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CLINICAL CHEMISTRY
SRI LANKA
MANUAL ON
STANDARD OPERATION PROCEDURES, SAMPLE COLLECTION
AND REFERENCE RANGES FOR
CLINICAL CHEMISTRY
ACKNOWLEDGEMENTS
We would like to acknowledge the WHO representative to Sri Lanka, Dr. Kan
Tun, for identifying the need for quality assurance in local laboratories and offering
us the opportunity to publish this handbook.
We are grateful to the Director of the MRI Dr. G. S. S. K. Colombage and the
Deputy Director of MRI Dr. Lulu Raschid, for all their support and
encouragement in bringing this project to fruition.
PREFACE
GENERAL INTRODUCTION
Standard Operating Procedure (SOP) manuals are required in the laboratory and
are a key element in internal Quality Control within the laboratory. Hence, assuring
quality health care, method manuals should include properly authenticated
methods, or methods recommended by relevant professional organisations. A
methods manual containing all methods and procedures needs to be authorised for
use in the laboratory. The manuals shall be available in the appropriate work areas
as bench copies and it is recommended that a master copy be maintained by the
head of the unit. These manuals should be review at least annually, by the
competent member of the technical staff and the head of the unit.
The first consideration is the selection of the test methods best suited to full fill the
function of the laboratory. Each test performed in the laboratory (for e.g.
screening, routine clinical testing, reference laboratory service) must be evaluated
and appropriate methods should be selected depending on the local needs and
resources. Each method should be evaluated in terms of sensitivity, specificity,
accuracy, simplicity, speed, reliability and economy.
The methods that are included in this manual are based on world health
organization recommended biochemical tests, adapted and evaluated by the editors
taking into consideration the clinical and technical experience, quality control
measures and the available resources. This standard operating procedure manual
includes the routine biochemical tests based on manual spectrophotometric
methods. The clinical significance of each analyte has been described in detail for
the benefit of the users. The precautions to be followed are included for each
analyte to maximize the performance. This is based largely on the experience of the
editors.
The manual contains the collection procedures of blood and urine samples. It
includes the method of collection, selection of containers and preservatives,
storage, transport and stability. There must be vigorous control of the procedures
involved in the collection and identification of specimens to ensure the quality of
the specimen to be examined in the laboratory. Copies of collection procedures
should be available at all collection areas. The collection manual should be updated
regularly as for laboratory procedure manual by the competent member of the
technical staff.
The manual includes a section for reference ranges. The reference ranges are given
for many biochemical analytes considering the age and the sex. Standard adult and
paediatric text books along with the information available on the internet were
taken into consideration in accepting the relevant reference ranges.
TITLE
Name of test or procedure.
INTRODUCTION
A brief, concise introduction of the analyte is recommended.
(In this manual a detailed description is included for the benefit of the user to prepare their
own local S.O.P)
CLINICAL SIGNIFICANCE
Give reasons as to why the test is usually requested and indicates the significance of low or
high results using the reference
PRINCIPLE
A short but informative statement which describe the basis of the method.
REFERENCE(S)
A list of primary reference(s) as well as modifications.
SPECIMEN
List the types of specimens which are appropriate and indicate stability limits if known
REAGENTS
State concentration or use descriptive name where appropriate (e.g. Biuret reagent) and
give detailed stepwise instructions for preparing each reagent. Specify chemical brand and
grade where it is known to be critical. Unusual reagents/components should have source
or company stated. Expiry of reagents should be specified.
PROCEDURE
Present a concise stepwise description of the method.
CALCULATIONS
Explain the use of formulae and indicate, briefly, the derivation of factors.
UNITS
Units used and abbreviations.
REFERENCE RANGE
Adult reference ranges for males and females and paediatric ranges where applicable.
QUALITY CONTROL
Quality control procedures, including documentation and methods of statistical analysis
NOTES
Elaborate on any particular points which may require further explanation
APPROVAL
Each procedure to be dated and signed at the bottom of the final page by the appropriate
senior authorised qualified staff. Any amendments should be authorised with date and
signature.
METHOD HISTORY
This should be in a separate sheet and include date notations of method changes and
review
REPORTING
Format and procedure of reporting, including procedures for urgent and especially
clinically significant results, must be described.
COPIES
Any copies of method for bench use are to be made in full from the master copy and
should include method history and approval sections
SUPPLEMENTARY INFORMATION
Certain information to be used at the bench to implement procedures may be extracted
from the procedure manual. This information may include flow diagrams, index cards, and
manufacturer product literature. Such supplementary information must be current and be
referenced to the procedure manual by date, procedure and reviewer’s initials.
2.1 INTRODUCTION
Albumin is the most abundant protein in human plasma from 20 weeks of gestation,
representing 40-60% of the total protein. It is synthesized exclusively in the liver. The rate
of synthesis is depended on protein intake and subject to feed back regulation by the
plasma albumin level. The half life of albumin is estimated at 15-19 days. Traces of
albumin can be found in almost all extra vascular body fluids. The loss of albumin via the
glomerular filtrate is very small as almost all the albumin is reabsorbed by the proximal
tubular cells. Albumin is catabolized in various tissues where it is taken up by cells by
pinocytosis. Its constituent amino acids are released by intracellular proteolysis and
returned to the body pool. Albumin has a molecular weight of approximately 66,000.
Albumin is an anion at pH 7.4 with more than 200 negative charges per molecule. The
chief biological functions of albumin are to transport and store a wide variety of ligands, to
maintain the plasma osmotic pressure and to serve as a source of endogenous amino acids.
The capacity of albumin to act as a binding protein is due to the large numbers of charges
of each molecule as well as very large no of molecules available. Albumin binds nonpolar
compounds such as bilirubin & long chain fatty acids. Albumin binds hormones such as
thyroxine, triiodothyronin, cortisol and aldosterone, thus act as a reservoir in which these
compounds are stored in inactive form but from which they are readily mobilised. Some
40% of serum calcium is bound to albumin. Many drugs such as phenylbutazone, warfarin
and salicylates are also strongly bounded to albumin. Albumin concentration is the major
determinant of plasma oncotic pressure, one of the factors that regulate partition of water
between intra and extra vascular compartments.
Hypoalbuminaemia is very common and may result due to the following factors
o Impaired synthesis: Diminished protein intake or liver disease
o Increased catabolism: Due to tissue damage and inflammation
o Reduced absorption of amino acids: Malabsorption syndromes or malnutrition.
o Protein loss in urine: Due to nephrotic syndrome,chronic glomerular nephritis,
diabetes,or systemic lupus erythromatosis
o Protein loss in faeces: Due to protein losing enteropathy
o Protein loss through the skin: Burns
o Altered distribution: Ascites(high pressure in the portal circulation drives albumin
into the peritoneal fluid)
Most severe hypoalbuminaemia is caused by protein loss by way of urine or faeces. When
plasma albumin levels are less than 2.0 g/l oedema is usually present.
Hyperalbuminemia: is of little diagnostic significance except in dehydration.
Albumin has more than 20 genetic variants, which are not associated with disease but
which cause two bands or a single band in the albumin region on electrophoresis. The
condition is called bisalbuminaemia. A transient form is sometimes caused by the intake of
drugs. Congenital absence of albumin or analbuminaemia is asymptomatic except for
occasional slight oedema.
The requirements of a dye binding method for albumin include specific binding of the dye
to albumin in the presence of other plasma or serum protein, high binding affinity between
dye and albumin so that small changes in ionic strength, pH or the presence of competing
Serum is preferred. Fasting specimen is not an absolute requirement but it may be desirable
because marked lipaemia interferes in the assay. Avoid, applying a tourniquet in specimen
collection because haemoconcentration due to venous stasis increases the apparent
concentration of albumin and other plasma proteins. Storage: Separate the serum within 2
hours of collection; separated serum in a tightly stoppered container is stable for 24 hours
at room temperature 25-30 0 C, for 1 week at 2-8 0C, for 3 months at -20 0C.
GLASSWARE:
Volumetric flasks (1 litre and 100 ml volumes)
Micro pipette (20 µl)
Graduated pipettes (10 ml in 0.1 ml)
Beakers (5ml, 50ml, 500 ml and 1 litre)
Amber colour reagent bottles (1 litre)
Test tubes (125 mm x 16 mm)
Measuring cylinders (1 litre)
CHEMICALS:
(All chemicals must be analytical grade)
Bromocresol green sodium salt, also called BCG, water soluble
Sodium azide caution: handle with care
Sodium chloride
Succinic acid
Sodium hydroxide pellets
Brij-35 (polyoxy7ethylene (23) lauryl ether) solid or solution 30% w/v
Standard buffers for pH meter
Bovine albumin or other available calibrator (fraction v powder)
REAGENTS
1. Succinic acid solution 50 g/l: weigh out 1.0 g of Succinic acid, dissolve and make up to
about 20 ml with distilled water. Prepare as required, discard after use.
2. Sodium hydroxide solution 10 g/l: Weigh out 1.0 g of sodium hydroxide in a glass beaker,
dissolve and make up to 100 ml with distilled water. This solution is stable for several
months at 20-25 C. Store in polypropylene bottle.
NOTE: The BCG working dye solution requires careful preparation. Some laboratories
may find it economical to purchase this solution. There are several different BCG reagents
available. Make sure you select a BCG reagent in Succinate buffer at pH 4.2.
In 10 mm light path cuvette (cell) the working dye solution should have the following
absorbance readings (zero the instrument with distilled water).
Spectrometer Colorimeter
Wavelength Absorbance Filter Absorbance
430 nm About 1.4 601 About 1.1
615 nm About 0.25 607 About 0.2
5. Succinate buffer solution: Prepare in exactly the same way as the working dye solution but
do not add any Bromocresol green. This solution is stable for several months at 2-8 0C
6. Albumin standard 40 g/l: Using a 5ml volumetric pipette dilute 5.0 ml of the bovine
albumin standard (80 g/l) with 5.0 ml of sodium chloride/sodium azide solution to
prepare an albumin standard containing 40 g/l. This standard is stable for 6 months at
2-8 0C. (Bovine albumin standard 80 g/l provide by the Department of Biochemistry,
MRI)
7. Sodium chloride /Sodium azide solution: Weigh out 9.0 g of sodium chloride and 1.0 g of
sodium azide, dissolve and make up to 1 litre with distilled water. This solution is
stable indefinitely at 20-25 0C (room temperature)
2.5 PROCEDURE
1. Pipette 4.0 ml of working dye solution into test tubes.
2. Add 20 µl of standard or control or test sample, mix and measure the absorbance
immediately (within 30 seconds).
3. Read the absorbance at 632 nm or filter No 607 after setting the instrument to zero
absorbance with the working dye solution.
Plot the absorbance of each tube against the concentrations of working standards solution
on the graph. The calibration graph should be linear up to 40 g/l. If the graph is linear then
a single standard (40g/l) may be used for routine analysis but linearity should be confirmed
for each new batch of working dye solution and at least once a month.
The recommended method proposes the use of a bovine albumin solution as a calibrator.
Alternative commercial bovine albumin solutions are available.
2.6 CALCULATION
Albumin concentration = T/S x 40 g/l
NOTE: Always include a standard (40 g/l) in each and every batch of samples.
QUALITY CONTROL
OPTIMAL CONDITIONS VARIANCE : A coefficient of variation of around 3%
should be attainable.
ROUTINE CONDITIONS VARIANCE : The value obtained for the RCV should not
exceed 6%
REFERENCE VALUES
New born : 25-50 g/l
1 Year : 35-50 g/l
2-3 Year : 36-50 g/l
4th Year and after: 37-50 g/l
Adult : 30-45 g/l
2.7 LIMITATIONS
If a serum sample is extremely lipaemic a serum blank should be used. (Moderate lipaemia
does not affect the results) The blank is prepared by adding 20 µl of sample to 4.0 ml of
Succinate buffer solution. The absorbance of this blank, with distilled water as reference is
subtracted from the test. Grossly haemolysed specimens are unsuitable for albumin
determination.
REFERENCES
Ann.clin.Biochem.14 (1977)105-115
Tietz text book of clinical chemistry
3. AMYLASE
3.1 INTRODUCTION
Amylases are a group of hydrolases that split complex carbohydrates constituted of α-D-
glucose units linked through carbon atoms 1 and 4 located on adjacent glucose residues.
Both straight-chain (linear) polyglucans, such as amylose, and branched polyglucans, such
as amylopectin and glycogen, are hydrolyzed, but at different rates. In the case of amylose,
the enzyme splits the chains at alternate α-1, 4-hemiacetal (-C-O-C-) links, forming maltose
and some residual glucose; maltose, glucose, and a residue of limit dextrins are formed if
branched –chain polyglucans are used as substrate. The α-1, 6- linkages at the branch
points are not attacked by the enzyme. Two types of amylases are recognized. Beta-amylase
(e.g. plant and bacterial exoamylase) acts only at the terminal-reducing end of polyglucan
chain; it splits off two glucose units (maltose) at a time. Animal amylases, including those
present in human tissues, are α-amylases. They are also called endoamylases because they
attack α-1, 4-linkages in a random manner anywhere along the polyglucan chain. Linear
starch chains in helical form react with molecular iodine to form the well-known deep blue
starch-iodine complex. The enzyme present in normal serum and urine is predominantly of
pancreatic (p-type) and salivary gland (S-type) origin. These isoenzymes are products of
two closely linked loci on chromosome 1. Each gene is allelic; thus there are 12 distinct
phenotypes for the salivary isoenzyme and 6 for the pancreatic isoenzyme.
GLASSWARE: CHEMICALS:
Volumetric flasks (1litre volume) Soluble starch pharmaceutical grade
Automatic micro pipette 20 µl Potassium iodide AR
Graduated pipettes (1ml, 5ml, 10 ml Potassium iodate AR
in 0.1 ml) Disodium hydrogen ortho-
Beakers (50ml, 1 litre) phosphate (anhydrous) AR
Graduated cylinders (100 ml and 1 Sodium chloride AR
litre) Benzoic acid AR
Amber colour reagents bottles (100 Hydrochloric acid (concentrated (37
ml and 1 litre) % w/v) caution: highly corrosive)
Buffers for pH meter
REAGENTS
1. Buffered starch substrate: Dissolve 26.6 g of anhydrous disodium hydrogen phosphate,
1.75 g of sodium chloride and 8.6 g of benzoic acid in about 500 ml of distilled water
in a large beaker. Heat to boil. In a 50 ml beaker, mix separately 0.4 g of soluble starch
in 10 ml of cold distilled water to form a paste. Add the paste with stirring to the
boiling mixture, rinsing the beaker with distilled water. Continue to boil for one
minute. Cool to room temperature, and transfer to volumetric flask and dilute to 1 litre
with distilled water. This solution is stable for at least one year at 20- 25 0C and should
have a pH of 6.9-7.1; the stability is monitored by noting the absorbance of the reagent
blank with each set of tests. We recommend that the solution is stored at 4-8 0C in the
refrigerator. Aliquot the required amount for daily use. If 1 litre substrate is excess
then prepare 500 ml for use.
2. Stock iodine solution 50 mmol/l: Dissolve 3.57 g of potassium iodate and 45 g of
potassium iodide in about 800 ml of water in a volumetric flask. Slowly and with
mixing add 9.0 ml of concentrated hydrochloric acid. Dilute to 1 litre with distilled
water. This solution should be stored in a dark bottle and is stable for a year at 4 -8 0C.
3. Working iodine solution: Dilute 10 ml of stock iodine solution with 90 ml distilled water
in a graduated 100 ml volumetric flask. This solution should be stored in a dark bottle
and is stable for 2 months at 2-8 0C.
3.6 PROCEDURE
1. Pipette 1.0 ml of buffered starch substrate into 150 x 16 mm test tubes. You will need
1 tube for each patient and control sample and 1 tube for a reagent blank.
2. Place all of the tubes in a water bath at 37 0C for 5 minutes to warm the contents.
3. Pipette 20 µl of patient’s or control serum into the bottom of the test tubes, mix and
incubate at 37 0C for exactly 7 minutes and 30 seconds. (No serum is added to the
reagent blank).
4. After 7 minutes and 30 seconds remove the test tubes from the water bath
immediately add 1.0 ml of working iodine solution to each tube (samples and reagent
blank) then add 8 ml of distilled water.
5. Mix the contents of each tube well then measure the absorbance without delay at 660
nm (red filter Ilford No. 608) setting the spectrometer to zero with distilled water.
3.7 CALCULATION
Amylase activity U/L = B-T x 1470
B
B = absorbance of reagent blank
T = absorbance of test
1470 = factor to express values in U/L
QUALITY CONTROL
A quality control sample with a value in the range 200 – 700 U/L should be analysed with
each batch of specimens. If single specimens are analysed a control specimen should
always be included.
REFERENCES
WHO Manual LAB/86.3
4.1 INTRODUCTION
The alkaline phosphatases (ALP) are a group of glycoprotein enzymes that act as
phosphotransferases by hydrolysing various types of monophosphate bonds at alkaline
pH. ALP activity is found in virtually all tissues, particularly bone, liver, kidney, intestine,
adrenal and placenta. The protein moieties comprise about 510 amino acid residues, to
which is attached various amounts of carbohydrate and sialic acid. Tissue-specific
posttranslational modifications occur to the carbohydrate content, leading to the formation
of isoforms, e.g. bone, liver and kidney ALP, each of which contains the same tissue non-
specific protein. ALP is found attached to the outer lipid bilayer of cell membranes by a
glycosyl-phosphatidylinositol group, in the case of liver ALP possibly as a tetramer of
identical subunits; if released from cell membranes, ALP is dimeric. Liver ALP is located in
the cell membranes of the hepatcoyte, and particularly in the outer layer of the cells
adjacent to the bile canaliculi and also in the cells lining the sinusoids. Adult intestinal ALP
lacks sialic acid and is found in the epithelial cells of the intestinal brush border. The
placental enzyme is formed by the syncitiotrophoblast cells lining the microvilli that
interface the placental and fetal blood circulations but the placental ALP does not cross
into the fetal circulation.
APPARATUS:
Water bath at 37 0C
pH meter
Spectrometer wavelength at 410 nm or Colorimeter with violet filter, Ilford 600 (410 nm)
REAGENTS
1. AMP Buffer pH 10.3: Dissolve 78.5 g of 2-amino-2-methyl-1-propanol (CH3)2 C (NH2)
CH2 OH in about 900 ml of distilled water. Adjust to pH 10.3 with concentrated
hydrochloric acid (about 18 ml) and make up to 1 litre with distilled water. Store in an
Amber colour reagent bottle. This solution is stable for 1 month at 20-25 0C.
Method done at MRI: Use the chemical with the specifications of 95 % (CH3)2C
(NH2)CH2OH with MW(89.14) and specific gravity of 0.93 g/ml (BDH). Add 84.4 ml
of the above liquid to 800 ml of distilled water.
2. Magnesium chloride solution 1.5mmol/l: Dissolve 300 mg of magnesium chloride
hexahydrate in water and make up to 1 litre. This solution is stable indefinitely at 20 -
250C. MgCl2.6H2O chemical and the solution (1.5mmol/l) are best stored in
refrigerator.
3. Substrate solution 225 mmol/l in the magnesium chloride solution: Dissolve 83.5 mg of
disodium 4 -Nitrophenyl phosphate hexahydrate (store the chemical in freezing
compartment) in 1.0 ml of magnesium chloride solution as required. This solution is
stable for one working day. It’s best to keep the solution in refrigerator since at room
temperature colour development may occur.
4. Sodium hydroxide solution 250 mmol/l: Dissolve 10 g of sodium hydroxide in distilled
water and make up to 1 litre. Store in a tightly Stopperd polyethylene bottle. This
solution is stable indefinitely at 20-25 0C. We have observed that this solution is stable
even at room temperature at 20-30 0C.
5. 4 – Nitrophenol stock solution 10.8 mmol/l: Weigh out 150 mg of 4 – Nitrophenol
accurately in a beaker & transfer the chemical from beaker to a 100 ml volumetric flask
using a funnel and wash any chemical remaining in the container into the volumetric
flask with distilled water. Make up to the mark with distilled water. This solution is
stable for about 6 months in an amber colour bottle at 4 0C.
6. 4 – Nitrophenol working solution 54 µmol/l: Pipette 0.5 ml of the 4 – Nitrophenol stock
solution into a 100 ml volumetric flask, make up to 100 ml with sodium hydroxide
solution (250mmol/l) Prepare this solution freshly before use.
4.6 PROCEDURE
1. Pipette 1.4 ml of AMP buffer into sufficient test tubes for patients’ samples, controls
and reagent blank and pre incubate in the water bath at 37 0C for about 5 minutes.
2. To each tube add 50 µl of serum, to the blank; add 50 µl of distilled water .Mix well.
3. To each tube in sequence add 100 µl of substrate solution at timed intervals. Mix well.
4. Incubate for exactly 15 minutes at 37 0C then add 4.0 ml of sodium hydroxide solution
(250mmol/l) to each tube in sequence, maintaining timed intervals. Mix each tube and
allow them to cool to room temperature.
5. Measure the absorbance of each test solution at 410 nm ( violet filter ,Ilford No :600)
setting the spectrometer to zero with the blank
6. If the absorbance is greater than the absorbance of a 400 U/L standard, then repeat
the procedures, but at step 4, incubate for exactly 5 minutes (instead of 15 minutes)
then add 4.0 ml sodium hydroxide solution (250mmol/l) and complete the procedure
described in steps 4 and 5. Multiply the activity by 3 before reporting the result.
4.7 CALCULATION
Read off the activities of alkaline phosphatase in the unknown and control samples from
the calibration graph. Multiply by 3 if you used 5 minute incubation instead of 15 minutes
incubation.
QUALITY CONTROL
At least two serum control specimens, having stated values in the range 20-350 U/L, one
of which is unknown to the operator should be included with each batch of specimens. If
single specimens are analysed a control specimen should always be included.
REFERENCES
LAB/86.3
A guide to diagnostic clinical chemistry By R.N. Walmsley and G.H. White -page 312
TIETZ TEXTBOOK OF Clinical chemistry page 831-832
5.1 INTRODUCTION
Aspartate Amino Transferase & Alanine Amino Transferase
The aminotransferases constitute a group of enzymes that catalyze the interconvertion of
amino acids and α- oxo-acids by transfer of amino groups. Aspartate amino transferase
(AST) and Alanine amino transferase (ALT) are the two enzymes that are of clinical
significance. Distinct isoenzymes of AST are present respectively in cytoplasm and
mytochondria of cells. The α- oxoglutarate/glutamate couple serves as one amino group
acceptor and donor pair in all amino-transfer reactions; the specificity of the individual
enzymes derives from the particular aminoacid that serves as the other donor of an amino
group.
AST
L-Aspartate + 2- Oxaglutarate Oxaloacetate + L –Glutamate
ALT
L-Alanine + 2- Oxaglutarate Pyruate + L –Glutamate
The reactions are reversible, but the equilibria of the AST and ALT reactions favour
formation of aspartate and alanine, respectively. Pyridoxal -5’ phosphate and its amino
analogue, pyridoxamine-5’ –phosphate function as coenzymes in the amino transfer
reactions. Transaminases are widely distributed in tissues. Both AST and ALT are normally
present in human plasma, bile, cerebrospinal fluid and saliva, but none is found in urine
unless a kidney lesion is present.
APPARATUS:
Water bath at 37 0C
pH meter
Visible spectrometer wavelength at 505 nm or Colorimeter with green filter Ilford No 604
(520 nm)
GLASSWARE:
Volumetric flasks (100 ml and 1 litre volumes)
Measuring cylinders (1 litre)
Automatic micro pipette (100 µl)
Graduated pipettes (1ml, 2 ml, 5ml, 10 ml in 0.1 ml)
Test tubes (150 x16 mm), Beakers (5ml, 10ml, 100ml and 1 litre)
Reagents bottles clear and amber coloured (250 ml and 1 litre)
Polypropylene bottle
CHEMICALS:
Disodium hydrogen ortho-phosphate-anhydrous, analytical grade
Potassium dihydrogen phosphate-anhydrous, analytical grade
Alpha – ketoglutaric acid -analar
DL- aspartic acid -analar, Sodium hydroxide pellets analar
2, 4- dinitrophenylhydrazine-AR; caution: may explode violently when dry,
Hydrochloric acid concentrated-AR (37% w/v), caution: highly corrosive
Sodium pyruvate (analytical grade)
REAGENTS
1. Phosphate buffer pH 7.4: Dissolve 11.9 g of disodium hydrogen phosphate (anhydrous)
and 2.2 g of potassium dihydrogen phosphate (anhydrous) in distilled water and make
up to 1 litre. Check the pH adjust to pH 7.4 if necessary using small amounts of the
appropriate phosphate (e.g. if the pH is more than 7.4 add potassium dihydrogen
phosphate). This solution is stable for about 2 months at 2-8 0C.
2. Sodium hydroxide solution 1 mol/l: Weigh 40 g of sodium hydroxide in beaker, slowly
dissolve in distilled water, transfer into a volumetric flask and make up to 1 litre. Store
in a tightly Stopperd polypropylene bottle. This solution is stable indefinitely at 20 -
250C, which can be achieved by air condition system. However we have observed that
this solution is stable in our room temperature (20-30 0C) for about 2 months.
3. Buffered substrate reagent: Weigh 29.2 mg of alpha- ketoglutaric acid 2.66 g of DL-aspartic
acid into a small beaker. Dissolve in 20 ml of sodium hydroxide solution (1 mol/l)
then adjust to pH 7.4 with more sodium hydroxide solution. Transfer to a 100 ml
volumetric flask and make up to 100 ml with phosphate buffer and mix well. Store the
reagent frozen in screw capped bottles; the volume should be the amount need for a
day’s analysis.
4. Hydrochloric acid 1 mol/l: Dilute 9 ml of hydrochloric acid (concentrated (37% w/v)) to
100 ml with distilled water.
5. Colour reagent 2,4 –dinitrophenylhydrazine 1 mmol/l: Dissolve the equivalent of 19.8 mg of
dry 2,4-dinitrophenylhydrazine in 100 ml of hydrochloric acid ( 1mol/l)and transfer
into a amber colour bottle; Note that the weight of 2, 4 – dinitrophenylehydrazine
must be adjusted to take into account the water content. e.g. If the label reads as 33%
by weight of water is added to ensure safety in transit the calculation is as
follows.100/67 x 19.8 = 29.55 mg. The prepared solution is stable for 2 months at 2 -
8 0C.
6. Sodium hydroxide solution 400 mmol/l: Dissolve 16.0 g of sodium hydroxide in distilled
water in a beaker and make up to 1 litre. Store in a tightly stoppered polypropylene
reagent bottle. This solution is stable indefinitely at 20- 25 0C. We have observed that
this solution is stable at our room temperature at 20-30 0 C for 2 months.
7. Pyruvate standard solution 4 mmol/l: Weigh out 44 mg of sodium pyruvate in a beaker,
transfer into a 100 ml volumetric flask and make up to the mark with phosphate
buffer. Mix well, divide into small portions (about 1ml) and store in the freezer
compartment of the refrigerator. The standard solution is stable for 6 months in the
freezer.
CALIBRATION
In this method the amount of pyruvate formed is calculated by comparing the absorbance
of the samples (Test- Blank absorbance) with that of the pyruvate standard (4mmol/l).
However alpha - ketoglutarate also contributes to the absorbance and the change in
absorbance is not linearly related to enzyme activity expressed in U/L. The table must
therefore be used to convert the amount of pyruvate formed into U/L.
5.7 CALCULATION
Use the table below to convert the amount of pyruvate into U/L (expressed as
µmol/minute/litre at 37 0C)
Calculated pyruvate AST result (U/L at Calculated pyruvate AST result (U/L at
(µmol/min/litre) 37 0C) (µmol/min/litre) 37 0C)
2 4 28 52
4 6 30 56
6 10 32 60
8 12 34 64
10 15 36 69
12 19 38 73
14 23 40 77
16 27 42 81
18 31 44 85
20 35 46 92
22 40 48 98
23 42 50 106
24 44 52 114
26 48 54 125
REFERENCES
Reitman, .S. & Frankel. S. (1957) Am. J. Clin.Pathol., 28, 56-63
REAGENTS
1. Phosphate buffer pH 7.4: Dissolve 11.9 g of disodium hydrogen phosphate (anhydrous)
and 2.2 g of potassium dihydrogen phosphate (anhydrous) in distilled water and make
up to 1 litre. Check the p H and adjust to pH 7.4 if necessary using small amounts of
the appropriate phosphate (e.g. if the pH is more than 7.4 add potassium dihydrogen
phosphate). This solution is stable for about 2 months at 2-8 0C.
2. Sodium hydroxide solution 1 mol/l: Slowly dissolve 40.0 g of sodium hydroxide in distilled
water in a beaker and make up to 1 litre. Store in a tightly stopperd polypropylene
bottle. This solution is stable indefinitely at 20-25 0C. we have observed this solution is
even stable at our room temperature 20-30 0C
3. Buffered substrate reagent: Weigh out 3.56 g of DL Alanine in a beaker and weigh 30 mg
of alpha ketoglutaric acid accurately in another small beaker. Transfer it to same
beaker by dissolving in the phosphate buffer. Add 0.5 ml of sodium hydroxide
(1mol/l) solution. Check the pH. The pH should be at 7.4.Make up to 100 ml with
phosphate buffer and mix. Divide the prepared substrate into small volumes and store
in the freezing compartment or in the freezer. Discard the remaining substrate after
use. Do not freeze the remaining substrate again.
6.4 PROCEDURE
1. Two test tubes are required for each serum or control sample( one for the “Test” and
one for a sample blank) one for a reagent blank and one for the standard
2. Transfer 0.5 ml buffered substrate to each tube and pre-incubate in the water bath (37
0C) for 5 minutes
3. Add 100 µl of patient’s or control serum to the “Test” tubes or 100 µl water(reagent
blank) or 100 µl pyruvate standard (standard) mix and incubate at 37 0C
4. After exactly 30 minutes add 0.5 ml colour reagent to each tube, mix and remove from
the water bath.
5. Add 100 µl of patient’s or control serum to the sample blank tubes.
6. Leave for 20 minutes at room temperature and then add 5.0 ml of sodium hydroxide
solution (400mmol/l) and mix thoroughly.
7. Leave the tubes at room temperature for at least 5 minutes, but not longer than 30
minutes, and then read the absorbance at 505 nm. Set the spectrometer to zero with
the reagent blank.
6.5 CALCULATION
Amount of pyruvate formed = (A Test-A Sample Blank) x 4x1x1000
(µmol/min/litre) A Standard 30
= A Test- A Sample Blank x 133
A Standard
With the reagent blank set to zero the spectrophotometer at 505 nm
A=absorbance
Calculated pyruvate ALT result (U/L at Calculated pyruvate ALT result (U/L at
(µmol/min/litre) 37 0C) (µmol/min/litre) 37 0C)
2 2 54 42
4 4 56 44
6 4 58 46
8 5 60 47
10 7 62 49
12 7 64 53
14 9 66 55
16 11 68 56
18 13 70 60
20 13 72 62
22 15 74 64
23 15 76 66
24 16 78 67
26 16 80 69
28 18 82 71
30 20 84 73
32 22 86 76
34 24 88 80
36 25 90 84
38 27 92 87
40 29 94 91
42 31 96 95
44 33 98 98
46 35 100 102
48 36 102 109
50 38
52 40
NOTE: When the activity of a sample exceeds 109 U/L the measurement should be repeated
with 10 minute incubation (instead of 30 minutes) and the results multiplied by 3. When the
activity is greater than 750 U/L, the serum should be diluted 1 in 10 with sodium chloride
solution (150mmol/l), an incubation time of 10 minutes should be used and the result
multiplied by 30.
QUALITY CONTROL
At least two serum control specimens, having stated values in the range 20-109 U/L, one
of which is unknown to the operator, should be included with each batch of specimens.
Even if single specimens are analysed a control specimen should always be included
REFERENCES
King and wooton- enzymology
7.1 INTRODUCTION
Haem, released from aged red cells and maturing cells during erythropoiesis, or from
degraded haemoproteins is converted to biliverdin in the reticuloendothelial system.
Biliverdin is reduced to bilirubin which is secreted into the plasma where it is transported
to the liver reversibly bound to albumin. The hepatocytes take the bilirubin up from the
plasma, conjugate it to glucuronic acid and excrete it in the bile. There are six steps in this
process namely; production, transport to the liver, hepatocyte uptake, conjugation, biliary
secretion, gut degradation and excretion.
The total bilirubin produced is around 250-350 mg/day (4 – 6 mmol/day). 15% to 20% is
derived from immature red cell and haemoproteins (early labelled fraction) whilst the
remainder comes from senescent red cells (1g of haemoglobin produces 620µmol of
bilirubin) Haem is converted to bilirubin in the reticuloendothelial system by two enzymes,
haem oxygenase and biliverdin reductase. Haem oxygenase breakes the alpha – CH bridge
of protoporphyrin IX to produce biliverdin IX α, which is then reduced to bilirubin IX α
by biliverdin reductase. Some cleavage occurs at the β, γ and δ bridges, but insignificant
amounts of these isomers are produced. Although bilirubin IX α has two polar propionic
acid side chains it is poorly soluble in water because of intramolecular hydrogen bonding
between the propionic acid residues and other parts of the molecule. This bonding may
also account for the necessity for conjugation with glucuronic acid prior to biliary
excretion. Bilirubin is transported to the liver reversibly bound to albumin. This protein
has one high – affinity binding site and an additional low – affinity site which is activated at
high concentrations of bilirubin. The amount of unbound bilirubin is low; approximately 4
nmol/l at a total plasma concentration of 20 µmol/L. Compounds such as free fatty acids,
sulphonamides, salicylate and ampicillin will displace bilirubin from its binding sites. This
species of bilirubin is called unconjugated bilirubin or indirect reacting bilirubin. The
bilirubin – albumin complex dissociates in the liver and the bilirubin is transported across
the hepatocyte membrane into the cell where it is reversibly bound to cytosolic proteins;
one such protein being ligandin. The function of these proteins appears to be prevention
of efflux of bilirubin from the cell. Bilirubin is conjugated in the endoplasmic reticulum
with glucuronic acid and to a lesser extent with glucose and xylose. The enzyme
responsible is UDP – glucuronosyltransferase which esterifies one or both propionic acid
side chains to produce di – and monoglucuronides. In man the product is predominantly
bilirubin diglucuronide with lesser amounts of the monoglucuronide. The bilirubin
conjugates are secreted into the biliary passages by an active process which is yet to be
clarified. This transport mechanism differs from that responsible for the biliary excretion
of bile acids. In the gut the bacterial flora reduce the bilirubin conjugates to urobilinogens
which are excreted in the faeces as such. Some of the urobilinogens are reabsorbed from
the gut to be re – excreted by the liver (enterohepatic circulation); as these compounds are
water soluble they will also be excreted by the kidney, but this is an unimportant excretory
route. Two points worthy of note are that conjugated bilirubin is more prone to reductive
processes than unconjugated bilirubin and that unconjugated bilirubin can be reabsorbed
from the gut. Thus, if bacterial flora are absent (e.g.: neonate, antibiotic therapy)
deconjugation of bilirubin glucuronides by intestinal mucosal ß – glucuronidase may occur
and reabsorption of the unconjugated compound result in an increase in the plasma
unconjugated bilirubin level. The total plasma bilirubin concentration in the normal subject
is usually less than 20 µmol/L. The clinical sign of jaundice appears when the plasma total
bilirubin rises beyond 40 µmol/L. When normal plasma is evaluated by high performance
liquid chromatography techniques four bilirubin fractions alpha, beta, gamma and delta are
obtained.
Delta fraction
The delta fraction, usually referred to as ‘delta’ bilirubin – not to be confused with bilirubin
IX delta –is an interesting compound with the following characteristics:
it is direct reacting
it is tightly bound to albumin
its plasma level is increased in diseases associated with high plasma levels of
conjugated bilirubin
It appears to be derived from conjugated bilirubin as it can be produced by incubating
bilirubin glucuronides with plasma albumin. It is thought that the bilirubin migrates from
the glucuronic acid residues and becomes covelantly bound to albumin. As noted above
this fraction increases in conditions associated with chronic conjugated
hyperbilirubinaemia (eg: cholestasis) and, as it is tightly bound to albumin, it has a t1/2
comparable to that of protein (20 days) .Thus, after formation delta bilirubin ‘hangs around
‘as it is not cleared by the liver or the kidney. The bile pigments that may be found in the
urine are urobilinogen and conjugated bilirubin. Unconjugated Bilirubin is water insoluble
and bound to albumin, and is thus not available for urinary excretion. The normal subject
excretes 1 – 4 mg (2 – 7 µmol) of urobilinogen daily (derived from the enterohepatic
circulation) Increased values occur in liver disease (inability to excrete the small amounts
reabsorbed from the gut) and in haemolytic disease (increased bilirubin production).
Decreased excretion occurs in bile duct obstruction. (cholestasis) Bilirubin appears in the
urine when the plasma level of conjugated bilirubin rises as in hepatocellular disease and
cholestasis.
GLASSWARE:
Volumetric flasks (100 ml, 500 ml and 1 litre volumes)
Beakers (5ml, 100 ml and 1 litre)
Automatic micro pipettes (50 and 100 µl)
Graduated pipettes (1ml and 10 ml in 0.1 ml)
Test tubes (100 x 13mm) & Rubber bulb
Reagent bottle, clear and amber coloured
REAGENTS
1. Caffeine- benzoate reagent: Dissolve 93 g of sodium acetate trihydrate, 56 g of sodium
benzoate and 1 g of disodium EDTA in approximately 500ml of distilled water. Add
38 g of caffeine. Dissolve and dilute to 1 litre in a volumetric flask. Mix well and filter.
This solution is stable for at least 6 months at 20-25 0C (Room temperature)
2. Sulfaninlic acid reagent: Add 2.5 g of sulfanilic acid to about 200 ml of distilled water in a
500 ml volumetric flask. Using a rubber bulb carefully pipette 7.5 ml of concentrated
hydrochloric acid into the flask. Dissolve and make up to 500 ml. This solution is
stable for up to 6 months at 20-25 0C (Room temperature)
Bilirubin working
(1) (2) (3) (4) (5) (6) (7)
Standard
Bilirubin standard
0 0.5 1 1 2 3 4
Solution 342µmol/l(ml)
Bovine serum Albumin (ml)
4 9.5 9 3 2 1 0
(BSA solution)
Concentration of Bilirubin
Working standard solution 0 17.1 34.2 85.5 171 256.5 342
(µmol/l)
A calibration graph is prepared from the Bilirubin working standards using the volumes of
standard and reagents described in the table below:
Tube No 1 (Blank) 2 3 4 5 6 7
Caffeine
benzoate 1.0 1.0 1.0 1.0 1.0 1.0 1.0
Reagent (ml)
Bilirubin
working 100 100 100 100 100 100 100
Standard (µl)
Mix well, protect the tubes from light then add
Diazo reagent
- 0.5 0.5 0.5 0.5 0.5 0.5
(ml)
Mix well and allow the solutions to stand at room temperature for 10 minutes. Protect the tubes from light.
Sulfanilic acid
0.5 - - - - - -
Reagent(ml)
Mix well
Alkaline
tartrate 1.0 1.0 1.0 1.0 1.0 1.0 1.0
Reagent (ml)
Mix well. Read the absorbance of each tube at 600 nm after setting the spectrometer to zero with the blank
(Tube No 1)
Plot the absorbance of the each tube on the vertical axis against the concentrations in
µmol/l of working standards on the horizontal axis
o The calibration graph should be prepared whenever the new batch of reagents are
prepared or any changes made in the spectrophotometers
o Freshly prepare all reagents and use clean glassware
o Measure the standards (each concentration) in duplicate
o Check the calibration graph by measuring a quality control serum
7.7 CALCULATION
If the calibration graph is linear, calculate the results using the following formula:
Concentration of Bilirubin (µmol/l) = T – TB x 342
S – SB
Where:
T =Absorbance reading of sample or control
TB =Absorbance reading of control or patient sample blank
S =Absorbance reading of Bilirubin standard (342 µmol/l)
SB =Absorbance reading of standard blank
If the calibration graph is not linear, then results should be read from a calibration curve
prepared using working standards 1, 2, 3, 5 and 6.
QUALITY CONTROL
At least two serum control specimens having stated values in the range 20 – 200 µmol/l,
one of which should be unknown to the operator, should be included with each batch of
specimens. If single specimens are analysed a control specimen should always be included.
ROUTINE CONDITIONS VARIANCE: The value obtained for the RCV should not exceed
12%
REFERENCE VALUES
Total bilirubin in adults: 3 – 21 µmol /l
Conversion from SI units into ‘old’ units: µmol/l x 0.0585=mg/dl
REFERENCES
WHO manual LAB /86.3
8.1 INTRODUCTION
Calcium is the fifth most common element and the most prevalent cation found in the
body. An average human body contains approximately 1kg (24.95 mol) of calcium.
Calcium is found in three main compartments: the skeleton, soft tissues, and the extra
cellular fluid. The skeleton contains 99% of the body’s calcium, predominantly as extra
cellular crystals of unknown structure with a composition approaching that of
hydroxyapatite. Soft tissues and extra cellular fluid contain about 1% of the body’s calcium.
In blood, virtually all of the calcium is in serum or plasma, which has a mean normal
calcium concentration of approximately 9.5mg/dL (2.37 mmol/L). Calcium exists in three
physiochemical states in plasma, of which approximately 50% is free or ionised. Another
40%is bound to plasma proteins, chiefly albumin. Because calcium binds to negatively
charged or anionic sties on albumin, it’s binding is pH-dependant. Alkalosis leads to an
increase in binding and a decrease in free calcium; conversely, acidosis leads to a decrease
in binding and an increase in free calcium. For each 0.1-unit change in pH, approximately
0.2-mg/dL (0.05mmol/L) of inverse change occurs in the serum free calcium
concentration. Approximately 20% of protein-bound calcium in serum is associated with
globulins. In some patients with multiple myeloma, the high concentrations of serum
globulin may bind sufficient calcium, about 10%, is complexed with small diffusible anions
including bicarbonate, lactate, phosphate and citrate. Calcium can be redistributed among
the three plasma pools, acutely of chronically, independently affecting the quantities of free
calcium and total calcium in the serum. The skeleton is a major storehouse for providing
calcium for the extra cellular and intracellular pool. Approximately 5g of calcium is rapidly
available from the skeletal exchangeable pool, which is accessible for maintaining normal
physiological functions. Intracellular calcium has many important physiological functions
within the cells, including muscle contraction, hormone secretion, glycogen metabolism
and cell division. Extra cellular calcium provides a source for maintenance of intracellular
calcium. In addition, it has an important role in providing calcium ions for bone
mineralization, coagulation cascade and maintaining plasma membrane potential. Calcium
stabilises the plasma membranes and influences permeability and excitability. A decrease in
serum free calcium concentration increases neuromuscular excitability and tetany. An
elevated free calcium concentration results in reduced neuromuscular excitability.
GLASSWARE:
Volumetric flask class A (100ml, 500ml and 1 litre volumes)
Automatic micro pipettes (50 µl)
Volumetric pipettes (5 ml, 10ml)
Graduated pipettes (1 ml, 2 ml, 5ml and 10 ml in 0.1ml)
Beaker (5ml, 250 ml)
Measuring cylinder (50ml), Test tubes (125 x 16 mm)
CHEMICALS:
Hydrochloric acid concentrated (37% w/v); caution: highly corrosive
O-cresolphthalein complexone-AR
Ethanediol-AR
2-amino-2-methyl-1-propanol-AR
8-hydroxyquinoline-AR
Calcium carbonate-AR
NOTES: All glassware must be thoroughly cleaned then soaked overnight in hydrochloric
acid (0.5mol/l) to remove traces of calcium then thoroughly rinsed with distilled or
deionised water and finally dried before use.
REAGENTS
1. Hydrochloric acid 0.5 mol/l: Adding the acid to the distilled water, dilute about 45 ml of
hydrochloric acid (concentrated) to 1 litre with distilled water. Use this solution for
soaking glassware as described above.
2. Stock CPC reagent: Add 38 ml ethanediol and 13 ml of 2- amino-2-methyl-1-propanol to
a 500 ml volumetic flask containing about 400 ml of distilled water. Weigh out 15 mg
of o-cresolphthalien complexone and add it to the volumetric flask, mix until the all
the chemicals are completely dissolved, make up to the mark with distilled water and
8.6 PROCEDURE
1. The analysis must be performed in duplicate. Transfer 50 µl of serum or plasma to
each of two clean tubes, add 5.0 ml working CPC reagent to each tube using a 5 ml
volumetric pipette and mix well.
2. Transfer 50 µl of working calcium standard (2.5mmol/l) to each of 2 clean tubes; add
5.0 ml working CPC reagent to each tube using 5 ml volumetric pipette and mix well.
3. Transfer 50 µl of distilled water to a clean tube, add 5.0 ml working CPC reagent, Mix
well. This is the reagent blank.
4. Set the spectrometer to zero at 575 nm with distilled water and measure the
absorbance of the reagent blank which should be about 0.2.
5. Measure the absorbance of the standards (2.5mmol/l) and serum sample
If the absorbance of duplicate readings varies by more than 0.015 then precision is
unsatisfactory. Check that the test tubes and pipettes are clean. Check the precision of the
50 µl volumetric pipette. Use a 5.0 ml volumetric pipette for the working CPC reagent.
Check that the spectrometer cuvettes (cells) are clean.
Tube Number 1 2 3 4 5
8.7 CALCULATION
Calculate the results using the following formula:
Concentration of calcium (mmol/l) = T-B x 2.5
S-B
Where:
T = Absorbance reading of sample or control
S = Absorbance reading of working calcium standard (2.5 mmol/l)
B = Absorbance reading of reagent blank
If the sample or control result is above the linearity of the method then repeat the analysis
after accurately diluting 200 µl of sample with 200 µl of distilled water in a clean tube. Use
50 µl of the diluted sample for the analysis. Remember to multiply the result by 2 to obtain
the calcium concentration of the sample.
QUALITY CONTROL
At least two serum control specimens, having stated values in the range 2.00-2.90 mmol/l
one of which is unknown to the operator, should be included with each batch of
specimens. If single specimens are analysed a control specimen should always be included.
ROUTINE CONDITIONS VARIANCE : The value obtained for the RCV should not
exceed 4%
REFERENCE VALUES
The reference interval for healthy ambulant adults is 2.25-2.60 mmol/l. Conversion of SI
units into “old” units: mmol/l x 4 = mg/dl.
The reference values are only appropriate if the patient has a normal serum albumin
concentration. If the patient has a low serum albumin then it may be helpful to report the
albumin corrected calcium as well as the measured calcium. The albumin corrected calcium
is calculated in this way:
(40 – Patient’s albumin) + measured calcium = albumin corrected calcium
40
For example if the patient has an albumin of 20 g/l and a measured calcium of 1.90
mmol/l, then the albumin corrected calcium is 2.40 mmol/l
(40-20) + 1.90 = 2.40 mmol/l
40
The albumin corrected calcium will be approximately 2.20-2.60 mmol/l if low measured
calcium is a consequence of a low serum albumin.
PRECAUTIONS
Use dry glass container which must be chemically cleaned and acid washed
Avoid use of plastic containers and use of rubber stoppers
Avoid use of acid etched glassware. It may lead either to calcium loss because
adsorption of calcium on to the damage surface or to contamination with calcium
because the etched areas cannot be cleaned thoroughly.
REFERENCES
LAB/86.3
CALCIUM IN URINE
SPECIMENS: a 24 hour urine collection in an acid washed 2.5 L bottle; 10 ml of 1 N HCl
is added as the preservative. Acid washed beaker and a funnel should be issued from the
laboratory along with collection bottle.
PROCEDURE:
Mix the 24 hour urine collection and measure the total urine volume
Follow the procedure as for serum calcium analysis
Urine samples with calcium concentration greater than 3.75 mmol/l should be diluted with
distilled water before analysis. Multiply the results by the dilution factor
CALCULATION:
Concentration of calcium (mmol/l) = T-B x 2.5
S-B
Concentration of calcium in 24 hour urine = T-B x 2.5 x (TV in litre) mmol/24 hour
S-B
Where:
T = Absorbance reading of sample or control
S = Absorbance reading of working calcium standard (2.5 mmol/l)
B = Absorbance reading of reagent blank
TV =24 hour urine total volume in litre
In children concentration of urinary calcium= T-B x 2.5 x TV in litre x 1000 µmol/kg/24 hours
S-B Body weight in kgs
REFERENCE VALUES:
New Born : 0-17.5 µmol/kg/24 hours
Infants : up to 1000 µmol/kg/24 hours
Older Children : up to100 µmol/kg/24 hours or 750-3750 µmol/24hours
Adults
Calcium in diet
Calcium free : 0.13-1.0 mmol/24 hours
Low –average : 1.25-3.75 mmol/24 hours
Average (800mg/day) : 2.5-7.5 mmol/24 hours
9.1 INTRODUCTION
Creatine is synthesized in the kidneys, liver and pancreas by two enzymatically mediated
reactions. In the first, transamidination of arginine and glycine forms guanidinoacetic acid:
the second methylation of guanidinoacetic acid occurs with S- adenosylmethionine as
methyl donor. Creatine is then transported in blood to other organs such as muscle and
brain, where it is phosphorylated to phosphocreatine a high energy compound.
Interconversion of phosphocreatine and creatine is a particular feature of metabolic
processes of muscle contration; some of the free creatine in muscle spontaneously converts
to creatinine, its anhydride. Between 1 and 2% of muscle creatine is converted to creatinine
daily. Because the amount of endogenous creatinine produced is propotional to muscle
mass, the production varies with age and sex; non obese men excrete about 1.5 g/day,
women 1.2 g/day. Daily excretion of creatinine can be 10% to 30% greater as a result of
dietary intake of creatine and creatinine in meats. On the whole however, dietary
fluctuations of creatinine intake cause only minor variation in daily creatinine excretion on
the same individual. The excretion rate in one individual, in the absence of renal disease, is
relatively constant and parallels endogenous production. Most of the interindividual
variations of creatinine excretion in healthy persons are attributable in the main to age, sex,
and lean body mass and intraindividual variation tends to be less than 15% from day to
day.
9.2 CLINICAL SIGNIFICANCE
As creatinine is endogenously produced and released into body fluids at a constant rate and
its plasma levels maintained within narrow limits, its clearance may be measured as an
indicator of glomerular filtration rate. However, a small quantity of creatinine is reabsorbed
by the tubules and a small quantity of creatinine appearing in the urine(7-10%)is due to
tubular secretion. As a result creatinine clearance (if creatinine is measured with an accurate
method) is approximately 7% greater than inulin clearance. Some methods for creatinine
used in clinical laboratories are nonspecific, however, and thus this difference is often
smaller. The creatinine clearance is performed by obtaining a 4-, 12- or 24-h urine
specimen and also a blood specimen within the period of urine collection. The volume of
the urine is measured, urine flow rate is calculated (millimetres per minute), and the assay
for creatinine is performed on plasma and urine to obtain the concentration in milligrams
per decilitre or per millilitre. Two factors influence measurement of creatinine clearance
and thus its interpretation. First, the most common methods for measuring creatinine use
the nonspecific alkaline picarate reaction, and thus noncreatinine chromogens in plasma
increase the apparent plasma concentration by as much as 30% if serum values are less
than 1.0mg/dL, and by approximately 10% is values exceed 1.0mg/dL. The percent
increase is progressively less with higher creatinine concentrations. (Urine contains
considerably fewer noncreatinine chromogens.) This overestimation of plasma creatinine
concentration results in underestimation of creatinine clearance and partially offsets the
apparent high clearance of creatinine that is due to tubular secretion. As a result, the
endogenous creatinine clearance agrees closely with the inulin clearance throughout a
substantial range of clearances. However, if accurate methods are used for assay of plasma
creatinine, the GFR estimated by creatinine clearance may not correlate with the GFR
estimated by inulin clearance. Secondly, GFR measured by creatinine clearance and GFR
measured by inulin clearance in the same patient progressively diverge as renal failure
progresses and plasma creatinine level rises. The greater apparent GFR found by creatinine
clearance may be due to an increase in tubular secretory activity for creatinine when plasma
levels increase much above normal and to the relatively smaller contribution of
noncreatinine chromogens in the nonspecific assay of plasma creatinine. In clinical
practice, it is now accepted that, by the time patients have lose one half to two thirds their
normal renal function, as demonstrated by creatinine clearance, it is more reliable and
APPARATUS:
Spectrophotometer at wavelength 500 nm or Colorimeter with blue green filter, Ilford 603)
GLASSWARE: CHEMICALS:
Volumetric flask (100 ml, 500 ml Sodium hydroxide pellets
and 1litre volumes) Picric acid; Note: water is added to
Automatic micro pipettes (50µl, picric acid to ensure safety in transit
100µl, 500 µl and 1000µl-5000µl) Creatinine anhydrous -AR
Graduated pipettes (5ml, 10 ml in Hydrochloric acid concentrated
0.1 ml) (37% w/v) caution: highly corrosive
Beakers (100ml, 500 ml) Sulphuric acid-AR
Test tubes (125 mm x 16 mm) Sodium tungstate dihydrate
Conical centrifuge tubes 15 ml Polyvinyl alcohol
Reagent bottles, clear and amber
coloured (500 ml), Rubber bulb
REAGENTS
1. Saturated picric acid solution: Picric acid is supplied as a moist chemical. Weigh out the
equivalent of 7 g of picric acid i.e. mix well the bottle and weigh out about 10.5 – 11 g
if your picric acid container states that 50 % by weight of water has been added. Add
11 g of moist picric acid to 500 ml distilled water, stir for several hours to ensure that a
saturated solution is produced. Transfer in to a brown bottle. This solution is stable
indefinitely at room temperature.
Note: the amount of picric acid weighed out will depend on the water content of the
chemical
2. Picric acid 0.036 mol/l: Measure 705 ml of saturated picric acid solution in to volumetric
flask and make up to 1 litre with distilled water store in a brown bottle. This solution is
stable indefinitely
3. Acid tungstate solution: Weigh out 11.1 g of sodium tungstate dihydrate (9.8 g anhydrous
salt) and dissolve in about 300 ml of distilled water in a 1 litre volumetric flask.
Dissolve 1 g of polyvinyl alcohol in about 100 ml of distilled water with heating (do
not boil) Allow to cool to room temperature then transfer into the volumetric flask
containing sodium tungstate. Measure 2.1 ml of concentrated sulphuric acid in to 300
ml of distilled water in a beaker mix well. Add this solution also-in to the same
volumetric flask containing tungstate solution. Dilute to 1 litre with distilled water
when the solution is cool to room temperature. Store in a brown bottle.
4. Sodium hydroxide solution 1.4mol/l: Dissolve 56 g of sodium hydroxide in distilled water
and dilute to 1 litre, store in a polypropylene bottle.
QUALITY CONTROL
OPTIMAL CONDITIONS VARIANCE: A coefficient of variation of around 4% should be
attainable.
ROUTINE CONDITIONS VARIANCE: This value should not exceed 8%
9.8 LIMITATIONS
Proteins give positive Jaffe reaction therefore
The supernatant fluid should be clear
Pipetting of 3 ml of supernatant should be done carefully without disturbing the
precipitate.
Sodium hydroxide solution should be added in timed intervals
Absorbance reading should be measured exactly 15 minutes after addition of Sodium
hydroxide
Interfering substances reacting like Creatinine are acetone, acetoacetate, pyruvate and
some cephalosporin antibiotics contribute to total colour production
Blood constituents such as glucose, ascorbate histidine and adrenaline may cause
disturbances to the colour development.
PRESERVATIVE
1 Normal hydrochloric acid (1 N)-10 ml or Thymol in Isopropanol (100g/l)-5 ml
10.4 PROCEDURES
1. Mix the 24 hours urine collection. Measure the total urine volume.
2. Pipette 1 ml of urine into a 100 ml volumetric flask. Make up to 100 ml with distilled
water. Mix well (1:100 dilution)
3. Three test tubes are required. One for the Test, one for a reagent blank, and one for
the standard
4. Transfer 3.0 ml of diluted urine into the Test. 3.0 ml of distilled water into the reagent
blank. 3 ml of distilled water and 0.1 ml of standard solution into the standard.
5. To each tube add 1.0 ml picric acid solution (0.036mol/l). Mix well.
6. Add 0.5 ml sodium hydroxide solution (1.4mol/l) to the first tube, mix well at 30
seconds intervals and add sodium hydroxide solution to the remaining tubes.
7. Exactly 15 minutes after adding sodium hydroxide read the absorbance against the
reagent blank at 500 nm. Read the tubes in sequence maintaining 30 seconds
intervals between readings.
10.5 CALCULATION
Same method as serum Creatinine but 3 ml of diluted urine (1:100 with distilled water) is
used instead of filtrate
No protein precipitation is needed
Concentration of standard = 1.32 mmol/l (1320 µmol/l)
Standard dilution is = 1 in 30 (i.e. 0.1 ml standard diluted with 3.0
ml of distilled water)
∴standard concentration = 1.32 mmol/l = 0.044 mmol/l (in assay condition)
30
urine dilution is = 1: 100
∴ urine Creatinine = Test x 0.044 x 100 mmol/l
Standard
24 hour Urine Creatinine = Test(T) x 4.4 x Total volume in litre
(mmol/24 hour) Standard(S)
CHILDREN
Urine Creatinine = T x 4.4 x Total volume in litre x 1000
(µmol/kg/24 hours) S x Body weight in kgs
Clearance varies with body size and is proportional to the body area (A) where this varies
much from the normal in adults and in all cases in children.
The determined clearance is corrected to a standard surface area of 1.73 m2 by multiplying
1.73/A. The A [Body surface area (in m2) can be calculated using a nomogram. From
height (in cm) and weight (in kg)]
REFERENCES
WHO manual
Varley’s practical clinical Biochemistry sixth edition
11. CHOLESTEROL
11.1 INTRODUCTION
Although every living organism examined has been found to contain sterols, cholesterol is
found almost exclusively in animals and humans, in which it is also the main
sterol.Virtually all cells and body fluids contain some cholesterol.Like other
sterols,cholesterol is a solid alcohol of high molecular weight and possesses the tetracyclic
perhydrocyclopentanophenanthrene skeleton. The molecule contains 27 carbon
atoms.Cholesterol is the initial starting point in many metabolic pathways. These include
vitamin D synthesis, steroid hormone synthesis, and bile acid metabolism.
Cholesterol is presented to the intestinal wall from three sources: the diet, bile and
intestinal secretions and cells. Animal products, especially meat, egg yolk, seafood, and
whole-fat dairy products, provide the bulk of dietary cholesterol. Cholesterol intake varies
considerably according to the dietary intake of animal products. A similar amount of
cholesterol is present in the gut from biliary secretion and the turnover of mucosal cells.
Practically all cholesterol in the intestine is present in the unesterified (free) form.
Esterified cholesterol in the diet is rapidly hydrolyzed in the intestine to free cholesterol
and free fatty acids (FFA) by cholesterol esterase in pancreatic and small intestinal
secretions.
Although portion of the body’s cholesterol is derived from dietary intake, most tissue and
plasma cholesterol is synthesized endogenously by the liver and other tissues from simpler
molecules, particularly acetate.
Once synthesized cholesterol is released into the circulation for transport in combination
with specific apoproteins, the apolipoproteins, in complexes known as lipoproteins.
Minimal cholesterol esterification occurs within the liver before its release, and cholesterol
is mainly esterified within the vascular compartment. Esterification is important because it
serves to enhance the lipid carrying capacity of the lipoproteins. The reaction is catalyzed
by the enzymes lecithin-cholesterol acyltransferase (LCAT) in the plasma and acyl-
cholesterol acyltransferase (ACAT) intracellularly.The intracellular ACAT pathway is the
major pathway in the liver, intestine, adrenal cortex, and probably in the arterial wall.
Once cholesterol enters the cell, the esters are hydrolyzed by the action of specific esterases
and enters into specific metabolic pathways.
Cholesterol reaching the liver is either secreted unchanged into bile or metabolized to bile
acids. Approximately one third of the daily production of cholesterol is catabolized into
bile acids. The first step in the bile acid synthesis involves the rate limiting step, 7 alpha
hydroxylation. Two bile acids, cholic and chenodeoxycholic, constitute the primary bile
acids.They are conjugated with either glycine or taurine and enter the bile canaliculi. Some
of the bile acids are deconjugated and converted by bacteria in the intestine to secondary
bile acids. Cholic acid is converted to deoxycholic acid, and chenodeoxycholic acid is
metabolized to lithocholic acid.Virtually all bile acids except lithocholic are reabsorbed in
the lower third of the ileum and returned to the liver via the portal vein, thus completing
the enterohepatic circulation.
APPARATUS:
Spectrophotometer at wavelength 500 nm
Water bath at 37 0C
Vortex mixer
Automatic micropipette 10 µl
Automatic pipette 1000µl
GLASSWARE:
Test tubes 100mm x 13mm
Semi micro cuvette (capacity =1ml)
REAGENTS:
There are various brands of commercially available reagents and standards for cholesterol
estimation. Evaluate the kits using quality control samples (low, high and normal values)
and consider the following to select the commercially available kits
1. Performance of the test
2. Reagent stability
3. Expiry date
4. Feasibility of test procedure
5. Interfering substances
6. cost
11.7 PROCEDURE
Following procedure is based on commercially available reagent kits. Test procedure may differ among
various products. Therefore strict to the test procedure provided with reagent kits
1. label tubes for Blank, Standard, Quality control and test
2. Add 10 µl of distilled water to Blank , 10 µl of standard solution to Standard, 10µl of
QC sample to Quality control and 10µl of patients serum to test
11.8 CALCULATION
Concentration = Absorbance of test x Standard concentration (mmol/l)
Absorbance of standard
QUALITY CONTROL
Include Quality Control sample for every batch of tests.
11.9 LIMITATION
The test is linear up to a cholesterol concentration of 750 mg/dl (19.3mmol/l). Dilute
samples with a higher cholesterol concentration 1+2 with physiological saline (0.9 %)
and repeat the determination. Multiply the result by 3.
Haemoglobin up to 200 mg/dl does not interfere with the test
Bilirubin >5mg/dl and ascorbic acid>10 mg/dl interfere the test
This limitation varies with kits.
PRECAUTIONS
Allow the samples, standard, QC and reagents to attain room temperature
Mix the thawed sample well.
Do not include more than 20 samples for a batch to maintain good quality
Verify the temperature of the water bath (at 37 0C) before the commencement of the
test.
As the final volume would be 1 ml, use appropriate tubes. (You may encounter
difficulties in pipetting with long tubes.)
Wipe outside of the pipette tip using a piece of gauze
Add serum to the bottom of the tube
Mix well in each step
Any colour change of the blank should be compared with previous blank readings
REFERENCES
Leaflets of commercially available kits
OTHER METHODS
Liebermann –Burchard method
Specific enzymatic methods (as described above) have replaced the chemical methods based on Libermann- Burchard and
Salkowski reactions.
12. GLUCOSE
12.1 INTRODUCTION
Glucose is the primary energy source for the human body. It is derived from the
breakdown of carbohydrates in the diet (grains, starchy vegetables and legumes) and in the
body stores (glycogen), as well as by endogenous synthesis from protein or the glycerol
moiety of triglycerides. When energy intake exceeds expenditure, the excess is converted to
fat and glycogen for storage in adipose tissue and liver or muscle respectively. When energy
expenditure exceeds caloric intake, endogenous glucose formation occurs from the
breakdown of carbohydrate stores and from non carbohydrate sources. (Amino acids,
lactate, and glycerol)
The glucose level in blood is maintained within a fairly narrow range under diverse
conditions (feeding, prolonged fasting or severe exercise) by regulatory hormones. These
include insulin, which decreases blood glucose, and the counter regulatory hormones
(glucagon, cortisol, noradrenaline and growth hormone) which increase blood glucose
levels.
Hypoglycemia is a blood glucose concentration below the fasting range, but it is difficult to
define specific limits. No symptoms are specific for hypoglycemia. A rapid decrease in
plasma glucose to hypoglycemic levels usually triggers a sympathetic response, with the
release of nor adrenaline, which produces classical signs and symptoms of hypoglycemia:
weakness. Sweating, nausea, rapid pulse, lightheadedness and hunger. The brain is totally
dependent on blood glucose, and very low levels of plasma glucose(less than 20 or 30 mg
/dl cause severe central nervous system dysfunction.
Neonatal blood glucose concentrations are much lower than adults and decline shortly
after birth when live glycogen stores are depleted. Glucose levels as low as 30 mg/dl in a
term infant and 20mg/dl in a premature infant may occur without any clinical evidence of
hypoglycemia. The more common causes in the neonatal period include prematurity ,
maternal diabetes and maternal toxemia. These are usually transient. Hypoglycemia with
onset in early infancy is usually less transitory and may be due to inborn errors of
metabolism or ketotic hypoglycemia, which usually develop after fasting or febrile illness.
SPECIMEN CONTAINERS
FASTING SPECIMEN
For adults the fasting time is usually 10-12 hours.
For children the fasting time is 6 hours unless longer time is indicated.
RANDOM SPECIMEN
Blood sample collected at any time regardless of food intake
STABILITY
Glucose stabilized up to 24 hour at room temperature when collected in an oxalate
and fluoride mixture.
Plasma should be separated soon after collection preferably within 1 hour
Separated plasma should not contain RBC or Leucocytes
BLOOD COLLECTION
VENOUS BLOOD
Avoid an intravenous (IV) infusion arm
Do not shake the blood but gently mix with the anticoagulant.(to prevent haemolysis)
Exact amount of an anticoagulant and blood should be mixed since sodium fluoride
inhibits the action of glucose oxidase and peroxidase in the assay.
GLASSWARE: CHEMICALS:
Volumetric flask (100 ml A grade Benzoic acid
and 500 ml volumes) 4 - amino phenazone/4 - amino anti
Graduated pipettes (0.5ml, 1 ml, 2 pyrine-AR
ml in 0.1 ml) Glucose oxidase lyophilized powder
Petri dish, watch glass or beaker from Aspergillus
Pasteur pipette Peroxidase lyophilized powder
Test tubes (100 mm x 13mm) Phenol crystals-AR
Reagent bottles clear and amber Tween 20
coloured D-Glucose anhydrous-AR
Rubber bulb Disodium hydrogen phosphate
dihydrate – AR (Na2HPO4.2H2O)
Potassium dihydrogen phosphate-
AR (KH2PO4)
Sodium azide AR
REAGENTS
1. Benzoic acid solution 1g/l: Weigh 1g of benzoic acid and transfer it to a 1 litre volumetric
flask. Add about 800 ml of distilled water and mix to dissolve the chemical completely.
Make up to 1 litre mark with distilled water and mix well. Transfer to a clean bottle,
label the bottle and store at room temperature. The solution is stable indefinitely.
Benzoic acid does not dissolve easily (Distilled water at 50 – 70 0C can be used –
Monica)
2. Stock glucose standard solution 1 g %( 55.55 mmol/l)
Use dry and clean glassware
Weigh 1.3 g of D-Glucose anhydrous (analytical grade) into a watch glass or Petri
dish or into a beaker. Spread the chemical over the bottom of the container and
keep in an oven at 60- 80 0C for 4 hours.
Allow to cool in a dessiccator and weigh out 1 g of dried glucose accurately
Transfer the chemical from the weighing container to a volumetric flask using a
funnel. Wash any chemical remaining in the container into the volumetric flask
with the benzoic acid solution (1 g/l). Always use a funnel to transfer the chemical
or solutions from any container to a flask
Half fill the volumetric flask with benzoic acid solution and mix until the chemical
is completely dissolved. Make the solution up to 100 ml with benzoic acid
solution. Make sure the bottom of the meniscus of the fluid is on the graduation
mark when viewed at eye level
Use a Pasteur pipette to add the final volume of the benzoic acid solution to the
flask.
Mix the solution well by inverting the flask for several times. Rinse the bottle with
small quantity of the standard solution, transfer in to the bottle and put the date of
the preparation on the label
The standard is stable for three months at 2- 8 0C
NOTE: The glucose standard solution should be kept at room temperature for 24
hours to α - β forms to reach in equilibrium after preparation (H&W- 6ht edition)
3. Working glucose standard 5.55 mmol/l:
Allow the stock glucose solution to attain room temperature.
Pipette accurately 10 ml of stock glucose solution using a volumetric pipette A
grade ( bulb pipette)
Carefully dispense into a 100 ml volumetric flask A grade
12.6 PROCEDURE
1. Label sufficient test tubes for the batch including standard (S) Quality controls (C1,C2)
and patients samples (1,2,3, etc)
2. pipette into the appropriately labelled 13 x 100 mm tubes as follows
S1 S2 C1, C2 1, 2, 3
Distilled water (ml) 1.9 1.8 1.9 1.9
Glucose standard 5.5 mmol/l (µl) 100 200 - -
Quality control/Patient’s plasma (µl) - - 100 100
Mix well
3. Label a second set of tubes including reagent blank (B), standard (S1, S2), Quality
controls (C1, C2 ) and patient’s samples (1, 2, 3 )
4. Pipette into the tubes as follows.
Blank S1 S2 C1, C2 1, 2, 3
Distilled water (µl) 100 - - - -
Diluted standards (µl) - 100 100 - -
Diluted patient’s sample(µl) - - - 100 100
Colour reagents(ml) 1.2 1.2 1.2 1.2 1.2
5. Mix all tubes well, incubate at 37 in a water bath for 15 minutes.
0C
6. Shake tubes two or three times during this period to ensure adequate aeration
7. Remove from the water bath, cool to room temperature and read the absorbance in a
spectrophotometer at 510 nm. Set the instrument to zero with the reagent blank (B).
8. Perform the standards in duplicate for greater accuracy and precision.
9. Calculate the results in mmol/l and check the quality control results.
NOTE: This procedure is designed for spectrophotometers that require a minimum
volume of reaction mixture in the cuvette of 1 ml. Economical use of reagents is possible
with this protocol, thus the cost per test can be kept to the minimum. However if the
laboratory employs a spectrophotometer requiring a large volume of the reaction mixture
for measurement, viz 5 ml, it is advisable to increase the volume of all reagents mentioned
under step 4 proportionately.
QUALITY CONTROL
Include QC sample for each batch of tests
REFERENCE VALUES
Random plasma Glucose level ≤7.8 mmol/l
Fasting plasma Glucose level 3.3-6.1 mmol/l
12.8 LIMITATION
Any sample that gives a glucose value >25 mmol/l should be diluted 1:2 with 0.9 %
Sodium chloride solution and the correct value obtained by multiplying the result by 3.
At high plasma levels of uric acid, glutathione and Bilirubin may interfere with the
assay by causing a decrease in glucose values. Ascorbic acid will decrease glucose
values by retarding colour development. Do not report results from specimens with
suspected interference. Inform the requesting physician of the problem.
13.1 INTRODUCTION
Phosphorus in the form of inorganic or organic phosphate is an important and widely
distributed element in the human body. An adult human has approximately 600g (19.4
mol) of phosphate expressed as phosphorus, of which about 85% is in the skeleton and
the rest principally in soft tissues. In the soft tissues most phosphate is cellular. Although
both inorganic and organic phosphate are present in cells, most is organic and
incorporated into nucleic acids, phospholipids and high energy compounds involved in
cellular integrity and metabolism.
Serum phosphate has a diurnal variation. It is higher in the afternoon and evening. The
serum phosphate level is dependent on meals and variation in the secretion of hormones
such as parathyroid hormone. Serum calcium and phosphate levels are regulated by the
kidneys.
The most important intracellular function of phosphate is the high energy bond in
adenosine triphosphate. These energy sources maintain many physiological functions such
as muscle contractility, neurological functions and electrolyte transport. Phosphate is a
constituent of cyclic adenine and guanine nucleotides as well as nicotinamide adenine
dinucleotide phosphate, which is important in many enzyme systems. It is an element in
phospholipid cell membranes, nucleic acids and phosphoproteins. It is also involved in the
regulation of intermediary metabolism of proteins, fats and carbohydrates, as well as in
gene transcription and cell growth.
Extra cellular phosphate maintains the critical intracellular concentration and provides
substrate for bone mineralization.
The skeleton serves as a store house for phosphate. The cellular demands for metabolic
function in bone cells are similar to those in other cells.
Intra cellular shift (A high carbohydrate diet stimulates insulin secretion, increasing the
transport of glucose and phosphate into the cell.)
Renal Phosphate wasting (Any cause of excessive parathyroid hormone secretion may
result in hypophosphataemia due to phosphaturia
Decreased intestinal phosphate absorption (Increased loss due to vomiting, diarrhoea
and phosphate binding antacids: Decreased absorption in malabsorption syndromes.)
Cellular phosphate loss (Acidosis results in catabolism of organic compounds within
the cell so that inorganic phosphate shifts into the plasma and excreted in the urine.
Hyperphosphatemia is usually due to acute or chronic renal failure because the kidneys fail
to excrete the amount taken in the diet. Lack of Parathyroid hormone and increased
growth hormone causes increased tubular reabsorption of phosphate resulting in increased
phosphate levels in blood. Increased phosphate intake, increased extra cellular phosphate
load in acidosis and any cause leading to cell lysis causes hyperphostaemia
APPARATUS:
Centrifuge
Spectrophotometer
Automatic pipette (500 µl, 1000 µl)
GLASSWARE: CHEMICALS:
Volumetric flasks (100 ml, 200ml, Ammonium molybdate-AR
500 ml) 1-Amino-2-napthol-4-sulphonic acid
Beakers (5ml, 200 ml, 500ml) Perchloric acid AR
Graduated pipette (0.5 ml, 1ml, 5ml, Sodium metabisulphite-AR (store in
10 ml) refrigerator)
Conical centrifuge tubes 15 ml Sodium sulphite-AR (store in
Test tubes (16 x 150 mm) refrigerator)
Potassium dihydrogen phosphate
KH2 PO4 - AR
Trichloro acetic acid - AR
REAGENTS
1. Reducing agent: Dissolve 12 g of sodium Meta bisulphite and 2.4 g of sodium sulphite in
about 80 ml of distilled water. Add 0.2 g of 1-amino-2-naphthol-4-sulphonic acid.
Dissolve and dilute to 100 ml in volumetric flask. Store in the refrigerator in a brown
bottle. The solution keeps up to 4 weeks. It is better prepare fresh reagent ( about 10
ml)
2. Trichloroacetic acid 10 %: Dissolve 10 g of Trichloroacetic acid in distilled water and
make up to 100 ml with distilled water. Store in the refrigerator.
3. Perchloric acid AR
4. Ammonium Molybdate 5%: Dissolve 5 g of Ammonium molybdate in distilled water and
make up to 100 ml with distilled water. Store at room temperature. Discard the
solution when a precipitate appears.
5. Stock phosphate standard solution 32mmol/l: Keep about 2.5 g of pure potassium
dihydrogen phosphate in a dessicator to dry, weigh exactly 2.194 g of potassium
dihydrogen phosphate and transfer into a 500 ml flask. Dissolve in distilled water and
make up to 500 ml with distilled water. Store at room temperature.
6. Working phosphate standard solution 0.128 mmol/l: Dilute 2 ml of stock phosphate
standard to 500 ml with distilled water. Store at room temperature
C 1, 2, 3
Trichloroacetic acid 10 % (ml) 9.0 9.0
Quality control serum (ml) 1.0 -
Patient’s sample (ml) - 1.0
3. Mix well and leave for about 10 minutes and centrifuge ( 2500-3000 rpm) for
about 10 minutes
4. Label a set of test tubes including reagent blank (B) Standard (S) Quality control (C1,
C2) and patient’s samples (1, 2, 3 …)
5. Pipette into the tubes as follows
B S C 1, 2, 3
Trichloro acetic acid 10% (ml) 5.0 - - -
Working standard solution (ml) - 5.0 - -
Supernatants from tubes above(ml) - - 5.0 5.0
Perchloric acid (ml) 0.4 0.4 0.4 0.4
Ammonium Molybdate 5% (ml) 0.4 0.4 0.4 0.4
Reducing reagent (ml) 0.2 0.2 0.2 0.2
NOTE: For inadequate specimen use appropriate volumes of serum and reagents.
The supernatant should be clear.
13.7 CALCULATION
Concentration of standard is 0.128 mmol/l, serum dilution is 1:10
∴Concentration of phosphorus = T x 0.128 x 10
S
= T x 1.28 mmol/l
S
13.8 LIMITATION
Glucose phosphate, CPK and other organic phosphates may also be hydrolyzed by assay
conditions, resulting in overestimation of inorganic specimens.
PRECAUTIONS
Glass ware should be properly cleaned and rinsed because phosphate is a common
component of many detergents
Discard Ammonium Molybdate solution when a precipitation formed at the bottom
of the container
Recommend to prepare only 10 ml of reducing agent at a time as the stability of the
reagent is one month.
14.1 INTRODUCTION
Urinary phosphate excretion varies with age,muscle mass,renal function,parathyroid
hormone level,the time of the day and diet.
14.4 PROCEDURE
1. Mix the 24 hours urine collection
2. Measure the total volume of urine
3. Pipette 1 ml of urine into a 100 ml volumetric flask. Make up to 100 ml with distilled
water.
4. Label 3 test tubes for Blank (B), Standard (S) and Test (T)
5. Pipette 5 ml of distilled water into the tube B, 5 ml of working standard into the tube S
and 5 ml of diluted urine into the tube T
6. Add 0.4 ml of Perchloric acid into each tube, Mix well
7. Then add 0.4 ml of Ammonium Molybdate 5% solution to each tube and mix well
8. Add 0.2 ml of reducing agent to each tube, Mix well and leave for 10 minutes at room
temperature
9. Read the absorbance at 700 nm, setting the spectrophotometer to zero with blank
solution
14.5 CALCULATION
Concentration of the Standard = 0.128 mmol/l
Urine phosphate = T x 0.128 x 100 mmol/l
S
= T x 12.8 mmol/l
S
24 hour urine phosphate = T x 12.8 x V (24 hour urine volume in litre) mmol/24 hrs
S
REFERENCES VALUES:
For an adult : 16-48 mmol/24 hours
For children : Refer appendix 3
GLOBULINS
Electrophoresis of normal serum performed on a carrier such as cellulose acetate reveals
four major non-albumin bands staining for protein/lipoprotein, designated by mobility as
α1, α2 β, γ
Band Concentration (g/L) Major components
α1-globulin 2-5 α1- antitrypsin, Apolipoprotein
α2 –globulins 4-10 Caeruloplasmin, α2 macroglobulins,
Haptoglobins
β-globulin 6-12 Transferrin β-lipoproteins
APPARATUS:
Spectrophotometer at wavelength 540 nm or colorimeter with yellow-green filter, Ilford
605(550 nm)
Automatic micro pipettes (50 µl)
GLASSWARE:
Volumetric flasks (500 ml and 1 litre volumes)
Graduated pipette (5 ml in 0.1 ml)
Measuring cylinders (100ml and 500 ml)
Test tubes (150 mm x16 mm)
Beaker (250 ml)
Polyethylene reagent bottles (I litre)
CHEMICALS:
Sodium chloride-AR
Sodium azide-AR caution: handle with care
Sodium hydroxide pellets-AR
Copper sulphate pentahydrate-AR
Potassium sodium tartrate tetrahydrate-AR
Potassium iodide-AR
Albumin bovine, a fraction v powder is suitable
REAGENTS
1. Sodium hydroxide solution 6 mol/l: Dissolve 120 g of sodium hydroxide little by little in
about 400 ml of distilled water. After cooling dilute to 500 ml. Store in a tightly- closed
polyethylene bottle. This solution is stable indefinitely at 20 -25 0C(room temperature)
2. Biuret reagent: Dissolve 3.0 g of copper sulphate in about 500 ml of distilled water. Add
9.0 g of potassium sodium tartrate and 5.0 g of potassium iodide. When they have
completely dissolved, add 100 ml of sodium hydroxide solution (6 mol/l) and make up
to 1 litre. The solution is stable indefinitely at 20 -25 0C(room temperature) store in a
tightly closed polyethylene bottle
3. Blank Biuret reagent: Dissolve 9.0 g of potassium sodium tartrate and 5.0 g of potassium
iodide in distilled water. Add 100 ml of sodium hydroxide solution (6mol/l) and make
up to 1 litre with distilled water. When kept in a tightly-stoppered polyethylene bottle
this solution is stable indefinitely at 20 -25 0C(room temperature)
4. Sodium chloride/Sodium azide solution: Weigh out 9.0 g of sodium chloride and 1.0 g of
sodium azide, dissolve and make up to 1 litre with distilled water. This solution is
stable indefinitely at 20 -25 0C (room temperature)
5. Protein standard 80 g/l: Weigh out about 4.3 g of bovine albumin powder and dry it in
the oven at about 60 C for 8-10 hours. After drying weigh out exactly 4.0 g of dried
15.6 PROCEDURE
1. For the standard and each patient or control sample, pipette into test tubes 2.5 ml of
Biuret reagent (standard and test) and 2.5 ml of blank Biuret reagent (standard blank
and test blank)
2. Add 50 µl of standard (80 g/l) or samples to each pair of tubes.
3. A reagent blank is set up for each batch and contains 2.5 ml of Biuret reagent and 50
µl of water.
4. Mix each tube and allow them to stand at room temperature for 30 minutes or at
370 C for 10 minutes.
NOTE: the same temperature must be used for standards and samples.
5. Measure the absorbance at 540 nm (yellow-green filter, Ilford No 605) setting the
spectrometer to zero with blank Biuret reagent. First read the absorbance of the
sample blanks, then the reagent blank, and then the tests.
6. If results are greater than 100 g/l then repeat the analysis using 20 µl of sample.
Multiply the result by 2.5 to obtain the protein concentration.
CALIBRATION
Although the measurement of total protein is simple, results of external quality assessment
programmes indicate that many laboratories have difficulty in producing accurate without
sample blank correction.
The recommended method proposes the use of a bovine albumin solution as a calibrator.
One alternative is to use lyophyilised serum calibrators. However many of these show
significant turbidity when they are reconstituted and sometimes it is difficult to know
whether the contribution that the turbidity makes to the absorbance at 540 nm has been
subtracted in the calculation of the total protein value. When possible use a calibrator with
a value assigned by a total protein method that incorporates a sample blank. A sample
blank for the standard (SB) is not necessary if a solution of bovine albumin is used.
A second alternative is to use an out – of-date of human serum albumin from a blood
transfusion or pharmacy department. Use the stated concentration of albumin for the total
protein value. One bottle will last for many months at 4-6 0C if small volumes (5-10 ml) are
withdrawn as required using a sterile syringe.
A calibration curve must be prepared as described below to check the linearity of the
spectrometer. If it is linear, then a single standard can be used routinely as described under
“Technique” .The calibration curve should be repeated at least once a month.
Working standard No 1 2 3 4
15.7 CALCULATION
S= A standard - A standard blank – A reagent blank
T= A sample - A sample blank- A reagent blank
QUALITY CONTROL
At least two quality control specimens, having stated values in the range 40-80 g/l, one of
which is unknown to the operator, should be included with each batch of specimens. If
single specimens are analysed a control specimen should always be included.
ROUTINE CONDITIONS VARIANCE: The value obtained for the RCV should not exceed
4%
REFERENCE VALUES
Approximate reference values for an adult 60-80 g/L
REFERENCES
Doumas, B.T. (1975) Clin. Chem, 21, 1159-1166.
16. UREA
DIACETYLE MONOXIME METHOD
16.1 INTRODUCTION
Urea is the major nitrogen containing metabolic product of protein catabolism in humans,
accounting for more than 75% of the non protein nitrogen eventually excreted. The
biosynthesis of urea from amino nitrogen-derived ammonia is carried out exclusively by
hepatic enzymes of the urea cycle.
More than 90% of urea is excreted through the kidneys, with losses through the
gastrointestinal tract and skin accounting for most of the remaining minor fraction. Urea is
neither actively reabsorbed nor secreted by the tubules but is filtered freely by the
glomeruli. In a normal kidney, 40 to 70% of the highly diffusible urea moves passively out
of the renal tubule and into the interstitium, ultimately to re-enter plasma. The back
diffusion of urea is also dependent on urine flow rate; with more entering the interstitium
in slow-flow states. Urea production is dependent on several non renal variables such as
diet and hepatic synthesis.
APPARATUS:
Water bath or heating block (temperature range 10- 110 0C)
Spectrophotometer wavelength 530 nm
Colorimeter green filter, Ilford 604 (520nm)
Rack for boiling tubes
GLASSWARE:
Amber coloured reagent bottles (500 ml volume)
Graduated pipettes (5 and 10 ml in 0.1 ml)
Graduated cylinders (50 ml, 100 ml and 500 ml)
Volumetric flasks (50 and 100 ml and 500 ml volumes)
Glass stoppered boiling tubes (160 x 18 mm)
Conical centrifuge tubes 15ml
CHEMICALS:
Benzoic acid-AR
Cadmium sulphate (as 3CdSO4.8H2O)-AR
Diacetyle monoxime-AR
Orthophosphoric acid (85% w/v)-AR caution: corrosive, handle with care
Sulphuric acid, concentrated (95 – 97 % w/v)-AR caution: corrosive, handle with care
Thiosemicarbazide-AR
Trichloroacetic acid-AR caution: corrosive, handle with care
Urea-AR
REAGENTS
1. Acid reagent : Add about 200 ml of distilled water to a 500 ml volumetric flask. Keep
the flask in basin containing water and then add slowly 44 ml of sulphuric
(concentrated) acid and 66 ml of orthophosphoric acid. Cool the solution to room
temperature but do not use ice water as a cooling bath. Add 50 mg thiosemicarbazide
and dissolve, then add 1.6 g cadmium sulphate and dissolve, add 1.5 ml of the urea
working standard solution (2.5mmol/l). Make up to 500 ml with distilled water.
Transfer to an amber coloured bottle. This reagent is stable for at least six months at
2- 8 0C.
NOTE: The presence of a small amount of urea in the reagent improves the linearity
of the calibration curve. The cadmium sulphate improves the stability of the final
coloured product.
2. Diacetyl monoxime reagent: Weigh out 2.0 g of diacetyl monoxime, dissolve in
distilled water and dilute to 500 ml with distilled water in a volumetric flask. This
solution is stable for at least six months at 2-8 0C
3. Colour reagent: Use a graduated cylinder and mix 50 ml of acid reagent with 50 ml of
diacetyl monoxime reagent in a small bottle. This amount is sufficient for 33 reactions.
This reagent must be prepared daily, therefore, the volume made up should depend on
the number of reactions anticipated; 3 ml is required for each reaction.
4. Benzoic acid solution 1 g/l: Weigh out 0.5 g of benzoic acid and transfer to a 500 ml
volumetric flask. Add distilled water, mix well to dissolve and make up to 500 ml with
distilled water. This solution is stable for several months at 20-250C (room
temperature)
5. Trichloroacetic acid solution 50 g/l: Weigh out 25 g of trichloroacetic acid in a beaker,
dissolve, transfer into 500 ml volumetric flask and make up to 500 ml with distilled
water. This solution is stable for several months at 20-25 0C. We recommend storing
the solution in the refrigerator.
6. Stock urea solution 125 mmol/l: Weigh out 1g of urea –AR in a beaker and keep in
the dessicator overnight. Weigh out 750 mg urea from the dessicator and transfer into
a 100 ml volumetric flask. Add 50 ml of benzoic acid solution; dissolve and dilute to
100 ml with benzoic acid solution. This reagent is stable for several months at 2-80 C
7. Working urea standards: Prepare working urea standards in 50 ml volumetric flasks
according to the table below;
Working standard No 1 2 3 4 5 6
Stock urea standard (ml) 1 2 3 4 6 8
Benzoic acid solution Up to 50 ml for each
Concentration of working standard 2.5 5.0 7.5 10 15 20
Solution (mmol/l)
These standards are stable for several months at 2-80 C
16.6 PROCEDURE
1. Pipette 0.5 ml of trichloroacetic acid solution using a rubber bulb into centrifuge tubes
for each standard, control serum and patient’s sample. Add 50 µl of standard, control
serum or patient’s sample to the appropriate tube, mix and leave at room temperature
for 5 minutes, then centrifuge to obtain a clear supernatant.
2. Label another set of tubes (18 x 160mm) and pipette 3.0 ml of colour reagent into test
tubes for each standard, blank, control serum and sample.
3. Add 100 µl of trichloroacetic acid solution to the blank tube, and 100 µl of supernatant
form the standard, control or samples to the appropriate tube.
4. Mix well and heat at 100 0C for 15 minutes exactly.
5. Cool the tubes to room temperature in a bowl of water (about 5 min) mix and read the
absorbance at 530 nm (green filter, Ilford No 604) first read the absorbance of the
blank against distilled water and note down the reading, then set to zero with the blank
and read the standards and unknowns. The absorbance measurements should be made
as soon as possible and not more than 30 minutes after the end of step 4.
16.7 CALCULATION
Read the results from the calibration curve or use the following formula if your calibration
graph is linear.
Concentration of urea in mmol/l = T x C
S
Where:
T = Absorbance reading of patient’s test
S = Absorbance reading working standard
C = Concentration of working standard (10 mmol/l)
If the result is greater than 20mmol/l, dilute 50 µl of supernatant from that sample with
100 µl of trichloroacetic acid solution. Mix well. Repeat the analysis using 3 ml of colour
reagent and 100 µl of the diluted supernatant. Remember to multiply the result by 3 to
obtain the urea concentration in the patient’s sample.
QUALITY CONTROL
At least two serum control specimens, having stated values in the range 3-20 mmol/l, one
of which is unknown to the operator, should be included with each batch of specimens. If
single specimens are analysed, a control specimen should always be included.
ROUTINE CONDITIONS VARIANCE: The value obtained for the RCV should not exceed
6%
REFERENCE VALUES
Approximate reference intervals for “healthy” ambulant adults: 2.5-6.6 mmol/l
Conversion of SI units into “old” units: mmol/l x 6 = mg/100 ml
Blood urea reference values are method dependable. Blood urea estimation by urease
method usually gives higher values
NOTE: The method recommended uses the reagents described in the above
references. However a protein precipitant has been included because the direct method
(No protein precipitation) gave results on patients’ samples which were significantly
higher than other methods.
16.8 LIMITATION
Cadmium sulphate has been included as recommended by Wybenga et al. In the absence
of cadmium ions, the absorbance decreased by about 7 % in 30 minutes; when the colour
reagent is prepared as described, then the absorbance decreased by about 4% in 30
minutes. Provided that the batch size is kept small, so that the absorbance readings can be
made over a period of a few minutes, the presence or absence of cadmium ions has little, if
any effect on the results.
REFERENCES
WHO Manual LAB/86.3
17.1 INTRODUCTION
Uric acid is the end product of purine metabolism (adenine,guanine) in the human. On a normal
diet some 5-6 mmol of urate is produced daily.Of this amount about 3-4 mmol is produced from
purines synthesized in the body (de novo synthesis), whilst the remaining 1-2 mmol are contributed
by dietary purine.Urate, the end product of purine base degradation, is formed from xanthine by the
enzyme xanthine oxidase.
Around 25-30% of the 5-6mmol of urate produced daily is eliminated via the gastrointestinal tract,
where it is degraded by bacterial uricases. The remainder is excreted by the kidney. In renal failure
the intestinal excretion can be markedly increased.
Some 98% of the filtered urate is reabsorbed in the proximal tubule. The 2% that escapes
reabsorption contributes around 20% of the total excreted, proximal tubular secretion accounting
for the remainder. Minor reabsorption also occurs in the distal nephron Renal clearance is
influenced by various drugs and metabolic products.
APPARATUS:
Water bath at 25 0C or Basin with water temperature maintained at 250C
Automatic micro pipette 500 µl
Quick fit complete assembly (round bottom flask and condenser) for refluxing
CHEMICALS:
Molybdate free sodium tungstate dihydrate-AR
Orthophosphoric acid-AR
Sodium carbonate-AR
Sulphuric acid-AR
Uric acid –AR
Formaldehyde 40% solution
REAGENTS
1. Stock Phosphotungstic acid reagent: Weigh out 50 g of molybdate free sodium tungstate dihydrate or
44.5 g anhydrous salt and dissolve in about 400 ml of distilled water in refluxing flask. Add 40
ml of Orthophosphoric acid (concentrated). Mix well and reflux gently for 2 hours. Allow the
solution to cool. Then transfer with rinsing to a 500 ml volumetric flask. Make up to 500 ml
with distilled water. Mix well. Transfer this solution into a clean dry brown bottle. This solution
is stable in the refrigerator for about one year.
2. Working Phosphotungstic acid reagent: Dilute 10 ml of stock phosphotungstic acid reagent to 100 ml
with distilled water. Transfer in to a clean, dry brown bottle. Stable in the Refrigerator for two
weeks.
3. Sodium carbonate 10g/dl: Weigh 10 g of Sodium carbonate anhydrous, dissolve in distilled water
and make up to 100 ml with distilled water. Store in a poly propylene bottle.
4. Sodium tungstate 10g/dl: Weigh out 10 g Sodium tungstate dihydrate, transfer into a 100ml
volumetric flask, dissolve and make up to 100 ml with distilled water. Store in a brown bottle.
5. Sulphuric acid (2/3 N) 0.34mol/l: Add slowly 3.7 ml of sulphuric acid concentrated in to 150 ml
distilled water in a beaker. Cool and stir well. Transfer in to a 200 ml volumetric flask and make
up to 200 ml distilled water. Transfer in to a brown bottle.
6. Acid tungstate reagent: To 80 ml of distilled water while mixing add 5 ml of sodium tungstate
solution, 0.05 ml of phosphoric acid (concentrated), 5 ml of 2/3 N sulphuric acid solution.
Transfer in to a brown bottle. Keep at room temperature.
7. Stock Uric acid standard solution 1.5mmol/l: Keep about 300 mg of uric acid analytical grade chemical
in a desiccator overnight. Weigh out 252.2 mg of uric acid and transfer in to a one litre
volumetric flask. Weigh out 150 mg of Lithium carbonate; dissolve in about 50 ml of distilled
water. Filter, heat the filtrate to 60 0C. Add this warm solution to the volumetric flask
containing uric acid. Mix until the uric acid completely dissolved. Allow the flask to cool. Then
add 20 ml formaldehyde 40 % solution. Add 500 ml of distilled water .Then add slowly 25 ml
of sulphuric acid solution. (Prepared by adding 1 ml of sulphuric acid (concentrated) to 35 ml
of water) Make up to one litre with distilled water. Mix and store in a brown bottle. This
solution is stable for about one year at 4- 6 0C.
8. Working uric acid standard: Allow the stock uric acid standard solution to attain the room
temperature. Pipette 2 ml of stock standard solution in to 100 ml volumetric flask. Make up to
100 ml with distilled water. This standard is equivalent to 300 µmol/l under the assay conditions
employed. Solution is stable for one week at 4-6 0C.
9. Working Uric acid standard series for calibration: Prepare working standard series by quantitative
transfer of 1ml, 2ml, 3ml and 4 ml of stock solution to each of four 100 ml volumetric flasks.
Dilute to 100 ml with distilled water. Mix well. These standards are equivalent to 150µmol/l,
300µmol/l, 450µmol/l and 600µmol/l under the employed assay condition.
17.7 CALCULATION
Calculate the uric acid concentration in the patient’s specimens from the absorbance of the standard
as follows
QUALITY CONTROL
Include Quality control sera for each batch of test
17.8 LIMITATION
Haemolysed sera interfere with the measurement of uric acid.
Supernatants should be clear as turbidity may interfere with colour development
18.2 PROCEDURE
1. Mix the urine collection well. Measure the total volume. Warm an aliquot of urine for a few
minutes to 600C to dissolve any urates in the deposit. Add 1ml of urine in to a 100ml
volumetric flask. Make up to 100ml with distilled water. Mix well. (Dilution 1 in 100)
2. Pipette 2.5 ml of distilled water to the tube marked B for blank , 2.5 ml of working standard to
the tube marked S as standard and pipette 2.5 ml of diluted urine in to the tube marked T for
test. Keep all the tubes in a water bath at 250C
3. Carry out the same procedure as for serum uric acid estimation from step 6.
18.3 CALCULATION
Calculate urine uric acid concentration in the sample using the absorbance of the standard as
follows.
19. ELECTROLYTES
DETERMINATION OF SERUM SODIUM AND POTASSIUM BY FLAME PHOTOMETRY [FLAME
EMISSION SPECTROSCOPY]
19.1 INTRODUCTION
SODIUM
In a normal adult the total body sodium is about 55mmol/kg of body weight; about 30% is tightly
bound in the crystalline structure of bone and thus is nonexchangeable. Thus only 40 mEq/kg is
exchangeable among the various compartments and accessible to measurements. The exchangeable
sodium is distributed primarily in the extra cellular space. About 97% to 98% of the exchangeable
sodium is found in the extra cellular water space and only 2% to 3% in the intracellular water space.
Approximately 16% of exchangeable sodium is in plasma, 41% is in interstitial fluid (ISF) that is
readily accessible to the plasma compartment, 17% is in ISF of dense connective tissue and
cartilage, 20% is in ISF of bone and 3% to 4% in the transcellular water compartment. Total bone
sodium (exchangeable and nonexchangeable) accounts for the 40% to 45% of the total body
sodium.
The amount of sodium in the body is a reflection of the balance between sodium intake and output.
Sodium intake depends on the quantity and type of food intake. Under normal conditions, the
average adults takes in about 50 – 200 mmol of sodium/ day. Sodium output occurs through three
primary routes; the gastrointestinal tract, the skin and the urine.
Under normal circumstances loss of sodium through the GIT is very small. Faecal water excretion is
only 100- 200 ml/day for a normal adult and faecal sodium excretion only 1 to 2 mmol/ day.
However, one should bear in mind that although losses of water and electrolytes are normally small,
the total volume of GIT fluid secreted is large, averaging about 8 L/day. Almost all this volume is
normally reabsorbed. However, with impaired GIT reabsorption, loss of water and electrolytes are
large. Thus with severe diarrhoea or with gastric or intestinal drainage tubes, sodium loss via the
GIT may exceed 100mmol/ day.
The sodium content of sweat averages about 50 mmol/ L but is somewhat variable. The sweat
sodium concentration is decreased by aldosterone and increased in cystic fibrosis. The rate of sweat
production is highly variable, increasing in hot environments, during exercise, and with fever. Under
extreme conditions sweat production can exceed 5 L/ day, accounting for a loss of more than 250
mmol of sodium .Under normal conditions, in a cool environment; sodium losses from the skin are
small. With extensive burns or exudative skin lesions there is great loss of sodium and water.
Major route of sodium excretion is through the kidney. Furthermore, urinary excretion of sodium is
carefully regulated to maintain body sodium homeostasis, which in turn is critical to control of extra
cellular volume.
Sodium is freely filtered by the glomerulus. Approximately 70% of the filtered sodium is reabsorbed
by the proximal tubule, about 15% by the loop of henle, 5% by the distal tubule, 5% by the cortical
collecting tubule, and another 5% by the medullary collecting duct; thus normally less than 1% of
the filtered sodium is excreted.
POTASSIUM
Approximately 98% of the total body potassium is found in the intracellular water space
(ICW),reaching a concentration there of about 150 to 160 mmol/ l .In the extra cellular water space
,the concentration of potassium is only 3.5 to 5 mmol/l . Total body potassium in an adult male is
about 50 mmol/kg of body weight and is influenced by age, sex, and very importantly muscle mass,
since most of the body’s potassium is contained in muscleThe amount of potassium in the body is a
reflection of the balance between potassium intake and output. Potassium intake depends on the
quantity and type of food intake. Under normal conditions the average adult takes in about 50 to
100 mmol/ day, about the same amount as sodium. Potassium output occurs through three primary
routes; the gastrointestinal tract, the skin and the urine.
Hypernatremia occurs when there is greater deficit of extra cellular water than of sodium. Greater
excess of sodium than of water rarely occurs.
Hypernatremia usually occurs as a chronic process secondary to loss of water in excess of sodium.
Symptoms are therefore those of dehydration.
POTASSIUM
POTASSIUM EXCESS
Potassium accumulates in the body when the intake of potassium exceeds output because of some
abnormality of the potassium homeostatic mechanism. It should be noted that under most
conditions the normal kidney is capable of excreting a great deal of potassium, and a high potassium
intake leads to potassium retention only when kidney function is compromised.
POTASSIUM DEPLETION
This occurs when potassium output exceeds intake. Only small amount of potassium is loss in the
faeces under normal conditions. GIT loss of potassium during diarrhoea and drainage of GIT
secretions can be large.
Alkalosis results in the total body potassium depletion. With alkalosis, potassium moves from the
extra cellular compartment to the intra cellular space. In the cells of the distal nephrone of the
kidney, this increase in intra cellular potassium stimulates potassium secretion and therefore
increases renal excretion of potassium.
REAGENTS
All glassware used to prepare standard solutions should be chemically cleaned and
finally rinsed with distilled water.
Weigh out separately into two watch glasses or into two Petri dishes about 15 g of analytical grade
Sodium chloride and about 1 g of Potassium chloride.
Dry for 6 hours at 100 0C in an oven and allowed to cool to room temperature in a desiccator.
1. Stock Sodium solution 1000 mmol/l: To prepare 200ml, weigh accurately 11.7 g of dried
Sodium chloride in a weighing bottle or in a beaker. Transfer it in to an ‘A grade’ 200 ml
volumetric flask using a funnel. Wash in any chemical remaining in the weighing bottle or in the
beaker in to the flask with a little amount of distilled water (glass distilled water) or deionised
water. Dissolve in about 150 ml of distilled water and make up to 200 ml with distilled water.
Use a pasture pipette or a wash bottle to add the final volume of distilled water to the flask. Mix
the solution well by inverting the flask for several times.
2. Stock Potassium solution 100 mmol/l: Weigh accurately 0.746 g of dried Potassium chloride
in a weighing bottle or beaker. Transfer it in to an ‘A grade’ 100 ml volumetric flask using a
funnel. Wash any remaining in the weighing container in to the flask with distilled water.
Dissolve in about 80 ml of distilled water. Make up to 100 ml with distilled water. Use a Pasture
pipette or a wash bottle to add the final volume of distilled water to the flask. Mix the solution
well by inverting the flask for several times.
3. Flame photometry deproteinising solution: The pack of deproteinising solution contains
deproteinising base solutions and Sachet of catalyst. For use add the catalyst into base solution
and mix thoroughly. This solution is stable for 4 weeks at 2-80 C.
4. Working Standard solution (Sodium-140 mmol/l, Potassium-5.0 mmol/l): Into a clean
500 ml ‘A grade’ volumetric flask, add 70 ml of stock solution-1(Stock sodium solution) and 25
ml of stock solution-2 (Stock potassium solution) Make up to 500 ml with good quality distilled
water or deionised water. Use a pasture pipette or a wash bottle to add the final volume of
distilled water to the flask. Mix the solution well by inverting the flask for several times. Rinse a
19.6 PROCEDURE
The details of the operation vary from one instrument to another. Following steps are related to
‘Corning 410’ clinical model flame photometer.
1. Sample dilution: Dilute each serum, quality control sample and working standard solution
1:200 with working diluent concentrate. Into 50 ml conical flasks pipette 19.9 ml of working
diluent concentrate and 0.1 ml of working standard solution or quality control sample or
patient’s serum and mix well.
2. Turn on the fuel supply at source
3. Depress the ‘power’ switch to switch on the instrument 410. The ‘power on’ LED will be
illuminated, the air compressor will start an ignition cycle will commence.
4. If the flame on LED is not illuminated at the end of the ignition cycle, (Refer the operator’s
manual available with the instrument) check that the air pressure gauge indicates a reading
between 11 and 13 psig, if it does not, lower the air regulator locking ring and adjust the
regulator for a reading of 12 psig on the air pressure gauge. Raise the locking ring to lock the air
regulator adjuster.
5. Set the filter selector to the required position. Non luminous blue flame with distinct cones can
be seen, if does not; adjust the fuel to get distinct blue cone flame.
6. Insert the Nebulizer inlet tube in a beaker containing approximately 100 ml of diluent and allow
15 minutes for the operating temperature to stabilize. This will ensure a stable burner
temperature when solutions are aspirated, after the warm up period.
7. While aspirating the diluent, adjust the ‘blank’ control so that the display reads zero
8. Aspirate a pre diluted standard solution
9. Allow 20 seconds for a stable reading and then adjust ‘coarse’ and ‘fine’ controls for a
convenient reading (if a 140 mmol/l Sodium standard is aspirated, set the display to 140)
10. Carefully adjust the ‘fuel’ control for a maximum reading on the display, ensuring that only
small adjustments are made, with a pause of several seconds between adjustments.
11. Remove the standard solution, wait 10 seconds, then aspirate a blank solution of diluent for 20
seconds. Adjust the ‘blank’ control for a zero reading. Remove the blank solution and wait 10
seconds.
12. Repeat paragraphs 8, 9 and 11 until the blank reading is zero (within ± 0.2) and the calibration
reading is within ± 1%.
13. Aspirate each of the unknown solutions for 20 seconds, then note the reading in mmol/l
14. Check the calibration frequently
15. When analyzing large batches of samples, recheck instrument calibration every 10 samples with
a single standard solution.
PRECAUTIONS
A diluent recommended by the manufacturer of instrument should be used. Deionised or high
quality distilled water should be used to prepare the diluent. Deionised or distilled water must be
free from contaminative elements (bacteria or moulds can cause inaccuracies by interrupting or
blocking the flow of sample through the Nebulizer always use the same batch of diluent for the
blank and the dilution of samples and standards.
Anticoagulants containing sodium or potassium salts must not be used. Use serum for
measurement of sodium and potassium
Dilute the sera with care. Good quality calibrated pipette or a sensitive diluter must be used.
Use the same pipette or dilution equipment for both standard and sample
Accuracy of the results depends on the accuracy and purity of the calibration standard. Always
use accurately prepared standards.
Both the accuracy and precision of results depends on maintenance and adherence to operating
instructions provided by the manufacturer. Careful cleaning of the atomizer-burner, cleanliness
of sample containers, the aspirating systems, proper adjustment of flame size, (blue flame with
distinct cone) aspiration rate, and geometry of the flame and uniform entry of atomized, diluted
sample into the flame are also critical for accuracy and precision. Thermal equilibrium must be
established before analysis of unknown samples. Warm up period is necessary because the
initial evaporation of water in the flame decreases the temperature of the burner and the entire
burner chamber.
Safety: Propane is highly inflammable and potentially explosive and commonly supplied as a
liquid under pressure in a cylinder for use with the 410. Cylinder should never be subjected to
heat or mechanical shock. Leakage of propane from tank, instrument fittings or from valves
may be detected with the aid of soap solutions.
Site conditions:
Never install the flame photometer beneath overhanging cupboards. There must be at least
1 metre of clear space above the 410 chimney
The environment must be clean and free from dust
The instrument must be placed on a strong, level work stop, free from vibration
Avoid the instrument to direct sunlight or draughts
QUALITY CONTROL
At least two serum control specimens, having stated values in the range 120-150 mmol/l for
sodium, 3.0-6.0 mmol/l for potassium, and one of which is unknown to the operator should be
included with each batch of specimens. If single specimens are analysed a control specimen should
always be included
OPTIMAL CONDITIONS VARIANCE: A coefficient of variation of around 1% for sodium and
1.5% potassium should be attainable
ROUTINE CONDITIONS VARIANCE: The value should not exceed 2% for sodium and 3% for
potassium
REFERENCE VALUES
Serum Sodium : 136mmol/l - 146mmol/l
Serum Potassium: 3.5mmol/l - 5.6mmol/l
REFERENCES
Operator’s Manual Corning 410
LAB/86.3
20. URINE SODIUM AND POTASSIUM
20.1 PRINCIPLE OF THE METHOD
Same as for serum sodium and potassium
20.1 PROCEDURE
Mix the 24 hour urine collection and measure the total volume using a clean measuring cylinder
Perform the test as for serum electrolytes
If the sodium level is low, dilute the neat sample 1:50 or 1:100 and divide the result by 4 or 2
respectively; dilution is depend on the level of the sodium
If the potassium level is higher than 10 mmol/l then dilute the urine 1:500 or 1:1000 and
multiply the result by 2.5 or 5 respectively. Dilution depends on the level of potassium
20.3 CALCULATION
Sodium (mmol/24 hour) = mmol/l x 24 hour urine volume in litre
Potassium (mmol/24 hour) = mmol/l x 24 hour urine volume in litre
REFERENCE VALUES
Urine Sodium : 40-220 mmol/24 hour
Urine Potassium : 25-150 mmol/24 hour
REFERENCES
Operator’s Manual Corning 410
LAB/86.3
VENIPUNCTURE
The phlebotomist should verify that the patient is fasting, if fasting be necessary to ensure
medically useful results. The patient should be comfortably seated or supine (if sitting is not
feasible.) and should be in that position for 20 minutes. The patients arm should be in a
straight line from the shoulder to the wrist. An with an inserted intravenous line should be
avoided,as should an arm with extensive scarring or haematoma at the intended collection
site .If a woman has had a mastectomy arm veins on that side of the body should not be
used since the surgery may have caused lymphostasis affecting the blood composition. The
median cubital vein in the antecubital fossa is the preferred site for collecting venous blood
in the adults. The area should be cleaned with alcohol swab.(Use benzylkonium chloride as
the cleaning during determinations of plasma alcohol.)When the skin is cleaned a tourniquet
is applied 10 tp 15 cms to obstruct the return of venous blood to the heart and to distend
the veins.(A tourniquet should be 2.5 cms wide and 38-40 cms in length. The appropriate
needle must also be selected. The larger the gauge size the smaller the bore. The usual
choice for an adult with normal veins is gauge 21 and they should be sharp, sterile and
without barbs. If blood is drawn for trace metals the needle should be stainless steel and
free from contaminants.
SKIN PUNCTURE
If only a small volume of blood is required for a test skin puncture is performed and
capillary blood is obtained. In an adult or grown child, blood may be obtained by
puncturing the tip of a finger. When the skin is dry after cleaning with alcohol the tip of the
finger is punctured by a sharp stab with a lancet.
ARTERIAL PUNCTURE
Arterial punctures require considerable skill and are usually performed by specially trained
medical officers. Femoral artery is the preferred site. The needle and syringe should be
flushed out with a heparin solution to ensure anticoagulation and to expel trapped air. If the
collected specimen is intended for blood gas analysis the nozzle of the syringe containing
the blood should be sealed and the syringe placed in ice water (melting ice) for immediate
transport to the laboratory. Analysis should be performed within 15 minutes.
URINE COLLECTION
The type of urine sample to be collected is dictated by the tests to be performed.
A clean early morning fasting specimen is generally the most concentrated and thus is
preferred for microscopic examination and for the detection of abnormal amounts of
constituents such as proteins, amino acids and β chorionic gonadotrophins.
A double voided specimen is the urine excreted during a timed period following the
complete emptying of the bladder. It is used for example to assess the glucose
excretion during a glucose tolerance test.
Timed urine specimens are used to minimize the influence of short term biological
variations. When specimens are to be collected over a specified period of time the
patient’s close adherence to instructions is important. The bladder should be emptied
at the time the collection is to begin, and this urine is discarded. Thereafter all urine
should be collected until the end of the scheduled time. The urine should be collected
in to a chemically clean container and should be transferred to the bottle using a
funnel. Any excess urine should be collected into a separate refrigerated container. At
the end of the collection period the last urine sample should be added into the bottle
and dispatched to the laboratory with out delay. Other relevant information (weight
Urine preservatives
One of the most satisfactory forms of preservation of urine specimens is refrigeration; it is
even more successful when combined with chemical preservation.
Gestational D.M
Any degree of clinical glucose intolerance with onset or first recognition during
pregnancy.
Symptoms
Polyuria
Polydypsia
Blurring of vision
Weight loss
OGTT is affected by
Metabolic stress( ↑ glucose secretion)
Major surgery, M.I, drugs (steroids)
Malabsorption
Vomiting
Gestational Diabetes
Diagnosis;
Fasting plasma glucose >7.0 mmol/L (126mg/dl)
Random plasma glucose >11.1 mmol/L (200mg/dl)
Lab diagnosis
One step approach 75g glucose-OGTT
Monitoring of Disease
Maintain Plasma glucose level as close as possible to normal levels during the day.
Use of the glucometer – only for monitoring not for the diagnosis
CALIBRATED glucometer
AFP Maternal 3-5 ml clotted blood 14 wks 46 µg/l Neural tube defects
15 wks 58 Downs syndrome
16 wks 64
17 wks 72
18 wks 84
19 wks 94
20 wks 108
21 wks 118
22 wks 120
Alkaline Phosphatase Isoenzymes 5 ml clotted blood Reference range available in text books Qualitative by Differentiation of increased
(Only analysed if patient has alkaline electrophoresis ALP
phosphatase >250 U/L) (Liver , Bone disorders)
ALP 3 ml clotted blood Males (age 20-60 Years) 20-90 IU/L 37C Bone and Liver diseases
( Alkaline Phosphatase) Females (age 15-60 Years) 20-90
Children (age 1-12 Years) up to 350
During the growth spurt of puberty up to 500
Alpha-1 Antitrypsin 4 -5ml Lithium Heparin or Newborn 1.45-2.7 g/L α 1- antitrypsin deficiency
(Phenotyping performed if <1.4 5 ml clotted blood Adult 0.78-2.00
g/L) >60 years 1.15-2.00
Adult
Male 2.6-7.2
Female 3-9.6
Apo- Lipoprotein B 3 ml clotted blood Male 0.63-1.33 g/L Investigation of lipid disorders
Female 0.60-1.26
Apo-Lipoprotein A-1 3ml clotted blood Male 0.94-1.78 g/L Investigation of lipid disorders
Female 1.01-1.99
AST 3 ml Clotted blood, avoid Newborn 10-75 IU/L 37C Hepatocellular disease
(Aspartate aminotransferase) haemolysis Children 10-45 Cardiac disease
Adult 4-42
Bilirubin (Urine) Random Urine Not normally detectable Qualitative Liver disease
Bilirubin (Total) 3 ml clotted blood Cord blood < 50 µmol/L Liver disease
Protect from light Cord blood premature infants < 58
First 24 h <103
2-5 days <205
>1 month 1.7-26
Adult 3-21
Bilirubin-Direct 3 ml clotted blood Adults and Children 0-3.4 µmol/L Neonatal jaundice
Protect from light Liver disease
CA 125 5 ml clotted blood < 35 kU/L Ovarian cancer
(Refer the analytical method)
CA 15-3 5 ml clotted blood Non pregnant < 28 kU/L Raised in cirrhosis
Pregnant Breast cancer
1 & 2 Trimester < 50
Calcium (Urine) 24 hours Urine Collected into New Born 0-17.5 µmol/kg/24 hours Disorders of calcium
acid washed bottle Infants up to 1000 µmol/kg/24 hours metabolism
Older Children up to100 µmol/kg/24 hours
Or 750-3750 µmol/24 hours
Adults
Calcium in diet
Calcium free 0.13-1.0 mmol/24 hours
Low –average 1.25-3.75 mmol/24 hours
Average( 800mg/day) 2.5-7.5 mmol/24 hours
Calcium ( Ionised) 3 ml Heparinised blood Cord blood 1.25-1.5 mmol/L Disorders of calcium
ISE (Ion Selective Electrode) Newborn 3-24 h 1.07-1.27 metabolism
24-48 h 1.00-1.17
Thereafter 1.12-1.23
Calcium ( Total) 3 ml clotted blood Collected Cord Blood 2.33-3.05 mmol/L Disorders of calcium
into acid washed bottle without metabolism
tourniquet New Born ( 1st week)
Bottle fed 1.85-2.75
Breast fed 2.05-3.05
Thereafter up to 12 years 2.20-2.75
Adults 2.25-2.60
CEA 5 ml clotted blood Non smokers <2.5 µg/L Tumour marker, especially of
(Cacino Embryonic Antigen) Smokers 2.6-5.0 colorectal, lung, breast and
pancreas
Ceruloplasmin 5 ml clotted blood 1 day- 3 months 5-18 mg/dl Wilson’s disease
6/12 months-1 year 33-43
1 year-7 years 24-56
>7 years , Adult 18-45
Chloride 5 ml clotted blood Cord blood 96-104 mmol/L Acid base disturbances
Newborn 0-30 days 98-113
After 1 month 98-107
Male Female
6 - 9 years 3.26-4.94 3.16-5.41 mmol/L
10-14 years 3.36-5.28 3.21-5.61
15-19 years 2.95-5.12 3.23-5.48
22-24 years 3.21-5.64 3.16-5.59
25-29 years 3.44-6.32 3.33-5.75
30-34 years 3.57-6.58 3.37-5.96
35-39 years 3.78-6.99 3.63-6.27
40-44 years 3.91-6.94 3.81-6.53
45-49 years 4.09-7.15 3.94-6.86
CK-MB 3 ml clotted blood < 12 or < 2.8 % of Total CK IU/L 37C Myocardial infarction
Adult
Male 10.99-21.98
Female 12.56-24.34
Cortisol-free (Urine) 24 hours urine collected into a Child 5.5-74.5 nmol/day Adrenocortical function
container with 10 g of Boric Adolescent 13.8-152
acid. Sample should be Adult 27.6-276
refrigerated during the
collection
Creatinine (Urine) 24 hours urine Under 12 years 44-354 µmol/kg/24 hours Marker of renal function
Adults 8.84-17.6 mmol/24 hours
Creatinine Clearance 24 hour urine and blood Newborn (up to 1 month) 40-65 ml/min/1.73 m2 Renal function
sample Male Female
( taken during the collection of Under 12 years 98-150 95-123 ml/min/1.73 m2
24 hour urine sample) 20-30 years 88-146 81-134
30-40 years 82-140 75-128
40-50 years 75-133 69-112
50-60 years 68-126 64-116
60-70 years 61-120 58-110
70-80 years 55-113 52-105
DHEA-Sulphate 3ml clotted blood Pre pubertal 0.25-1.00 µg/ml Adrenocortical function
preferred
Faecal Fat Minimum 3 day collection- Infant breast fed <1 g/day Gastrointestinal malabsorption
patient must be on a normal 0-6 years <2
diet(Collected between two Adult <7
markers) Adult (fat free diet) <4
Faecal Reducing substances Fresh faeces (send to lab Undetectable Qualitative test Gut sugar malabsorption
within 20 minutes or freeze)
Adult
Male 20-250
Female 10-120
5 12.8-17.3 y 2.6-11.0
Adult 2.0-9.2
Adult
Follicular 1.8-11.2
Mid cycle 6.0-35.0
Luteal 1.8-11.2
Post menopausal 30-120
Adult
Male ≤50
Female ≤30
Gastrin 3 clotted blood. (12 hour Child < 10-125 ng/L Zollinger-Ellison syndrome
fasting) Serum should be Adult 16-60 y Male <100
centrifuged, separated & Female <75
frozen at -20 C without delay.
Samples must not be >60 y <100
haemolysed and lipaemia
should be avoided
Glucose (CSF) 1 ml of CSF into Fluoride/ 70 % of plasma glucose mmol/L Meningitis (Bacterial/Viral)
Oxalate bottle (accompanied
with the blood sample into
fluoride oxalate)
Growth Hormone (serum) 3 ml clotted blood Newborn 1st day 5-53 ng/ml Pituitary function
1 week 5-27
1 month – 1 year 2-10
Child fasting at rest 0.7-6.0
Adult 0.7-6.0
Hydroxy progesterone(17 OHP) Serum 3 ml clotted Blood Male, Puberty stage -1 0.1-2.7 nmol/L Adrenal status
Adult 1.5-7.5
Female, Puberty stage -1 0.1-2.5
Follicular 0.6-3.0
Luteal 3.0-15.5
Postmenopausal ≤2.1
Congenital Adrenal
Neonates <30
Hyperplasia
>1 month 6
Hydroxyindoleacetic Acid 24 hours urine Adult 10.4-31.2 µmol/24 hours Carcinoid syndrome
(5-HIAA) Children 7-70
Insulin (12 hour fasting ) 5 ml clotted serum should be Newborn 21-139 pmol/L Insulinoma
centrifuged , separated & Adult 13-174 Insulin overdose
frozen at -20 C within 2 hours >60 years 42-243
Iron 5 ml clotted blood collected New Born up to 1 month 17.9-44.75 µmol/L Iron status
into acid washed bottle Infant (1month-1 year) 7.16-17.9
Child (1 year-12 year) 8.95-21.48
Adult
Male 11.64-30.43
Female 8.95-30.43
(Strongly method dependent)
Iron Binding Capacity , Total 5 ml clotted blood collected Infant 17.9-71.6 µmol/L Iron status
(TIBC) into acid washed bottle Thereafter 44.5-80.55 µmol/L
1 <9.2 y 0.02-0.18
2 9.2-13.7 y 0.02-4.7
3 10-14.4 y 1.0-12.0
4-5 10.7-15.6 y 0.4-11.7
Adult
Follicular 29
Mid cycle 18-49
Luteal 2-11
Magnesium 3 ml clotted bottle collected Newborn 2-4day 0.6-0.9 mmol/L Electrolyte status
into acid washed bottle 5 months – 6year 0.70-0.95
(preferably without tourniquet) 6-12 years 0.70-0.86
12-20 years 0.70-0.91
Adult 0.66-1.07
Potassium 3 ml clotted blood (avoid <2 month 3-7 mmol/L Electrolyte status
haemolysis & do not 2-12 month 3.5-6.0
refrigerate) > 12 month 3.5-5.0
Adult 3.5-5.6
Pregnancy Test(Urine) Random Urine Qualitative Not applicable Pregnancy
Progesterone 5 ml clotted Pre pubertal child (1-10 y) 0.2-1.7 nmol/L Ovulatory status
Puberty
Tanner Male Female
1 <0.3-1.0 <0.3-1.0
2 <0.3-1.0 <0.3-1.7
3 <0.3-1.5 .3-14.3
4 <0.3-3.4 <0.3-41.3
5 0.7-2.6 0.3-30.2
Adult
Male 0.4-3.1
Female
Follicular 0.5-2.2
Luteal 6.4-79.5
Pregnancy 7-13 wk 32.6-139.9
4-37 wk 62-262.4
30-42 wk 206.7-728.2
Children
Tanner Male Female
1 <10 3.6-12
2-3 <6.1 2.6-18
4-5 2.8-11 3.2-20
Adult
Male 3-14.7
Female 3.8-23.2
Pregnancy 3rd trimester 95-473
Prostate Specific Antigen (PSA) 5 ml clotted blood <4 µg/L Prostate cancer
Protein (CSF), Lumbar CSF in plain container Premature 15-130 mg/dl Inflammatory conditions of the
Full term newborn 40-120 meninges
<1 month 20-80
Thereafter 15-40
Protein(Total) 3 ml clotted blood New borne 46-77 g/L Malnutrition , liver disease and
1 Year 56-73 protein losing conditions
2-3 Year 58-76
4th Year and after 60-80
Adult 60-80
Body Fluid (Transudates) <3
(Exudates) >3
Renin 5 ml blood taken into a bottle 0-3 y <16.6 µg/L/hr Hypertensive states
containing EDTA ,must be 3-6 y <6.7
separated at room temperature 6-9 y <4.4
and frozen at -20 C or 9-12 y <5.9
lower(Contact the reference lab 12-15 y <4.2
before collection of blood) 15-18 y 4.3
Normal sodium diet
Supine 0.2-1.6
Upright (4 hours) 0.7-3.3
Low sodium diet
Supine 1.0-5.4
Upright (4 hours) 0-19.0
31-33 1.0-3.8
34-36 1.2-4.4
3 10-14.4 y 0.15-0.35
4 10.7 -15.6 y 0.13-0.32
5 11.8 -18.6 y 0.20-0.38
Adult 0.1-0.5
Thyroglobulin 5ml Clotted blood 3.0- 42 µg/L Carcinoma of thyroid
Vitamin D (25 OHD3) 5ml Clotted blood Children Vitamin D metabolism and
(25 Hydroxy cholecalciferol) 1-30 d 1.9-33.4 ng/ml Calcium metabolism
31d - 1 y 7.4-53.3
Adult 14-60
Vitamin D(1,25 OHD3) 5ml Clotted blood 43-154 pmol/L Vitamin D metabolism and
(1,25-dihydroxy cholecalciferol) Calcium metabolism
Vitamin E 5ml Clotted blood 11.6-46.4 µmol/L Nutritional status
REFERENCES:
1. Teitz text book of clinical chemistry by Carl A.Burtis, Edward R.Ashwood; 2nd and 3rd Edition
2. Clinical chemistry – theory , analysis, correlation by Lawrence A.Kaplan, Amadeo J. Pesce; 3rd Edition 1996
3. WHO Guidelines on standard operating procedures for clinical chemistry, Sep 2000
4. Nelson text book of paediatrics by Behrman, Kliegman, Jenson; 16th Edition 2000
5. Forfar and Arneil’s Text book of paediatrics
6. Biochemical basis of paediatric disease by Steven J Soldin,Nader Rifai,Jocelyn M. Hicks ;3rd Edition 1998
WE SINCERELY THANK THE FOLLOWING COLLEAGUES / MEMBERS OF MRI AND WHO COUNTRY OFFICE
Administrative staff
Ms. Kumuduni Ragel
Secretary WHO country office Sri Lanka
Mrs. G. Subramanium
Accountant MRI
Mr. A. Ravichandran
Financial staff
Support staff
Mr. N. A. H. H. Nissanka
Mr. J. M. Wijesinghe
Mr. D. L. Upasena
Mr. T. V. Anton
Ms. W. Pushpika Perera