 400 375 350 300 200 --
1 x 1 mL Sera sample positive for antithrombin III, IgA, IgG, IgM, transferrin, C3, C4, Factor 0 0.0625 0.125 0.25 0.5 1.0
α1-antitrypsin and α-1 acid glycoprotein.
The concentration is stated on the insert. The method described above is only indicative, as it should be specifically adapted to each
REAGENT PREPARATION autoanalyzer.
700 - 1600 mg/dL
B. Goat antiserum anti-human IgG in buffered saline pH 7.4 with stabilizers and preservatives. assaying procedures.
The antiserum reagent, the buffer solution, the calibrator and control are stable up to the
Immunology
expiry date indicated on the label when kept at 2-8ºC. using an autoanalyzer.
Do not freeze the antiserum or the buffer solution.
Sensitivity, as detection limit: 22 mg/dL
Signs of reagent deterioration: Reaction range: 2650 mg/dL. Samples that give higher concentration should be diluted in
Presence of particles in the reagent. Quality control values out of the acceptation range. saline NaCl 0.9% (1+1) and the final result has to be multiplied by 2.
ADDITIONAL EQUIPMENT Accuracy: 105.9%
General laboratory equipment. Repetitivity, as CV%: 1.75%
Spectrophotometer or photometer thermostatable at 37ºC. Cuvette: 1 cm light-path. Reproducibility, as CV%: 2.41%
Waste products must be handled as per local regulations. No interference was observed by hemoglobin (1000mg/dL), bilirubin (20mg/dL),
triglycerides (2500 mg/dL), heparin (50 mg/dL), and RF (150 UI/mL). Hemolyzed sera and
SAMPLE other substances could interfere (see references).
Recent serum or plasma (heparin or EDTA plasma) kept at 2-8ºC no longer than 48h If No cross reaction under test conditions with IgA or IgM has been observed.
the test is to be performed later, it is recommended to freeze the sample. Avoid repeated Samples that give an Abs. higher than that obtained for the last point in the standard curve
freezing and thawing. Discard hemolyzed or contaminated samples. should be diluted in saline (NaCl 0.9%) and the determination repeated.
QUALITY CONTROL Lipemic or hemolyzed samples could give false results.
Control serum should be included in each test series. Each particular laboratory should AUTOANALYZERS
establish its own control program. Adaptations to different autoanalyzers are available on request.
REFERENCES
AACCPress.
Young, D.S.; Effects of drugs on Clinical Laboratory Tests. 4th Ed. AACCPress
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IMMUNOGLOBULIN M (IgM)
PRINCIPLE PROCEDURE
The reagent allows to quantify the level of human IgM present in the sample by comparing Technique
concentrations of human IgM.
The reagent is an antiserum anti-human IgM that reacts with the IgM of the serum giving 1. Shake gently the reagents before use. Use the sera samples without dilution.
Immunoglobulin M is the first Ig to appear in response to an antigenic stimulus such as Reaction temperature: 37º C
an infectious agent. In many cases, the antigen specific IgM level subsequently falls and
remains low as the IgG response appears. 2. Mix well; start the stopwatch and read the Abs (Absi) and after 3 min. of reaction (Absf)
at 340nm.
High levels of serum IgM are found in response to chronic infections.
IgM deficiency is associated with immunodeficiencies acquired or congenital. CALCULATIONS
Single test result could not be used to make a clinical diagnosis. It should integrate clinical Determine the ΔAbs (Absorbance increase) as (Absf – Absi) for each sample, standard or
and laboratory data. control. Immunoglobulin M concentrations are obtained by the interpolation of the ∆Abs
REAGENTS value in the calibration curve.
A. 1 x 20.0 mL Buffer solution Ref. 99 90 88 Calibration curve
B. 1 x 4.0 mL anti-human IgG serum Ref. 99 90 48 Prepare the curve by serially diluting the calibrator (Protein Calibrator Ref. 99 99 39).
To obtain the concentration of each dilution, multiply the calibrator concentration by the
Protein Calibrator. Ref. 99 99 39 corresponding factor indicated in the table:
1 x 1 mL Pool of sera samples calibrated in antithrombin III, IgA, IgG, IgM,transferrin, C3,
C4, α1-antitrypsin and α-1 acid glycoprotein. 0 1 2 3 4 5
The concentration is stated on insert.
CAL (μL) -- 25 50 100 200 400
Control of Proteins Ref. 99 66 86 Saline sol. (μL) 400 375 350 300 200 --
1 x 1 mL Sera sample positive for antithrombin III, IgA, IgG, IgM, transferrin, C3, C4, α1-
antitrypsin and α-1 acid glycoprotein. Factor 0 0.0625 0.125 0.25 0.5 1.0
REAGENT PREPARATION The method described above is only indicative, as it should be specifically adapted to each
Reagents are ready to use. autoanalyzer.
REFERENCES VALUES
REAGENT COMPOSITION 40 - 230 mg/dL
A. Sodium phosphate/saline buffer pH 7.4 with stabilizers and preservatives. Each particular laboratory should establish its own normal range, obtained from samples
B. Goat antiserum anti-human IgM in buffered saline pH 7.4 with stabilizers and of a representative population, using its own instrumentation, blood collection methods and
preservatives. assaying procedures.
The antiserum reagent, the buffer solution and the protein controls are stable up to the calculate these data for each particular test protocol. These results have been obtained
expiry date indicated on the label when kept at 2-8ºC. using an autoanalyzer.
Do not freeze the antiserum or the buffer solution.
Sensitivity, as detection limit: 4 mg/dL
Signs of reagent deterioration: Reaction range: 400 mg/dL. Samples that give higher concentration should be diluted in
Presence of particles in the reagent. Quality control values out of the acceptation range. saline NaCl 0.9% (1+1) and the final result has to be multiplied by 2.
ADDITIONAL EQUIPMENT Accuracy: 103%
PRÉCAUTIONS Trueness: Results obtained with this reagent did not show systematic differences when
Les indications de sécurité sont sur l’étiquette des produits. Il est conseillé de consulter la compared with the reference reagent.
fiche de données de sécurité avant de manipuler le réactif. Les sérums humains utilisés Details of the reagent performance are available on request.
pour la préparation des calibrateurs et des contrôles ont donné un résultat négatif lors
de la réaction avec l’Ag HBs et l’HIV I/II. Malgré cela, ils doivent être manipulés avec INTERFERENCES
précaution. Par ailleurs, tous les réactifs contiennent de l’azide de sodium à 0,09% en tant No interference was observed by hemoglobin (250mg/dL), bilirubin (8 mg/dL), triglycerides
que conservateur. (5000 mg/dL), heparin (50 mg/dL) and RF (150 UI/mL). Hemolyzed sera and other
On conseillé de consulter la fiche des données de sécurité avant de manipuler le réactif. substances could interfere (see references). No cross reaction under test conditions with
L’élimination des déchets doit être effectuée conformément aux normes en vigueur. IgG or IgA have been observed. Samples that give an Abs. higher than that obtained
SAMPLE for the last point in the standard curve should be diluted in saline (NaCl 0.9%) and the
Fresh serum or plasma (heparin or EDTA plasma) kept at 2-8ºC no longer than 48h If determination repeated.
the test is to be performed later, it is recommended to freeze the sample. Avoid repeated Lipemic or haemolysed sample could give false results.
freezing and thawing. Discard hemolyzed or contaminated samples.
AUTOANALYZERS
QUALITY CONTROL Adaptations to different autoanalyzers are available on request.
REFERENCES
AACCPress.
Young, D.S.; Effects of drugs on Clinical Laboratory Tests. 4th Ed. AACCPress.
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MICROALBUMIN
PRINCIPLE PROCEDURE
The reagent is a solution of goat anti-human albumin, which reacts with the albumin present Technique
in the urine producing antigen-antibody aggregates. To be used in clinical chemistry analyzers.
The formation of these aggregates produces a change in the Abs. system directly
proportional to the protein concentration of the sample. By means of a calibration curve, 1. Shake gently the reagents before use.
where absorbance is related to the albumin concentration, it is possible to quantify the The sample shall be processed without dilution.
sample concentration. Conditions:
Volume of buffer solution: 300 μL
DIAGNOSTIC USE Volume of antibody solution: 50 μL
Microalbuminuria is the term used to describe an increase in urinary excretion of albumin Volume of sample: 10 μL
above the reference interval for healthy nondiabetic patients (0-20 mg/L). It is considered Reaction temperature: 37º C
microalbuminuria when the urinary albumin concentration falls between 20 and 200 mg/L.
The increase in urinary albumin loss is considered a clinical indicator of a failure of the renal 2. Mix well; start the stopwatch and read the Abs (Absi) and after 3 min. of reaction (Absf)
function in diabetic patients. at 340 nm
Single test result can not be used to make a clinical diagnosis. It should integrate clinical
and laboratory data. CALCULATIONS
REAGENTS Determine the ΔAbs (Absorbance increase) as (Absf – Absi) for each sample, standard or
Kit (Ref. 99 38 00). Contents: control. Microalbumin concentration is obtained by the interpolation of the ∆Abs value in the
A. 1 x 24,0 mL Buffer solution Ref. 99 38 44 calibration curve.
B. 1 x 4,0 mL anti-human albumin Ref. 99 38 41
C. 1 x 1 mL Microalbumin Standard Ref. 99 38 45 Calibration curve
D. 1 x 1 mL Microalbumin Control Ref. 99 38 47 Prepare the curve by serially diluting the standard. To obtain the concentration of each
dilution, multiply the calibrator concentration by the corresponding factor indicated in the
REAGENT PREPARATION table:
The components of the kits are ready to use.
REAGENT COMPOSITION 0 1 2 3 4 5
A. Sodium phosphate/saline buffer pH 7.4.
STD (μL) -- 25 50 100 200 400
B. Goat anti-human albumin in buffered saline pH 7.4.
C and D. Pool of plasma samples calibrated in microalbumin. Concentrations are stated on Saline sol. (μL) 400 375 350 300 200 --
the vials label.
Factor 0 0.0625 0.125 0.25 0.5 1.0
When stored at 2-8º C, the kit will remain stable until the expiration date stated on the label. In some instruments, calibration curve is linear in the range 0 – 50mg/L. In such a case, it is
Do not freeze the antiserum nor the buffer solution. possible to perform a single point calibration (Diluting the standard 1/10 with saline solution).
However, to achieve a higher accuracy, it is recommended the use of a calibration curve.
Presence of particles in the reagent. Quality control values outside the acceptance range. The method described above is only indicative, as it should be specifically adapted to each
autoanalyzer.
General laboratory equipment. REFERENCES VALUES
Spectrophotometer or photometer thermostatable at 37ºC. Cuvette: 1 cm light-path. 0 - 20 mg/L
Immunology
CAUTION assaying procedures.
manipulation Human serum used in the control preparation have been found negative in PERFORMANCE CHARACTERISTICS
the reaction to HBsAg and HIV I/II. However they should always be handled with care. All The performance characteristics depend on the method used. It is recommended to
reagents contain sodium azide 0.09% as preservative. It is recommended to read SDS calculate these data for each particular test protocol.
before reagent manipulation. These results below, have been obtained in autoanalyzer.
Waste products must be handled as per local regulations. Linearity: up to 50 mg/L
SAMPLE Sensitivity, as detection limit: 3.3 mg/L
24 hours or random first morning urine. Sample can be stored at 2-8ºC for no longer than Reaction range: 400 mg/L. Samples with higher concentration should be diluted in saline
48h. If the test is to be run later, it is better to freeze the sample. The use of centrifugated NaCl 0.9% (1+1) and the final result multiplied by 2.
urine is highly recommended. Accuracy: 102.9%
Control serum should be included in each test series. Each particular laboratory should Reproducibility, as CV%: 2.15%
establish its own control program. No prozone effect was observed up to 1550 mg/L
INTERFERENCES
No interference was observed by hemoglobin (500 mg/dL), bilirubin (20 mg/dL), triglycerides
(5000 mg/dL), heparine (50 mg/dL), RF (150 UI/mL). Hemolyzed sera and other substance
may interfere.
Samples with an Abs. higher than that of the last point of the calibration curve should be
diluted in saline (NaCl 0.9%) and assayed once again.
AUTOANALYZERS
REFERENCES
Tietz Textbook of Clinical Chemistry and Molecular Diagnostics, 5th ed. Burtis, C.A.;
Ashwood, E.R.; Bruns, D. E.; Philadelphia (1999).
Miller, W.G.; et al., Clin. Chem., 2009, 55, 24-38.
Young, D.S. Effects of Preanalytical Variables on Clinical Laboratory Tests. 3rd Ed.
AACCPress.
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TRANSFERRIN
PRINCIPLE PROCEDURE
The reagent allows quantifying the level of Transferrin present in the sample by Technique
comparing the turbidimetric response produced with that obtained from the standard To be used in clinical chemistry analyzers.
curve of known human Transferrin concentrations.
The reagent is an antiserum anti-Transferrin which reacts with serum Transferrin 1. Shake gently the reagents before using it. Use the sera sample without dilution.
giving protein aggregates. This aggregation process produces an increase in the Abs. Conditions:
of the system. Volume of buffer solution: 350 μL
Transferrin is a serum β-glubulin responsible for transport of iron. Hypotransferrinemia Reaction temperature: 37ºC
of variable degree can occur in chronic diseases and hemolytic anemia. Elevated
levels occur as the result of severe iron deficiency or hepatitis. 2. Mix well; start the stopwatch and read the Abs (Absi) and after 5 min. of reaction
Single test result can not be used to make a clinical diagnosis. It should integrate (Absf) at 340 nm.
REAGENTS CALCULATIONS
Kit (Ref. 99 06 04). Contents: Determine the ΔAbs (Absorbance increase) as (Absf – Absi) for each sample,
A. 1 x 28.0 mL Buffer solution Ref. 99 06 22 standard or control. Transferrin concentrations are obtained by the interpolation of the
B. 1 x 2.8 mL anti-human Transferrin serum Ref. 99 06 12 ∆Abs value in the calibration curve.
Protein Calibrator Ref. 99 99 39 Calibration curve

1 x 1 mL Pool of sera samples calibrated in Antithrombin III, IgA, IgG, IgM,Transferrin, Prepare the curve by serially diluting the calibrator (Protein Calibrator Ref. 99 99 39).
C3, C4, α1-antitrypsin and α-1 acid Glycoprotein. To obtain the concentration of each dilution, multiply the calibrator concentration by
The concentration is stated on the insert. the corresponding factor indicated in the table:
Positive Control of Proteins Ref. 99 66 86

0 1 2 3 4 5
1 x 1 mL Sera sample positive for Antithrombin III, IgA, IgG, IgM,Transferrin, C3, C4,
α1-antitrypsin and α-1 acid Glycoprotein. CAL (μL) -- 25 50 100 200 400
Saline sol. 400 375 350 300 200 --
REAGENT PREPARATION (μL)
Factor 0 0.0625 0.125 0.25 0.5 1.0
REAGENT COMPOSITION
A. Sodium phosphate/saline buffer pH 7.4 with stabilizers and preservatives. The method described above is only indicative, as it should be specifically adapted
B. Goat antiserum anti-human Transferrin in buffered saline pH 7.4 with stabilizers to each autoanalyzer.
and preservatives. REFERENCES VALUES
170 - 340 mg/dL
STORAGE AND STABILITY Each particular laboratory should establish its own normal range, obtained from
The components of the kit are stable up to the expiry date indicated on the label when samples of a representative population, using its own instrumentation, blood collection
kept at 2-8ºC. methods and assaying procedures.
Do not freeze antisera or buffer solution. PERFORMANCE CHARACTERISTICS
Signs of reagent deterioration: The performance characteristics depend on the method used. It is recommended
Presence of particles in the reagent. Quality control values outside acceptation range. to calculate these data for each particular test protocol. These results have been
ADDITIONAL EQUIPMENT obtained using an autoanalyzer.
Spectrophotometer or photometer thermostatable at 37ºC. Cuvette: 1 cm light-path. Sensitivity, as detection limit: 2.2 mg/dL
Reaction range: 500 mg/dL. Samples that give higher concentration should be diluted
CAUTION in saline NaCl 0.9% (1+1) and the final result has to be multiplied by 2.
The safety statements are on the label. It is recomended to read SDS before reagent Accuracy: 96.6%
manipulation Human serum used in the control preparation have been found negative Repetitivity, as CV%: 0.92%
in the reaction to HBsAg and HIV I/II. However they should always be handled with Reproducibility, as CV%: 3.0%
care. All reagents contain sodium azide 0.09% as preservative. It is recommended to No prozone effect was observed up to 2050 mg/dL
read SDS before reagent manipulation. Trueness: Results obtained with this reagent did not show systematic differences
Waste products must be handled as per local regulations. when compared with the reference reagent.
SAMPLE
Recent serum or plasma kept at 2-8ºC no longer than 48h. If the test is to be performed
later, it is recommended to freeze the sample. Avoid repeated freezing and thawing.
INTERFERENCES
Discard hemolyzed or contaminated samples.
No interference was observed by Hemoglobin (1000 mg/dL), Bilirubin (20 mg/
QUALITY CONTROL dL), Heparine (50 mg/dL),Triglycerides (5000 mg/dL), RF (150 UI/mL). Lipemic or
Control serum should be included in each test series. Each particular laboratory hemolyzed sera and other substances could interfere (see references).
should establish its own control program. Serum samples that give an Abs. higher than that obtained for the last point in
the standard curve should be diluted in saline (NaCl 0.9%) and the determination
repeated.
AUTOANALYZERS
REFERENCES
Dati, F. et. al (1989) Lab. Med., 13, 87.
(2012).
AACCPress.
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PROTEINS POSITIVE CONTROL
FOR AT III, IgA, IgG, IgM, TRANSFERRIN
PRODUCT DESCRIPTION
Pool of human plasma for quality control for protein determinations in sera or
plasma by immunoturbidimetry.
REAGENTS
Contents:
REAGENT PREPARATION
Controls are ready to use
REAGENT COMPOSITION
Pool of human plasma with stabilizers and preservatives.
MATERIAL NECESARIO NO SUMINISTRADO

Pipetas volumétricas.

ASSIGNED VALUES
The concentration value for each parameter is indicated in the table and they are
all traceable to the reference standard ERM-DA470k/IFCC.
The value and expected range for each parameter are derived from interlaboratory
data and they are given for orientation only; each laboratory should establish its
own acceptation range.
CAUTION
Each individual plasma used in the preparation of this control has been found to
be negative in the reaction against the HBsAg and HIV I/II.
However, the reagent should be handled with care.
Immunology
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PROTEIN CALIBRATOR
FOR AT III, IGA, IGG, IGM AND TRANSFERRIN
PRODUCT DESCRIPTION
Pool of human plasma for the preparation of the standard curve for protein
determinations in sera or plasma by immunoturbidimetry.
REAGENTS
Contents:
REAGENT PREPARATION
REAGENT COMPOSITION
Pool of human plasma with stabilizers and preservatives.

ASSIGNED VALUES
The table below shows the concentration values for each parameter which are
traceable to the reference standard ERM-DA470k/IFCC.
CAUTION
Each individual plasma used in the preparation of this control has been found to
be negative in the reaction against the HBsAg and HIV I/II.
However, the reagent should be handled with care.
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ANTITHROMBIN III (AT III)
PRINCIPLE PROCEDURE
The reagent allows quantifying the level of human AT III present in the sample by Technique
comparing the turbidimetric response produced with that obtained from standard To be used in clinical chemistry analyzers.
curve of known human AT III concentrations.
The reagent is an antiserum anti-human AT III which reacts with the AT III of the serum 1. Shake gently the reagents before using it. Use the sera sample without dilution.
giving protein aggregates. This aggregation process produces an increase in the Abs. Conditions:
of the system. Volume of buffer solution: 280 μL
The primary function is to inactivate the proteolytic enzyme thrombin, preventing Reaction temperature: 37ºC
uncontrolled clot formation. AT III also functions to inhibit activated proteases such as
coagulation factors IX, X, XI and XII. 2. Mix well; start the stopwatch and read the Abs (Absi) and after 5 minutes of reaction
Low levels of serum AT III are found in individuals suffering from severe hepatic (Absf) at 340nm.
disorders and as the result of protein-losing syndrome, particularly in the protein-
losing nephropathies. CALCULATIONS
Diseases accompanied by inflammation may show elevated serum levels of Determine the ΔAbs (Absorbance increase) as (Absf – Absi) for each sample,
Antithrombin III. standard or control. Antithrombin III concentrations are obtained by the interpolation
Single test result can not be used to make a clinical diagnosis. It should integrate of the ∆Abs value in the calibration curve.
REAGENTS Calibration curve
Kit (Ref. 99 66 06). Contents: Prepare the curve by serially diluting the calibrator (Protein Calibrator Ref. 99 99 39).
A. 1 x 23.0 mL Buffer solution Ref. 99 66 46 To obtain the concentration of each dilution, multiply the calibrator concentration by
B. 1 x 2.8 mL anti-human AT III serum Ref. 99 66 16 the corresponding factor indicated in the table:

0 1 2 3 4 5
1 x 1 mL Pool of sera samples calibrated in Antithrombin III, IgA, IgG, IgM,Transferrin,
C3, C4, α1-antitrypsin and α-1 acid Glycoprotein. CAL (µL) -- 25 50 100 200 400
Saline sol. 400 375 350 300 200 --
(µL)
1 x 1 mL Sera sample positive for Antithrombin III, IgA, IgG, IgM,Transferrin, C3, C4, Factor 0 0.0625 0.125 0.25 0.5 1.0
α1-antitrypsin and α-1 acid Glycoprotein.
The concentration is stated on the insert. The method described above is only indicative, as it should be specifically adapted
to each autoanalyzer.
REAGENT PREPARATION
23-39 mg/dL
REAGENT COMPOSITION Each particular laboratory should establish its own normal range, obtained from
A. Sodium phosphate/saline buffer pH 7.4 with stabilizers and preservatives. samples of a representative population, using its own instrumentation, blood collection
B. Goat antiserum anti-human Antithrombin III in buffered saline pH 7.4 with stabilizers methods and assaying procedures.
and preservatives. PERFORMANCE CHARACTERISTICS
STORAGE AND STABILITY to calculate these data for each particular test protocol. These results have been
The antiserum reagent, the buffer solution, calibrator and control are stable up to the obtained using an autoanalyzer.
expiry date indicated on the label when kept at 2-8ºC.
Do not freeze antisera or buffer solution. Sensitivity, as detection limit: 1.3 mg/dL
Signs of reagent deterioration: Reaction range: 75 mg/dl. Samples that give higher concentration should be diluted in
Presence of particles in the reagent. Quality control values outside acceptation range. saline NaCl 0.9% (1+1) and the final result has to be multiplied by 2.
ADDITIONAL EQUIPMENT Accuracy: 98%
Haemostasia
The safety statements are on the label. It is recomended to read SDS before reagent when compared with the reference reagent.
manipulation Human serum used in the control preparation have been found negative Details of the comparison experiments are available on request.
in the reaction to HBsAg and HIV I/II. However they should always be handled with
care. All reagents contain sodium azide 0.09% as preservative. It is recommended to INTERFERENCES
read SDS before reagent manipulation. No interferences were observed by heparin (25 mg/dL), hemoglobin (500 mg/dL),
Waste products must be handled as per local regulations. bilirrubin (20 mg/dl), triglycerides (5000 mg/dL), RF (100 UI/mL). Other substances
could interfere. (see references)
SAMPLE
Recent plasma or kept at 2-8ºC for no longer than 48h. If the test is to be performed QUALITY CONTROL
later, it is recommended to freeze the serum. Avoid repeated freezing and thawing. Control serum should be included in each test series. Each particular laboratory
Discard hemolyzed or contaminated samples. should establish its own control program.
AUTOANALYZERS
REFERENCES
Davie, EW., Fujikawa, K., (1975) Annu. Rev. Biochem., 44, 799-829.
Griffith, MJ., Carraway, T., White, GC., Dombrose, FA., (1983) Blood, 61, 111-118.
Moll.S. Antithrombin Deficiencye (NATT), 2006.
(2012).Young, D.S.; Effects of Preanalytical Variables on Clinical Laboratory Tests.
3rd Ed. AACCPress.
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COAGULATION CONTROLS
PRODUCT DESCRIPTION
Pool of human plasma for the quality control of the coagulation tests.
REAGENTS
Set of Controls Ref. 99 25 50
Contents:
A. 1 x 1 mL Coagulation Control. Low level. Ref. 99 00 09
Freeze-dried.
B. 1 x 1 mL Coagulation Control. High level. Ref. 99 00 46
Freeze-dried.
REAGENT PREPARATION
Rehydrate the vial with 1 mL of deionized water.
Mix gently and let stand at room temperature for 15 min. Do not invert nor
vigorously mix the vial. Swirl gently prior to each test.
REAGENT COMPOSITION
Pool of human plasma with a known concentration of coagulation factors, added
with stabilizers.
Low level: Coagulation factors into the normal range
High level: Coagulation factors into the pathological range.

stated on the label. Once rehydrated, the reagent is stable for 8 hours, when
stored at 2-8ºC.
ASSIGNED VALUES
The stated values for each level are for guidance. Results depend on reagent lot
number, device and technique used. To determine these expected values QCA
reagents and a mechanical instrument have been used.
It is recommended that each laboratory establish its own normal range as well
as the limits of quality control acceptability.
QUALITY CONTROL
Control serum should be included in each test series. Each particular laboratory
PROCEDURE
The control shall be processed as any other plasma sample, according to the
corresponding test instructions of each reagent.
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FIBRINOGEN
CLAUSS METHOD
PRINCIPLE PROCEDURE
The fibrinogen determination by clotting time is based on the method originally described by
Clauss. In the presence of an excess of thrombin, the fibrinogen is transformed into fibrin 1. Plasma dilution
and the time to clot formation is inverselly proportional to the concentration of fibrinogen
present in the plasma sample. Dilute plasma and control plasma 1:10 with buffer (reagent B) (1 plasma + 9 buffer)
The QCA Thrombin reagent placed in contact with the plasma of the patient, acts on the
fibrinogen to be converted into fibrin. This fibrin polymerizes to form a dense network 2. Method
which contributes to the clot formation. The training time can be measured manually or
automatically. a) Preincubate thrombin (reagent A) at 37ºC/10-15min
b) Dispense 0.2 mL of diluted plasma into a test tube and incubate at 37ºC during
DIAGNOSTIC USE 2min
Fibrinogen is a plasma glycoprotein which is involved in the coagulation processes. c) Add 0.1 mL of thrombin (reagent A) at 37ºC and start the stopwatch.
Fibrinogen increases in inflammatory processes and tissue diseases (infarction, leukaemia d) Record the time for the clot to be formed.
and lupus). Fibrinogen decreases in severe hepatic diseases.
Single test result could not be used to make a clinical diagnosis. It should integrate clinical 3. Calibration curve
a) Rehydrate fibrinogen calibrator vial as described above.
REAGENTS b) Prepare dilution series as following:
KIT Ref. 99 94 30
Content: 150% 100% 50% 25%
A. 4 x 2 mL Thrombin Ref. 99 94 10
B. 1 x 50 mL Buffer Ref. 99 94 11 Buffer (mL) 0.85 0.90 1.90 3.90
C. 1 x 1 mL Fibrinogen calibrator Ref. 99 94 12
Calibrator (mL) 0.15 0.10 0.10 0.10
c) Determine the coagulation time of each sample dilution according to the
Reagent A: rehydrate one vial with 2 mL of distilled water. Mix gently by inversion and wait
described method.
10 min for complete homogenization. Once open and rehydrated is stable 7 days at 2-8ºC.
Reagent B: imidazol buffer is ready to use. d) Coagulation time obtained can be plotted on a graph vs each concentration
Reagent C: rehydrate one vial with 1 mL of distilled water. Mix gently by inversion and wait in the calibration curve. Calibration curve allows to change into fibrinogen
20 min for complete homogenization. Once open and rehydrated is stable 8h at room tem- concentration (%) the plasma coagulation times of the patients samples by
perature (≤ 25ºC) or 2 days at -20ºC. The concentration is stated on the vial label. interpolation.
Calibration curve must be repeated at every fibrinogen lot change.
A: Freeze-dried bovine thrombin buffered at pH 7.4 with preservatives and stabilizers. CALCULATION
B: Imidazole buffer solution 15mM at pH 7.4, which contains NaCl 125mM with preserva- Each particular laboratory should establish its own calibration curve as described above,
tives and stabilizers and then determine by interpolation the sample concentration. Duplicate coagulation
C: Freeze-dried pool of human plasma, standardized and assayed for fibrinogen. time determinations are recommended, and then calculate the mean value of double
determinations of each plasma sample before to interpolate in the calibration curve.
When stored at 2-8ºC, reagents are stable until the expiration date stated on the label. REFERENCES VALUES
Do not use reagents over the expiration date. Do not freeze. Each particular laboratory should establish its own normal range, obtained from samples
Signs of reagent deterioration: assaying procedures.
Presence of particles in the reagent B. Quality control values outside acceptation range. It is advisable not to establish the mean value with less than 20 individuals. However and as
a guideline, the values of a certain ambulatory population are related herewith:
ADDITIONAL EQUIPMENT 200-400 mg/dL (2–4 g/L)
Coagulometer or bath 37ºC and stopwatch. PERFROMANCE CHARACTERISTICS
SAMPLE calculate these data for each particular test protocol. These results have been obtained
Mix carefully 9 parts of fresh blood with 1 part of trihydrated trisodium citrate 3.2% (0.109M, using mechanical coagulometer.
Ref. 99 59 59). Centrifuge at 3.000 rpm/5 min and separate the supernatant plasma. Store
at 2-8ºC until prior to assay. (Not more than 2 hours) Sensitivity, as minimum detectable concentration: 5mg/dL
Accuracy: 99.5%
CAUTION Repetitivity, as CV%: 5.25%
Haemostasia
The safety statements are on the label. Handle the reagent with care. Reproducibility, as CV%: 5.9%
It is advisable to read the SDS before the reagent manipulation. Trueness: Results obtained with this reagent did not show systematic differences when
The disposal of the residues has to be made according to local regulations. compared with reference reagent.
INTERFERENCES
QUALITY CONTROL Important interferences have been observed in samples with degradation fibrinogen
Control plasma should be included in each test series. products (FDP or D-Dimer).
Acute inflammatory reactions can increase circulating fibrinogen (factor I).
Set of controls Ref. 99 25 50. Contents: Hemolysis can cause clotting factor activation and end point detection interference.
1 x 1 mL Coagulation Control, Low Level Drugs and substances consumption may interfere (See references)
1 x 1 mL Coagulation Control, High Level
Pool of human plasma with standardized levels of coagulation factors. REFERENCES
Clauss, A. Acta Haemat., 1957, 17, 237-246.
Each particular laboratory should establish its own control program and correctives actions NCCLS, H30-A2, 2nd Ed., 2001 21(18).
of measure deviations. Kitchen, S.; McCraw, A.; Echenagucia, M.; Diagnosis of Hemophilia and Other Bleeding
Disorders, 2nd Ed.; 2010, 55-57.
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HEMOSCANN®
REAGENT FOR APTT DETERMINATION
For “in vitro” Diagnostic
PRINCIPLE PROCEDURE
The reagent is formed by a phospholipid obtained from a chloroform extract of rabbit brain,
that performs as a platelet substitute and ellagic acid which is used as the activator of the 1. Preincubate the CaCl2 0.025M solution at 37ºC.
coagulation mechanism. 2. Dispense 0.1 mL of plasma into a test tube.
Once optimal activation is achieved by incubation of plasma with the reagent (at 37ºC/3min),
the mixture was recalcified and the coagulation time is determined. 3. Mix, by gentle inversion, the Hemoscann® reagent and add 0.1 mL to the plasma.
DIAGNOSTIC USE 4. Incubate the mixture plasma-reagent during 3 min. (time of activation) at 37ºC.
The determination of Activated Partial Thromboplastin Time (APTT) is mainly used to detect 5. After this period of time, add 0.1 mL of the CaCl2 solution at 37ºC and start the
deficiencies in the first stage of the coagulation mechanism, principally factors VIII, IX, XI, stopwatch.
XII and Prekallikrein (Fletcher Factor). It is also sensitive to deficiencies of the rest of factors
except Factor VII. 6. Record the time for the clot to be formed.
It is commonly used for monitoring heparin anticoagulant therapy.
Levels approximately 40% of the normal value of Factors VIII, IX, XI and XII are sufficient RESULTS
to produce normal values of APTT. For levels lower than 40%, the APTT will be longer. The results shall be expressed as clotting time in seconds. Calculate the mean value of
Heparin, in the presence of adequate level of Antithrombin III, will also prolong the clotting duplicate samples and controls.
time value.
Single test result could not be used to make a clinical diagnosis. It should integrate clinical REFERENCES VALUES
and laboratory data. Each particular laboratory should establish its own normal range, obtained from samples
REAGENTS assaying procedures.
1 x 4 mL (Ref. 99 05 89) Contents: It is recommended a minimum of 20 individual samples to establish the mean value. The
1 x 4 mL Reagent for APTT determination Ref. 99 01 87 values obtained in samples from healthy individuals are indicated as a guideline
1 x 10 mL (Ref. 99 36 34) Contents: Manual 20 - 35s
1 x 10 mL Reagent for APTT determination Ref. 99 02 87 Mechanical 22 - 36s
Kit 4 x 10 m (Ref. 99 26 89) Contents:
4 x 10 mL Reagent for APTT determination Ref. 99 02 87 PERFORMANCE CHARACTERISTICS
Kit 10 x 4 m (Ref. 99 40 30) Contents: The performance characteristics depend on the method used. It is recommended to
10 x 4 mL Reagent for APTT determination Ref. 99 01 87 calculate these data for each particular test protocol. These results have been obtained
using mechanical coagulometer.
Reagent is ready to use. Sensitivity to Heparin:
REAGENT COMPOSITION
Heparin
Extract of rabbit phospholipid with ellagic acid as activator, stabilizers and preservatives. 0.0 0.1 0.2 0.3 0.4 0.6 0.7 0.8
concentration U/mL
STORAGE AND STABILITY Coagulation time sec. 24.6 27.7 33.5 39.6 49.4 68.9 88.5 108
When stored at 2-8ºC, the reagent is stable until the expiration date stated on the label if the
bottle is properly closed and preserved from contamination. Repetitivity, as CV%: 1.8%
After a long period of storage, yellow sediment may appear. In such a case, mix by Reproducibility, as CV%: 3.4%
gentle inversion to assure a proper activator suspension. Trueness: Results obtained with this reagent did not show systematic differences when
Do not use reagents over the expiration date. Do not freeze. compared with reference reagent.
Signs of reagent deterioration: Details of the performance studies are available on request.
Presence of particles in the reagent after suspension. Quality control values outside
acceptation range. INTERFERENCES
Turbid, icteric, lipemic or grossly haemolysed samples will give erroneous results.
ADDITIONAL EQUIPMENT APTT values can be shortened as a result of the response to an acute inflammatory reaction
General laboratory equipment. in which the elevation of Factor I (Fibrinogen) is very common.
Coagulometer or bath 37ºC and stopwatch. Contraceptives, estrogens, pregnancy, coumarin derivatives, heparin, asparaginase and
naloxone, have been reported to influence the APTT values.
SAMPLE CAUTION
Mix carefully 9 parts of fresh blood with 1 part of trihydrated trisodium citrate 3.2% (0.109M, The safety statements are on the label. Handle the reagent with care.
Ref. 99 59 59). Centrifuge at 3.000 rpm/5min. and separate the supernatant plasma. Store It is advisable to read the SDS before the reagent manipulation.
at 2-8ºC until prior to assay. (Not more than 2 hours). The disposal of the residues has to be made according to local regulations.
QUALITY CONTROL
Control plasma should be included in each test series. REFERENCES
Langdell, R. D., Wagner, R. H., Brinkhous, K. M. (1953). J. Lab. Clin. Med., 41, 637.
Set of controls Ref. 99 25 50. Contents: Ratnoff, O. D., Crum, S. D. (1964). J. Lab. Clin. Med., 63, 359.
1 x 1 mL Coagulation Control, Low Level Brandt, J.T., Triplett, D.A. (1981). Amer. J. Clin. Path., 76, 530.
1 x 1 mL Coagulation Control, High Level Procedure for the handling and processing of blood specimens, H18-A3, NCCLS, 24(38),
Pool of human plasma with standardized levels of coagulation factors. 2004.
One-Stage Prothrombin Time Test and Activated Partial Thromboplastine Time Test,
Each particular laboratory should establish its own control program and correctives actions H47-A2, CLSI, 28(20), 2008.
of measure deviations.
ISO 9001 / ISO 13485

110
Tel. ++ 34 (977) 70. 62. 30 Fax ++ 34 (977) 70. 30. 40
PLASMASCANN®
CALCIUM THROMBOPLASTIN (ISI 1.40 - 1.60)
PRINCIPLE
Prothrombin time or Quick time is the time, in seconds, necessary for plasma to clot at 37ºC, once an CALCULATIONS
external coagulation factor (thromboplastin) and Ca2+ has been added. Long coagulation times with regard INR Calculation
to those obtained for healthy people indicate a deficiency in some of the components of the coagulation The International Normalized Ratio, INR is a standardized ratio to express the Prothrombin Time results
system. (PT), that allows a better control of patients treated with oral anticoagulants.
The INR would be the prothrombin ratio obtained if the International Reference Preparation of
DIAGNOSTIC USE Thromboplastin had been used as reagent.
Plasmascann® is a calcium thromboplastin, freeze-dried, that allows the determination of variations in The INR is calculated by use of the International Sensitivity Index, ISI, that relates PT ratios obtained with
those coagulation factors (especially factors VII and X) that have been depressed during the treatment home Thromboplastin and Reference Thromboplastin:
with coumarinic derivatives, in vitamin K deficiency, hepatopathy or due to a hereditary abnormally in the INR = RISI
coagulation system.
The reagent has been obtained from a rabbit brain extract, and it is standardized from lot to lot according R: PT ratio: ratio of patient prothrombin time to the mean prothrombin time of normal individuals
to the technique of Hills-Ingram. ISI: Slope of the linear relationship between the logarithms of the prothrombin time results (in seconds)
Single test result could not be used to make a clinical diagnosis. It should integrate clinical and laboratory obtained with the International Reference Thromboplastin (y axis) and those determined with the home
data. Thromboplastin (x axis).
REAGENTS The ISI of Plasmascann® has been obtained following the guidelines given by WHO as it is stated in the
Kit 10 x 2 mL (Ref. 99 70 14). Contents: recommended methodology of Community Bureau of Reference-BCR.
A. 10 vials of calcium thromboplastin. Ref. 99 70 30 Plasmascann® has an ISI (1.40-1.60). ISI of each batch is stated in the label on the vial.
Kit 10 x 4 mL (Ref. 99 62 21). Contents:
A. 10 vials of calcium thromboplastin. Ref. 99 70 40 Results
Determine prothrombin times for patient plasma and calculate R values as follows:
Optionally, 1 x 500 mL trihydrated trisodium citrate, 3.2% (Ref. 99 59 59).
Patient PT (sec.)
Ready to use. R=
Normal PT (sec.)
Rehydrate the vial with the volume of deionized water stated on the label, mix gently and wait 10 min. for It is suggested that each laboratory should establish its Normal Prothrombin Time, using its own
the homogenisation of the reagent. determination method. Guide values of normal PT with Plasmascann® as reagent are 10.5 – 15 sec.
The INR is obtained from the table using the R values calculated and the ISI for the Plasmascann® batch
WORKING REAGENT COMPOSITION employed in the test.
Thromboplastin: Sufficient activity to promote the coagulation of plasma in the assay conditions.
Example: R=2.5 and ISI=1.50, the INR obtained is 3.95
CaCl2 0.015M
Stabilizers and preservatives. The INR could be calculated from the expression:
STORAGE AND STABILITY INR = RISI

The lyophilized reagent is stable, when kept at 2-8ºC, until the expiration date stated on the label. Once or,
rehydrated will remain stable for one week at the same temperature conditions. log INR = ISI x log R
Do not use reagents over the expiration date. Do not freeze.
In the above mentioned example: log INR = 1.50 x log 2.5. The corresponding antilogarithm is the INR
Signs of reagent deterioration: value.
Presence of particles in the reagent after its preparation. Quality control values outside acceptation range.
REFERENCE VALUES
ADDITIONAL EQUIPMENT In percentage of activity: 80-100% for normal plasma samples.
General laboratory equipment. Therapeutic interval is 20-35% approximately (oral anticoagulants).
Coagulometer or bath 37ºC and stopwatch. It is advisable that each laboratory obtains its own therapeutic interval for the methodology used.
SAMPLE PERFROMANCE CHARACTERISTICS

Mix carefully 9 parts of fresh blood with 1 part of trihydrated trisodium citrate 3.2% (0.109M, Ref. 99 59 59). The performance characteristics depend on the method used. It is recommended to calculate these data for
Centrifuge at 3.000 rpm/5min. and separate the supernatant plasma. Store at 2-8ºC until prior to assay. each particular test protocol. These results have been obtained using mechanical coagulometer.
(Not more than 2 hours).
Sensitivity to Factor VII:
CAUTION
The safety statements are on the label. It is recommended to read MSDS before reagent manipulation. Factor VII % 100% 60% 50% 40% 30% 20% 10%
The control plasma and patient plasma must be handled with care. Coagulation time (seconds) 12.2 12.4 13 13.7 14.3 15.8 19.4
PROCEDURE Trueness: Results obtained with this reagent did not show systematic differences when compared with
reference reagent.
1. Rehydrate a Plasmascann® vial, mix gently and wait 10 min. for the homogenisation of the reagent.
2. Incubate the reagent at 37ºC/10–15 min. Details of the performance studies are available on request.
3. Dispense 0.1 mL of the plasma sample into a tube.and incubate at 37ºC/2.5 min.
4. Add 0.2 mL of Plasmascann® reagent (at 37ºC). INTERFERENCES
5. Start the stopwatch immediately and measure the time for the clot to be formed (Quick time).
Haemostasia
Traces of detergent or blood in the glassware can give false results. Likewise, a wrong reaction temperature,
inadequate proportions in the blood-citrate mixture or the use of aged samples, will give rise to erroneous
Calibration curve values in the determination.
From a pool of citrated plasmas, of at least five healthy male donors (18-40 years old), make the Discard plasma samples from the ESR.
following dilutions: In the case the venous puncture became arduous, the risk of an undesirable tissue thromboplastin
aspiration is run.
Dilution -- 1/2 1/3 1/4 1/10
Citrated Plasma (mL) 1 1 1 1 1
QUALITY CONTROL
Sol. 0.9% NaCl (mL) -- 1 2 3 9
% Coagulation 100% 50% 33% 25% 10% Control serum should be included in each test series. Each particular laboratory should establish
Quick time is determined by the above mentioned technique. These values can be plotted on graph
vs the inverse of dilution and the plasma times of the patients will be interpolated for having % of REFERENCES
coagulability. Sultan, C., Priolet, G., Beuzart, Y., Rosa, R., Josso, F. (1979). Técnicas en hematologia, 1a. Ed, 186-188.
Hills, M., Ingram, I.C. (1973). Brit. J. Hematol., 25, 445-451
RESULTS Heckermann, H.J. (1979). Thromb. Res., 15, 769-780
Can be expressed in seconds, in percentages of activity or in INR. Procedure for the handling and processing of blood specimens, H18-A3, NCCLS, 24(38), 2004.
The percentage of activity can be obtained with the aid of the attached Table or the calibration curve. One-Stage Prothrombin Time Test and Activated Partial Thromboplastine Time Test, H47-A2, CLSI, 28(20),
2008.
Note WHO Expert Comittee on biological standardization: Thirty-third report. WHO Technical report series 687.
For automated systems refer to the appropriate instruction manual. Geneve (1983) 87-107.
van der Besselaar, A.M.H.P. The certification of the second reference material for rabbit thromboplastin.
CRM 1491. Commission of the European Communities, BCR Information. Report EUR 11686 (1988).
Loeliger, E.A. , ICSH/ICTH. Recommendations for Reporting Prothrombin Time in Oral Anticoagulant
Control. Thromb.Haemostas. (1985), 53, 155 - 156.
Kirkwood, T.B.C. Calibration of reference thromboplastins and standardization of the phrotrombin time
ratio. Thromb. Haemostas. (1983), 49, 238 - 244.
ISO 9001 / ISO 13485

111
Tel. ++ 34 (977) 70. 62. 30 Fax ++ 34 (977) 70. 30. 40
PLASMASCANN® LIQUID
PRINCIPLE CALCULATIONS
Prothrombin or Quick time is the time, in seconds, necessary for plasma to clot at 37ºC, once an external INR Calculation
coagulation factor (thromboplastin) and Ca2+ has been added. Long coagulation times with regard to those The International Normalized Ratio, INR is a standardized ratio to express the Prothrombin Time results
obtained for healthy people indicate a deficiency in some of the components of the coagulation system. (PT), that allows a better control of patients treated with oral anticoagulants.
Plasmascann® Liquid is a calcium thromboplastin, that allows the determination of variations in those The INR is calculated by use of the International Sensitivity Index, ISI, that relates PT ratios obtained with
coagulation factors (especially factors VII and X) that have been depressed during the treatment with home Thromboplastin and Reference Thromboplastin:
coumarinic derivatives, in vitamin K deficiency, hepatopathy or due to a hereditary abnormally in the
coagulation system. INR = RISI
The reagent has been obtained from a rabbit brain extract, and it is standardized from lot to lot according R or PT ratio: ratio of patient prothrombin time to the mean prothrombin time of normal individuals
to the technique of Hills-Ingram. ISI: Slope of the linear relationship between the logarithms of the prothrombin time results (in seconds) obtained
Single test result could not be used to make a clinical diagnosis. It should integrate clinical and laboratory with the International Reference Thromboplastin (y axis) and those determined with the home Thromboplastin
data. (x axis).
REAGENTS The ISI of Plasmascann® Líquid has been obtained following the guidelines given by WHO as it is stated
Kit 4 x 4 mL (Ref. 99 70 54). Contents: in the recommended methodology of Community Bureau of Reference-BCR.
A. 4 vials of calcium liquid thromboplastin. Ref. 99 70 60 Plasmascann® Liquid has an ISI between 1.40-1.60. The ISI of each batch is stated in the label on
the vial.
Optionally, 1 x 500 mL trihydrated trisodium citrate, 3.2% (Ref. 99 59 59).
Ready to use.
Results
Determine prothrombin times for patient plasma and calculate R values as follows:
REAGENT PREPARATION
Plasmascann® liquid is ready to use. Patient PT (sec.)
Note: If a slight precipitate appears in the vial, shake gently and use it as usual. R=
Normal PT (sec.)
REAGENT COMPOSITION
It is suggested that each laboratory should establish its Normal Prothrombin Time, using its own
Thromboplastin: Sufficient activity to promote the coagulation of plasma in the assay conditions.
determination method. Guide values of normal PT with Plasmascann® Líquid as reagent are 10.5 – 15 sec.
CaCl2 0.015M
The INR is obtained from the table using the R values calculated and the ISI for the Plasmascann® Líquid
Stabilizers and preservatives.
batch employed in the test.
Example: R=2.5 and ISI=1.50, the INR from the table is 3.95
Unopened reagent is stable, when kept at 2-8ºC until the expiration date stated on the label. Once opened
will remain stable for 15 days at 2-8ºC, one week at 25ºC or 37ºC.
The INR could be calculated from the expression:
Do not use reagents over the expiration date. Do not freeze
Signs of reagent deterioration: Presence of particles in the reagent after its homogenization. Quality
INR = RISI
control values outside acceptation range.
or,
log INR = ISI x log R
In the above mentioned example: log INR = 1.50 x log 2.5. The corresponding antilogarithm is the INR
Coagulometer or bath 37ºC and stopwatch.
value.
SAMPLE
REFERENCE VALUES
Mix carefully 9 parts of fresh blood with 1 part of trihydrated trisodium citrate 3.2% (0.109 M. Ref.99 59 59).
In percentage of activity: 80-100 % for normal plasma samples.
Centrifuge at 3.000 rpm/5 min. and separate the supernatant plasma. Store at 2-8ºC until prior to assay.
Therapeutic interval is 20-35 % approximately (oral anticoagulants).
(Not more than 2 hours).
It is advisable that each laboratory obtains its own therapeutic interval for the methodology used.
CAUTION
PERFROMANCE CHARACTERISTICS
The safety statements are on the label. It is recommended to read MSDS before reagent manipulation.
The performance characteristics depend on the method used. It is recommended to calculate these data for
The control plasma and patient plasma must be handled with care.
each particular test protocol. These results have been obtained using mechanical coagulometer.
Sensitivity to Factor VII:
QUALITY CONTROL
Control serum should be included in each test series. Each particular laboratory should establish Factor VII % 100% 60% 50% 40% 30% 20% 10%
its own control program. Coagulation time (sec.) 11.9 12.0 12.5 13.1 13.7 14.5 17.9

PROCEDURE
1. Mix gently a Plasmascann® liquid vial until complete homogenization Trueness: Results obtained with this reagent did not show systematic differences when compared with
2. Incubate the reagent at 37ºC/10–15min. reference reagent.
3. Dispense 0.1 mL of the plasma sample into a tube.and incubate at 37ºC/2.5min.
4. Add 0.2 mL of Plasmascann® Líquid reagent (at 37ºC). Details of the performance studies are available on request.
INTERFERENCES
Calibration curve Traces of detergent or blood in the glassware can give false results. Likewise, a wrong reaction temperature,
From a pool of citrated plasmas, of at least five healthy male donors (18-40 years old), make the inadequate proportions in the blood-citrate mixture or the use of aged samples, will give rise to erroneous
following dilutions: values in the determination.
Discard plasma samples from the ESR.
Dilution -- 1/2 1/3 1/4 1/10 In the case the venous puncture became arduous; the risk of an undesirable tissue thromboplastin
Citrated plasma (mL) 1 1 1 1 1 aspiration is run.
Sol. 0.9% NaCl (mL) -- 1 2 3 9
% Coagulation 100% 50% 33% 25% 10%
REFERENCES
Clotting times obtained with the above dilutions are plotted against the reciprocal of the dilution. In Sultan. C., Priolet. G., Beuzart. Y., Rosa. R., Josso. F. (1979). Técnicas en hematologia. 1a. Ed. 186-188.
the line obtained results are interpolated to convert patients clotting times in % activity. Hills. M., Ingram. I.C. (1973), Brit. J. Hematol., 25. 445-451
Heckermann. H.J. (1979), Thromb. Res., 15, 769-780
RESULTS Procedure for the handling and processing of blood specimens, H18-A3, NCCLS, 24(38), 2004.
Can be expressed in seconds, in percentages of activity or in INR. One-Stage Prothrombin Time Test and Activated Partial Thromboplastine Time Test. H47-A2. CLSI. 28(20),
The percentage of activity can be obtained with the aid of the Table or the calibration curve. 2008.
WHO Expert Comittee on biological standardization: Thirty-third report. WHO Technical report series 687,
Note: For automated systems refer to the appropriate instruction manual. Geneve (1983) 87-107.
van der Besselaar. A.M.H.P. The certification of the second reference material for rabbit thromboplastin,
CRM 1491, Commission of the European Communities, BCR Information, Report EUR 11686 (1988).
Loeliger. E.A. , ICSH/ICTH. Recommendations for Reporting Prothrombin Time in Oral Anticoagulant
Control, Thromb.Haemostas, (1985), 53, 155 - 156.
Kirkwood. T.B.C. Calibration of reference thromboplastins and standardization of the phrotrombin time
ratio, Thromb. Haemostas, (1983), 49, 238 - 244.
ISO 9001 / ISO 13485

112
Tel. ++ 34 (977) 70. 62. 30 Fax ++ 34 (977) 70. 30. 40
PLASMASCANN® / PLASMASCANN® LIQUID
(ISI 1,4-1,6) For “in vitro” diagnostic
VALUES OF CLOTTING TIMES – % COAGULATION CAPACITY –RATIO – INR Calculation
INR
TP normal values (sec.) % coag R
ISI 1,40 ISI 1,45 ISI 1,50 ISI 1,55 ISI 1,60
10,5 11,5 12,0 12,5 13,0 13,5 14,0 15,0 100 1,00 1,0 1,0 1,0 1,0 1,0
11,0 12,1 12,6 13,1 13,7 14,2 14,7 15,8 87,0% 1,05 1,07 1,07 1,08 1,08 1,08
11,6 12,7 13,2 13,8 14,3 14,9 15,4 16,5 77,0% 1,10 1,14 1,15 1,15 1,16 1,16
12,1 13,2 13,8 14,4 15,0 15,5 16,1 17,3 69,1% 1,15 1,22 1,22 1,23 1,24 1,25
12,6 13,8 14,4 15,0 15,6 16,2 16,8 18,0 62,7% 1,20 1,29 1,30 1,31 1,33 1,34
13,1 14,4 15,0 15,6 16,3 16,9 17,5 18,8 57,3% 1,25 1,37 1,38 1,40 1,41 1,43
13,7 15,0 15,6 16,3 16,9 17,6 18,2 19,5 52,8% 1,30 1,44 1,46 1,48 1,50 1,52
14,2 15,5 16,2 16,9 17,6 18,2 18,9 20,3 49,0% 1,35 1,52 1,55 1,57 1,59 1,62
14,7 16,1 16,8 17,5 18,2 18,9 19,6 21,0 45,6% 1,40 1,60 1,63 1,66 1,68 1,71
15,2 16,7 17,4 18,1 18,9 19,6 20,3 21,8 42,7% 1,45 1,68 1,71 1,75 1,78 1,81
15,8 17,3 18,0 18,8 19,5 20,3 21,0 22,5 40,2% 1,50 1,76 1,80 1,84 1,87 1,91
16,3 17,8 18,6 19,4 20,2 20,9 21,7 23,3 37,9% 1,55 1,85 1,89 1,93 1,97 2,02
16,8 18,4 19,2 20,0 20,8 21,6 22,4 24,0 35,9% 1,60 1,93 1,98 2,02 2,07 2,12
17,3 19,0 19,8 20,6 21,5 22,3 23,1 24,8 34,1% 1,65 2,02 2,07 2,12 2,17 2,23
17,9 19,6 20,4 21,3 22,1 23,0 23,8 25,5 32,4% 1,70 2,10 2,16 2,22 2,28 2,34
18,4 20,1 21,0 21,9 22,8 23,6 24,5 26,3 30,9% 1,75 2,19 2,25 2,32 2,38 2,45
18,9 20,7 21,6 22,5 23,4 24,3 25,2 27,0 29,6% 1,80 2,28 2,35 2,41 2,49 2,56
19,4 21,3 22,2 23,1 24,1 25,0 25,9 27,8 28,3% 1,85 2,37 2,44 2,52 2,59 2,68
TP patient plasma (seg.)
20,0 21,9 22,8 23,8 24,7 25,7 26,6 28,5 27,2% 1,90 2,46 2,54 2,62 2,70 2,79
20,5 22,4 23,4 24,4 25,4 26,3 27,3 29,3 26,1% 1,95 2,55 2,63 2,72 2,82 2,91
21,0 23,0 24,0 25,0 26,0 27,0 28,0 30,0 25,1% 2,00 2,64 2,73 2,83 2,93 3,03
22,1 24,2 25,2 26,3 27,3 28,4 29,4 31,5 23,4% 2,10 2,83 2,93 3,04 3,16 3,28
23,1 25,3 26,4 27,5 28,6 29,7 30,8 33,0 21,9% 2,20 3,02 3,14 3,26 3,39 3,53
24,2 26,5 27,6 28,8 29,9 31,1 32,2 34,5 20,5% 2,30 3,21 3,35 3,49 3,64 3,79
25,2 27,6 28,8 30,0 31,2 32,4 33,6 36,0 19,3% 2,40 3,41 3,56 3,72 3,88 4,06
26,3 28,8 30,0 31,3 32,5 33,8 35,0 37,5 18,3% 2,50 3,61 3,78 3,95 4,14 4,33
Haemostasia
27,3 29,9 31,2 32,5 33,8 35,1 36,4 39,0 17,3% 2,60 3,81 4,00 4,19 4,40 4,61
28,4 31,1 32,4 33,8 35,1 36,5 37,8 40,5 16,5% 2,70 4,02 4,22 4,44 4,66 4,90
28,9 31,6 33,0 34,4 35,8 37,1 38,5 41,3 16,1% 2,75 4,12 4,34 4,56 4,80 5,05
30,5 33,4 34,8 36,3 37,7 39,2 40,6 43,5 15,0% 2,90 4,44 4,68 4,94 5,21 5,49
32,0 35,1 36,6 38,1 39,7 41,2 42,7 45,8 14,1% 3,05 4,76 5,04 5,33 5,63 5,95
34,1 37,4 39,0 40,6 42,3 43,9 45,5 48,8 13,0% 3,25 5,21 5,52 5,86 6,21 6,59
36,2 39,7 41,4 43,1 44,9 46,6 48,3 51,8 12,0% 3,45 5,66 6,02 6,41 6,82 7,25
36,8 40,3 42,0 43,8 45,5 47,3 49,0 52,5 11,8% 3,50 5,78 6,15 6,55 6,97 7,42
37,8 41,4 43,2 45,0 46,8 48,6 50,4 54,0 11,4% 3,60 6,01 6,41 6,83 7,28 7,76
39,9 43,7 45,6 47,5 49,4 51,3 53,2 57,0 10,7% 3,80 6,48 6,93 7,41 7,92 8,47
42,0 46,0 48,0 50,0 52,0 54,0 56,0 60,0 10,1% 4,00 6,96 7,46 8,00 8,57 9,19
44,1 48,3 50,4 52,5 54,6 56,7 58,8 63,0 9,5% 4,20 7,46 8,01 8,61 9,25 9,94
46,2 50,6 52,8 55,0 57,2 59,4 61,6 66,0 9,0% 4,40 7,96 8,57 9,23 9,94 10,70
48,3 52,9 55,2 57,5 59,8 62,1 64,4 69,0 8,5% 4,60 8,47 9,14 9,87 10,65 11,49
49,4 54,1 56,4 58,8 61,1 63,5 65,8 70,5 8,3% 4,70 8,73 9,43 10,19 11,01 11,89
50,4 55,2 57,6 60,0 62,4 64,8 67,2 72,0 8,1% 4,80 8,99 9,72 10,52 11,37 12,30
52,5 57,5 60,0 62,5 65,0 67,5 70,0 75,0 7,7% 5,00 9,52 10,32 11,18 12,12 13,13
Plasmascann® Liquid has a ISI (1.4-1.6). ISI of each batch is stated in the label on the vial.
Mean values obtained with different PLASMASCANN LIQUID lots. It is suggested that each laboratory should
establish its Normal Prothrombin Time and INR calculation, using its own determination method.
ISO 9001 / ISO 13485

113
Tel. ++ 34 (977) 70. 62. 30 Fax ++ 34 (977) 70. 30. 40
PLASMASCANN® HS
PRINCIPLE CALCULTIONS
Prothrombin or Quick time is the time, in seconds, necessary for a plasma to clot at 37ºC, once an external INR Calculation
coagulation factor (thromboplastin) and Ca2+ has been added. Long coagulation times with regard to those The International Normalized Ratio, INR is a standardized ratio to express the Prothrombin Time results
obtained for healthy people indicate a deficiency in some of the components of the coagulation system. (PT), that allows a better control of patients treated with oral anticoagulants.
Plasmascann®HS is a calcium thromboplastin, freeze-dried, that allows the determination of variations The INR is calculated by use of the International Sensitivity Index, ISI, that relates PT ratios obtained with
in those coagulation factors (especially factors VII and X) that have been depressed during the treatment home Thromboplastin and Reference Thromboplastin:
with coumarinic derivatives, in vitamin K deficiency, hepatopathy or due to a hereditary abnormally in the
coagulation system. INR = RISI
The reagent has been obtained from a rabbit brain extract, and it is standardized from lot to lot according R = PT ratio: ratio of patient prothrombin time to the mean prothrombin time of normal individuals
to the technique of Hills-Ingram. ISI: Slope of the linear relationship between the logarithms of the prothrombin time results (in seconds)
Single test result could not be used to make a clinical diagnosis. It should integrate clinical and laboratory obtained with the International Reference Thromboplastin (y axis) and those determined with the home
data. Thromboplastin (x axis).
REAGENTS The ISI of Plasmascann® HS has been obtained following the guidelines given by WHO as it is stated in
Kit 10 x 2 mL (Ref. 99 75 80). Contents: the recommended methodology of Community Bureau of Reference-BCR.
A. 10 vials of calcium thromboplastin. Ref. 99 70 80 Plasmascann® HS has a ISI between1.10 - 1.35. The ISI of each batch is stated in the label on the vial.
Optionally, 1 x 500 mL trihydrated trisodium citrate, 3.2% (Ref. 99 59 59). Results

Ready to use. Determine prothrombin times for patient plasma and calculate R values as follows:
REAGENT PREPARATION Patient PT (sec.)

R=
Plasmascann® HS vial once rehydrated with the volume of deionised water stated on the label, mix gently Normal PT (sec.)
and wait 10 min. for the homogenisation of the reagent.
It is suggested that each laboratory should establish its Normal Prothrombin Time, using its own
WORKING REAGENT COMPOSITION determination method. Guide values of normal PT with Plasmascann® HS as reagent are 10.5 – 15 sec.
Thromboplastin: Sufficient activity to promote the coagulation of plasma in the assay conditions. The INR is obtained from the table using the R values calculated and the ISI for the Plasmascann® HS
CaCl2 0.015M batch employed in the test.
Stabilizers and preservatives.
Example: R=2.9 and ISI=1.20, the INR obtained is 3.59
The lyophilized reagent is stable, when kept at 2-8ºC, until the expiration date stated on the label. Once The INR could be calculated from the expression:
rehydrated will remain stable for one week at the same temperature conditions.
INR = RISI
Do not use reagents over the expiration date. Do not freeze.
or,
log INR = ISI x log R
Presence of particles in the reagent after its preparation. Quality control values outside acceptation
range. In the above mentioned example: log INR = 1.20 x log 2.9. The corresponding antilogarithm is the INR
value.
General laboratory equipment. REFERENCE VALUES
Coagulometer or bath 37ºC and stopwatch. In percentage of activity: 80-100% for normal plasma samples.
Therapeutic interval is 20-35% approximately (oral anticoagulants).
SAMPLE It is advisable that each laboratory obtains its own therapeutic interval for the methodology used.
Mix carefully 9 parts of fresh blood with 1 part of trihydrated trisodium citrate 3.2% (0.109 M, Ref.99 59 59).
Centrifuge at 3.000 rpm/5min. and separate the supernatant plasma. Store at 2-8ºC until prior to assay. PERFORMANCE CHARACTERISTICS
(Not more than 2 hours). The performance characteristics depend on the method used. It is recommended to calculate these data for
each particular test protocol. These results have been obtained using mechanical coagulometer.
CAUTION
The safety statements are on the label. It is recommended to read MSDS before reagent manipulation. Sensitivity to Factor VII:
The control plasma and patient plasma must be handled with care.
Waste products must be handled as per local regulations. Factor VII % 100% 60% 50% 40% 30% 20% 10%
Coagulation time sec. 11.9 12.9 13.2 14.3 15.5 16.9 20.5
QUALITY CONTROL
Control serum should be included in each test series. Each particular laboratory should Repetitivity, as CV%: 2.7%
establish its own control program. Reproducibility, as CV%: 4.1%
reference reagent.
PROCEDURE
1. Rehydrate a Plasmascann® HS vial, mix gently and wait 10 min. for the homogenisation of the
reagent.
2. Incubate the reagent at 37ºC/10–15 min.
INTERFERENCES
3. Dispense 0.1 mL of the plasma sample into a tube.and incubate at 37ºC/2.5 min.
Traces of detergent or blood in the glassware can give false results. Likewise, a wrong reaction
4. Add 0.2 mL of Plasmascann® HS reagent (at 37ºC).
temperature, inadequate proportions in the blood-citrate mixture or the use of aged samples, will give
rise to erroneous values in the determination.
Discard plasma samples from the ESR.
Calibration curve
In the case the venous puncture became arduous; the risk of an undesirable tissue thromboplastin
From a pool of citrated plasmas, of at least five healthy male donors (18-40 years old), make the following
dilutions:
aspiration is run.
Dilution -- 1/2 1/3 1/4 1/10 REFERENCES

Citrated Plasma (mL) 1 1 1 1 1 Sultan, C., Priolet, G., Beuzart, Y., Rosa, R., Josso, F. (1979). Técnicas en hematologia, 1a. Ed, 186-188.
Sol. 0.9% NaCl (mL) -- 1 2 3 9 Hills, M., Ingram, I.C. (1973). Brit. J. Hematol., 25, 445-451
% Coagulation 100% 50% 33% 25% 10% Heckermann, H.J. (1979). Thromb. Res., 15, 769-780
Procedure for the handling and processing of blood specimens, H18-A3, NCCLS, 24(38), 2004.
Quick time is determined by the above mentioned technique. These values can be plotted on graph One-Stage Prothrombin Time Test and Activated Partial Thromboplastine Time Test, H47-A2, CLSI, 28(20),
vs the inverse of dilution and the plasma times of the patients will be interpolated for having % of 2008.
coagulation capacity. WHO Expert Comittee on biological standardization: Thirty-third report. WHO Technical report series 687.
Geneve (1983) 87-107.
RESULTS van der Besselaar, A.M.H.P. The certification of the second reference material for rabbit thromboplastin.
Can be expressed in seconds, in percentages of activity or in INR. CRM 1491. Commission of the European Communities, BCR Information. Report EUR 11686 (1988).
The percentage of activity can be obtained with the aid of the Table or the calibration curve. Loeliger, E.A. , ICSH/ICTH. Recommendations for Reporting Prothrombin Time in Oral Anticoagulant
Control. Thromb.Haemostas. (1985), 53, 155 - 156.
Note Kirkwood, T.B.C. Calibration of reference thromboplastins and standardization of the phrotrombin time
For automated systems refer to the appropriate instruction manual. ratio. Thromb. Haemostas. (1983), 49, 238 - 244.
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PLASMASCANN® HS
(ISI 1,20 - 1,35) Para diagnóstico “in vitro”
VALUES OF CLOTTING TIMES – % COAGULATION CAPACITY –RATIO – INR Calculation
INR
TP valores normales (seg.) % coag R
ISI 1,10 ISI 1,20 ISI 1,25 ISI 1,30 ISI 1,35
10,5 11,5 12,0 12,5 13,0 13,5 14,0 15,0 100 1,00 1,0 1,0 1,0 1,0 1,0
11,0 12,1 12,6 13,1 13,7 14,2 14,7 15,8 90,5% 1,05 1,06 1,06 1,06 1,07 1,07
11,6 12,7 13,2 13,8 14,3 14,9 15,4 16,5 82,6% 1,10 1,11 1,12 1,13 1,13 1,14
12,1 13,2 13,8 14,4 15,0 15,5 16,1 17,3 76,0% 1,15 1,17 1,18 1,19 1,20 1,21
12,6 13,8 14,4 15,0 15,6 16,2 16,8 18,0 70,4% 1,20 1,22 1,24 1,26 1,27 1,28
13,1 14,4 15,0 15,6 16,3 16,9 17,5 18,8 65,5% 1,25 1,28 1,31 1,32 1,34 1,35
13,7 15,0 15,6 16,3 16,9 17,6 18,2 19,5 61,3% 1,30 1,33 1,37 1,39 1,41 1,43
14,2 15,5 16,2 16,9 17,6 18,2 18,9 20,3 57,6% 1,35 1,39 1,43 1,46 1,48 1,50
14,7 16,1 16,8 17,5 18,2 18,9 19,6 21,0 54,3% 1,40 1,45 1,50 1,52 1,55 1,57
15,2 16,7 17,4 18,1 18,9 19,6 20,3 21,8 51,4% 1,45 1,50 1,56 1,59 1,62 1,65
15,8 17,3 18,0 18,8 19,5 20,3 21,0 22,5 48,7% 1,50 1,56 1,63 1,66 1,69 1,73
16,3 17,8 18,6 19,4 20,2 20,9 21,7 23,3 46,4% 1,55 1,62 1,69 1,73 1,77 1,81
16,8 18,4 19,2 20,0 20,8 21,6 22,4 24,0 44,2% 1,60 1,68 1,76 1,80 1,84 1,89
17,3 19,0 19,8 20,6 21,5 22,3 23,1 24,8 42,2% 1,65 1,73 1,82 1,87 1,92 1,97
17,9 19,6 20,4 21,3 22,1 23,0 23,8 25,5 40,4% 1,70 1,79 1,89 1,94 1,99 2,05
18,4 20,1 21,0 21,9 22,8 23,6 24,5 26,3 38,8% 1,75 1,85 1,96 2,01 2,07 2,13
18,9 20,7 21,6 22,5 23,4 24,3 25,2 27,0 37,3% 1,80 1,91 2,02 2,08 2,15 2,21
TP plasma paciente (seg.)
19,4 21,3 22,2 23,1 24,1 25,0 25,9 27,8 35,9% 1,85 1,97 2,09 2,16 2,22 2,29
20,0 21,9 22,8 23,8 24,7 25,7 26,6 28,5 34,6% 1,90 2,03 2,16 2,23 2,30 2,38
20,5 22,4 23,4 24,4 25,4 26,3 27,3 29,3 33,4% 1,95 2,08 2,23 2,30 2,38 2,46
21,0 23,0 24,0 25,0 26,0 27,0 28,0 30,0 32,2% 2,00 2,14 2,30 2,38 2,46 2,55
22,1 24,2 25,2 26,3 27,3 28,4 29,4 31,5 30,2% 2,10 2,26 2,44 2,53 2,62 2,72
23,1 25,3 26,4 27,5 28,6 29,7 30,8 33,0 28,4% 2,20 2,38 2,58 2,68 2,79 2,90
24,2 26,5 27,6 28,8 29,9 31,1 32,2 34,5 26,8% 2,30 2,50 2,72 2,83 2,95 3,08
25,2 27,6 28,8 30,0 31,2 32,4 33,6 36,0 25,4% 2,40 2,62 2,86 2,99 3,12 3,26
26,3 28,8 30,0 31,3 32,5 33,8 35,0 37,5 24,1% 2,50 2,74 3,00 3,14 3,29 3,45
27,3 29,9 31,2 32,5 33,8 35,1 36,4 39,0 22,9% 2,60 2,86 3,15 3,30 3,46 3,63
28,4 31,1 32,4 33,8 35,1 36,5 37,8 40,5 21,9% 2,70 2,98 3,29 3,46 3,64 3,82
Haemostasia
28,9 31,6 33,0 34,4 35,8 37,1 38,5 41,3 21,4% 2,75 3,04 3,37 3,54 3,73 3,92
30,5 33,4 34,8 36,3 37,7 39,2 40,6 43,5 20,0% 2,90 3,23 3,59 3,78 3,99 4,21
32,0 35,1 36,6 38,1 39,7 41,2 42,7 45,8 18,8% 3,05 3,41 3,81 4,03 4,26 4,51
34,1 37,4 39,0 40,6 42,3 43,9 45,5 48,8 17,4% 3,25 3,66 4,11 4,36 4,63 4,91
36,2 39,7 41,4 43,1 44,9 46,6 48,3 51,8 16,3% 3,45 3,90 4,42 4,70 5,00 5,32
36,8 40,3 42,0 43,8 45,5 47,3 49,0 52,5 16,0% 3,50 3,97 4,50 4,79 5,10 5,43
37,8 41,4 43,2 45,0 46,8 48,6 50,4 54,0 15,5% 3,60 4,09 4,65 4,96 5,29 5,64
39,9 43,7 45,6 47,5 49,4 51,3 53,2 57,0 14,5% 3,80 4,34 4,96 5,31 5,67 6,06
42,0 46,0 48,0 50,0 52,0 54,0 56,0 60,0 13,7% 4,00 4,59 5,28 5,66 6,06 6,50
44,1 48,3 50,4 52,5 54,6 56,7 58,8 63,0 12,9% 4,20 4,85 5,60 6,01 6,46 6,94
46,2 50,6 52,8 55,0 57,2 59,4 61,6 66,0 12,3% 4,40 5,10 5,92 6,37 6,86 7,39
48,3 52,9 55,2 57,5 59,8 62,1 64,4 69,0 11,7% 4,60 5,36 6,24 6,74 7,27 7,85
49,4 54,1 56,4 58,8 61,1 63,5 65,8 70,5 11,4% 4,70 5,49 6,40 6,92 7,48 8,08
50,4 55,2 57,6 60,0 62,4 64,8 67,2 72,0 11,1% 4,80 5,62 6,57 7,10 7,68 8,31
52,5 57,5 60,0 62,5 65,0 67,5 70,0 75,0 10,6% 5,00 5,87 6,90 7,48 8,10 8,78
Plasmascann® has a ISI (1.20-1.35). ISI of each batch is stated in the label on the vial.
Mean values obtained with different PLASMASCANN HS lots. It is suggested that each laboratory should
establish its Normal Prothrombin Time and INR calculation. using its own determination method.
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ANTICOAGULANTS. ANCILLARY PRODUCTS
FOR COAGULATION TESTS
EDTA K3
1 x 25mL Ref. 99 68 21
PRODUCT DESCRIPTION
Anticoagulant for use in hematology and biochemistry. Specially
recommended for the determination of glycohemoglobin and for
hematologic recounting. The blood collected with EDTA shows stability
with celular components and no singns of hemolysis for up to 8 days
from collection. One drop is enough to inhibit coagulation of 5mL of total
blood. The use of higher amounts of anticoagulant may yield falsely
decreased hematocrit values.
REAGENT COMPOSITION
EDTA solution (0,738 mol/L) pH 8,4
Store at Room Temperature. The reagent will remain stable until the
REAGENT PREPARATION
CAUTION
SODIUM CITRATE (3.2%)

1 x 500mL Ref. 99 59 59
PRODUCT DESCRIPTION
Anticoagulant of choice for routine coagulation tests. Generally used for
the determination of the VSG and the prothrombin time, the antithrombin
III and the fibrinogen. The adequate blood/anticoagulant ratio is 9 parts
of blood and 1 part of sodium citrate. Other citrate concentrations than
the one specified will affect coagulation time.
REAGENT COMPOSITION
sodium Citrate solution 3,2%
Store at 4-30ºC. The reagent will remain stable until the expiration date
REAGENT PREPARATION
CAUTION
Contains sodium azide (0.09%). Waste products must be handled as
per local regulations.
CALCIUM CHLORIDE (0.025M)

1 x 100mL Ref. 99 88 02
PRODUCT DESCRIPTION
Product specially recommended for the Activated Partial Thromboplastin
Time (APTT) and Prothrombin Time (PT) determinations.
REAGENT COMPOSITION
Calcium chloride solution 0,025 mol/L
Store at Room Temperature. The reagent will remain stable until the
REAGENT PREPARATION
CAUTION
Contains sodium azide (0.09%). Waste products must be handled as
per local regulations.
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Microbiology
ISO 9001 / ISO 13485

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GRAM PVP KIT
For in vitro diagnostic
PRINCIPLE
The Gram stain was created in 1884 by Christian Gram, and is an essential factor in microbial identification. Although Gram observed what is now known as the “Gram reaction”, he did not
recognise the taxonomic value of his technique. The Gram stain makes it possible to differentiate bacteria into two groups, according to the colour of the cell after staining. It is a system
with two simple, successive stains, separated by a destaining phase. It makes it possible to differentiate the bacteria which retain the first colour, and appear blue (Gram-positive) from
those that do not retain the colour and are stained pink (Gram-negative). The difference is due to the cell wall. The key is peptidoglycan, the material which provides rigidity to the bacterial
cell wall. Gram-positive bacteria have a much higher proportion of peptidoglycan than gram-negative bacteria. The cell wall must be intact for correct staining to take place. During the
staining, the cell wall of the gram-positive cells is dehydrated by the alcohol of the decolourising agent and it losses permeability. It therefore retains the compound formed by the primary
stain (Crystal violet) and the iodine (mordant). By contrast, the gram-negative cells, which have a wall with a higher lipid content, become more permeable when destained with alcohol and
lose the primary stain. They are subsequently stained with the counterstain (safranin or fuchsin).
This kit differs from the classic Gram kit as it contains iodine stabilised with L-polyvinylpyrrolidone, to offer a more stable iodine compound which does not lose its efficacy as a mordant
over time.
DIAGNOSTIC USE STORAGE AND STABILITY
Gram stain dyes are used to perform the staining of microorganisms, cultures or samples Reagents which are stored at 15-30ºC and protected from light are stable until the expiry
using the Gram differential method. date stated on the label. Containers must always be kept tightly closed. Over time, a light
REAGENTS precipitate may form in some reagents. This does not affect their functionality.
Kit 4 x 500 mL Ref. 99 87 02 PRECAUTIONS
Contains: The products are for professional use. All the reagents must be handled with care by trained
A. 1 x 500 mL Reagent No. 1 Crystal violet Ref. 99 52 02 technicians. The safety data sheet should be consulted before using the reagents.
B. 1 x 500 mL Reagent No. 2 Iodine PVP Ref. 99 55 83 Waste disposal must be carried out according to the local regulations in force.
C. 1 x 500 mL Reagent No. 3 Gram decolorizer Ref. 99 04 95
D. 1 x 500 mL Reagent No. 4 Safranin Ref. 99 99 21
SAMPLE
Kit 4 x 250 mL Ref. 99 88 00 Smears of bacterial cultures. Samples of various body fluids: sputum, lung fluid, urine
Contains: sediment, cerebrospinal fluid, tissue, etc.
A. 1 x 250 mL Reagent No. 1 Crystal violet Ref. 99 87 10
B. 1 x 250 mL Reagent No. 2 Iodine PVP Ref. 99 78 15 Spread the sample with an inoculating loop onto a slide to obtain a uniform and thin smear.
C. 1 x 250 mL Reagent No. 3 Gram decolorizer Ref. 99 64 21 Air dry and heat fix passing the slide through a low flame 2 or 3 times. Leave to cool before
D. 1 x 250 mL Reagent No. 4 Safranin Ref. 99 61 11 performing the staining.
Kit 4 x 100 mL Ref. 99 88 35 Handle the samples with care due to their potentially infectious nature.
Contains:
A. 1 x 100 mL Reagent No. 1 Crystal violet Ref. 99 05 64 PROCEDURE
B. 1 x 100 mL Reagent No. 2 Iodine PVP Ref. 99 05 65
C. 1 x 100 mL Reagent No. 3 Gram decolorizer Ref. 99 05 66
D. 1 x 100 mL Reagent No. 4 Safranin Ref. 99 05 67 1. Cover the fixed smear with the primary stain, Crystal violet or Carbol gentian
violet. Leave to work for 1 minute.
Also available: 2. Rinse with running tap water. Remove excess water.
Primary stain 3. Cover the smear with the mordant, Iodine PVP. Leave to work for 1 minute.
a) Crystal violet 1 x 500 mL Ref. 99 52 02 4. Rinse with running tap water. Remove excess water.
1 x 1000 mL Ref. 99 45 00 5. Destain the smear with the Gram decolorizer until no more stain is removed from
1 x 5000 mL Ref. 99 98 02 the preparation. Approximately 15-30 seconds, depending on the thickness of
6 x 250 mL Ref. 99 99 13 the smear.
b) Carbol gentian violet 1 x 500 mL Ref. 99 79 28 6. Rinse with water. Remove excess water.
1 x 1000 mL Ref. 99 79 41 7. Stain the smear for 1 minute with the counterstain, Safranin or Gram fuchsin.
1 x 5000 mL Ref. 99 65 57 8. Rinse with running tap water and air dry.
Iodine PVP solution 1 x 500 mL Ref. 99 55 83 9. Examine under the microscope with the immersion lens.
1 x 1000 mL Ref. 99 58 81
1 x 5000 mL Ref. 99 65 02
6 x 250 mL Ref. 99 60 11
Gram decolorizer 1 x 1000 mL Ref. 99 04 89 QUALITY CONTROL
1 x 5000 mL Ref. 99 11 10 Following the quality control practices defined by the CLSI (formerly NCCLS) is
6 x 250 mL Ref. 99 76 70 recommended. For this purpose, carry out controls with ATCC microorganisms, or known
Counterstain acid-fast or non acid-fast microorganism cultures.
a) Safranin 1 x 500 mL Ref. 99 99 21
1 x 1000 mL Ref. 99 87 15 RESULTS
1 x 5000 mL Ref. 99 65 01 Gram-positive bacteria: dark violet
6 x 250 mL Ref. 99 98 04 Gram-negative bacteria: pink-red
Gram fuchsin: 1 x 1000 mL Ref. 99 72 17
NOTES
REAGENT COMPOSITION
The result of the staining should be treated as a guide and must be confirmed with additional
Crystal violet
tests. The technique outlined above may be modified in accordance with the technician’s
Crystal violet stain 0.40%
preferences in order to obtain variations in the staining intensity. This entails modifications
Ethanol 15%
to the staining, destaining and rinsing times, etc.
Phenol < 1%
Using cultures which are a maximum of 18-24 hours old and fresh smears is recommended,
Carbol gentian violet
as old cultures and preparations may produce erroneous results.
Carbol gentian violet stain 0.45%
It is important to control the heat fixation as excess heat may result in inaccurate staining
Ethanol 13%
since it may alter the cell wall.
Phenol 1%
Antibiotic treatment prior to sample collection may produce alterations in the staining, giving
Iodine PVP solution
a gram-negative result in gram-positive bacteria.
Iodine 1%
If running tap water is used for the rinsing, please be aware that strongly chlorinated water
Potassium iodide 2%
may weaken the contrast staining.
PVP as a stabiliser
Gram decolorizer REFERENCES
Ethanol 70% Clark, G. (1981) “Staining Procedures”, 4th ed., Williams & Willkins.
Acetone 30% Bartholomew, J. M., Mittwer, T. (1952), Bacteriol. Rev., 16, 1-29.
Safranin CLSI Guidelines and Standards, CLSI, Wayne, P.A
Safranin 0.4% Young D.S., Effect of drugs on Clinical Lab. Test, 5th Ed. AACC Press (2000).
Ethanol 20%
Gram fuchsin:
Basic fuchsin 0.02%
MATERIAL REQUIRED (NOT SUPPLIED)
General-purpose laboratory material.
Slides and coverslips, Bunsen burner, inoculating loop, staining rack, filter paper,
microscope with immersion lens.
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STAINS FOR GRAM STAINING
PRINCIPLE
The Gram stain was created in 1884 by Christian Gram, and is an essential factor in microbial identification. Although Gram observed what is now known as the “Gram reaction”, he did not
recognise the taxonomic value of his technique. The Gram stain makes it possible to differentiate bacteria into two groups, according to the colour of the cell after staining. It is a system
with two simple, successive stains, separated by a destaining phase. It makes it possible to differentiate the bacteria which retain the first colour, and appear blue (Gram-positive) from
those that do not retain the colour and are stained pink (Gram-negative). The difference is due to the cell wall. The key is peptidoglycan, the material which provides rigidity to the bacterial
cell wall. Gram-positive bacteria have a much higher proportion of peptidoglycan than gram-negative bacteria. The cell wall must be intact for correct staining to take place. During the
staining, the cell wall of the gram-positive cells is dehydrated by the alcohol of the decolourising agent and it losses permeability. It therefore retains the compound formed by the primary
stain (Crystal violet) and the iodine (mordant). By contrast, the gram-negative cells, which have a wall with a higher lipid content, become more permeable when destained with alcohol and
lose the primary stain. They are subsequently stained with the counterstain (safranin or fuchsin).
DIAGNOSTIC USE PRECAUTIONS

Stains for gram stain are used to perform the staining of microorganisms, cultures or The products are for professional use. All reagents must be handled with care by trained
samples using the Gram differential method. technicians. The safety data sheet should be consulted before using the reagents.
The Gram decolorizer is an irritant and is highly flammable.
Waste disposal must be carried out according to the local regulations in force.
REAGENTS
Primary stain SAMPLE
a) Crystal violet 1 x 500 mL Ref. 99 52 02 Smears from bacterial cultures, preferably 18-24 hours old. Samples from various body
1 x 1000 mL Ref. 99 45 00 fluids: exudates, pulmonary fluid, urine sediment, etc.
1 x 5000 mL Ref. 99 98 02
6 x 250 mL Ref. 99 99 13 Spread the sample with an inoculating loop onto a slide directly, or mix it with saline solution,
Composition to obtain a thin, uniform smear. Air dry and heat fix passing the slide through a low flame 2
Crystal violet stain 0.40% or 3 times. Leave the slide to cool before performing the staining.
Ethanol 15%
Phenol < 1% Handle the samples with care due to their potentially infectious nature.
b) Carbol gentian violet 1 x 500 mL Ref. 99 79 28 QUALITY CONTROL

1 x 1000 mL Ref. 99 79 41 Following the quality control practices defined by the CLSI (formerly NCCLS) is
1 x 5000 mL Ref. 99 65 57 recommended. For this purpose, carry out controls with ATCC microorganisms, or known
Composition acid-fast or non acid-fast microorganism cultures.
Carbol gentian violet stain 0.45%
Ethanol 3%
Phenol 1% PROCEDURE
Iodine Lugol solution 1 x 500 mL Ref. 99 30 85

1. Cover the fixed smear with the primary stain, Crystal violet or Carbol gentian
1 x 1000 mL Ref. 99 88 42
violet. Leave to work for 1 minute.
Composition
2. Rinse with running tap water. Remove excess water.
Iodine 0.4%
3. Cover the smear with the mordant, Lugol’s Iodine. Leave to work for 1 minute.
Potassium iodide 0.8%
4. Rinse with running tap water. Remove excess water.
5. Destain the smear with the Gram decolorizer until no more stain is removed from
Gram decolorizer 1 x 1000 mL Ref. 99 04 89
the preparation. Approximately 15-30 seconds, depending on the thickness of
1 x 5000 mL Ref. 99 11 10
the smear.
6 x 250 mL Ref. 99 76 70
6. Rinse with water. Remove excess water.
Composition
7. Stain the smear for 1 minute with the counterstain, Safranin or Gram fuchsin.
Ethanol 70%
8. Rinse with running tap water and air dry.
Acetone 30%
9. Examine under the microscope with the immersion lens.
Counterstain
a) Safranin 1 x 500 mL Ref. 99 99 21
1 x 1000 mL Ref. 99 87 15
1 x 5000 mL Ref. 99 65 01 RESULTS
6 x 250 mL Ref. 99 98 04 Gram-positive bacteria: dark violet
Composition Gram-negative bacteria: pink-red
Safranin 0.4%
Ethanol 20%
NOTES
b) Gram fuchsin: 1 x 1000 mL Ref. 99 72 17 The result of the staining should be treated as a guide and must be confirmed with
appropriate additional tests.
Composition The technique outlined above may be modified in accordance with the technician’s
Basic fuchsin 0.02% preferences in order to obtain variations in the staining intensity. This entails modifications
to the staining, destaining and rinsing times, etc.
Using cultures which are a maximum of 18-24 hours old and fresh smears is recommended,
STORAGE AND STABILITY as old cultures and preparations may produce erroneous results.
Reagents which are stored at 15-30ºC and protected from light are stable until the expiry It is important to control the heat fixation as excess heat may result in inaccurate staining
Microbiology
date stated on the label. Containers must always be kept tightly closed. since it may alter the cell wall.
Antibiotic treatment prior to sample collection may produce alterations in the staining, giving
Over time, a light precipitate may form in some reagents. This does not affect their a gram-negative result in gram-positive bacteria.
functionality. If running tap water is used for the rinsing, please be aware that strongly chlorinated water
may weaken the contrast staining.

General-purpose laboratory material. REFERENCES
Slides and coverslips, Bunsen burner, inoculating loop, staining cuvettes, blotting paper, Clark, G. (1981) “Staining Procedures”, 4th ed., Williams & Willkins.
microscope with immersion lens. Bartholomew, J. M., Mittwer, T. (1952), Bacteriol. Rev., 16, 1-29.
ISO 9001 / ISO 13485

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TB ZIEHL-NEELSEN KIT
PRINCIPLE
Ziehl–Neelsen staining is used to differentiate acid-fast microorganisms. These microorganisms have a special lipid cell wall, which contains mycolic acid, amongst other substances. This
enables the wall to resist destaining with acid-alcohol after staining with basic dyes such as fuchsine. The cell wall appears a reddish-pink colour, while the remainder of the microorganisms
are stained with the counterstain, normally Methylene Blue. Classic staining requires heat in order for the stain to penetrate the bacterial wall. It appears that intact cells are required in order
for the acid-fast property to be demonstrated. It is assumed that permeability through intact membranes may be an important component of the mechanism involved in the phenomenon
of resistance to destaining by the acid-alcohol solution indicated above.
Although the mechanism of differentiation is not fully established, the method is fully accepted as a procedure for early diagnosis, as well as to provide information on the number of bacilli
Acid-fast microorganisms are basically mycobacteria, although there are other microorganisms which also present this characteristic, such as the genus Nocardia and some parasites,
such as Cryptosporidium.
DIAGNOSTIC USE SAMPLE

Stains for Ziehl–Neelsen staining are used to perform staining of cultures or samples which Smears of bacterial cultures. Samples of various body fluids: sputum, lung fluid, urine
are suspected of containing mycobacteria, for early diagnosis of mycobacterial infection and sediment, cerebrospinal fluid, tissue, etc.
its characterisation.
Spread the sample with an inoculating loop onto a slide to obtain a uniform and thin smear.
REAGENTS Air dry and heat fix passing the slide through a low flame 2 or 3 times. Leave to cool before
Reagents performing the staining.
Kit 3 x 500 mL Ref. 99 61 04
Contains: Handle the samples with care due to their potentially infectious nature.
A. 1 x 500 mL Reagent No. 1 Carbol fuchsin Ref. 99 16 28
B. 1 x 500 mL Reagent No. 2 TB Decolorizer Ref. 99 71 24 PROCEDURE
C. 1 x 500 mL Reagent No. 3 Kühne’s methylene blue Ref. 99 37 40
1. Place filter paper on top of the preparation and cover with Fenicade Fuchsine.
Kit 3 x 250 mL Ref. 99 08 79
Leave for approximately 5 - 10 minutes.
Contains:
During this time, heat occasionally until the stain begins to form vapour. Prevent the
A. 1 x 250 mL Reagent No. 1 Carbol fuchsin Ref. 99 99 10
preparation from drying out and do not boil.
B. 1 x 250 mL Reagent No. 2 TB Decolorizer Ref. 99 20 84
2. Rinse in gently running tap water. Remove excess water.
3. Remove the paper and cover with the KB destaining agent, rock gently and then
rinse with water. If red staining continues to appear in the sample, repeat the
Also available:
destaining process.
Carbol fuchsin 1 x 500 mL Ref. 99 16 28
5. Cover the preparation with Methylene Blue for 1 minute.
1 x 1,000 mL Ref. 99 30 23
6 x 250 mL Ref. 99 36 14
Composition
Basic fuchsine 0.2%
Phenol 4.6%
RESULTS
Acid-fast bacilli: dark red to pink
TB Decolorizer 1 x 1,000 mL Ref. 99 71 15
Non acid-fast bacilli: blue
6 x 250 mL Ref. 99 20 90
Composition
Ethanol 97% QUALITY CONTROL
HCl 3% Following the quality control practices defined by the CLSI (formerly NCCLS) is
recommended. For this purpose, carry out controls with ATCC microorganisms, or known
acid-fast or non acid-fast microorganism cultures.
Kühne’s methylene blue 1 x 500 mL Ref. 99 37 40
1 x 1,000 mL Ref. 99 89 77
Composition NOTES
Methylene blue 0.5% The result of the staining should be treated as a guide. Positive staining provides
Phenol 1% presumptive evidence of the presence of mycobacteria in the sample and should be
Ethanol 30% confirmed with additional tests (culture, molecular tests, etc.). Negative staining does not
necessarily indicate that the sample is negative for mycobacteria in the culture.
The technique outlined above may be modified in accordance with the technician’s
Löffler’s Methylene Blue 1 x 500 mL Ref. 99 09 62 preferences in order to obtain variations in the staining intensity. This entails modifications
Composition to the staining, destaining and rinsing times, etc. If running tap water is used for the
Methylene blue 0.45% rinsing, please be aware that strongly chlorinated water may weaken the contrast staining.
Ethanol 35% Excessive rinsing after adding the Fuchsine may produce false negatives. Excessive rinsing
after using the counterstain may reduce the staining of the non acid-fast microorganisms.
Reagents which are stored at 15-30ºC and protected from light are stable until the expiry
date stated on the label. Containers must always be kept tightly closed.
REFERENCES
Gurr, E. (1965). “The rational use of dyes in Biology”. Leonard Hill, London.
Over time, a light precipitate may form in some reagents. This does not affect their
Clark, G. (1981) “Staining Procedures”, pp.380-382, 4th ed. Williams & Willkins
functionality.
PRECAUTIONS
The products are for professional use. All the reagents must be handled with care by trained
technicians. The safety data sheet should be consulted before using the reagents.
Carbol fuchsin, as well as Kühne’s methylene blue may be harmful if ingested or if they
come into contact with the skin. Carbol fuchsin is corrosive. The TB Decolorizer, Kühne’s
methylene blue and Löffler’s Methylene Blue are flammable.

Slides and coverslips, Bunsen burner, inoculating loop, staining rack, filter paper, microscope
with immersion lens.
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TB ZIEHL-NEELSEN TENSOACTIVE
PRINCIPLE
are stained with the counterstain, normally Methylene Blue. It is assumed that permeability through intact membranes may be an important component of the mechanism involved in the
phenomenon of resistance to destaining by the acid-alcohol solution indicated above.
While the classic Ziehl-Neelsen staining procedure specifies the use of heat in the primary staining phase, the addition of a surface-active agent to a more concentrated Fuchsine dye
makes it possible to perform cold staining. The remaining phases in the procedure are the same as those in the original Ziehl method.
DIAGNOSTIC USE MATERIAL REQUIRED (NOT SUPPLIED)

Stains for Ziehl–Neelsen staining are used to perform staining of cultures or samples which General-purpose laboratory material.
are suspected of containing mycobacteria, for early diagnosis of mycobacterial infection and Slides and coverslips, Bunsen burner, inoculating loop, staining rack, microscope with
its characterisation. immersion lens.
REAGENTS SAMPLE
Reagents Smears of bacterial cultures. Samples of various body fluids: sputum, lung fluid, urine
Kit 3 x 500 mL Ref. 99 73 21 sediment, cerebrospinal fluid, tissue, etc.
Contains:
A. 1 x 500 mL Reagent No. 1 Carbol Fuchsin tensoactive Ref. 99 77 68 Spread the sample with an inoculating loop onto a slide to obtain a uniform and thin smear.
B. 1 x 500 mL Reagent No. 2 TB Decolorizer Ref. 99 71 24 Air dry and heat fix passing the slide through a low flame 2 or 3 times. Leave to cool before
C. 1 x 500 mL Reagent No. 3 Kühne’s methylene blue Ref. 99 37 40 performing the staining.
Kit 3 x 250 mL Ref. 99 82 72 Handle the samples with care due to their potentially infectious nature.
Contains:
A. 1 x 250 mL Reagent No. 1 Carbol Fuchsin teonsoactive Ref. 99 68 81 PROCEDURE
1. Cover with the surfactant Fenicade Fuchsine. Leave for approximately 5 minutes.
Also available: DO NOT HEAT
Carbol Fuchsin tensoactive 1 x 1,000 mL Ref. 99 93 99 3. Cover with the KB destaining agent, rock gently and then rinse with water. If red
Composition staining continues to appear in the sample, repeat the destaining process.
Basic fuchsine 1.5% 4. Rinse in gently running tap water. Remove excess water.
Phenol 4.5% 5. Cover the preparation with Methylene Blue for 1 minute.
Ethanol 15% 6. Rinse with running tap water and air dry.
Surface-active agents 15% 7. Examine under the microscope with the immersion lens.
Wetting agents 15%
TB Decolorizer 1 x 1,000 mL Ref. 99 71 15 RESULTS

6 x 250 mL Ref. 99 20 90 Acid-fast bacilli: dark red to pink
Composition Non acid-fast bacilli: blue
Ethanol 97%
HCl 3%
QUALITY CONTROL
Kühne’s methylene blue 1 x 500 mL Ref. 99 37 40 Following the quality control practices defined by the CLSI (formerly NCCLS) is
1 x 1,000 mL Ref. 99 89 77 recommended. For this purpose, carry out controls with ATCC microorganisms, or known
Composition acid-fast or non acid-fast microorganism cultures.
Methylene blue 0.5%
Phenol 1%
Ethanol 30% NOTES
The result of the staining should be treated as a guide. Positive staining provides
Löffler’s Methylene Blue 1 x 500 mL Ref. 99 09 62 presumptive evidence of the presence of mycobacteria in the sample, and should be
Composition confirmed with additional tests (culture, molecular tests, etc.). Negative staining does not
Methylene blue 0.45% necessarily indicate that the sample is negative for mycobacteria in the culture.
Ethanol 35% The technique outlined above may be modified in accordance with the technician’s
preferences in order to obtain variations in the staining intensity. This entails modifications
to the staining, destaining and rinsing times, etc. If running tap water is used for the
rinsing, please be aware that strongly chlorinated water may weaken the contrast staining.
STORAGE AND STABILITY Excessive rinsing after adding the Fuchsine may produce false negatives. Excessive rinsing
Reagents which are stored at 15-30ºC and protected from light are stable until the expiry after using the counterstain may reduce the staining of the non acid-fast microorganisms.
Over time, a light precipitate may form in some reagents. This does not affect their REFERENCES
Microbiology
functionality. Gurr, E. (1965). “The rational use of dyes in Biology”. Leonard Hill, London.
Clark, G. (1981) “Staining Procedures”, pp.380-382, 4th ed. Williams & Willkins
PRECAUTIONS CLSI Guidelines and Standards, CLSI, Wayne, P.A
The products are for professional use. All the reagents must be handled with care by trained Young D.S., Effect of drugs on Clinical Lab. Test, 5th Ed. AACC Press (2000).
The Carbol Fuchsin teonsoactive, as well as Kühne’s methylene blue may be harmful if
ingested or if they come into contact with the skin. The Carbol Fuchsin teonsoactive is
corrosive. The TB Decolorizer, Kühne’s methylene blue and Löffler’s Methylene Blue are
flammable.
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TB KINYOUN KIT
PRINCIPLE
Kinyoun staining is a variant of the classic Ziehl-Neelsen staining procedure which uses basic fuchsine with high concentrations of phenol as its primary stain. This allows cold staining,
whereas the classic Ziehl-Neelsen staining procedure specifies the use of heat in the primary staining phase. The remaining phases in the procedure are the same as those in the original
Ziehl method. Brilliant Green is used as a counterstain, although Methylene Blue may also be used.
are stained with the counterstain. It is assumed that permeability through intact membranes may be an important component of the mechanism involved in the phenomenon of resistance
to destaining by the acid-alcohol solution indicated above.

Stains for Kinyoun staining are used to perform the staining of cultures or samples which Smears of bacterial cultures. Samples of various body fluids: sputum, lung fluid, urine
are suspected of containing mycobacteria, for early diagnosis of mycobacterial infection and sediment, cerebrospinal fluid, tissue, etc.
its characterisation.
Spread the sample with an inoculating loop onto a slide to obtain a uniform and thin smear.
REAGENTS Air dry and heat fix passing the slide through a low flame 2 or 3 times. Leave to cool before
Kit 3 x 250 mL Ref. 99 25 05 performing the staining.
Contains:
A. 1 x 250 mL Reagent No. 1 Carbol Fuchsin Kinyoun Ref. 99 25 55 Handle the samples with care due to their potentially infectious nature.
C. 1 x 250 mL Reagent No. 3 Brilliant green Ref. 99 56 68 PROCEDURE
Carbol Fuchsin Kinyoun

Composition 1. Cover with Kinyoun Fenicade Fuchsine. Leave for approximately 5 minutes. DO
Basic fuchsine 1.5% NOT HEAT
Phenol 5% 2. Rinse in gently running tap water. Remove excess water.
Isopropanol 20% 3. Cover with the KB destaining agent, rock gently and then rinse with water. If red
staining continues to appear in the sample, repeat the destaining process.
Brilliant green 4. Rinse in gently running tap water. Remove excess water.
Composition 5. Cover the preparation with Brilliant Green or Methylene Blue for 1 minute.
Brilliant green 0.2% 6. Rinse with running tap water and air dry.
Also available:
RESULTS
TB Decolorizer 1 x 1,000 mL Ref. 99 71 15 Acid-fast bacilli: dark red to pink
6 x 250 mL Ref. 99 20 90 Non acid-fast bacilli: pale green or blue, depending on the stain used
Composition
Ethanol 97%
HCl 3% QUALITY CONTROL
Following the quality control practices defined by the CLSI (formerly NCCLS) is
Kühne’s Methylene Blue 1 x 500 mL Ref. 99 37 40 acid-fast or non acid-fast microorganism cultures.
1 x 1,000 mL Ref. 99 89 77
Composition
Methylene blue 0.5% NOTES
Phenol 1% The result of the staining should be treated as a guide. Positive staining provides
Ethanol 30% presumptive evidence of the presence of mycobacteria in the sample, and should be
confirmed with additional tests (culture, molecular tests, etc.). Negative staining does not
necessarily indicate that the sample is negative for mycobacteria in the culture.
STORAGE AND STABILITY preferences in order to obtain variations in the staining intensity. This entails modifications
Reagents which are stored at 15-30ºC and protected from light are stable until the expiry to the staining, destaining and rinsing times, etc. If running tap water is used for the
date stated on the label. Containers must always be kept tightly closed. rinsing, please be aware that strongly chlorinated water may weaken the contrast staining.
Excessive rinsing after adding the Fuchsine may produce false negatives. Excessive rinsing
Over time, a light precipitate may form in some reagents. This does not affect their after using the counterstain may reduce the staining of the non acid-fast microorganisms.
functionality.
REFERENCES
MATERIAL REQUIRED (NOT SUPPLIED) Gurr, E. (1965). “The rational use of dyes in Biology”. Leonard Hill, London.
General-purpose laboratory material. Clark, G. (1981) “Staining Procedures”, pp.380-382, 4th ed. Williams & Willkins
Slides and coverslips, Bunsen burner, inoculating loop, staining rack, microscope with CLSI Guidelines and Standards, CLSI, Wayne, P.A
immersion lens. Young D.S., Effect of drugs on Clinical Lab. Test, 5th Ed. AACC Press (2000).
Somoskövi, A., Hotaling, J.E., Fitzgerald, M., O’Donnell, D., Parsons, L.M., Salfinger, M.
(2001), Chest, 120, 250-257.
PRECAUTIONS Good, R.C., Silcox, V, Kilborn, C., ch.40, 679-686 in Balows, A., Hausler, W., Diagnostic
The products are for professional use. All the reagents must be handled with care by trained procedures for bacterial, mycotic and parasitic infections. (1981), 6th ed. Washington D.C.
technicians. The safety data sheet should be consulted before using the reagents. Ederer, G.M., Lund, M.E., ch.46, 806-811 in Balows, A., Hausler, W., Diagnostic procedures
The surfactant Fenicade Fuchsine, as well as Kühne’s Methylene Blue may be harmful if for bacterial, mycotic and parasitic infections. (1981), 6th ed. Washington D.C.
ingested or if they come into contact with the skin. The surfactant Fenicade Fuchsine is Lennette, Spaulding & Truant. Manual of Clinical Microbiology. 3rd ed., 1974, ASM.
corrosive. The TB Decolorizer and Kühne’s Methylene Blue are flammable.
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TB AURAMINE (MORSE) KIT
PRINCIPLE
Mycobacteria are acid-fast microorganisms that have a special lipid cell wall, which contains mycolic acid, amongst other substances. This enables the wall to resist destaining with acid-
alcohol after staining with basic dyes such as fuchsin. The cell wall appears a reddish-pink colour, while the remainder of the microorganism is stained with the counterstain, normally
Methylene Blue. The technique for detecting acid-fast microorganisms by fluorescence is similar to the Ziehl classic staining method. The fenicade fuchsine is replaced by a fluorescent
dye with added phenol - in this case auramine, according to the Morse technique. The auramine binds to the mycolic acids of the wall and the free dye is removed by rinsing with the acidic
destaining agent. The counterstain, potassium permanganate or thiazine red, removes the non-specific background fluorescence.
Compared to the classic staining methods, fluorescence staining provides the advantage of greater visibility of the fluorescent microorganisms on a dark background, making it easier to
work with lower magnification lenses, increasing the field of vision and reducing the time required to evaluate the preparation.
DIAGNOSTIC USE
The kits are used to perform staining of cultures or samples which are suspected of containing PROCEDURE
mycobacteria, for early diagnosis of mycobacterial infection and the characterisation thereof.
REAGENTS 1. Cover the preparation with Reagent No. 1 Auramine.

Kit 3 x 250 mL Ref. 99 38 10 Leave for approximately 15 minutes.
Contains: 2. Rinse in gently running tap water.
A. 1 x 250 mL Reagent No. 1 Auramine Ref. 99 38 42 3. Destain with Reagent No. 2, MT Decolorizer, for 30-60 seconds.
B. 1 x 250 mL Reagent No. 2 MT Decolorizer Ref. 99 68 30 4. Rinse in gently running tap water.
C. 1 x 250 mL Reagent No. 3 Potassium permanganate Ref. 99 43 60 5. Cover the preparation with Reagent No. 3, Potassium Permanganate, for
approximately 2 minutes. Thiazine Red can also be used.
Also available: 6. Rinse with water and air dry.
7. Examine the preparation under a fluorescence microscope at a magnification
Auramine 1 x 1000 mL Ref. 99 38 55 of 25X with an appropriate emission and absorption filter (absorption filter
6 x 250 mL Ref. 99 38 76 460 nm and emission filter 550 nm).
Composition
Auramine stain 0.2%
Phenol 0.4%
RESULTS
Ethanol 25%
Acid-fast bacilli: fluorescent yellow-green
Glycerin 30%
Background: black with permanganate or red with thiazine red and other bacteria may
appear slightly fluorescent.
MT Decolorizer 1 x 1000 mL Ref. 99 68 60
6 x 250 mL Ref. 99 68 96
Composition QUALITY CONTROL
Isopropanol 70% Following the quality control practices defined by the CLSI (formerly NCCLS) is
HCl 0.05% recommended. For this purpose, carry out controls with ATCC microorganisms, or known
Potassium permanganate 1 x 1000 mL Ref. 99 43 70
6 x 250 mL Ref. 99 43 95
Composition NOTES
KMnO4 0.5% The result of the staining should be treated as a guide. Positive staining provides
presumptive evidence of the presence of mycobacteria in the sample and should be
Thiazine Red 1 x 1000 mL Ref. 99 39 45 confirmed with additional tests (culture, molecular tests, etc.). Negative staining does not
Composition necessarily indicate that the sample is negative for mycobacteria in the culture.
Thiazine Red 0.1% The technique outlined above may be modified in accordance with the technician’s
Phenol 5% preferences. This entails modifications to the staining, destaining and rinsing times, etc. If
running tap water is used for the rinsing, please be aware that strongly chlorinated water
STORAGE AND STABILITY may alter the staining. Once the fluorescent microorganisms have been detected, confirming
Reagents which are stored at 15-30ºC and protected from light are stable until the expiry the results with higher magnification lenses (100X) is recommended. This second review
date stated on the label. Containers must always be kept tightly closed. makes it possible to distinguish between non-cellular fluorescent particles and fluorescent
bacterial cells, which may be confused under low magnification.
Indications of any changes in the reagents:
functionality.
REFERENCES
Somoskövi, A., Hotaling, J.E., Fitzgerald, M., O’Donnell, D., Parsons, L.M., Salfinger, M.
(2001), Chest, 120, 250-257.
MATERIAL REQUIRED (NOT SUPPLIED) Good, R.C., Silcox, V, Kilborn, C., ch.40, 679-686 in Balows, A., Hausler, W., Diagnostic
General-purpose laboratory material. procedures for bacterial, Mycotic and parasitic infections. (1981), 6th ed. Washington D.C.
Slides and coverslips, Bunsen burner, inoculating loop, staining rack, fluorescence Ederer, G.M., Lund, M.E., ch.46, 806-811 in Balows, A., Hausler, W., Diagnostic procedures
microscope. for bacterial, Mycotic and parasitic infections. (1981), 6th ed. Washington D.C.
Lenette, Spaulding and Truant. Manual of Clinical Microbiology (1974), 3rd ed., ASM.
PRECAUTIONS
Truant, Brett, Thomas, fluorescent microscopy acid-fast procedure 1962, 382-383 in Clark,
The products are for professional use. All reagents must be handled with care by trained
G., Staining procedures (1981), 4th ed. W&W.
Microbiology
The reagents are irritants of the skin, eyes and mucosa, and harmful if ingested or inhaled.
The MT Decolorizer contains isopropyl alcohol which is flammable.
SAMPLE
Smears from bacterial cultures. Samples from various body fluids: sputum, pulmonary fluid,
urine sediment, cerebrospinal fluid, tissue, etc.
Spread the sample with an inoculating loop onto a slide to obtain a thin, uniform smear. Air
dry and heat fix passing the slide through a low flame 2 or 3 times. Leave to cool before
performing the staining.
Handle the samples with care due to their potentially infectious nature.
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TB AURAMINE-RHODAMINE (TRUANT) KIT
PRINCIPLE
Mycobacteria are acid-fast microorganisms that have a special lipid cell wall, which contains mycolic acid, amongst other substances. This enables the wall to resist destaining with acid-
alcohol after staining with basic dyes such as fuchsin. The cell wall appears a reddish-pink colour, while the remainder of the microorganism is stained with the counterstain, normally
Methylene Blue. The technique for detecting acid-fast microorganisms by fluorescence is similar to the Ziehl classic staining method. The fenicade fuchsine is replaced by a fluorescent
dye with added phenol - in this case Auramine-Rhodamine, according to the Truant technique. The Auramine-Rhodamine binds to the mycolic acids of the wall and the free dye is removed
by rinsing with the acidic destaining agent. The counterstain, Potassium Permanganate or Thiazine Red, removes the non-specific background fluorescence.
Compared to the classic staining methods, fluorescence staining provides the advantage of greater visibility of the fluorescent microorganisms on a dark background, making it easier to
work with lower magnification lenses, increasing the field of vision and reducing the time required to evaluate the preparation.

Stains for Auramine-rhodamine staining are used to perform the staining of cultures
or samples which are suspected of containing mycobacteria, for early diagnosis of
mycobacterial infection and the characterisation thereof. 1. Stir Reagent No. 1, Auramine-rhodamine and cover the preparation.
Leave for approximately 20-25 minutes.
REAGENTS 2. Rinse in gently running tap water.
Kit 3 x 250 mL Ref. 99 93 20 3. Destain with Reagent No. 2, MT decolorizer, for 2-3 minutes.
Contains: 4. Rinse in gently running tap water.
A. 1 x 250 mL Reagent No. 1 Auramine-rhodamine Ref. 99 93 25 5. Cover the preparation with Reagent No. 3, Potassium Permanganate, for
B. 1 x 250 mL Reagent No. 2 MT decolorizer Ref. 99 68 30 approximately 4-5 minutes. Thiazine Red can also be used.
C. 1 x 250 mL Reagent No. 3 MT Potassium permanganate Ref. 99 43 60 6. Rinse with water and air dry.
7. Examine the preparation under a fluorescence microscope at a magnification
Also available: of 25X with an emission and absorption filter, following the Truant indications
(absorption filter 540 nm and emission filter 625 nm).
Auramine-rhodamine 6 x 250 mL Ref. 99 93 56
Composition
Auramine 1.2%
Rhodamine 0.6% RESULTS
Phenol 8% Acid-fast bacilli: fluorescent red-orange
Glycerol 60% Background: black with permanganate or red with thiazine red and other bacteria may
appear slightly fluorescent.
MT decolorizer 1 x 1000 mL Ref. 99 68 60

QUALITY CONTROL
6 x 250 mL Ref. 99 68 96
Composition
Isopropanol 70%
HCl 0.05%
NOTES
Potassium permanganate 1 x 1000 mL Ref. 99 43 70 The result of the staining should be treated as a guide. Positive staining provides
6 x 250 mL Ref. 99 43 95 presumptive evidence of the presence of mycobacteria in the sample and should be
Composition confirmed with additional tests (culture, molecular tests, etc.). Negative staining does not
KMnO4 0.5% necessarily indicate that the sample is negative for mycobacteria in the culture.
Thiazine Red 1 x 1000 mL Ref. 99 39 45 preferences. This entails modifications to the staining, destaining and rinsing times, etc. If
Composition running tap water is used for the rinsing, please be aware that strongly chlorinated water
Thiazine Red 0.1% may alter the staining. Once the fluorescent microorganisms have been detected, confirming
Phenol 5% the results with higher magnification lenses (100X) is recommended. This second review
makes it possible to distinguish between non-cellular fluorescent particles and fluorescent
STORAGE AND STABILITY bacterial cells, which may be confused under low magnification.
REFERENCES
Over time, a light precipitate may form in some reagents. This does not affect their Lenette, Spaulding and Truant. Manual of Clinical Microbiology (1974), 3rd. ed., ASM.
functionality. Truant, Brett, Thomas, fluorescent microscopy acid-fast procedure 1962, 382-383 in Clark,
G., Staining procedures (1981), 4th ed. W&W.
MATERIAL REQUIRED (NOT SUPPLIED) Somoskövi, A., Hotaling, J.E., Fitzgerald, M., O’Donnell, D., Parsons, L.M., Salfinger, M.
General-purpose laboratory material. (2001), Chest, 120, 250-257.
Slides and coverslips, Bunsen burner, inoculating loop, staining rack, fluorescence Good, R.C., Silcox, V, Kilborn, C., ch.40, 679-686 in Balows, A., Hausler, W., Diagnostic
microscope. procedures for bacterial, Mycotic and parasitic infections. (1981), 6th ed. Washington D.C.
Ederer, G.M., Lund, M.E., ch.46, 806-811 in Balows, A., Hausler, W., Diagnostic procedures
PRECAUTIONS for bacterial, Mycotic and parasitic infections. (1981), 6th ed. Washington D.C.
The products are for professional use. All reagents must be handled with care by trained CLSI Guidelines and Standards, CLSI, Wayne, P.A
technicians. The safety data sheet should be consulted before using the reagents. Young D.S., Effect of drugs on Clinical Lab. Test, 5th Ed. AACC Press (2000).
The reagents are irritants of the skin, eyes and mucosa, and harmful if ingested or inhaled.
The MT destaining agent contains isopropyl alcohol which is flammable.
SAMPLE
Smears from bacterial cultures. Samples from various body fluids: sputum, pulmonary fluid,
urine sediment, cerebrospinal fluid, tissue, etc.
Spread the sample with an inoculating loop onto a slide to obtain a thin, uniform smear. Air
dry and heat fix passing the slide through a low flame 2 or 3 times. Leave to cool before
performing the staining.
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LACTOPHENOL BLUE
PRINCIPLE
Lactophenol blue is a stain used to detect yeasts and fungi. It contains phenol, lactic acid and methyl blue. There is a high concentration of phenol, which promotes the inactivation of
hydrolytic enzymes, thereby preventing cell lysis. Lactic acid preserves the fungal structures and methyl blue binds to the chitin of the cell walls, leaving the fungal hyphae stained blue.
Lactophenol blue is used in direct clinical samples as a mounting solution and a stain for the preparation of slides for observation under the microscope, as the blue staining of fungal
structures makes it easier to view and study them.

For the staining of fungal structures.
1. Place a drop of stain in the centre of a clean slide.

2. Take a fragment of the microorganism colony using an inoculating needle or
REAGENTS adhesive tape.
Lactophenol blue 1 x 100 mL Ref. 99 49 70 3. Place the fragment in the drop of stain and disperse gently.
4. Place a coverslip loosely over it, avoiding the formation of bubbles.
Composition 5. Stain for 3 – 4 minutes.
Methyl blue stain 1.0 g/L 6. Examine under the microscope with the 10X or 40X lens.
Phenol 250 g/L
Lactic Acid 250 g/L
Glycerol 500 g/L
Stabilisers and preservatives RESULTS
The fungal elements (yeast, mycelia and spore-producing bodies) appear blue in a slightly
paler blue background.

Reagents which are stored at 15-30ºC and protected from light are stable until the expiry NOTES
date stated on the label. Containers must always be kept tightly closed. The technique described is indicated for the staining of colonies grown in culture media.
The tissue samples or biological fluids must be treated beforehand in a basic medium
before staining. It is recommended that each laboratory establishes its own method which
MATERIAL REQUIRED (NOT SUPPLIED) is adapted to its most common type of samples.
Slides and coverslips, inoculating loop, staining cuvettes, inoculating needle, adhesive tape, Staining with Lactophenol blue is useful to establish a presumptive identification of the
blotting paper, microscope. microorganism from its characteristics.
However, performing biochemical and serological tests in isolated colonies and a study of
its morphology is recommended for a complete identification.
PRECAUTIONS
REFERENCES
Waste disposal must be carried out according to the local regulations in force. Balows A., Hauslesr, W.J., “Diagnostic Procedures for Bacterial, Mycotic and Parasitic
Infections”, Am. Pub. Health Ass. 6th ed., (1981) p.846 - 850.
Clark, G. (1981) “Staining Procedures”, 4th ed., Williams & Willkins.
SAMPLE Bartholomew, J. M., Mittwer, T. (1952), Bacteriol. Rev., 16, 1-29.
Samples from bacterial cultures or samples from various body fluids: exudates, pulmonary CLSI Guidelines and Standards, CLSI, Wayne, P.A
fluid, urine sediment, nails, etc. Young D.S., Effect of drugs on Clinical Lab. Test, 5th Ed. AACC Press (2000).
QUALITY CONTROL
Microbiology
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MALACHITE GREEN
PRINCIPLE
The formation of endospores is a survival strategy of certain bacteria. Endospores are resistant forms which are created in unfavourable environmental conditions (extreme temperatures,
lack of nutrients, toxic compounds, etc.). The spore can survive for years until the environment is favourable, germinating to create a new vegetative form.
Staining with Malachite Green enables the presence of microorganisms with spores to be detected. The positioning of the spores, as well as the proportion of spores to vegetative cells,
provides information about the microorganism being studied.
The thick keratin external wall of the spore is resistant to heat and to staining with various stains. Heat is used to force staining with Malachite Green. Once the stain has penetrated the
inside of the spore, withdraw the preparation from the heat source and remove the stain from the vegetative cells by simply rinsing with water. The spore retains the dye and is stained
green. Safranin is used as a counterstain to stain vegetative cells.
Performing biochemical and serological tests is recommended for a complete identification.

The Malachite Green stain is used for the staining of endospores in microorganisms. Some Smears of bacterial cultures, preferably 18-24 hours old. Samples of various body fluids:
of the endospore-producing bacteria, including Clostridium and Bacillus, are pathogenic to exudate, lung fluid, urine sediment, etc.
humans, making their study and observation of great interest. The position and morphology
of the endospore inside bacterial cells is a useful taxonomic character to differentiate Spread the sample with an inoculating loop onto a slide directly, or mix it with saline solution,
species within the same genus. to obtain a uniform and thin smear. Air dry and heat fix passing the slide through a low flame
2 or 3 times. Leave the slide to cool before performing the staining.
REAGENTS Handle the samples with care due to their potentially infectious nature.
Kit 200 mL Ref. 99 89 89
Contains: PROCEDURE
Reagent A. Malachite green 1 x 100 mL Ref. 99 09 89
Composition
Malachite Green stain 4.5% 1. Cover the smear with Reagent A, Malachite Green. Using wooden forceps,
hold the sample over the Bunsen flame for 5-7 minutes, in order for the stain
Reagent B. Safranin O 1 x 100 mL Ref. 99 09 90 to evaporate. Do not boil the sample. Add more stain if it all evaporates. It is
Composition important that the sample does not dry up.
Safranin O stain 0.25% 2. Rinse with running tap water. Remove excess water.
Ethanol 10% 3. Stain the smear for 1 minute with Reagent B, Safranin, which is the counterstain.

date stated on the label. Containers must always be kept tightly closed. RESULTS
Endospores: Green
Vegetative cells: pink-red
Slides and coverslips, Bunsen burner, inoculating loop, staining cuvettes, blotting paper, NOTES
filter paper, microscope with immersion lens. The result of the staining should be treated as a guide and must be confirmed with
appropriate additional tests.
PRECAUTIONS preferences in order to obtain variations in the staining intensity. This entails modifications
The products are for professional use. All the reagents must be handled with care by trained to the staining and rinsing times, etc.
REFERENCES
QUALITY CONTROL Clark, G. (1981) “Staining Procedures”, 4th ed., Williams & Willkins.
Following the quality control practices defined by the CLSI (formerly NCCLS) is Bartholomew, J. M., Mittwer, T. (1952), Bacteriol. Rev., 16, 1-29.
recommended. For this purpose, carry out controls with ATCC microorganisms, or known CLSI Guidelines and Standards, CLSI, Wayne, P.A
acid-fast or non acid-fast microorganism cultures. Young D.S., Effect of drugs on Clinical Lab. Test, 5th Ed. AACC Press (2000).
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HELICOBACTER
DETECTION OF UREASE ACTIVITY IN GASTRIC BIOPSIES
PRINCIPLE PROCEDURE
Helicobacter pylori has a great capacity to produce urease, an enzyme capable
of breaking urea into ammonium and bicarbonate. The high local ammonium Technique
concentration enables the microorganism to survive in the very low pH gastric
environment. 1. Prepare the same number of test tubes as biopsies.
2. Observe the color of the reagent. If the reagent has changed to
(NH2)2 CO+ 2H2O + H+ 2NH4+ + HCO3- magenta discard it.
3. Insert one or more samples of the same patient into the test tube and
The reagent contains an optimum concentration of urea in a reaction medium close it again.
prepared to maximize the sensitivity and simplicity of the test. 4. Shake for approx 5 seconds. Check that the samples are completely
When the reaction is positive, the pH change produced by the ammoniun ion immersed in the reagent.
makes the indicator turn from yellow to magenta. 5. Read the results after 10 min. Repeat at 60 minutes.
DIAGNOSTIC USE RESULTS

Helicobacter pylori is associated with a variety of gastro-intestinal diseases, NEGATIVE:The reagent remains yellow after 60 minutes.
such as chronic-active gastritis, gastric and duodenal ulcers and gastric POSITIVE: The reagent changes to red-magenta. The time needed for the
adenocarcinoma. change of the color is related to the microorganism concentration in the
About 90% of patients suffering from duodenal or gastric ulcers are infected with sample.
H. pylori.
The infection can be detected by different methods. The urea breath test is If the color change occurs within 30 seconds after the addition of the
considered as the reference method. sample due to the presence of blood or alkali, repeating the test with a
The detection of the microorganism in gastric biopsies by evidentiating its urease new sample.
activity provides for a reliable and specific method for the diagnostic of patients
undergoing endoscopy. PERFORMANCE CHARACTERISTICS
Single test result could not be used to make a clinical diagnosis. It should The performance characteristics depend on the method used. It is recommended
integrate clinical and laboratory data. to calculate these data for each particular test protocol.
REAGENTS Specificity, as the ratio of true negatives, is 100%.

Kit for 20 tests Ref. 99 56 15 Sensitivity, as the ratio of true positives, is 97.5%
Contents 20 monotest tubes There is a correlation of 98.1% with the results obtained with histological
methods and culture techniques.
REAGENT PREPARATION The analytical sensitivity was evaluated with suspensions of Helicobacter pylori
Reagent is ready to use. with different concentrations. At levels of microorganisms higher than 106, the
reagent changes to magenta at 10 minutes; at lower levels of bacteria the
STORAGE AND STABILITY change of color is produced between 10 and 60 minutes.
The components of the kit will remain stable until the expiration date stated on
the label, when stored at 4-30ºC. Do not freeze. INTERFERENCES
False negative results can arise when the microorganism’s concentration in the
ADDITIONAL EQUIPMENT sample is below the detection limit of the test, or there is a non-homogeneous
General laboratory equipment. distribution of the H. pylori in the gastric mucose.
A therapy with antibiotics or inhibitors of the protonic pump can affect the
SAMPLE sensitivity of the test.
Gastric mucosal biopsies. It is recommended to consult the procedures
described by the Workshop on the Histopathology of Gastritis (Houston 1994). The method is a screening test to be used only for the qualitative detection of
Patients should not take antibiotics or bismuth derivatives during the Helicobacter pylori in gastric biopsies.
three weeks preceding the endoscopy. The incomplete erradication of the As in all screening tests the result must be considered within a complete clinical
microorganism can give false negative results. evaluation.
Samples must be processed immediately after obtained.
Handle all specimens as if they contain infectious agents. REFERENCES
Goodwin, C.S.; Mendall, M.M.; Northfield, T.C. Lancet (1997); 349: 265 – 9.
CAUTION Axon, A.T.; Gut (1990), 45 Suppl 1: 11 - 14 IARC Monograph on the Evaluation
The safety statements are on the label. It is recomended to read SDS before reagent manipulation of Carcinogenic Risk to Humans (Vol 61). Lyon: IARC, (1994), 177 – 240.
Human serum used in the control preparation have been found negative in the reaction to HBsAg Pajares-García J.M., Ital. J. Gastroenterol. Hepatol. (1998), 30 Suppl 3: S320
and HIV I/II. However they should always be handled with care. All reagents contain sodium azide – 3.
Waste products must be handled as per local regulations. Dixon, M. F.; Genta, R.; Yardley, J.H.; Correa, P. International Workshop on the
Histopathology of Gastritis, Houston 1994. Am. J. Surg. Pathol. (1996 Oct),
20(10): 1161 – 81.
Microbiology
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BRILLIANT CRESYL BLUE
PRINCIPLE
Reticulocytes are red blood cells which have not reached full maturity. They are anucleate cells which are the predecessors of red blood cells. The difference is that they have ribosomal
granules, RNA and some mitochondria. These structures are present in the form of a network of filaments and granules which are stained in the blood smear, thereby distinguishing the
reticulocytes from the mature red blood cells. Brilliant cresyl blue is basophilic and has affinity with the mitochondria and ribosomes, resulting in a blue precipitate in the shape of granules
or filaments inside the red blood cells. The greater the degree of immaturity of the cell, the greater the precipitated material.
Reticulocytes have a half-life of two days in the bone marrow. They then pass into the circulation where they become mature red blood cells after a day. During this period, 20% of the
haemoglobin contained in red blood cells is synthesised.
Counting reticulocytes makes it possible to evaluate the effective production of red blood cells by the bone marrow, and to discover its regenerative capacity. Reticulocyte counts which
are greater than normal values indicate an increase in erythropoiesis. This is a situation which occurs as a response to haemorrhages, haemolytic anaemia and during the treatment of
nutritional anaemia (iron-deficiency anaemia and megaloblastic anaemia). Low reticulocyte counts suggest defective erythropoiesis, such as in cases of aplastic anaemia, aplastic crisis
of haemolytic anaemia and in infiltration of the bone marrow by tumour cells.

Counting reticulocytes is an essential procedure in haematological diagnosis to determine 1. Mix 20 µl of whole blood with 20 µl of stain in a haemolysis tube.
anaemia and other diseases. The formation of reticulocytes is involved in the production of 2. Keep the mixture at room temperature for 30 minutes and mix again.
red blood cells in the bone marrow. 3. Prepare the blood smear in the normal manner.
4. Leave to air dry.
REAGENTS
Brilliant cresyl blue 1 x 100 mL Ref. 99 36 60 Observe under the microscope with the immersion lens.
WORKING REAGENT COMPOSITION RESULTS
Composition Reticulocytes: The reticular filaments are stained a deep blue, in contrast with the
Brilliant cresyl blue stain 3.0 g/L rest of the cell which is stained blue – pale grey.
NaCl 150 mM Leukocytes: Blue nuclei.
Stabilisers and preservatives Red blood cells: Not stained
PREPARATION OF THE WORKING REAGENT:

Ready for use. CALCULATIONS
The number of reticulocytes is compared with the total number of red blood cells and
STORAGE AND STABILITY it is reported as a percentage of reticulocytes:
Reagents which are stored at 15-30ºC and protected from light are stable until the expiry Reticulocytes (%) = [number of reticulocytes /Total number of red blood cells] x 100
Normal values in adults range between 0.5 and 1.5%.
functionality. Filtering before use is recommended.
MATERIAL REQUIRED (NOT SUPPLIED) NOTES
General-purpose laboratory material The staining intensity is proportional to the staining time. To obtain results [...] The technique
Slides for blood smears described above may be modified to adapt it to the laboratory’s standard procedures. The
Manual or automated staining system new method must be validated, as any variation may affect the staining capacity.
Microscope and immersion oil Each user may apply the different versions of this procedure, both manual and automated,
adapting it to their standard method.
PRECAUTIONS
The products are for professional use. All the reagents must be handled with care by trained Some authors recommend a shorter blood-stain interaction time, incubating the mixture at
technicians. The safety data sheet should be consulted before using the reagents. 37ºC for 15 minutes. If this option is chosen, the mixing tube must be kept tightly closed to
Waste disposal must be carried out according to the local regulations in force. prevent evaporation.
SAMPLE REFERENCES
Air-dried blood smears. It is recommended that they are thin and homogeneous in order to Clark, G. (1981) “Staining Procedures”, 4th ed., Williams & Willkins.
obtain a better fixation of the dye without overstaining. Krafts Woronzoff-Dashkoff, Kristine, Clinics in Laboratory Medicine. Vol. 22 (2002).
Staining should be carried out during the two hours following preparation in order to obtain Biological Stains (1977) 9th ed. Ed. by R. D. Lillie; Williams & Willkins.
good results. Old smears may stain irregularly. CLSI Guidelines and Standards, CLSI, Wayne, P.A
The ideal sample is capillary blood, but if venous blood is used then EDTA should be used
as an anticoagulant. The use of Heparin is not advised.
QUALITY CONTROL
recommended. The staining method indicated is the one which is used routinely in our
laboratories.
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GIEMSA STAIN
PRINCIPLE
The Giemsa stain belongs to the Romanowsky group of stains. It is named after Gustav Giemsa, the German chemist and bacteriologist who developed it. Romanowsky stains consist
of Methylene Blue and its oxidised derivatives, basic dyes and the acidic dye, Eosin. Basic dyes bind to the acidic components of cells, nucleic acids, granules in neutrophils and acidic
proteins which are stained a relatively deep red-purple colour, while eosin binds to haemoglobin, basic components of cell structures and eosinophil granules.
The balance between Methylene Blue and its oxidised derivatives, and between these and Eosin, provides an essentially blue shade and a greater or lesser staining intensity, which are
characteristics of each type of stain - Giemsa, May-Grünwald or Wright. This stain may be used alone or combined with the May-Grünwald stain.
Its use allows the differential staining of blood cells. Some authors use the Giemsa stain or the May-Grünwald-Giemsa combination for the staining of parasites in blood. The result of the
staining may be influenced by several factors, such as the fixation, the staining time and the pH value of the staining solution and the buffer solution. If the pH is too basic, the staining will
be more blue; and if the pH is too acidic, then the staining will be more pink.
PROCEDURE
DIAGNOSTIC USE
For the differential staining of peripheral blood and bone marrow smears.
1. The Giemsa stain is diluted 1/10 with Phosphate Buffer pH 7.2 (previously
REAGENTS diluted 1/10) before use.
Giemsa 1 x 500 mL Ref. 99 84 40 2. The air-dried smear is fixed with methyl alcohol for 3 minutes.
1 x 1000 mL Ref. 99 09 39 3. Decanting the methanol and without rinsing the preparation, cover the smear
with the diluted stain, and leave to work for 8-20 minutes, depending on the
Phosphate buffer pH 7.2 8 x 25 mL Ref. 99 65 13 desired staining intensity.
4. Then rinse abundantly with diluted Phosphate Buffer pH 7.2 and leave to
Kit 2 x 50 mL Ref. 99 43 05 air dry.
Contents: 5. Examine under the microscope.
A. 1 x 50 mL Giemsa Ref. 99 04 88
B. 1 x 45 mL Phosphate buffer pH 7.2 Ref. 99 05 88
REAGENT COMPOSITION RESULTS
Giemsa Red blood cells: Pink or pink-orange.
Eosin-methylene blue according to Giemsa 7.0 g/L Platelets: Pale violet or purple.
Methanol 50% Neutrophils: Dark violet nucleus. Pink cytoplasm with red-violet granulations.
Glycerol 50% Eosinophils: Violet nucleus. Blue cytoplasm with red or red-orange granules.
Phosphate Buffer pH 7.2 Basophils: Dark blue or purple nucleus. Purple, almost black, granules.
Sodium phosphate 33 mM Lymphocytes: Purple nucleus. Sky blue cytoplasm.
Sodium azide 0,9 g/L Monocytes: Very pale violet nucleus. Sky blue cytoplasm.

Giemsa: Dilute 1/10 with the previously diluted Phosphate Buffer pH 7.2 (as indicated NOTES
below). The staining intensity is proportional to the staining time. The stain must be kept away from
Phosphate Buffer: Dilute in the ratio 1/10 with distilled water. Once diluted, use as indicated moisture in order to obtain optimal results.
by the technique. Using buffered water instead of running tap water is recommended for the rinsing
temperature (≤ 25ºC). processes. Running tap water has a varied pH and salt content, which, along with the high
concentrations of chlorine, may alter the results of the staining and produce inconsistent
STORAGE AND STABILITY results.
Reagents which are stored at 15-30ºC and protected from light are stable until the expiry Each user may apply the different versions of this procedure, both manual and automated,
date stated on the label. Containers must always be kept tightly closed. adapting it to their standard method.
The results indicated should be treated as a guideline as the intensity and shade of the
Over time, a light precipitate may form in some reagents. This does not affect their colour could vary with the pH used in the staining.
functionality. In general, staining with the Giemsa reagent results in redder stains when the pH of the
buffered water used is reduced.
Avoid excessive cold which may cause precipitation of the stain. If this has occurred, it is
recommended that the stain is filtered before use. Longer staining times may be required in PRECAUTIONS
this case due to loss of the stain by precipitation. The products are for professional use. All reagents must be handled with care by trained
The working stain diluted 1/10 with Phosphate Buffer pH 7.2 is stable for 3 hours at room
temperature (≤ 25ºC). The stain is toxic and flammable due to its methanol content.
General-purpose laboratory material. QUALITY CONTROL
Slides for blood smears. Following the quality control practices defined by the CLSI (formerly NCCLS) is
Manual or automated staining system. recommended. The staining method indicated is the one which is used routinely in our
Microscope. laboratories.
SAMPLE REFERENCES
Air-dried blood smears. It is recommended that they are thin and homogeneous in order to Clark, G. (1981) “Staining Procedures”, 4th ed., Williams & Willkins.
obtain a better fixation of the dye without overstaining. Krafts Woronzoff-Dashkoff, Kristine, Clinics in Laboratory Medicine. Vol. 22 (2002).
Staining should be carried out during the two hours following preparation in order to obtain Biological Stains (1977) 9th ed. Ed. by R. D. Lillie; Williams & Willkins.
good results. Old smears may stain irregularly. CLSI Guidelines and Standards, CLSI, Wayne, P.A
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HAYEM
For in vitro diagnostics
PRINCIPLE
Hayem’s reagent is an isotonic haematology solution composed of different salts. This con-
centration of salts causes lysis of the leukocytes while erythrocytes remain intact.
DIAGNOSTIC USE
The regular blood count forms the basis of haematologic diagnostics. Hayem’s reagent is
used for diluting blood samples in red blood cell counts.
REAGENT
Hayem 1 x 500 mL Ref. 99 15 28
Composition
NaCl 0,8%
KCl 0,04%
Na2PO4H 0,08%
CaCl2 0,001%

The reagent stored at 15-30ºC will remain stable until the expiration date stated on the label.
The bottles must be kept tightly closed.
Neubauer, Bürker or Fuchs-Rosenthal counting chamber, microscope and calibrated erythro-
cyte pipette (red Thoma pipette).
CAUTION
Professional use only. Handle with care. The safety statements are on the label. It is
advisble to look at the SDS before using the reagent.
SAMPLE
Venous blood with EDTA.
Effective measures must be taken to protect against infection.
PROCEDURE
1.-Fill the red Thoma pipette up to the 0.5 mark
2.-Clean
3.-Then fill with Hayem’s reagent up to the 101 mark. A blood dilution of 1:200 is obtained.
Micropipettes can also be used
4.-Shake the filled pipette for 3 min. until complete mixing
5.-Put the glass cover on the counting chamber central area
6.-Discard the first 4 or 5 drops and fill the counting chamber. Release the reagent slowly
watching how the liquid enters the chamber uniformly, being absorbed by capillarity
7.-Let stand for 3 min to allow sedimentation of the erythrocytes
8.-Using a 40X objective, count the erythrocytes in central squares subdivided in 16 small
squares. Four of the squares placed in the corners and the central square (80 of the smallest
or basic squares). Check that the erythrocytes are evenly distributed. In case of the appea-
rance of bubbles, or that the glass cover has moved, repeat the operation
RESULTS
Erythrocyte count/mm3 = N X 200 X 10 X 400 = N X 10 000

80
N= number of erythrocytes counted in 80 of the smallest squares

200= Dilution factor
10= correction factor for depth of the chamber
NORMAL RANGE
Newborn babies 4.0-7.0 X 106 cells/dl

Children 4.0-7.0 X 106 cells/dl
Women 4.0-5.5 X 106 cells/dl
Men 4.0-6.0 X 106 cells/dl
REFERENCES
Henry, JB. (1979) “Clinical Diagnosis and management”, 16th ed., Saunders.
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MAY-GRÜNWALD
PRINCIPLE
The May-Grünwald stain belongs to the Romanowsky group of stains. Romanowsky stains consist of Methylene Blue and its oxidised derivatives, basic dyes and the acidic dye, Eosin.
Basic dyes bind to the acidic components of cells, nucleic acids, granules in neutrophils and acidic proteins which are stained a relatively deep red-purple colour, while eosin binds to
haemoglobin, basic components of cell structures and eosinophil granules.
The balance between Methylene Blue and its oxidised derivatives, and between these and Eosin, provides an essentially blue shade and a greater or lesser staining intensity, which are
characteristics of each type of stain - Giemsa, May-Grünwald or Wright. This stain may be used alone or combined with the Giemsa stain.
Its use allows the differential staining of blood cells. Some authors use the May-Grünwald stain or the May-Grünwald-Giemsa combination for the staining of parasites in blood. The result
of the staining may be influenced by several factors, such as the fixation, the staining time and the pH value of the staining solution and the buffer solution. If the pH is too basic the staining
will be more blue, and if the pH is too acidic, the staining will be more pink.

The reagent is used together with the Giemsa stain in the May-Grünwald/Giemsa
combined stain.
REAGENTS 1. Cover the air-dried and non-fixed smear with a known volume (approx. 2 mL)
May-Grünwald 1 x 500 mL Ref. 99 66 54 of May-Grünwald stain and leave to work for 3 minutes.
1 x 1,000 mL Ref. 99 97 18 2. Then, pour off the stain, tilting the slide, and, without rinsing, cover with
Giemsa recently diluted (1/10) with buffered water (pH 7.0 - 7.2) or Phosphate
Composition buffer pH 7.2 (previously diluted 1/10) before use.
Eosin-methylene blue according to May-Grünwald 2.7 g/L 3. Leave to work for 8-20 minutes depending on the desired staining intensity.
Methanol > 99% 4. Then rinse thoroughly with buffered water (pH 7.0-7.2) or diluted Phosphate
buffer pH 7.2 and leave to air dry.
5. Examine under the microscope.

Red blood cells: Pink or pink-orange.
Over time, a light precipitate may form in some reagents. This does not affect their Platelets: Pale violet or purple.
functionality. Neutrophils: Dark violet nucleus. Pink cytoplasm with red-violet granulations.
Eosinophils: Violet nucleus. Blue cytoplasm with red or red-orange granules.
Avoid excessive cold which may cause precipitation of the stain. If this occurs, the stain Basophils: Dark blue or purple nucleus. Purple, almost black, granules.
should be filtered before use. Longer staining times may be required in this case due to loss Lymphocytes: Purple nucleus. Sky blue cytoplasm.
of the stain by precipitation. Monocytes: Very pale violet nucleus. Sky blue cytoplasm.
MATERIAL REQUIRED (NOT SUPPLIED) NOTES

General-purpose laboratory material. The staining intensity is proportional to the staining time. The stain must be kept away from
Slides for blood smears. moisture in order to obtain optimal results.
Manual or automated staining system. Using buffered water instead of running tap water is recommended for the rinsing
Buffered water. processes. Running tap water has a varied pH and salt content, which, along with the high
Phosphate buffer pH 7.2 (Ref. 99 65 13) concentrations of chlorine, may alter the results of the staining and produce inconsistent
Giemsa (Ref. 99 84 40 or Ref. 99 09 39) results.
Microscope.
The results indicated should be treated as a guideline as the intensity and shade of the
colour could vary with the pH used in the staining.
PRECAUTIONS In general, staining with the May-Grünwald Giemsa reagent results in redder stains
The products are for professional use. All the reagents must be handled with care by trained when the pH of the buffered water used is reduced.
The stain is toxic and flammable due to its methanol content.

REFERENCES
Clark, G. (1981) “Staining Procedures”, 4th ed., Williams & Willkins.
Krafts Woronzoff-Dashkoff, Kristine, Clinics in Laboratory Medicine. Vol 22 (2002).
Biological Stains (1977) 9th ed. Ed. by R.D. Lillie; Williams & Willkins.
SAMPLE
Air-dried blood smears. It is recommended that they are thin and homogeneous in order to
obtain a better fixation of the dye without overstaining.
Staining should be carried out during the two hours following preparation in order to obtain
good results. Old smears may stain irregularly.
QUALITY CONTROL
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QUICK PANOPTIC
PRINCIPLE
The stains which make up the Quick Panoptic stain combine polychromy and the quality of classic haematology staining methods (May-Grünwald, Giemsa, Wright) with a very quick
execution time of just 15 seconds. The technique is performed by immersion in the staining solutions.
As with the other Romanowsky stains, the basic dyes bind to the acidic components of cells, nucleic acids, granules in neutrophils and acidic proteins which are stained a relatively deep
red-purple colour, while the acid dyes bind to haemoglobin, basic components of cell structures and eosinophil granules.
Its use allows the differential staining of blood cells. The result of the staining may be influenced by several factors, such as the fixation, the staining time and the pH value of the rinsing
water. If the pH is too basic the staining will be more blue, and if the pH is too acidic, the staining will be more pink.

For the differential staining of peripheral blood and bone marrow smears. It can also be used
for the staining of semen samples.
HAEMATOLOGY STAIN
REAGENTS 1. Leave to air dry.
Kit 3 x 500 mL Ref. 99 30 90 2. Set out stains No. 1, No. 2 and No. 3 in Wertheim (or similar) cuvettes.
Contains 3. Immerse the rack with the slides for 5 seconds (5 immersions lasting 1
A. Quick panoptic nr. 1 1 x 500 mL Ref. 99 16 81 second each) in staining solution No. 1 and drain.
B. Quick panoptic nr. 2 1 x 500 mL Ref. 99 42 39 4. Immerse for another 5 seconds (5 immersions lasting 1 second each) in
C. Quick panoptic nr. 3 1 x 500 mL Ref. 99 24 26 staining solution No. 2 and drain again.
5. Immerse for another 5 seconds (5 immersions lasting 1 second each) in
Also available: staining solution No. 3.
A. Quick panoptic nr. 1 6. Rinse with tap water and leave to dry.
1 x 500 mL Ref. 99 16 81
1 x 1,000 mL Ref. 99 85 66 SPERM CELLS’ STAIN
1 x 5,000 mL Ref. 99 26 12 8. Prepare a smear with 15 µl of fresh semen on a standard slide and leave
Composition: Methanol solution of triarylmethane dye to air dry for at least 10 minutes.
B. Quick panoptic nr. 2 9. Set out stains No. 1, No. 2 and No. 3 in Wertheim (or similar) cuvettes.
10. Immerse the rack with the slides for 5 seconds (5 immersions lasting 1
1 x 500 mL Ref. 99 42 39 second each) in staining solution No. 1 and drain.
1 x 1,000 mL Ref. 99 48 78 11. Immerse for another 5 seconds (5 immersions lasting 1 second each) in
1 x 5,000 mL Ref. 99 38 86 staining solution No. 2 and drain again.
Composition: Buffered aqueous solution of xanthene 12. Immerse for another 5 seconds (5 immersions lasting 1 second each) in
C. Quick panoptic nr. 3 staining solution No. 3.
13. Rinse with tap water and leave to dry.
1 x 500 mL Ref. 99 24 26 14. Examine under the microscope.
1 x 1,000 mL Ref. 99 00 91
1 x 5,000 mL Ref. 99 61 16
Composition: Buffered aqueous solution of dyes derived from thiazine.
RESULTS
Red blood cells: Pale or deep pink.
All the reagents are ready for use.
Platelets: Pale violet or purple.
Neutrophils: Dark blue nucleus. Pink cytoplasm with red-violet granulations.
Eosinophils: Blue nucleus. Blue cytoplasm with red or red-orange granules.
Basophils: Dark blue or purple nucleus. Purple, almost black, granules.
Lymphocytes: Violet nucleus. Sky blue cytoplasm.
Monocytes: Very pale violet nucleus. Sky blue cytoplasm.
functionality.
Head of sperm cell: Dark violet.
MATERIAL REQUIRED (NOT SUPPLIED) Acrosome of sperm cell: Violet, but a lighter shade of violet than the head.
General-purpose laboratory material. Mid-piece and tail of the sperm cell: Dark violet.
Slides for blood smears. Background: Pale pink.
Manual or automated staining system.
Microscope. NOTES
The staining intensity is proportional to the staining time. The stain must be kept away from
PRECAUTIONS moisture in order to obtain optimal results. The staining intensity can be varied by modifying
The products are for professional use. All the reagents must be handled with care by trained the number of immersions in stains No. 2 and No. 3, depending on whether it is wished to
technicians. The safety data sheet should be consulted before using the reagents. emphasise the pink or blue shades.
Stain No.1 is a methanol solution and is therefore flammable and toxic if ingested or inhaled. The cuvettes containing the stains, especially that of stain No. 1, should be kept covered,
Waste disposal must be carried out according to the local regulations in force. in order to prevent excessive evaporation, which could lead to colour deviations compared
to normal staining.
SAMPLE Before the content of the cuvette is depleted, add new quantities of the staining solution on
Air-dried blood smears. It is recommended that they are thin and homogeneous in order to a daily basis in order to maintain an adequate level. Renew all the content from time to time.
obtain a better fixation of the dye without overstaining. Using buffered water instead of running tap water is recommended for the rinsing
Staining should be carried out during the two hours following preparation in order to obtain processes. Running tap water has a varied pH and salt content, which, along with the high
good results. Old smears may stain irregularly. concentrations of chlorine, may alter the results of the staining and produce inconsistent
The ideal sample is capillary blood, but if venous blood is used then EDTA should be used results.
as an anticoagulant. The use of Heparin is not advised. Each user may apply the different versions of this procedure, both manual and automated,
Handle the samples with care due to their potentially infectious nature. adapting it to their standard method.
QUALITY CONTROL
REFERENCES
Gurr, E. (1965) “The rational use of dyes in Biology”, p. 115. Leonard Hill, London.
Gurr, E. (1971) “Synthetic dyes in Biology, Medicine and Chemistry”. Academic Press.
London & New York.
Maree, L.; du Plessis, S.S.; Menkvels, R. and van der Horst, G. Human Reproduction, 25
(6), 1369 – 1382 (2010).
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QUICK PANOPTIC CONCENTRATED (1:10)
PRINCIPLE
The stains which make up the Quick Panoptic stain combine polychromy and the quality of classic haematology staining methods (May-Grünwald, Giemsa, Wright) with a very quick
execution time of just 15 seconds. The technique is performed by immersion in the staining solutions.
As with the other Romanowsky stains, the basic dyes bind to the acidic components of cells, nucleic acids, granules in neutrophils and acidic proteins which are stained a relatively deep
red-purple colour, while the acid dyes bind to haemoglobin, basic components of cell structures and eosinophil granules.
Its use allows the differential staining of blood cells. The result of the staining may be influenced by several factors, such as the fixation, the staining time and the pH value of the rinsing
water. If the pH is too basic the staining will be more blue, and if the pH is too acidic, the staining will be more pink.

For the differential staining of peripheral blood and bone marrow smears. It can also be
used for the staining of semen samples.
HAEMATOLOGY STAIN
REAGENTS 1. Leave to air dry.
Kit 3 x 100 mL Ref. 99 03 45 2. Set out stains No. 1, No. 2 and No. 3 in Wertheim (or similar) cuvettes.
Contains: 3. Immerse the rack with the slides for 5 seconds (5 immersions lasting 1
A.- Reagent No. 1 1 x 10 mL Ref. 99 03 50 second each) in staining solution No. 1 and drain.
B.- Reagent No. 2 1 x 100 mL Ref. 99 03 52 4. Immerse for another 5 seconds (5 immersions lasting 1 second each) in
C.- Reagent No. 3 1 x 100 mL Ref. 99 03 54 staining solution No. 2 and drain again.
staining solution No. 3.
Reagent No. 1: Dilute the vial to 1,000 mL with methanol.
6. Rinse with tap water and leave to dry.
Reagent No. 2: Dilute the vial to 1,000 mL with deionised water.
Reagent No. 3: Dilute the vial to 1,000 mL with deionised water.
Once diluted to 1,000 mL, the 3 reagents are ready for use.
SPERM CELLS’ STAIN
REAGENT COMPOSITION 8. Prepare a smear with 15 µl of fresh semen on a standard slide and leave
A. Aqueous solution of hexamethyl-p-rosaniline to air dry for at least 10 minutes.
B. Buffered aqueous solution of xanthene. 9. Set out stains No. 1, No. 2 and No. 3 in Wertheim (or similar) cuvettes.
C. Buffered aqueous solution of thiazine 10. Immerse the rack with the slides for 5 seconds (5 immersions lasting 1
second each) in staining solution No. 1 and drain.
STORAGE AND STABILITY staining solution No. 2 and drain again.
Reagents which are stored at 15-30ºC and protected from light are stable until the expiry 12. Immerse for another 5 seconds (5 immersions lasting 1 second each) in
date stated on the label. Containers must always be kept tightly closed. Once diluted they staining solution No. 3.
are stable at room temperature (15 - 30ºC), for at least three months, if contamination and 13. Rinse with tap water and leave to dry.
excessive evaporation is prevented. 14. Examine under the microscope.
functionality.
General-purpose laboratory material. RESULTS
Slides for blood smears. Red blood cells: Pale or deep pink.
Manual or automated staining system. Platelets: Pale violet or purple.
Microscope. Neutrophils: Dark blue nucleus. Pink cytoplasm with red-violet granulations.
Eosinophils: Blue nucleus. Blue cytoplasm with red or red-orange granules.
PRECAUTIONS Basophils: Dark blue or purple nucleus. Purple, almost black, granules.
The products are for professional use. All the reagents must be handled with care by trained Lymphocytes: Violet nucleus. Sky blue cytoplasm.
technicians. The safety data sheet should be consulted before using the reagents. Monocytes: Very pale violet nucleus. Sky blue cytoplasm.
SAMPLE Head of sperm cell: Dark violet.
Air-dried blood smears. It is recommended that they are thin and homogeneous in order to Acrosome of sperm cell: Violet, but a lighter shade of violet than the head.
obtain a better fixation of the dye without overstaining. Mid-piece and tail of the sperm cell: Dark violet.
Staining should be carried out during the two hours following preparation in order to obtain Background: Pale pink.
The ideal sample is capillary blood, but if venous blood is used then EDTA should be used NOTES
as an anticoagulant. The use of Heparin is not advised. The staining intensity is proportional to the staining time. The stain must be kept away from
Handle the samples with care due to their potentially infectious nature. moisture in order to obtain optimal results. The staining intensity can be varied by modifying
the number of immersions in stains No. 2 and No. 3, depending on whether it is wished to
QUALITY CONTROL emphasise the pink or blue shades.
Following the quality control practices defined by the CLSI (formerly NCCLS) is The cuvettes containing the stains, especially that of stain No. 1, should be kept covered,
recommended. The staining method indicated is the one which is used routinely in our in order to prevent excessive evaporation, which could lead to colour deviations compared
laboratories. to normal staining.
Before the content of the cuvette is depleted, add new quantities of the staining solution on
a daily basis in order to maintain an adequate level. Renew all the content from time to time.
Using buffered water instead of running tap water is recommended for the rinsing
processes. Running tap water has a varied pH and salt content, which, along with the high
concentrations of chlorine, may alter the results of the staining and produce inconsistent
results.
Each user may apply the different versions of this procedure, both manual and automated,
REFERENCES
Gurr, E. (1965) “The rational use of dyes in Biology”, p. 115. Leonard Hill, London.
Gurr, E. (1971) “Synthetic dyes in Biology, Medicine and Chemistry”. Academic Press.
London & New York.
Maree, L.; du Plessis, S.S.; Menkvels, R. and van der Horst, G. Human Reproduction, 25
(6), 1369 – 1382 (2010).
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TÜRK
For in vitro diagnostics
PRINCIPLE
Türk’s reagent is an hypotonic haematology solution composed by acetic acid and a dye. The
erythrocytes are hemolyzed by the acetic acid and the leukocytes are stained by the dye.
DIAGNOSTIC USE
The regular blood count forms the basis of haematologic diagnostics. Türk’s reagent is used
for diluting blood samples in white cell counts.
REAGENT
Türk 1 x 500 mL Ref. 99 69 72
Composition
Methylene Blue < 0,1%
Acetic Acid 1%

The reagent stored at 15-30ºC will remain stable until the expiration date stated on the label.
The bottles must be kept tightly closed.
Neubauer, Bürker or Fuchs-Rosenthal counting chamber, microscope and calibrated leuko-
cytes pipette (white Thoma pipette).
CAUTION
Professional use only. HandLe with care. The safety statements are on the label. It is advis-
able to look at the SDS before using the reagent.
SAMPLE
Venous blood with EDTA.
Effective measures must be taken to protect against infection.
PROCEDURE
1.-Fill the white Thoma pipette up to the 0.5 mark
2.-Clean
3.-Then fill with Türk’s reagent up to the 11 mark. A blood dilution of 1:20 is obtained. Micro-
pipettes can also be used
4.-Shake the filled pipette for 3 min. until complet mixing
5.-Put the glass cover on the counting chamber central area
6.-Discard the first 4 or 5 drops and fill the counting chamber. Release the reagent slowly
watching how the liquid enters the chamber uniformly, being absorbed by capillarity
7.-Let stand for 3 min to allow sedimentation of the leukocytes
8.-Using a 10X objective, count the leukocytes in in the 4 large corner squares. Check that
the leukocytes are evenly distributed. In case of the appearance of bubbles, or that the glass
cover has moved, repeat the operation
RESULTS
N X 10 X 20
number of leukocytes/mm3 = = N X 50
4
N= number of leukocytes counted in 4 of the large corner squares

20= Dilution factor
10= correction factor for depth of the chamber
NORMAL RANGE
Newborn babies 10.000-30.000 cells/dL

Infants 7.000-17.000 cells/dL
Children 5.000-15.000 cells/dL
Adults 4.000-9.000 cells/dL
REFERENCES
Henry, JB. (1979) “Clinical Diagnosis and management”, 16th ed., Saunders.
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WRIGHT
PRINCIPLE
The Wright’s stain belongs to the Romanowsky group of stains. It is named after the scientist who developed it, James Homer Wright. He created it in 1902 by modifying the Romanowsky
stain. Romanowsky stains consist of methylene blue and its oxidised derivatives, basic dyes and the acidic dye, eosin. Basic dyes bind to the acidic components of cells, nucleic acids,
granules in neutrophils and acidic proteins which are stained a relatively deep red-purple colour, while eosin binds to haemoglobin, basic components of cell structures and eosinophil
granules.
The balance between methylene blue and its oxidised derivatives, and between these and eosin, provides an essentially blue shade and a greater or lesser staining intensity, which are
characteristics of each type of stain - Giemsa, May-Grünwald or Wright.
Its use allows the differential staining of blood cells. The result of the staining may be influenced by several factors, such as the fixation, the staining time and the pH value of the staining
solution and the buffer solution. If the pH is too basic the staining will be more blue, and if the pH is too acidic, the staining will be more pink.

1. Cover the air-dried and non-fixed smear with a known volume (approx. 1 mL) of
REAGENTS stain, and leave to work for 5 minutes.
Wright 1 x 500 mL Ref. 99 79 39 2. Then add an equal volume of phosphate buffer or buffered water (pH 6.8 or 7.2)
1 x 1,000 mL Ref. 99 79 79 drop by drop, swirling lightly with the pipette to obtain a homogeneous mixture.
3. After 5 minutes, pour off the stain, rinse with phosphate buffer or buffered water
Composition (pH 6.8 or 7.2) and leave to air dry.
Eosin-methylene blue according to Wright 2.5 g/l 4. Examine under the microscope.
Methanol min. 99.9%
Preparation of the working reagent:

Ready for use. RESULTS
Red blood cells: Pink or pink-orange.
Platelets: Pale violet or purple.
STORAGE AND STABILITY Neutrophils: Dark violet nucleus. Pink cytoplasm with red-violet granulations.
Reagents which are stored at 15-30ºC and protected from light are stable until the expiry Eosinophils: Violet nucleus. Blue cytoplasm with red or red-orange granules.
date stated on the label. Containers must always be kept tightly closed. Basophils: Dark blue or purple nucleus. Purple, almost black, granules.
Lymphocytes: Purple nucleus. Sky blue cytoplasm.
Over time, a light precipitate may form in some reagents. This does not affect their Monocytes: Very pale violet nucleus. Sky blue cytoplasm.
functionality.
Avoid excessive cold which may cause precipitation of the stain. If precipitation has NOTES
occurred, it is recommended that the stain is filtered before use. Longer staining times may The staining intensity is proportional to the staining time. The stain must be kept away from
be required in this case due to loss of the stain by precipitation. moisture in order to obtain optimal results.
Using buffered water instead of running tap water is recommended for the rinsing
processes. Running tap water has a varied pH and salt content, which, along with the high
MATERIAL REQUIRED (NOT SUPPLIED) concentrations of chlorine, may alter the results of the staining and produce inconsistent
General-purpose laboratory material. results.
Slides for blood smears.
Manual or automated staining system. The results indicated should be treated as a guideline as the intensity and shade of the
Phosphate buffer or buffered water. colour could vary with the pH used in the staining.
Microscope. In general, staining with the Wright reagent results in redder stains when the pH is reduced.
PRECAUTIONS
The products are for professional use. All the reagents must be handled with care by trained REFERENCES
technicians. The safety data sheet should be consulted before using the reagents. Clark, G. (1981) “Staining Procedures”, 4th ed., Williams & Willkins.
Krafts Woronzoff-Dashkoff, Kristine, Clinics in Laboratory Medicine. Vol 22 (2002).
The stain is toxic and flammable due to its methanol content. Biological Stains (1977) 9th ed. Ed by R.D. Lillie; Williams & Willkins.
SAMPLE
Air-dried blood smears. It is recommended that they are thin and homogeneous in order to
QUALITY CONTROL
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EOSIN-NIGROSIN
PRINCIPLE
The study of the morphology, mobility, concentration and integrity of the spermatozoon membrane is an important component in the evaluation of semen quality. Fertility is reduced when
the percentage of abnormal cells increases.
The membranes of dead spermatozoa, or ones that are in the process of dying, are permeable to the stains. For this reason, this property is used to determine live spermatozoa. Given
that immobile spermatozoa may be live, only supravital staining, such as that of eosin-nigrosin, may establish this difference.
Eosin penetrates through the membrane of the dead spermatozoa staining them pink, while the live spermatozoa remain colourless. Nigrosin, on the other hand, outlines the profile of the
live spermatozoa in dark blue.

Determination of live spermatozoa and their morphology in sperm samples.
1. Once the semen has liquefied (at least 30 minutes after ejaculation) mix
REAGENTS two drops of sperm with two drops of Eosin solution in a haemolysis tube.
Kit 2 x 10 mL Ref. 99 65 18 2. After 30 seconds, add two drops of Nigrosin solution. Shake gently.
3. Place a drop of the above mixture on the slide and cover.
Contains: 4. Examine under the microscope counting the number of dead spermatozoa
compared to the number of live spermatozoa.
A. 1 x 10 mL Eosin solution Ref. 99 65 20
B. 1 x 10 mL Nigrosin solution Ref. 99 65 25

All the reagents are ready for use.
RESULTS
Live spermatozoa: white
Dead spermatozoa: pink
date stated on the label. Containers must always be kept tightly closed. NOTES
Over time, a light precipitate may form in some reagents. This does not affect their The smears must be kept in a dry place. Moisture may cause deterioration of the tests.
functionality.
The stains must be kept at approximately 30°C (thermal plate or oven) during use, to
prevent thermal shock producing erroneous results.
MATERIAL REQUIRED (NOT SUPPLIED) Each user may apply the different versions of this procedure, adapting it to their standard
General-purpose laboratory material. method.
Slides for semen smears
Heat source
Microscope.
REFERENCES
Heinke, E. (1951). Z. Haut. u. Geschl. Kr., 10, 254 – 255.
PRECAUTIONS
SAMPLE
Semen smears.
QUALITY CONTROL
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ALCIAN BLUE
PRINCIPLE
Alcian blue is a basic stain composed of a series of tetramethyl-thiouronium isomers. It was synthesised for the first time in the 1940s for industrial use, and in 1950 Steedman used it for
the first time as a selective stain for mucins. It is used mainly to detect carbohydrates in histochemical processes in animal tissues, and for the staining of primary cell walls in plants. The
blue colour is due to copper in the molecule.
Acid mucopolysaccharides are polysaccharides which do not have alcoholic groups in the 1,2 glycol position, but instead have carboxylic or sulphuric acid groups. It has been proven
that alcian blue stains sulphated and carboxylated acid mucopolysaccharides in pH conditions of approximately 2.4 to 2.6. At a very low pH, of approximately 1, only the sulphated
mucopolysaccharides are stained. The carboxylated mucopolysaccharides do not stain. The staining selectivity increases if the pH is reduced to 0.5, when only the strongly sulphated
mucopolysaccharides are stained.
The mucin in a tumour can be used to assess its malignancy. Carcinomas derived from epithelial tissue generally contain mucins, while melanomas, lymphomas and sarcomas rarely
contain high levels of these. The detection of sulfomucins in gastric mucosa may help in the detection and characterisation of intestinal metaplasia, a lesion associated with gastric cancer.

For the detection of acid mucopolysaccharides in the histological examination of human
samples.
1. Dewax the sections and rehydrate them in alcohols with a decreasing
REAGENTS alcohol content, according to the standard method
Alcian blue 1 x 250 mL Ref. 99 32 85 2. Rinse with distilled water for 1 minute after the last immersion in Ethanol
3. Immerse in Alcian blue for 5 minutes
4. Rinse with running tap water for 3 minutes
Composition
5. Rinse with distilled water
Aqueous solution of:
6. Stain with Nuclear Fast Red for 10 minutes
Alcian blue stain 1.0%
7. Rinse with running tap water for 3 minutes
Acetic Acid 3.0%
9. Dehydrate with the series of alcohols with an increasing alcohol content
10. Immerse in xylol
11. Mount the preparation
The stain is ready for use.

Acid mucins / mucosubstances: Blue
Over time, a light precipitate may form in some reagents. This does not affect their Nuclei: Red-pink
functionality. The stain should be filtered before use.
Indications of reagent deterioration NOTES

Abundant precipitate The staining intensity is proportional to the staining time. The stain must be kept away from
moisture in order to obtain optimal results.
pH ≥ 4.0
The indicated method, which is normally used in our laboratory, is simply a guide. The stains
may be used in the different variants of the technique, both manual and automated, used
in different laboratories.
General-purpose pathological anatomy laboratory material
Dye for staining nuclei: Using the Nuclear Fast Red dye is advised
Different concentrations of Ethanol
REFERENCES
Xylene or Xylene Substitute
Theory and Practice of Histological techniques, Ed. by J. D. Bancroft and M. Gamble 6th
Mounting medium
Ed. (2008). Churchill Livingstone
Microscope
Viguer, J.M. y Garcia del Moral, R. Laboratorio y Atlas de Citología [Laboratory and
Cytology Atlas]
McGraw-Hill/Interamericana (1995)
PRECAUTIONS
SAMPLE
Histological sections fixed in formalin and treated with paraffin or cell smears. Thickness of
the paraffin sections: between 3 and 5 µm.
Samples must be handled in accordance with the established protocols in each laboratory
for the preparation of samples for staining with the Alcian blue stain.
QUALITY CONTROL
recommended. The staining method indicated is the one used routinely in our laboratories.
Suitable controls must be performed for each set of stains to avoid inaccurate results. Using
samples from the colon or small intestine is recommended.
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MASSON’S TRICHROME STAINING
PRINCIPLE
Trichrome techniques enable the differential staining of collagen, muscle fibres, fibrin and red blood cells. Three different stains are used which selectively stain the different cell structures,
nucleus, cytoplasm and collagen fibres. First, the samples are stained with an acid dye, such as Biebrich scarlet. All the acidophilus elements of the tissue (cytoplasm, muscle and collagen)
will bind to the acid dye. Then the samples are treated with phosphotungstic and/or phosphomolybdic acid. As cytoplasm is much less permeable than collagen, phosphotungstic and
phosphomolybdic acids enable Biebrich scarlet to disseminate from the collagen but not from the cytoplasm. Phosphotungstic and phosphomolybdic acids have numerous acid groups
which probably act as a means of binding the discoloured collagen and the aniline blue, which is the collagen stain. The pH of the phosphotungstic/phosphomolybdic solution probably also
increases staining, aiding the diffusion of the dye through the collagen.
Trichrome stains make it possible to differentiate between collagen and smooth muscle in tumours, and to identify increases in collagenous tissues in diseases such as cirrhosis of the liver.
DIAGNOSTIC USE QUALITY CONTROL

For the detection of collagen fibres and connective tissue in general in tissue samples. Following the quality control practices defined by the CLSI (formerly NCCLS) is
REAGENTS
Kit 5 x 250 mL Ref. 99 57 00
PROCEDURE
Reagent A. Weigert’s Haematoxylin A 1 x 250 mL Ref. 99 03 42
Composition: 1. Rehydrate the sample by rinsing with absolute alcohol, alcohol 95° and
Haematoxylin in alcohol 70°
Ethanol 1.0% 2. Rinse with distilled water
3. Stain with the Haematoxylin working solution for 10 minutes
Reagent B. Weigert’s Haematoxylin B 1 x 250 mL Ref. 99 03 56 4. Rinse with running tap water and then with distilled water
5. Stain with Reagent C, Acid fuchsin, for 10–15 minutes
Composition: 6. Rinse with distilled water
Ferric chloride 2.0% 7. Differentiate with Reagent D, phosphomolybdic acid, for 10–15 minutes, or
Hydrochloric Acid 1.0% until the collagen has lost its colour
8. Without rinsing, stain the samples with Reagent E, methyl blue, for 5–10
Reagent C. Acid fuchsin 1 x 250 mL Ref. 99 03 63 minutes
Composition: 10. Differentiate with 1% acetic acid for 2–5 minutes
Acid fuchsin 0.5% 11. Rinse with distilled water
Acetic Acid 0.5% 12. Dehydrate quickly with alcohol (95% and absolute); rinse with xylene and
mount the preparation
Reagent D. Phosphomolybdic Acid 2 x 250 mL Ref. 99 03 81 13. Examine under the microscope
Composition:
Phosphomolybdic Acid 1.0%
RESULTS:
Reagent E. Methyl blue 1 x 250 mL Ref. 99 03 92 Collagen: Blue
Nuclei: Black
Composition: Cytoplasm, muscle and keratin: Pinkish-red
Methyl blue 2.0%
Acetic Acid 2.5%
NOTES
Mix reagent A (Weigert’s Haematoxylin A) and reagent B (Weigert’s Haematoxylin B) in
Although any fixative is valid, some authors advise using the Bouin’s fixative in order to
equal parts, just before use.
obtain optimal results. If the tissue has not been fixed with Bouin, immersing the samples
in Bouin solution, which acts as an etchant, for 24 hours at room temperature or for 1 hour
at 56-60°C is recommended.
PRESERVATION AND STABILITY The step involving alcohol should only last a few seconds, always checking that the parts
Reagents which are stored at 15-30°C and protected from light are stable until the expiry stained with scarlet-acid fuchsin solution turn appropriately to a pinker and lighter colour, but
date stated on the label. Containers must always be kept tightly closed. should not last too long as it would discolour the parts stained with reagent E too much. It is
essential to carry out this step properly in order to obtain a good contrast.
functionality.
REFERENCES
Theory and Practice of Histological Techniques., ed. by Bancroft, J.D.; Gamble, M. Churchill
General-purpose anatomical pathology laboratory material.
Livingstone, (2008), pg 135 -150
Different concentrations of Ethanol.
Mounting medium
Microscope
PRECAUTIONS
SAMPLE
Histological samples.
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MYELOPEROXIDASE
PRINCIPLE
Myeloperoxidase is an enzyme which is widely distributed in the body and its main sources are found in leukocytes (neutrophils and monocytes) and macrophages. This enzyme combines
in vivo with hydrogen peroxide, and is transformed into a powerful bactericidal, virucidal and fungicidal agent. The cytochemical demonstration of this enzyme is based on its reaction with
pyronin in the presence of alpha-naphthol and hydrogen peroxide, producing a bright red compound which remains in the cell after rinsing with water. Mayer’s haematoxylin is used to
stain the nuclei.
Various pathological processes are linked to an increase in myeloperoxidase activity, as is the case in infectious diseases, inflammatory diseases, such as rheumatoid arthritis, and
ischaemia.
It is used in haematology for diagnosing leukaemia, as it enables myeloid and lymphoid cells to be differentiated. A deficiency of myeloperoxidase in mature polymorphonuclear cells, except
in cases of congenital deficiency, should be interpreted as a sign of dysgranulopoiesis.

For the differential staining of myeloperoxidase granulations in peripheral blood and bone Following the quality control practices defined by the CLSI (formerly NCCLS) is
marrow smears. recommended. For this purpose, carry out controls with ATCC microorganisms, or known
REAGENTS
Kit 330 mL Ref. 99 38 16
PROCEDURE
Reagent A . Fixative solution 1 x 100 mL Ref. 99 08 16
Composition
Formalin (37% - 40%) 10% 1. Fix the smear in reagent A for 1 minute
Ethanol 90% 2. Rinse with tap water and leave to air dry
3. Stain with the working reagent for 3 minutes
Reagent B. Alpha-Naphtol 1 x 25 mL Ref. 99 08 17 4. Rinse with tap water and leave to air dry
Composition 5. Stain with reagent E, Mayer’s Haematoxylin, for 3 minutes
Alcoholic solution of 6. Rinse with tap water for 3 minutes and leave to air dry
Alpha-Naphtol 0.5% 7. Examine under the microscope
Reagent C. Pyronin Y 1 x 100 mL Ref. 99 08 18

Composition RESULTS:
Tris buffer pH 7.5 20 mM Peroxidase granulations: bright red
NaCl 500 mM Leukocyte nuclei: blue
Pyronin Y 0.15% Red blood cells: light beige
Reagent D. Hydrogen peroxide 1 x 5 mL Ref. 99 08 19 NOTES

Reagent E. Mayer’s Haematoxylin 1 x 100 mL Ref. 99 08 20 adapting it to their standard method.
Composition If running tap water is used for the rinsing, please be aware that strongly chlorinated water
Haematoxylin C.I. no. 75 290 0.5% may weaken the staining.
Aluminium Ammonium Sulphate 5%
Sodium iodate 0.02%
Glycerol 30% REFERENCES
PREPARATION OF THE WORKING REAGENT: Raimundo García de Moral. Laboratorio de Anatomía Patológica. (1993) McGraw-Hill /
Mix, just before use, the required quantity in the proportion: 1 mL of reagent B + 4 mL of Interamericana de España SAU
reagent C + one drop (50 µl) of reagent D.
PRESERVATION AND STABILITY

Reagents which are stored at 15-30°C and protected from light are stable until the expiry
functionality.

Microscope.
PRECAUTIONS
SAMPLE
Air-dried blood smears. It is recommended that they are thin and homogenous in order to
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PERLS’ STAIN
PRINCIPLE
The detection of iron using the Perls’ reaction was described in 1867. It is based on releasing ferric ions from their bond with proteins using hydrochloric acid, which produces a greenish
blue precipitate of ferric ferrocyanide when it reacts with potassium ferrocyanide. This reaction only detects deposits of ferric iron, which are insoluble in water (haemosiderin), and not the
hydrosoluble ferritin.
The study of medullary iron deposited as haemosiderin in macrophages and in erythroblasts has great value in the analysis of anaemia, if this is complemented with the determination of
iron, the fixation capacity of transferrin and the saturation index.
The dye, Nuclear Fast Red, is used to stain the nuclei.

For staining free iron (Fe(III)) in cells, and in blood, bone marrow and tissue samples.
1. Fix the smear in methanol. In the case of histological sections, these

REAGENTS should be dewaxed and rehydrated with the descending alcohol series.
Kit 200 mL Ref. 99 67 68 2. Stain the samples in the working reagent: 5-10 minutes for the smears (20
minutes for bone marrow) and 30 minutes for the histological sections.
Reagent A. Potassium Ferrocyanide 1 x 50 mL Ref. 99 07 68 3. Rinse with running tap water for 5 minutes
4. Rinse with deionised water
Composition 5. Stain with Reagent C, Nuclear Fast Red, for 5 minutes
Aqueous solution of 6. Rinse with running tap water for 10 minutes
Potassium Ferrocyanide 4.5% 7. Rinse with deionised water
8. In the case of histological sections, these should be dehydrated with a
series of increasing alcohol concentrations. Rinse in xylene.
Reagent B. Hydrochloric acid 4.5% 1 x 50 mL Ref. 99 07 69 9. Mount the preparation
10. Examine under the microscope
Reagent C. Nuclear Fast Red 1 x 100 mL Ref. 99 07 70
Composition RESULTS:
Aqueous solution of Iron deposits: blue
Nuclear Fast Red 0.1% Nuclei: red
Background: pink

Mix reagent A (Potassium Ferrocyanide) and reagent B (hydrochloric acid) in equal parts,
just before use. NOTES
PRESERVATION AND STABILITY If running tap water is used for the rinsing, please be aware that strongly chlorinated water
Reagents which are stored at 15-30°C and protected from light are stable until the expiry may weaken the staining.
Over time, a light precipitate may form in some reagents. This does not affect their REFERENCES
functionality. CLSI Guidelines and Standards, CLSI, Wayne, P.A
Theory and Practice of Histological Techniques. 6th Edition (2008); pg 235-236,
Churchill Livingstone.
MATERIAL REQUIRED (NOT SUPPLIED) Ed by J.D. Brancroft and M. Gamble
Microscope.
PRECAUTIONS
SAMPLE
Air-dried blood smears. Histological sections. It is recommended that they are thin and
homogenous in order to obtain a better fixation of the dye without overstaining.
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SHORR’S STAIN
PRINCIPLE
It is a staining technique which makes it possible to obtain excellent results in the hormonal evaluation of vaginal cytology. Sex hormones cause characteristic changes in the vaginal
epithelium during the course of the menstrual cycle.
The Shorr’s stain makes it possible to distinguish keratinised cells (eosinophils) from intermediate and parabasal cells (basophils). The ratio of eosinophils to basophils makes it possible
to evaluate the effects of follicle-stimulating hormone and the corpus luteum. Keratinised eosinophils increase under the hormonal action of follicle-stimulating hormone, while the non-
keratinised cells increase under the action of the corpus luteum.
The nuclei are stained using Haematoxylin.
It also makes it possible to clearly distinguish blood cells present (leukocytes and red blood cells), and bacteria or sperm cells.

For the hormonal evaluation of vaginal cytology.
REAGENTS 1. Fix the preparation in 96% ethanol or in 50% alcohol-ether.

Shorr’s stain 1 x 500 mL Ref. 99 05 80 2. Hydrate in alcohols with a decreasing alcohol content.
3. Rinse with distilled water for 1 minute after the last immersion in ethanol.
Composition 4. Immerse in Harris’s Haematoxylin for 1 minute.
50% Ethanol solution of 5. Rinse with water.
Biebrich Scarlet 0.5% 6. Rinse in 70% ethanol.
Orange G 0.25% 7. Rinse with 96% ethanol.
Fast Green FCF 0.75% 8. Stain with the Shorr’s stain for 1 minute.
Phosphotungstic Acid 0.5% 9. Dehydrate with 96% ethanol and absolute ethanol.
Phosphomolybdic Acid 0.5% 10. Immerse in xylol.
Acetic Acid 1.0% 11. Mount the preparation

RESULTS
Cytoplasm of keratinised cells: bright orange
STORAGE AND STABILITY Cytoplasm of non-keratinised cells: greenish blue
Reagents which are stored at 15-30ºC and protected from light are stable until the expiry Nuclei: blue-violet
date stated on the label. Containers must always be kept tightly closed. Bacteria: dark blue
NOTES
The staining intensity is proportional to the staining time. The stain must be kept away from
MATERIAL REQUIRED (NOT SUPPLIED) moisture in order to obtain optimal results.
General-purpose pathological anatomy laboratory material.
Dye for staining nuclei: Using the Harris’s haematoxylin stain is advised, (References: 1 x The indicated method, which is normally used in our laboratory, is simply a guide. The stains
500 mL 99 22 32 or 1 x 1,000 mL 99 59 60) may be used in the different variants of the technique, both manual and automated, used
Different concentrations of ethanol. in different laboratories.
Mounting medium
Microscope REFERENCES
Boon, M.E., Drijver, J.S., Routine Cytological staining techniques, Macmillan Education Ltd,
1st. ed (1986).
PRECAUTIONS Viguer, J.M. y Garcia del Moral, R. Laboratorio y Atlas de Citología [Laboratory and Cytology
The products are for professional use. All the reagents must be handled with care by trained Atlas]
technicians. The safety data sheet should be consulted before using the reagents. McGraw-Hill/Interamericana (1995)
SAMPLE
Gynaecological cytology samples.
Handling of the samples must be carried out in accordance with the established protocols in
each laboratory for the preparation of samples for staining with the Shorr’s stain.
QUALITY CONTROL
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SUDAN BLACK
PRINCIPLE
It is based on the ability of Sudan Black dye for the specific staining of lipid substances, both neutral fats, such as phospholipids, and sterols, inside cells. The dye is insoluble in a cellular
aqueous medium, but becomes soluble in the lipid medium of certain granules, which become stained a dark green, almost black, colour.
The black lipid granules appear on all granulocytes, intensifying as they change from myeloblasts to mature granulocytes. Lipid granules are also present in Auer bodies and in monocytes,
in which they appear like a thin cytoplasmic layer. Lymphocytes, lymphoblasts and erythroblasts are negative to Sudan Black.
It is used in haematology for diagnosing leukaemia, as it enables differentiation between monocytic and granulocytic leukaemia.

For staining lipids in blood, bone marrow and tissue samples.
1. Fix the smear in air In the case of histological sections, these should be
REAGENTS dehydrated beforehand with propylene glycol for 2–3 minutes
Kit 2 x 100 mL Ref. 99 45 02 2. Stain the tissue sections for 5–7 minutes and the smears for up to 10
minutes
Reagent A. Sudan Black, 1 x 100 mL Ref. 99 02 88 3. Differentiate with propylene glycol or xylene for 2–3 minutes
4. Leave to air dry
Composition: 5. Rinse with tap water for 3–5 minutes for histological sections and for about
Sudan Black dye in Ethanol 0.36% 30 seconds for smears and leave to air dry
6. Examine under the microscope
Reagent B. Buffered phenol 1 x 100 mL Ref. 99 03 07
Composition
Phenol 9.8%
Ethanol 40% RESULTS:
Disodium phosphate 1.5 % Lipid compounds: Dark green-black staining
Cytoplasm: not stained

Mix reagent A (Sudan Black) and reagent B (buffered phenol) in equal parts, just before use. NOTES
If running tap water is used for the rinsing, please be aware that strongly chlorinated water
PRESERVATION AND STABILITY may weaken the staining.
Reagents which are stored at 15-30°C and protected from light are stable until the expiry
REFERENCES
Over time, a light precipitate may form in some reagents. This does not affect their CLSI Guidelines and Standards, CLSI, Wayne, P.A
functionality. Clark, G., Staining Procedures, Williams and Willkins, 1981, 4th Ed., 193-194

Microscope.
PRECAUTIONS
SAMPLE
Air-dried blood smears. Histological sections. It is recommended that they are thin and
homogenous in order to obtain a better fixation of the dye without overstaining.
QUALITY CONTROL
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HAEMATOXYLIN FOR HISTOCYTOLOGY
PRINCIPLE
The Papanicolaou test, which owes its name to Dr Georgios Papanicolaou, a pioneer in cytology and cancer, is performed to diagnose cervical cancer or cancer in other organs. It makes it
possible to detect changes in cells which may be cancer precursors. The first step in this technique is the staining of nuclei with haematoxylin. The second step is the staining of cytoplasm
with an orange solution, which stains the mature cells and the keratinised cells with different intensities. The so-called polychrome solution, which is a mixture of eosin, light green SF
and Bismarck brown, is used in the third step. The differentiation of simple squamous epithelium is shown with the polychrome solution. The Haematoxylin and Eosin stain is the most
commonly used stain in histology.
Haematoxylin stains are basically formed from metallic complexes of oxidised haematoxylin with divalent and trivalent ions. The mechanism of staining is carried out between the covalent
bonds of the metal-haematoxylin compound and the anionic radicals of the tissue. The anionic groups of phosphoric acid in DNA will intervene if there is nuclear material in the reaction.
The selectivity of the stain increases with the acid medium of the dye, since it hinders the staining of amphoteric elements, such as the amino acids of proteins. The nuclei appear blue to
dark violet.
Progressive and regressive staining can be performed with haematoxylin. The structures of the nuclei show greater differentiation in regressive staining and are more easily seen.

For the staining of nuclei in histocytology. PAPANICOLAOU STAIN
1. The smears which have been spray-fixed should be immersed in water for
REAGENTS 5-10 minutes so that the carboxymethyl cellulose (CMC) that they contain is
Harris’s Haematoxylin completely dissolved
1 x 500 mL Ref. 99 22 32 2. Immerse in Harris’s Haematoxylin for 1 minute
1 x 1,000 mL Ref. 99 59 60 3. Rinse with water for 15 seconds
1 x 5,000 mL Ref. 99 12 97 4. Rinse with ethanol-HCl (0.25%) for 10 seconds
Composition 5. Rinse with water for 15 seconds
Haematoxylin C.I. No. 75 290 0.5% 6. Rinse with ammonia water (0.05% in NH3) for 5 seconds
Aluminium Sulphate 6% 7. Rinse with water for 15 seconds
Ethylene glycol 15% 8. Immerse in ethanol 96º for 10 seconds (twice, using a different container
each time)
Carazzi’s Haematoxylin 9. Immerse in OG-6 stain for 3 minutes
1 x 1,000 mL Ref. 99 16 43 10. Rinse in ethanol 96º for 15 seconds (twice, using a different container each
1 x 5,000 mL Ref. 99 41 75 time)
Composition 11. Immerse in EA 50, EA 36 or EA 65 stain for 3 minutes
Haematoxylin C.I. No. 75 290 0.1% 12. Rinse in ethanol 96º for 15 seconds (twice, using a different container each
Aluminium/Potassium Sulphate 6% time)
Glycerin 20% 13. Immerse in absolute ethanol for 15 seconds (twice, using a different container
each time)
14. Immerse in ethanol/xylol (50%) for 15 seconds
Mayer’s Haematoxylin
15. Immerse in xylol for 15 seconds
1 x 1,000 mL Ref. 99 77 50 16. Immerse in xylol for 2 minutes. Mount the preparation
1 x 5,000 mL Ref. 99 06 38
HAEMATOXYLIN AND EOSIN STAIN
Composition
1. Dewax the preparation
Haematoxylin C.I. No. 75 290 0.5%
2. Rinse with distilled water for 1 minute after the last immersion in ethanol
Aluminium Ammonium Sulphate 5%
3. Immerse in Haematoxylin for 8 minutes
Glycerin 30%
4. Rinse with water for 1 minute
5. Rinse with ethanol-HCl (0.5%) for 13 seconds
6. Rinse with water for 15 seconds
7. Rinse with ammonia water (0.05% in NH3) for 30 seconds
9. Immerse in Eosin 1% for 30 seconds
10. Immerse in ethanol 96º for 6 seconds (twice, using a different container each
time)
11. Immerse in absolute ethanol for 6 seconds (three times, using a different
container each time)
12. Immerse in xylol/eucalyptol for 6 seconds (twice, using a different container
each time)
General-purpose pathological anatomy laboratory material.
Dye for staining cytoplasm: EA 50, EA 36 or EA 65 stains, Orange G-6 stain, Alcoholic or
aqueous solution of eosin at 1% MAYER’S HAEMATOXYLIN AND EOSIN STAIN
Different concentrations of ethanol. 1. Dewax the preparation
Xylene or Xylene Substitute 2. Rinse with distilled water for 5 minutes after the last immersion in ethanol
Mounting medium 3. Immerse in Mayer’s Haematoxylin for 10 minutes
Microscope 4. Rinse with water for 5 minutes
5. Immerse in alcoholic Eosin 1% for 2 minutes
PRECAUTIONS 6. Immerse in ethanol 96º for 10-15 seconds (twice, using a different container
The products are for professional use. All the reagents must be handled with care by trained each time)
technicians. The safety data sheet should be consulted before using the reagents. 7. Immerse in absolute ethanol for 10-15 seconds (twice, using a different
Waste disposal must be carried out according to the local regulations in force. container each time)
8. Immerse in xylol (three times, using a different container each time)
SAMPLE 9. Mount the preparation
Cytological samples of different origins: gynaecological, sputum, liquid biopsies.
Histological samples. RESULTS
Handling of the samples must be carried out in accordance with the established protocols in Nuclei: blue-violet
each laboratory for the preparation of samples for staining with the Papanicolaou method or Acidophilic cytoplasm: reddish
with the Haematoxylin and Eosin stain. Basophilic cytoplasm: blue
Handle the samples with care due to their potentially infectious nature. Keratinised cytoplasm: orange
QUALITY CONTROL NOTES

Following the quality control practices defined by the CLSI (formerly NCCLS) is The staining intensity is proportional to the staining time. The stain must be kept away from
recommended. moisture in order to obtain optimal results.Each user may apply the different versions of this
procedure, both manual and automated, adapting it to their standard method.
Suitable controls must be carried out for each set of stains to avoid inaccurate results.
REFERENCES
Marshall, P.N., Galbrait, W., Bacus, J.W., (1979). Anal. Quant. Cytology, 1, 169-178.
Baker, J.R., (1962), Quart. J. Micr. Sci., 103, 493-517.
Anapath
Boon, M.E., Drijver, J.S., Routine Cytological staining techniques, 1st. ed (1986)
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STAINS FOR HISTOCYTOLOGY. CITOPLASM STAINING
PRINCIPLE
The Papanicolaou test, which owes its name to Dr Georgios Papanicolaou, a pioneer in cytology and cancer, is performed to diagnose cervical cancer or cancer in other organs. It makes it
possible to detect changes in cells which may be cancer precursors. The first step in this technique is the staining of nuclei with haematoxylin. The second step is the staining of cytoplasm
with an orange solution, which stains the mature cells and the keratinised cells with different intensities. The so-called polychrome solution, which is a mixture of eosin, light green SF
and Bismarck brown, is used in the third step. The differentiation of simple squamous epithelium is shown with the polychrome solution. The Haematoxylin and Eosin stain is the most
commonly used stain in histology.
The dyes for the staining of the cytoplasm are fixed in the extracellular components of the tissue and enable its differentiation. Eosin is the main component of this group. The stain spreads
easily between the tissue structures and is fixed, due to its acidic nature, to the basic radicals present in the tissue proteins. The Orange G stain is also an acid stain which has greater
penetration capacity than eosin in dense structures, therefore enabling a greater staining of keratinised cells. Stains from the EA group combine the staining capacity of an acidic dye, such
as Eosin, with the staining obtained using the Light Green stain, which, due to its less acidic nature, adds staining shades to the cytoplasmic structure.

For cytoplasmic staining in histocytology. Following the quality control practices defined by the CLSI (formerly NCCLS) is
REAGENTS acid-fast or non acid-fast microorganism cultures.
Alcoholic eosin 1% 1 x 1000 mL Ref. 99 14 15
Composition PROCEDURE
Eosin 1%
PAPANICOLAOU STAIN
Eosin 1% 1 x 1000 mL Ref. 99 30 06 1. The smears which have been spray-fixed should be immersed in water for
Composition 5-10 minutes so that the carboxymethyl cellulose (CMC) that they contain is
Eosin aqueous solution 1% completely dissolved
2. Immerse in Harris’s Haematoxylin for 1 minute
Papanicolaou OG-6 1 x 500 mL Ref. 99 46 45 3. Rinse with water for 15 seconds
1 x 1000 mL Ref. 99 70 10 4. Rinse with ethanol-HCl (0.25%) for 10 seconds
1 x 2500 mL Ref. 99 76 02 5. Rinse with water for 15 seconds
1 x 5000 mL Ref. 99 74 20 6. Rinse with ammonia water (0.05% in NH3) for 5 seconds
Orange G 0.25% 8. Immerse in ethanol 96º for 10 seconds (twice, using a different container
Ethanol 90% each time)
9. Immerse in OG-6 stain for 3 minutes
Papanicolaou EA-36 1 x 1000 mL Ref. 99 69 89 10. Rinse in ethanol 96º for 15 seconds (twice, using a different container each
1 x 5000 mL Ref. 99 55 36 time)
Composition 11. Immerse in EA 50, EA 36 or EA 65 stain for 3 minutes
Eosin 0.20% 12. Rinse in ethanol 96º for 15 seconds (twice, using a different container each
Light Green 0.1% time)
Methanol 90% 13. Immerse in absolute ethanol for 15 seconds (twice, using a different container
each time)
Papanicolaou EA-50: 1 x 500 mL Ref. 99 19 07 14. Immerse in ethanol/xylol (50%) for 15 seconds
1 x 1000 mL Ref. 99 70 12 15. Immerse in xylol for 15 seconds
1 x 2500 mL Ref. 99 83 34 16. Immerse in xylol for 2 minutes. Mount the preparation
1 x 5000 mL Ref. 99 19 22
Composition
Eosin 0.20% HAEMATOXYLIN AND EOSIN STAIN
Light Green 0.05% 1. Dewax the preparation
Ethanol 80% 2. Rinse with distilled water for 1 minute after the last immersion in ethanol
Methanol 10% 3. Immerse in Haematoxylin for 8 minutes
Papanicolaou EA-65: 1 x 1000 mL Ref. 99 69 64 5. Rinse with ethanol-HCl (0.5%) for 13 seconds
Eosin 0.15% 7. Rinse with ammonia water (0.05% in NH3) for 30 seconds
Light Green 0.02% 8. Rinse with water for 1 minute
Methanol 90% 9. Immerse in Eosin 1% for 30 seconds
10. Immerse in ethanol 96º for 6 seconds (twice, using a different container each
time)
11. Immerse in absolute ethanol for 6 seconds (three times, using a different
container each time)
12. Immerse in xylol/eucalyptol for 6 seconds (twice, using a different container
each time)
RESULTS
MATERIAL REQUIRED (NOT SUPPLIED) Nuclei: blue-violet
General-purpose pathological anatomy laboratory material. Acidophilic cytoplasm: reddish
Haematoxylin stain. Basophilic cytoplasm: blue
Different concentrations of ethanol. Xylene or Xylene Substitute. Mounting medium Keratinised cytoplasm: orange
Microscope
PRECAUTIONS NOTES
The products are for professional use. All the reagents must be handled with care by trained The staining intensity is proportional to the staining time. The stain must be kept away from
technicians. The safety data sheet should be consulted before using the reagents. moisture in order to obtain optimal results.
Waste disposal must be carried out according to the local regulations in force. Each user may apply the different versions of this procedure, both manual and automated,
SAMPLE
Cytological samples of different origins: gynaecological, sputum, liquid biopsies. REFERENCES
Histological samples. Marshall, P.N., Galbrait, W., Bacus, J.W., (1979). Anal. Quant. Cytology, 1, 169-178.
Handling of the samples must be carried out in accordance with the established protocols in Baker, J.R., (1962), Quart. J. Micr. Sci., 103, 493-517.
each laboratory for the preparation of samples for staining with the Papanicolaou method or Boon, M.E., Drijver, J.S., Routine Cytological staining techniques, 1st. ed (1986)
with the Haematoxylin and Eosin stain. CLSI Guidelines and Standards, CLSI, Wayne, P.A
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Auxiliars
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QCA-BIO 100
AUTOANALYZER - RANDOM ACCESS
System Software
• Principle: Colorimetry, turbidimetry • Host communication:Ethernet LAN,
• Speed: Up to 150 tests/hour single Standard ASTM ASCII,
reagent • Quality control: 3 level / test,
• Working modes: Random Access, urgencies 1-month monitoring
• Methodology: Fixed time, Endpoint,
Kinetic, Bichromatic, Sample
Optical System
blank
• Lamp: Halogen with extension
Sampling UV
• Photometer: 10 filters (340, 405, 505,
• Reagents: 20 total positions 546, 578, 600, 650, 700
38 mL or 15 mL nm) 1 free position y 1
removable basket solid position for dark
• Samples: 10 total positions, Tubas 12- :reading, photoelectric
13 mm, cups 5-7 mU of 1 mL detector
removable tray • Reading: Direct in reaction cuvettes
• Refrigeration: Reagent ≈ 12 °C unter room Tª• Rank: 0.1-1.5 Abs ±1% CV
• Working temp.: Between T. room and 42 °C
• Samples volume: 2-300 µL
• Reagent volume: 2-350 µL
Options / Requirements
• Reaction volume: 300-500 µL (min/max) • Computer: External PC through
• Dilution: In needle, automatic predilu- USB
tion • Operating system: Windows 7 / 8 / 10
• Needle: 2 sampling, 110 mm stroke
liquid level detection automa-
tic cleaning in/out
• Diluter: Long life plunger precision 1.5
CV% at2 µL; 1 CV% at 4 µL
• Wash station: Optional 3’” peristaltic pump
+ aspiration needle to empty
reaction cuvettes
Specifications
• Power: 100-240 V - 50/60 Hz

• Consumption: < 150 VA
• Dimensions: 40 x 60 x 38 cm
• Weight: 20 Kg
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QCA-BIO 200
System Software
• Speed: Up to 180 tests/hour single Standard ASTM ASCII,
reagent • Quality control: 3 level / test,
Kinetic, Bichromatic, Sample Optical System
blank
UV
Sampling • Photometer: 10 filters (340, 405, 505,
546, 578, 600, 650, 700
• Reagents: 30 total positions
nm) 1 free position y 1
38 mL or 15 mL
solid position for dark
removable basket
reading, photoelectric
• Samples: 46 total positions, cups 3.0 mL
detector
removable tray
• Reading: Direct in reaction cuvettes
• Refrigeration: Reagent ≈ 12 °C unter room Tª
• Rank: 0-2.5 Abs ±1%
• Working temp.: Between T. room y 42 °C
• Reagent volume: 5-350 µL
• Reaction volume: 210-350 µL (min/max) • Computer: External PC through
• Dilution: In needle, automatic predilu- USB
tion • Operating system: Windows 7 / 8 / 10
• Cuvette reading: 80 washable BIOMEX®
• Needle: Liquid level detection automa-
tic cleaning in/out
• Diluter: Long life plunger precision
±0.14 µL to 368 µL
• Wash station: Needles: 4 dispensing,
5 aspiration, 1 cleaning
Specifications
• Power: 100-240 V - 50/60 Hz
• Humidity: 10-80% HR
• Weight: 32 Kg
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QCA-BIO 300
System Software
• Speed: 200 tests/h double reagent Standard ASTM ASCII,
300 tests/h single reagent • Quality control: 3 level / test,
Kinetic, Bichromatic, Sample Optical System
blank
UV
Sampling
• Reagents: 30+30 total positions 546, 578, 600, 650, 700
55 mL or 24 mL nm) 1 free position y 1
2 removable trays solid position for dark
• Samples: 60 total positions, Tubas 12- reading, photoelectric
13 mm, cups 5-7 mU of 0.5- detector
1.5 mL. Removable tray • Reading: Direct in reaction cuvettes
• Refrigeration: Reagent ≈ 12 °C unter room Tª• Rank: 0.1-1.5 Abs ±1% CV
• Working temp.: Between T. room and 42 °C
• Reagent volume: 5-350 µL Options / Requirements
• Reaction volume: 210-350 µL (min/max)
• Computer: External PC through
• Dilution: In needle, automatic predilu-
USB
tion
• Operating system: Windows 7 / 8 / 10
• Needle: 2 sampling, 110 mm stroke
liquid level detection automa-
tic cleaning in/out
• Diluter: Long life plunger
precision 1.5 CV% at2 µL; 1
CV% at 4 µL
• Wash station: Needles:6 dispensing,
5 aspiration, 1 cleaning
Specifications
• Power: 100-240 V - 50/60 Hz
• Consumption: < 200 VA
• Weight: 55 Kg
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QCA PLUS II / BT 1500
System Software
• Principle: Colorimetry, turbidimetry and • Host communication::RS232 Bidireccional /
ISE optional LAN
• Speed: 250 tests/hour • Quality control: Statistical analysis,
• Working modes: Random Access, Batch, Stat interpolation ad-
• Methodology: Endpoint, Fixed time, Kinetic justments
ISE (opcional), mono- or
bireactive biochemistry, mo- Optical System
nochromatic and bichromatic,
sample blank, sample starter • Lamp: Halogen and dichroic
reflect
• Photometer: 10 filtros (340, 380, 405,
Sampling 436, 480, 510, 566, 578,
• Reagents: 48 total positions 630 y 700 nm)
24 positions of 50mL • Rank: ±1% (0-2 O.D), ±2,5%
24 positions of 10 or 20 mL (2-3 O.D)
• Samples: 78 total positions
62 samples and emergencies
16 standard and controls Options / Requirements
• Refrigeration: Peltier system (5-15 °C)
• Working temp.: 30, 32, 37 °C and room Tª. • Computer: External PC
• Sample volume: 1.8-100 µL • Operating system: Windows 7 / 8 / 10
• Reagent volume: 180-300 µL • UPS: UPS online
• Reading cuvettes: 32, quartz self-washing • Barcode: Identification of samples
• Needle: liquid level detection, automa- and reagent
tic cleaning in/out • Module ISE: Integrated for Na+, K+,
• Dilution: ceramic plunger without main- Cl- in serum and urine
tenance, accuracy 0.1 µL ±1%
Specifications
• Power: 300 W (100-240 V - 50/60 Hz)
• Humidity: 10-80% RH
• Weight: 53 Kg
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BT 3500
System Software
• Principle: Colorimetry, turbidimetry and • Bar code: Identification of samples
ISE optional and reagent
• Speed: 350 tests/hour • Module ISE: Integrated for Na+, K+,
• Working modes: Random Access, Batch, Stat Cl- in serum and urine
• Methodology: Endpoint, Fixed time, Kinetic
ISE (opcional), mono- or
bireactive biochemistry, mo-
Optical System
nochromatic and bichromatic,
• Lamp: Halogen and dichroic
sample blank, sample starter
reflect
Sampling 436, 480, 510, 566, 578,
630 y 700 nm)
• Reagents: 80 total positions • Rank: ±1% (0-2 O.D), ±2,5%
40 positions of 50-80 mL (2-3 O.D)
40 positions of 10-20 mL
• Samples: 78 total positions: Options / Requirements
62 samples and urgencies
16 standard and control • Computer: Internal PC
• Refrigeration: Peltier (5-15 °C) system • Operating system: Windows 7 / 8 / 10
• Working temp.: 30, 32, 37 °C and T. room • Host communication: RS232 Bidireccional /
• Samples volume: 1,8-100 µL LAN
• Reagent volume: 180-400 µL • Software: Quality control. statisti
• Reading cuvettes: 45 quartz self-washing cal analysis, interpola
• Needle: Liquid level detection, auto tion adjustments
matic cleaning in/out
• Diluter: Ceramic plunger without
maintenance
accuracy 0.1 µL ±1%
Specifications
• Power: 300 W (100-240 V - 50/60 Hz)
• Weight: 95 Kg
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BT 4500
System Optical System

• Principle: Colorimetry, turbidimetry and • Lamp: UV dichroic halogen
ISE • Photometer: 14 filters (340-800 nm)
• Speed: 450 tests/hour 1 free position and 1 posi
250 test/hour ISE module tion for background rea
• Working modes: Random Access, Batch, Stat ding, photoelectric detec
tor.
Sampling • Reading: Direct in reaction cuvette
• Rank: 0-3 Abs ±1%
• Reagents: 40 + 40 total positions
50 o 80 mL / 10 o 20 mL
• Samples: 100 sectional positions Options / Requirements
• Refrigeration: Reagents, standards and con
trols • Computer: External PC via USB
• Working temp.: Peltier system 30, 32, 37 °C • Operating system: Windows 7 / 8 / 10
and T. room • Host communication: Ethernet LAN, RS232
• Samples volume: 1,8-100 µL • Quality control: 3 known levels, 3 unk
• Reagent volume: 180-400 µL nown levels
• Sampling arm: 1 for samples, standards and
:controls, 2 for reagents
• Reading cuvettes: Self-washable optical quartz,
non disposable
• Needle: Liquid level detection
Automatic cleaning in/out
• Diluter: 3 units of 340 µL
:accuracy 0.1 µL ±1%
• Wash: Automatic
Specifications
• Power: 100-240 V - 50/60 Hz

• Weight: 120 Kg
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QCA-COAG I
Coagulometer 1 Channels

• Principle: Turbodensitometric • Lamp: Solid state led emitter
• Measuring Channel:1 channel • Photometer: 3 filters (460, 510, and
• Reading cuvettes: Microcuvettes 630 nm)
• Determination: PT, APTT, FIB • Accuracy: High linearity ±0,5% F.S.
Sampling Software/Harware
• Reagents: 4+2 stations • Languages: Multilanguages on re -

• Samples: 16 stations quest
• Station Temp.: Digital controlled • Calibration curve: Until 4 points
• Stations: Reagent 4 stations: 37 °C • Calculations: Sec, INR, mg/dl, %, Ra-
Reagent 2 stations: room tio
temp. • Control panel: Alphanumeric keyboard
Sample 16 stations: 37 ºC • Display: LCD Graphic module
• Samples volume: > 50 µL 128x64 Pixels
• Reaction volume: 150-450 µL • Processor: Internal 30 mips
Specifications Options / Requirements
• Power: Input 12V DC power supply • Printer: Optional external printer

• Temperature: 37 °C ±0,4°C working temp. • Interface: Optional bluetooth
• Dimensions: L21 x W19 x H8 cm • Bar code: No present
• Weight: 1 Kg
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QCA-COAG II
Coagulometer 2 Channels

• Measuring Channel:2 channels • Photometer: 3 filters (460, 510, and
• Determination: PT, APTT, FIB • Accuracy: High linearity ±0,5% F.S.
Sampling Software/Harware
• Reagents: 2+2 stations • Languages: Multilanguages on re-

• Samples: 16 stations quest
• Station Temp.: Digital controlled • Calibration curve: Until 4 points
• Stations: Reagent 4 stations: 37 °C • Calculations: Sec, INR, mg/dl, %, Ra-
Reagent 2 stations: room tio
temp. • Control panel: Alphanumeric keyboard
Sample 16 stations: 37 ºC • Display: LCD Graphic module
• Samples volume: > 50 µL 128x64 Pixels
• Reaction volume: 150-450 µL • Processor: Internal 30 mips
Specifications
• Power: Input 12V DC power supply
• Temperature: 37 °C ±0,4°C working temp. • Printer: Optional external printer
• Dimensions: L21 x W19 x H8 cm • Interface: Optional bluetooth
• Weight: 1 Kg • Bar code: No present
Auxiliars
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QCA-COAG AUTO
Coagulometer Automatic

• Measuring Channel:Automatic • Photometer: 3 filters (460, 510, and
• Determination: PT, APTT, FIB, AT-II, • Accuracy: High linearity ±0,5% F.S.
PRO-C, PRO-S
Software/Harware
Sampling
• Languages: Multilanguages on re-
• Reagents: Reagent 6 stations: 37 °C quest
Reagent 5 stations: room • Calibration curve: Multipoint or regression
temp curve
• Samples: 18 primary plasma tubs • Calculations: Sec, INR, mg/dl, %, Ra-
• Reaction: 36 plastic cuvettes tio
• Arm: 1 for sample and reagents
• Throughput: Up to 120 PT test/h
• Samples volume: > 50 µL Options / Requirements
• Reaction volume: 150-450 µL
• Sampling: 10-250 µL (High Precision) • Computer: Optional External Win 7
• Refrigerator: Optional • Interface: USB 2.0
• Bar code: Optional internal
• Washing: Solution A 1L & B
Specifications 250mL
• Power: Input 100/240 VAC
• Temperature: 37 °C ±0,4°C working temp.
• Dimensions: L45 x W65 x H40 cm
• Weight: 25 Kg
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QCA MICRO
AUTOMATIC STAINING SYSTEM
Specifications QCA Stains

• Programs Chipcard::4 technics per card Microbiology
• Reagent volume: 250 mL per cuvettes
• Samples number: 25 slides per rack • Crystal Violet
• Stations: 7 positions in total • Gram Decolorizer
5 cuvettes per reagent • Iodine PVP
1 loading/drying cuvette • Safranin
1 washing cuvette • Gram Fuchsin
• Carbol Fuchsin Tensoactive
Performance • TB Decolorizer
• Kühne’s Methylene Blue
• Process: 1 rack per routine • Auramine
• Extraction vapors: Activated carbon filter • Auramine-Rhodamine
• Cuvette protection: Covers with automatic ope • MT decolorizer
ning • Potassium Permanganate
• Residue discarding:Pump system • Thiazine Red
• Water renewal: Botton cuvette draining
Technical aspects Haematology

• Power: 220 ± 10% V / 50 Hz • Giemsa
• Temperature: 10-40 °C working Tª • May-Grünwald
• Humidity: 10-80% RH • Phosphate buffer - Giemsa
• Dimensions: 500 x 450 x 450 mm • Quick panoptic nº 1
• Weight: 12 Kg • Quick panoptic nº 2
• Quick panoptic nº 3
Accessories and Consumables
• Reagent cuvettes
• Wash water and drying cuvettes
• Racks
• Basket racks
• Activated carbon filter
• Reusable absorbent pad (x 10 u.)
Auxiliars
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QCA STAINER
AUTOMATIC STAINING SYSTEM
Specifications QCA Stains
• Programs: Up to 20 programs Microbiology

• Steps: Up to 50 steps per program
• Reagent volume: 300 mL per cuvette • Crystal Violet
• Samples number: 30 slides per rack • Gram Decolorizer
• Reagents: 52 in memory • Iodine PVP
• Programmable stations: 20 total stations up to 18 • Safranin
cuvettes per reagent • TB Decolorizer
1, 2 or 3 loading cuvettes • Gram Fuchsin
1 or 2 unloading cuvettes • Carbol Fuchsin Tensoactive
from 1 to 3 washing cuvettes • Kühne’s Methylene Blue
1 drying cuvette • Auramine
• Auramine-Rhodamine
• MT decolorizer
Performance • Potassium Permanganate
• Process: 5 racks simultaneously • Thiazine Red
• Extraction vapors: Activated carbon filter
• Cuvette protection: Covers for preservation
Haematology
• Immersion sample: Range from 1 s to 59 min
• Giemsa
Technical aspects • May-Grünwald
• Phosphate buffer - Giemsa
• Power: 100-240 V / 50-60 Hz • Quick panoptic nº 1
• Internal battery: Lithium-ion. 2 h autonomy • Quick panoptic nº 2
• Temperature: 10-40°C working Tª • Quick panoptic nº 3
• Dimensions: 1200 x 440 x 368 mm
• Weight: 65 Kg Anatomical Pathology
• Harris’ Hematoxilyn
Accessories and Consumables • Papanicolaou EA 50
• Papanicolaou OG 6
• Reagent cuvettes • Eosin 1%
• Wash water and drying cuvettes • Eosin Alcoholic 1%
• Baskets
• Racks
• Activated carbon filter
• Cuvettes’ individual covers
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ISE / TARGA
WASHING SOLUTION FOR ELECTRODES
REAGENTS
Kit 10 x 10 mL. (Ref. 99 08 15) Contents:
A. 5 x 10 mL Washing solution Ref. 99 12 20
B. 5 x 10 mL Active pepsin Ref. 99 68 28

Just before use add the contents of the washing solution vial to the
active pepsin vial.
Afterwards pass this solution through the electrodes according to the
instrument’s instructions.
Electrolytes
CONSERVATION AND STABILITY
The kit components, when kept refrigerated at 2-8ºC, are stable up to
the expiry date indicated in the label.
Once the pepsin has been rehydrated, it must be used before 48h at
room temperature. Kept at 2-8ºC, the product is stable up to 15 days.
CAUTION
The disposal of the residues has to be made according to local
regulations.
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I.S.E. / TARGA PLUS
ION SELECTIVE ELECTRODE REAGENTS (Na+ / K+ / Cl-)
REAGENTS
Kit 530 mL. (Ref. 99 91 30). Contents: PROCEDURE
A. 2 x 250 mL Conc. reference solution Ref. 99 82 20 Sample volume: 30 µL
B. 3 x 10 mL Conc. buffer solution Ref. 99 82 35 Units: mmol/L / mEq/L
C. 3 x 10 mL Calibrator. Low level Ref. 99 98 06 Serum dilution: 1:14
D. 3 x 10 mL Calibrator. High level Ref. 99 98 47 Reagent dilution: 1:9
E. 3 x 10 mL Cleaning solution Ref. 99 84 10 Nr. of washes: 1
Incubation time: 6
REAGENT PREPARATION Reading time: 4
All the solutions are ready for use Urine dilution (µL): 400 µL
REAGENT COMPOSITION
The concentrations in the solutions are: REFERENCES VALUES
Each particular laboratory should establish its own reference value
A. Reference solution ranges as these can vary according to sex, age, diet,…
Borate bufffer pH 8.6 220 mM However, as an orientation only, values of the following order can be
Sodium chloride 9 mM expected:
Potassium chloride 0.3 mM
Sodium bicarbonate 2 mM Sample K+ Na+ Cl-
Serum 3.5-5.1(*) 136-146 98-106
B. Buffer solution.
Borate bufffer pH 8.6 220 mM Urine, 24 h 25-150 40-220 110-250
(*) All the table values are in mmol/L.
CONSERVATION AND STABILITY
The kit components, when stored at room temperature (≤ 25ºC), are stable
until the expiry date state on the label.
LINEALITY
General laboratory equipment. Sample K+ Na+ Cl-
Spectrophotometer or photometer thermostatable at 37ºC.
Serum 2 - 200(*) 20 - 400 40 - 400
SAMPLE
Serum or urine. Urine 2 - 200 20 - 400 40 - 400
Serum: Must be quickly separated from the globular pack.
Urine: Collect 24h urine. If the test must be delayed, the sample should best be (*) All the table values are in mmol/L
stored under refrigeration (2 – 8ºC).
CALIBRATORS
Slope ranges
The calibrators are aqueous solutions of great stability and with a great
reproducibility. Minimum Maximum
Let both the calibrators and the samples reach the room temperature before
K+ 30 75
proceeding to effect the tests.
The values are stated on the label of each and every corresponding vial. Na+ 40 80
Cl- -70 -25
CALIBRATION
For the calibration of the instrument both, the low level calibrator and the high
level calibrator in the kit, must be used. QUALITY CONTROL
It is convenient to calibrate the equipment every 4 - 5 h, or when the controls Control serum, Seriscann Normal (Ref. 99 41 48) and Seriscann Anormal (Ref.
used be out of range. 99 46 85) should be included in each test series. Each particular laboratory
Avoid possible evaporation. should establish its own control program.
The calibration slopes must be within range, if not this could be indicative that
the electrode’s life is coming to an end or that either the buffer solution, the LIMITATIONS OF THE METHOD
reference solution or the standards are damaged. False results can be obtained with haemolyzed samples.
All the reagents of the ISE / TARGA kit are very sensitive to air contact. In
consecuence they must be handled with care. When ending the work, the
buffer solution must be closed soonest possible.
The cleaning solution must be used daily, before turning the equipment off.
To add either buffer or reference solution implies that the instrument must be
calibrated again.
If seric proteins obstruct the electrodes, specially the Cl-, they must be manually
cleaned.
CAUTION
The safety statements are on the label. It is advisable to look at the MSDS
before using the reagent. The disposal of the residues has to be made according
to local regulations.
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EXTRA WASH KIT
FOT AUTOMATIC ANALYZERS
Reagents
Kit. (Ref. 99 01 86) Contents:
A. 1 x 250 mL Alkaline Solution Ref. 99 02 00

B. 1 x 100 mL Acid Solution Ref. 99 03 05
Working reagent
Reagents are ready to use
Composition
Concentrations of solutions are:
A Alkaline Solution
Sodium hypochlorite ≥ 9.0 g Cl2 / L
B. Acid Solution
HCl 0.1M
Procedure
Insert the corresponding washing solution, as it is stated on the
cleaning planning, into the container used for the extra wash solution.
Wash the cuvettes as it is indicated on the analyzer manual, provided
by the manufacturer.
Once finished the washing procedure, wait for 10 min for the reagent
to work.
Wash again the cuvettes with deionised water as to eliminate all the
washing reagent used.
Cleaning planning
It is determined by the number of samples processed in a day
a) ≤ 100 samples/day
Wash using A solution (basic solution) every 30 days
Wash using B solution (acid solution) every 3 months
b) > 100 samples/day

Wash using A solution (basic solution) every 15 days
Wash using B solution (acid solution) every 2 months
Storage and stability

The kit components, when kept at room temperature are stable up to
the expiry date indicated in the label.
Caution
Handling of reagents must be done by trained laboratory staff with
knowledge of analyzer operations and chemical reagents use.
It is necessary to wear protective gloves. Avoid the inhalation of
reagents.
It can irritate the eyes, the respiratory tract and the skin. In case of
contact with the eyes wash with abundant water and consult the
physician.
See MSDS.
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WASHING SOLUTION PS-200
For automatic analyzers
UTILITY
For the preparation of washing solutions used in the cleaning of
hydraulic circuits of the automatic analyzers.
REAGENTS
Kit. (Ref. 99 99 50) Contents:
10 x 10 mL Washing solution Ref. 99 27 69
WORKING REAGENT
Pour one vial (10 mL) into 5 L of deionized water.
COMPOSITION
Concentration of the working solution is:
Tensoactives 2%
PROCEDURE
Use as described in the analyzer’s operation manual.

The kit, when stored at room temperature (< 25ºC) is stable until the
expiry date stated on the label.
CAUTION
The disposal of the residues has to be made according to local
regulations.
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LABWARE
Format Reference
STAINING GLASS JARS (Box) (Box)
Material: Soda-lime glass
Coplin type
Up to 5 slides (cap included)
1 x 20 un. 44 09 00
Hellendalh type
1 x 20 un. 44 09 01
Schiefferdecker type
1 x 20 un. 44 09 02
STAINING JAR SETS

1 glass jar with cap + 1 Steel rack of slides
Staining Jar Set nr. 1
Up to 30 slides
1 x 20 un. 44 09 03
Staining Jar Set nr. 2

Up to 60 slides
1 x 10 un. 44 09 05
SPARE STAINING PARTS

Rack for 30 slides
Material: Steel
1 x 50 un. 44 09 04
Rack for 60 slides

Material: Steel
1 x 50 un. 44 09 06
Staining glass jar, up to 30 slides

Material: Glass
1 x 20 un. 44 09 07
Staining glass jar, up to 60 slides

Material: Glass
1 x 10 un. 44 09 08
Auxiliars
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ANCILLARY PRODUCTS
For Histocytology and general laboratory use
VASELINE OIL CITOSCANN 96 METHANOL

1 x 1000 ml Ref. 99 57 11 1 x 1000 ml Ref. 99 95 25 1 x 1000 ml Ref. 99 14 01
For general laboratory use 1 x 5000 ml Ref. 99 77 11 1 x 5000 ml Ref. 99 05 10
Dehydrating agent for histological techniques. Water For general laboratory use
Specifications: contents: 4 %.
Density (15ºC): 0.835 - 0.860 g/ml Specifications:
Freezing point: - 15ºC Composition: Purity: ≥ 99.9%
Ethanol ≥ 84.5 % Density (20ºC): 0.7910 g/ml
Store at: Room temperature (15-30ºC) Isopropyl Alcohol ≥8% Water: < 0.1% w/w
Store at: Room temperature (15-30ºC) Ethanol: < 0.005 ppm
Chloride: < 1 ppm
Store at: Room temperature (15-30ºC)

IMMERSION OIL
1 x 100 ml Ref. 99 81 02 CITOSCANN 90
For microscopy 1 x 5000 ml Ref. 99 41 90
Dehydrating agent for histological techniques. Water
contents: 10 %. PARAFFIN PEARLS
Specifications:
1 x 1000 g Ref. 99 46 46
nD = 1.477 – 1.481 (20º C) Composition: 6 x 2000 g Ref. 99 05 05
Ethanol ≥ 79 % Imbibing media for histological samples. Auxiliary reagent
Store at: Room temperature (15-30ºC) Isopropyl Alcohol ≥8% for histological samples used for diagnostic purposes
Specifications:
Melting point 56 - 58ºC
Oil contents ≤ 0.9 %
Viscosity at 100ºC: 3.6 – 5.5 cSt
ACETONE *
1 x 1000 ml Ref. 99 02 92 CITOSCANN 70
1 x 5000 ml Ref. 99 49 07 1 x 5000 ml Ref. 99 41 40
For general laboratory use. Dehydrating agent for histological techniques.
Water contents: 30 %.
Specifications: Composition:
Purity: > 99.5% Ethanol ≥ 62 %
Density (20ºC): 0.790 - 0.792 g/ml ISOPROPANOL
Isopropyl Alcohol ≥ 6.3 %
Water: < 0.3% w/w 1 x 5000 ml Ref. 99 60 13
Methanol: < 1000 ppm For general laboratory use
Acidity: < 0.002% (w/w)
Specifications:
Store at: Room temperature (15-30ºC) Purity: ≥ 99.9%
Destilation Range: 82 - 83ºC
Water: < 0.1% p/p
CITOSCANN 50 Relative Density (20ºC): 0.784 – 0.788 kg/dm3
1 x 5000 ml Ref. 99 41 50
Dehydrating agent for histological techniques.
DEIONISED WATER Water contents: 50 %. Store at: Room temperature (15-30ºC)
1 x 1000 ml Ref. 99 60 54
Composition:
1 x 5000 ml Ref. 99 89 53
Ethanol ≥ 44 %
1 x 10000 ml Ref. 99 72 25
Isopropyl Alcohol ≥ 4.5 %
Highly purified water, with very low soluble salts levels. For
general laboratory use Store at: Room temperature (15-30ºC) NOTE
Specifications: The products listed above have different levels of

No sterile dangerousness.
Conductivity: < 2 µS / cm It is advisable to read the MSDS prior to use.
Resistivity: ≥ 0.5 MΩ x cm GLYCERIN
Basicity: ≤ pH 7.5 (Bromothymol Blue indicator) 1 x 1000 ml Ref. 99 59 67 MSDS are available upon request or at www.qca.es
Acidity: ≥ pH 4.4 (Methyl Red indicator) For general laboratory use
Store at: Room temperature (15-30ºC) Specifications:

Purity: > 99.5%
Density (20ºC): 1.2620 - 1.2636 g/ml
Water: ≤ 0.5% w/w
Acidity: < 0.04% meq/g
CITOSCANN
1 x 1000 ml Ref. 99 03 00 Store at: Room temperature (15-30ºC)
1 x 5000 ml Ref. 99 97 01
Dehydrating agent for histological techniques.
Composition:
Ethanol ≥ 88 %
Isopropyl Alcohol ≥9%
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BONE MARROW BIOPSY KIT
For Histological and Histopathological Laboratories
PRINCIPLE USE. PERFORMANCE CHARACTERISTICS

Tissue fixation is mainly carried out by the immersion of the tissues into chemical In the fixative pathway, due to the low rate of penetration, it is recommended
agents (fixatives) in order to interrupt the degradation processes that occur after to use samples thinner than 4 mm and the fixation time should be lower than 4
the cell death, and thus, it preserves the structures and composition of the hours.
tissue. Fixing agents have the ability to stop the enzyme activity, the process of It is recommended 90-120 minutes of fixation, depending on the sample
tissue autolysis and the protein denaturation. thickness.
Some fixatives can be used, likewise, as preservatives, but not in all cases can
be ensured a long term preservation. It is recommended to rinse the sample with running tap water during 30 minutes.
The decalcification process consists in the removal of calcium salts from the Decalcification is performed after washing process. Since it is a fast decalcifier,
tissues once they have been fixed to prepare the samples for further histological the end point should be more precisely determined so as to avoid an
examination. unnecessarily extended process.
A good decalcifier should remove completely the calcium deposits without
affecting the tissues nor generating any artefact in the tissues and not interfering It is recommended 45 minutes of decalcification to allow the conservation of the
in the further staining processes. cellular structure and achieve a good sample decalcification.
The procedure described above is the one used in our laboratories and the
Bone Marrow Biopsy Kit contents specific fixative solution and decalcifier for stated methods are for guidance. Each particular laboratory should establish its
bone marrow biopsy processing which allow the preservation of the cellular own method.
structure and the performance of immunohistochemical or enzyme staining.
General considerations of the tissue decalcification process
REAGENTS
Kit 2 x 250 ml (Ref. 99 56 05). Contents: 1. The fixation process must be completed prior to initiate the decalcification
A. 1 x 250 ml. Fixative B5 Fast Decalcifier Ref. 99 04 45 in order to avoid the possible adverse effects of the decalcifying treatment.
B. 1 x 250 ml. Fast Decalcifier Ref. 99 56 42 2. Enough volume of the decalcifying solution shall be used. It is generally
used a ratio 1/20 (piece volume/ liquid volume).
WORKING REAGENT PREPARATION 3. Temperatures higher than 25ºC should be avoided. Although the
Reagents B is ready-to-use. process is accelerated, the risk of sample alteration is also increased.
4. To eliminate the decalcifier used, the sample should be rinsed with
A working reagent should be prepared for reagent A by adding 1 volume of tap water or with an aqueous solution of lithium carbonate or sodium
concentrated Formaldehyde solution (37 – 40%) (Ref. 99 13 10) to 10 volumes hydrogen carbonate.
of reagent. 5. The decalcification end point should be accurately controlled to avoid
The working solution A must be prepared at the moment of use because of its an overexposure of the tissue to the acid solution. For this purpose the
unstability. different physical or chemical procedures elsewhere described may be
used.
Fixative B5
Mercury Chloride 6%, Sodium acetate 1.3%
BIBLIOGRAPHY
Fast Decalcifier Theory and Practice of Histological Techniques. Ed by J.D. Bancroft; M. Gamble.
HCl 12% 6thEd. (2008). Churchill LivingstonLaboratorio de Anatomía Patológica.
Stabilizers and tissue protectors. Raimundo Gómez del Moral (1993) ; McGraw-Hill/Interamer. de España , S.A.U.
Newell Ken J.; Sawka, Barry W; Rudrick Brian F.; Driman David K. Arch.
CAUTION Pathol. Lab. Med. (2001) 125: 642-645.
Fixative and Decalcifier contain substances that may be harmful for the persons
or the environment. Safety statements can be found on the product label. It is
advisable to read the SDS before using the product. The waste disposal should
be performed according to the local regulations.

Fixative and decalcifier are stable at room temperature (15º - 25 ºC) until the
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QCA-PBS
PHOSPHATE BUFFER SALINE pH 7.2 - 150 mM
UTILITY
For general laboratory use.
REAGENT
Kit 10 x 1000 mL. Ref. 99 10 95
Contents:
A.10 x 1000 mL PBS powder Ref. 99 10 97

Dissolve the contents of a vial in deionized water so as to obtain 1
litre of solution.

Sodium chloride 7.650 g
Monopotassium phosphate 0.210 g
Disodium phosphate 0.724 g

The powder, when stored at room temperature (between 15º-25ºC),
will remain stable until the expiration date stated on the label.
The working reagent solution should be kept in the refrigerator

at 2º-8ºC. Stored in these conditions, will remain stable for 2
months. AVOID CONTAMINATION.

Should the solution be turbid or show signs of bacterial contamination,
it must be rejected immediately.
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TISSUE FIXATIVES
PRINCIPLE Bouin’s Fixative

Tissue fixation is mainly done by the immersion of the tissues into chemical Picric acid 1%; Formaldehyde (soln. 37-40 % p/v) 25 %, Acetic acid 5%
agents (fixatives) in order to interrupt the degradation processes that occur Reference Packing size
after the cell death, and thus, it preserves the structures and composition 99 04 33 1 x 1000 mL
of the tissue. Fixing agents have the ability to stop the enzyme activity, the
process of tissue autolysis and protein denaturation. Some fixatives can be
used, likewise, as a preservative, but not in all cases can be ensured a long USE AND PERFORMANCE CHARACTERISTICS
term preservation. Fixation is not synonymous with tissue preservation. Bouin’s fixative is intended for delicate tissues’ fixation (skin, endocrine
Fixation types: The fixation processes can be classified in two kinds organs, testis, embryonic tissues) for which it’s recommended to avoid an
depending on the results obtained in regards the tissue structure and its excessive hardening that could make difficult the tissue processing, cutting
biochemical composition. and staining. It’s not recommended for renal biopsies.
1. Histological fixation: it basically preserves the tissue structures, B5 fixative

regardless if it produces or not biochemical changes. Mercury Chloride 6%, Sodium acetate 1.3%
2. Histochemical fixation: it’s intended to preserve the maximum possible
the molecular composition. Reference Packing size
99 04 41 1 x 1000 mL
It must be noted that it doesn’t exist a multi - purpose fixation agent suitable
for all tissues and assays. Each fixation agent is appropriate for certain USE AND PERFORMANCE CHARACTERISTICS
types of tissues and techniques and they can’t provide suitable results B5 fixative is recommended for the observation of nuclear details. Ideal for
with another tissues and assays. Each particular assay requires a different lymphoid and hematopoietic tissue. Likewise, it’s the fixative of choice for
fixation agent. kidneys, liver, connective tissue, and fibrin studies.
TISSULAR FIXATION SYSTEMS The working solution –unstable– must be prepared at the moment of use
1. Simple Fixatives by adding 1 volume of concentrated Formaldehyde solution (37 – 40%) to
Formaldehyde Solution 4.0 % p/v (Formalin 10 % aprox) 10 volumes of reagent.
Buffered pH 7.0 Stabilized with methanol It’s a fixing agent with a low rate of penetration thus it’s recommended to
Reference Packing size use samples thinner than 4 mm and the fixation time shouldn’t exceed 4
99 10 05 1 x 1000 mL hours.
99 10 76 1 x 5000 mL
99 10 85 1 x 10,000 mlL B5 fixative is not a good preservative agent, thus after fixation, the samples
must be kept in Ethanol 70 – 80 %.
Formaldehyde Solution (Formalin) 30 – 35 % p/v
GEWF Fixative
Buffered pH 7.0 Stabilized with methanol
Glacial Acetic acid 6.5%, Ethanol 60%, Water, Formaldehyde (Soln. 37-40
Reference Packing size % p/v) 10 %
99 13 10 1 x 1000 mL
Reference Packing size
99 80 33 1 x 5000 mL
99 18 27 1 x 5000 mL
USE AND PERFORMANCE CHARACTERISTICS USE AND PERFORMANCE CHARACTERISTICS

The buffered formaldehyde solutions 4 % p/v, also known as “10% GEWF is particularly recommended for the fixation and selection of lymph
formalin”, are recommended for fixing samples in routine pathology as nodes from oncological surgical pieces, from breast, digestive system and
they are compatible with most staining techniques. The buffered solutions several nodal dissections.
prevent osmotic shock and sedimentation that occurs at pH below 6.0. Its composition provide a fixation that makes easy the selection of lymph
Characteristics: it’s also a disinfecting agent which ensures a proper nodes in adipose tissue, improving the identification and significantly
preservation and storage of biopsies and surgical specimens. It yields a increasing the number of nodes isolated for the study, without
low tissue retraction with a middle rate of tissue penetration that can be affecting the performance on routine stainings, both histochemical or
accelerated by increasing the temperature during the fixation process immunohistochemical ones.
without endangering the final result.
Buffered formaldehyde solutions 4% p/v are the first choice for nervous
CAUTIONS
tissue and argentic fixation. Good results with fat and lipids in general.
Simple fixatives and fixative mixtures contain compounds that can
be harmful to people or the environment. Please, read carefully the
2. Fixative Mixtures
corresponding Material Safety Data sheet before use. The waste disposal
Formalin Substitute
should be done according to the local regulations.
Glyoxal <5%, Ethanol <20%, Acetic acid <1%
Reference Packing size STORAGE AND STABILITY
99 02 50 1 x 5000 mL The fixatives are stable at room temperature (15 - 30 ºC) until the expiration
date stated on the label. Formalin Substitute 990250 must be kept at 4 -
USE AND PERFORMANCE CHARACTERISTICS 30ºC.
It’s recommended for fixing routine samples in pathology with the advantage
of avoiding the inconvenience of toxic formaldehyde fumes. BIBLIOGRAPHY
Theory and Practice of Histological Techniques. Ed by J.D. Bancroft; M.
Gamble. 6thEd. (2008). Churchill LivingstonLaboratorio de Anatomía
Patológica. Raimundo Gómez del Moral (1993); McGraw-Hill/
Interamericana de España , S.A.U.
Pathol. Lab. Med. (2001) 125: 642-645.
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TISSUE DECALCIFIERS
Principle DECALCIFIER
The decalcification process consists in the removal of calcium salts from the
tissues once they have been fixed to prepare the samples for further histological Configuration Reference
examination. 1 x 2500 ml 99 86 40
It is carried out in mineralized tissues (bones) as well as in that material with
calcifications that make difficult the microscopical observation. Composition
HCl 8%
A good decalcifier should remove completely the calcium deposits without EDTA 1%
affecting the tissues nor generating any artifact in the tissues and not interfering
in the further staining processes. Use. Performance characteristics
It is a fast decalcifier, adapted to process bone marrow samples. It can also be
The decalcifying agents can be classified: used with small bone pieces.
Since it is a fast decalcifier, the end point should be more precisely determined
1.Strong inorganic acids: Nitric or hydrochloric acid solutions. The
decalcification process is fast but some tisular antigens necessary for the FAST DECALCIFIER
immunohistochemistry staining may be altered.
2.Organic acids: Formic or acetic acid solutions. The acetic acid may cause
some alterations in the tissue and it is usually used in mixtures with fixative Configuration Reference
substances for those samples with a minimum calcification. 1 x 1000 ml 99 56 40
Formic acid, whether buffered or not, is highly recommended for those cases
where the labile tisular antigens should remain unaltered, as it acts more slowly Composition
than the strong acids. HCl 12%
3.Chelating agents: EDTA solutions. It is the decalcifier of choice for Stabilizers and tissue protectors
immunohistochemical or enzymatic stainings, The process, however, is slow.
Use. Performance characteristics
It is a fast decalcifier, adapted to process bone marrow samples. It can also be
General considerations of the tissue decalcification process used with small bone pieces.
Since it is a fast decalcifier, the end point should be more precisely determined
1. The fixation process must be completed prior to initiate the decalcification in so as to avoid an unnecessarily extended process.
order to avoid the possible adverse effects of the decalcifying treatment. The presence of tissue stabilizers keeps a good staining capacity of the sample.
2. Enough volume of the decalcifying solution shall be used. It is generally used
a ratio 1/20 as the ratio of the volume of the piece and the liquid volume. It is Caution
recommended to frequently replace the liquid to accelerate the process. Decalcifiers contain substances that may be harmful for the persons or the
3. Keep stirring the decalcifier with the piece in the center of the vessel. In this environment. Safety statements can be found on the product label. It is advisable
manner high local concentrations of calcium are avoided around the sample. to read the SDS before using the product. The waste disposal should be done
4. Temperatures higher than 25ºC should be avoided. Although the process is according to the local regulations.
accelerated, the risk of sample alteration is also increased.
5. To eliminate the decalcifier used, the sample should be rinsed with tap water Storage and stability
or with an aqueous solution of lithium carbonate or sodium hydrogen carbonate. The decalcifiers are stable at room temperature (15º - 25 ºC) until the expiration
6. The decalcification end point should be accurately controlled to avoid an date stated on the label.
overexposure of the tissue to the acid solution. For this purpose the different
physical or chemical procedures elsewhere described may be used. Bibliography
Theory and Practice of Histological Techniques. Ed by J.D. Bancroft; M. Gamble.
6thEd. (2008). Churchill LivingstonLaboratorio de Anatomía Patológica.
Raimundo Gómez del Moral (1993) ; McGraw-Hill/Interamer. de España , S.A.U.
Pathol. Lab. Med. (2001) 125: 642-645.
ISO 9001 / ISO 13485

166
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WATER: CHLORINE
DPD METHOD
PRINCIPLE
N,N-diethyl-p-phenylendiamine (DPD), reacts with FREE CHLORINE Procedure
to produce a pink colour. In the same manner, BOUND CHLORINE, Prior to assay rinse the reaction cuvette several times with the
produces the same colour reaction when potassium iodide is added. water to be tested and proceed as follows:
A) FREE CHLORINE
1. Fill the cuvette with the water to be assayed up to the upper
mark (10 mL).
UTILITY 2. Add 10 drops of Reagent (A).
Disinfection of water (drinking, domestic use, swimming-pools...) by 3. Add 1 drop of Reagent (B).
chlorination, promotes the production of different chlorinated species. 4. Cover and mix.
The contents in chlorine and hypochlorous acid is known as FREE 5. Compare, after 1 min, the colour developed with the
CHLORINE. Chloramines are the main constituents of what is called colour chart and determine the corresponding chlorine
BOUND CHLORINE. TOTAL CHLORINE, then, is the sum of the concentration value in ppm (mg/L).
above two fractions.
On the other hand, the use of pH monitoring allows the knowledge of B) TOTAL CHLORINE
which are the main constituents in the assayed water. 6. Add 3 drops of Reagent (C) to the solution obtained in A) 5.
Thus, at a pH value between 7.2 - 7.8, monochloramine concentration 7. Cover and mix.
is higher than the one of trichloramine that shows a lower desinfecting 8. Compare with the colour chart the TOTAL CHLORINE
power. Likewise, the FREE CHLORINE stability is higher. contents in the water sample.
In swimming-pools, pH values under 7.0 can damage filters and other
installations. C) BOUND CHLORINE
It is determined from the difference between TOTAL and
FREE CHLORINE.
REAGENTS
Kit (Ref. 99 00 99) for 150 chlorine tests. Interpretation of the results
Contents:
A) FREE CHLORINE
A. 3 x 20 mL Reagent (A) Ref. 99 48 08 For drinking water, values should range at about 0.1 ppm.
Phosphate buffer solution For swimming-pools, between 0.3 and 0.4 ppm.
DPD solution B) TOTAL CHLORINE
C. 1 x 15 mL Reagent (C) Ref. 99 63 03 For drinking water, values should range around 0.3 ppm.
KI solution For swimming-pools, a maximum of 1.0 ppm.
D. Reaction cuvette
E. Colour chart C) BOUND CHLORINE
Values are obtained by substracting the FREE from the TOTAL
CHLORINE.

The components of the kit, when stored at room temperature (15º-
25ºC), will remain stable until the expiration date stated on the label.
REFERENCES
REMARK Rodier,J.(1978). Lánalyse de l’eau, eaux naturelles, eaux residuaires,
Relatively high amounts of free chlorine inhibit the reaction. In eau de mer. Dunod, Paris. APHA, AWWA, WPCF,(1985). Standard
such a case, dilute the sample (1:2 or 1:4) ith deionized water methods for the examination of water and wastewater. Washington.
and run the test once again. Journal officiel des Communautés européennes. (30.08.80), nº L
229/11-29.
Auxiliars
ISO 9001 / ISO 13485

167
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WATER: CHLORINE / pH
DPD AND PHENOL RED METHODS
PRINCIPLE
N,N-diethyl-p-phenylendiamine (DPD), reacts with FREE CHLORINE
to produce a pink colour. In the same manner, BOUND CHLORINE, PROCEDURE
produces the same colour reaction when potassium iodide is added. Prior to assay rinse the reaction cuvette several times with the
On the other hand, pH monitoring is carried out by the addition, to the water to be tested and proceed as follows:
water to be examined , of a Phenol Red indicator that changes from
yellow to reddish violet in the pH range of 6.2 - 8.2. A) FREE CHLORINE
1. Fill the cuvette with the water to be assayed up to the upper
mark (10 mL)
2. Add 10 drops of Reagent (A)
UTILITY 3. Add 1 drop of Reagent (B)
Disinfection of water (drinking, domestic use, swimming-pools...) by 4. Cover and mix
chlorination, promotes the production of different chlorinated species. 5. Compare, after 1 min, the colour developed with the
The contents in chlorine and hypochlorous acid is known as FREE colour chart and determine the corresponding chlorine
CHLORINE. Chloramines are the main constituents of what is called concentration value in ppm (mg/L)
BOUND CHLORINE. TOTAL CHLORINE, then, is the sum of the
above two fractions. B) TOTAL CHLORINE
On the other hand, the use of pH monitoring allows the knowledge of 6. Add 3 drops of Reagent (C) to the solution obtained in A) 5
which are the main constituents in the assayed water. 7. Cover and mix
Thus, at a pH value between 7.2 - 7.8, monochloramine concentration 8. Compare with the colour chart the TOTAL CHLORINE
is higher than the one of trichloramine that shows a lower desinfecting contents in the water sample
power. Likewise, the FREE CHLORINE stability is higher.
In swimming-pools, pH values under 7.0 can damage filters and other C) BOUND CHLORINE
installations. It is determined from the difference between TOTAL and
FREE CHLORINE
REAGENTS D) pH
Kit (Ref. 99 27 47) for 150 chlorine / 100 pH tests. Contents: 1. Rinse the reaction cuvette with the water to be assayed and
A. 3 x 20 mL Reagent (A) Ref. 99 24 48 fill it up to the upper mark (10 mL)
Phosphate buffer solution 2. Add 5 drops of Reagent (D). Cover and mix
B. 1 x 7 mL Reagent (B) Ref. 99 21 63 3. Compare with the colour chart and determine the pH value
DPD solution
C. 1 x 15 mL Reagent (C) Ref. 99 29 00
KI solution INTERPRETATION OF RESULTS
D. 1 x 15 mL Reagent (D) Ref. 99 25 06
Phenol Red solution A) FREE CHLORINE
E. Reaction cuvette For drinking water, values should range at about 0.1 ppm.
F. Colour chart For swimming-pools, between 0.3 and 0.4 ppm
B) TOTAL CHLORINE
For drinking water, values should range around 0.3 ppm.
STORAGE AND STABILITY For swimming-pools, a maximum of 1.0 ppm
The components of the kit, when stored at room temperature (15º -
25ºC), will remain stable until the expiration date stated on the label. C) BOUND CHLORINE
Values are obtained by substracting the FREE from the TOTAL
CHLORINE
D) pH
REMARK For drinking water, values should range between 6.5 -
Relatively high amounts of free chlorine inhibit the reaction. 6.8.
In such a case, dilute the sample (1:2 or 1:4) ith deionized For swimming-pools, between 7.2 - 7.8. In the latter, higher or
water and run the test once again. lower values will require the addition of an acid or alkalinizing
agent respectively.
REFERENCES
Rodier,J.(1978). Lánalyse de l’eau, eaux naturelles, eaux residuaires,
eau de mer. Dunod, Paris. APHA, AWWA, WPCF,(1985). Standard
methods for the examination of water and wastewater. Washington.
Journal officiel des Communautés européennes. (30.08.80), nº L
229/11-29.
ISO 9001 / ISO 13485

168
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DEXTROSE SOLUTION
Glucose solution ready for use for oral administration. Nutritional information
• Available in 2 flavors: Orange, Lemon Solution 50 per 100 mL

• Available in concentrations of 50, 75, and 100 grams Energetic value 418 kj
• Packaged in 30 x 200ml PET bottle 100 Kcal
• Made in EU Carbohydrates 25 g
• Quality control and analysis certificate
Proteins 0g
Lipids 0g
Ingredients (Orange flavor)
Water, Glucose, Orange Essence, Acidulant (E-330), Flavo-
Solution 75 per 100 mL
ring (E-954), Preservative (E-202), Coloring (E-110, E-102).
It contains no gluten. Energetic value 627 kj
150 Kcal
Ingredients (Lemon flavor) Carbohydrates 37,5 g
Water, Glucose, Lemon Essence, Acidulant (E-330), Flavo- Proteins 0g
ring (E-954), Preservative (E-202), Coloring (E-X). It conta- Lipids 0g
ins no gluten.
Solution 100 per 100 mL
It is recommended to drink cold. Energetic value 836 kj
Once opened, it should be consumed immediately. 200 Kcal
Storage temperature between (+ 15 ° C to + 25 ° C). Carbohydrates 50 g
Proteins 0g
Lipids 0g
Auxiliars
ISO 9001 / ISO 13485

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Una empresa al servicio del biodiagnóstico - A company in the service of biodiagnostics - Une entreprise au service du bio-diagnostique.

Desde 1976 - Since 1976 - Depuis 1976

ISO 9001 / ISO 13485

ISO 9001 / ISO 13485

ISO 9001 / ISO 13485

ISO 9001 / ISO 13485

ISO 9001 / ISO 13485

DIAGNOSTIC USE PROCEDURE

REAGENTS Sample 0.2 0.1

WORKING REAGENT CONCENTRATIONS CALCULATIONS

ISO 9001 / ISO 13485

DIAGNOSTIC USE PROCEDURE

ISO 9001 / ISO 13485

STORAGE AND STABILITY

ISO 9001 / ISO 13485

ISO 9001 / ISO 13485

DIAGNOSTIC USE PROCEDURE

STORAGE AND STABILITY Vt x 106

ISO 9001 / ISO 13485

REAGENTS Sample 0.10 0.10

WORKING REAGENT CONCENTRATIONS Semimicromethod

ISO 9001 / ISO 13485

thiocholine + DTNB* 2-nitro-mercapto-benzoate

DIAGNOSTIC USE PROCEDURE

REAGENTS Reagent A 3.00 1.50

Details of the performance studies are available on request.

ISO 9001 / ISO 13485

ADDITIONAL EQUIPMENT CK-MB activity

ISO 9001 / ISO 13485

ISO 9001 / ISO 13485

DIAGNOSTIC USE PROCEDURE

ISO 9001 / ISO 13485

DIAGNOSTIC USE PROCEDURE

ISO 9001 / ISO 13485

DIAGNOSTIC USE PROCEDURE

REAGENTS Incubate at 37ºC 60 min. 30 min.

STORAGE AND STABILITY Note

ISO 9001 / ISO 13485

DIAGNOSTIC USE PROCEDURE

WORKING REAGENT PREPARATION Reading

Signs of reagent deterioration:

ISO 9001 / ISO 13485

DIAGNOSTIC USE PROCEDURE

WORKING REAGENT PREPARATION Reading

ISO 9001 / ISO 13485

DIAGNOSTIC USE PROCEDURE

WORKING REAGENT PREPARATION Reading

ISO 9001 / ISO 13485

DIAGNOSTIC USE PROCEDURE

Sample 0.2 0.1

Kit 1 x 250 mL. (Ref. 99 92 00). Contents: Sample 0.2 0.1

ISO 9001 / ISO 13485

DIAGNOSTIC USE PROCEDURE

ISO 9001 / ISO 13485

DIAGNOSTIC USE Sample --- 0.01 ---

ISO 9001 / ISO 13485

Kit 3 x 100 mL. (Ref. 99 72 58). Contents: Where:

ISO 9001 / ISO 13485

C. Potassium tartrate 0.80 M Reading

STORAGE AND STABILITY CALCULATIONS

Signs of reagent deterioration: Where: Abs SA= Sample absorption

ISO 9001 / ISO 13485

DIAGNOSTIC USE Saline 1.0 1.0