TRAINING FILE

ON

MEDICAL LABORATORY

TECHNOLOGY

Submitted By

PRAVEEN YADAV
Roll No-170661900023

Training coordinator Submitted To

MR. RAJ KUMAR MRS. ARCHANA SISODIA

HR HOD

RAGHAV DIAGNOSTIC CENTRE DPG POLYTECHNIC COLLEGE

 INDEX
BIO-CHEMISTRY

S. NAME OF THE EXPERIMENT PAGE

NO. NUMBER

1. ESTIMATION OF SGOT

2 ESTIMATION OF SGPT

3. ESTIMATION ALKALINE PHOSPHATE

4. ESTIMATION OF BILIRUBIN

5 ESTIMATION OF HDL
 INDEX
SEROLOGY
S. NAME OF THE EXPERIMENT PAGE
NO. NUMBER

1. WIDAL TEST

2 DENGUE NS1

3. MALARIA PARASITE
 INDEX
HAEMATOLOGY
S. NAME OF THE EXPERIMENT PAGE
NO. NUMBER

1. BLOOD GROUP

2 BLEEDING TIME

3. CLOTTING TIME
BIO-CHEMISTRY
 Experiment No. 1
Aim of Experiment: - ESTIMATION OF SGOT
Name of method: - Reitman’s and Frankels Method
Principle: - the buffered substrate reagent contains aspartate and 2 ketoglutarate,
SGOT reacts with the substrate and transfer the amino group from aspartate to 2
ketoglutarate to form oxaloacetate and glutamate. The oxaloacetate reacts with 2, 4
dinitrophenylhydrazine and produce 2, 4 dinitrophenylhydrazone at alkaline pH.

Requirement: -
Test tube, test tube stand, pipette, incubator, colorimeter and test kit.

Procedure: -
 Take 4 test tube and marks as test, standard, control and blank.
 Then add reagent 1 in all test tube 0.5 micro liter.
 Then add distilled water 0.1 ml in blank test tube.
 Then add serum sample 0.1 micro liter in test marked tube.
 Then add standard 0.1 ml in standard test tube.
 Incubate the tube at room temperature for 60 minutes.
 Add reagent 2, 0.5 micro liters in all test tube.
 Then add serum sample 0.1 micro liter in control marked tube
 Incubate the tube at room temperature for 20 minutes.
 Add reagent 3, 3.0 micro liters in all test tubes.
 Mix well and read the observance.

Calculation: -
OD of test\OD of standard*concentration of standard.
 Experiment No. 2
Aim of Experiment: - ESTIMATION OF SGPT
Name of method: - Reitman’s and Frankels Method
Principle: - the buffered substrate reagent contains aspartate and 2 ketoglutarate,
SGOT reacts with the substrate and transfer the amino group from aspartate to 2
ketoglutarate to form oxaloacetate and glutamate. The oxaloacetate reacts with 2, 4
dinitrophenylhydrazine and produce 2, 4 dinitrophenylhydrazone at alkaline pH.

Requirement: -
Test tube, test tube stand, pipette, incubator, colorimeter and test kit.

Procedure: -
 Take 4 test tube and marks as test, standard, control and blank.
 Then add reagent 1 in all test tube 0.5 micro liter.
 Then add distilled water 0.1 ml in blank test tube.
 Then add serum sample 0.1 micro liter in test marked tube.
 Then add standard 0.1 ml in standard test tube.
 Incubate the tube at room temperature for 60 minutes.
 Add reagent 2, 0.5 micro liters in all test tube.
 Then add serum sample 0.1 micro liter in control marked tube
 Incubate the tube at room temperature for 20 minutes.
 Add reagent 3, 3.0 micro liters in all test tubes.
 Mix well and read the observance.

Calculation: -
OD of test\OD of standard*concentration of standard.
 Experiment No. 3
Aim of Experiment: - ESTIMATION ALKALINE PHOSPHATE
Name of method: - Para-nitro phenyl phosphate method
Principle: -
The determination of alkaline phosphates using an optimized substrate concentration
and 2-amino-2-methyl-1-propanol as buffer, plus the cations magnesium and zinc. The
assay described below meets the recommendations of the IFCC. Colorimetric assay in
accordance with a standardized method. In the presence of magnesium and zinc ions, p-
nitro phenyl phosphate is cleaved by phosphates into phosphate and p-nitro phenol.
Alkaline Phosphates Reagent 1 Composition Concentration 2-Amino-2-methyl-1-
propanol (pH 10.44) 1.724 mol/l Magnesium acetate 3.830 mol/l Zinc sulfate 0.766 mol/l
N-(2-hydroxyethyl)-ethylenediamine triacetic acid 3.830 mol/l Reagent 2 Composition
Concentration Catalog Number p-Nitro phenyl phosphate 132.8 mol/l 10 270 857 103
Preservative Test principle: Enzymatic colorimetric 4-Nitrophenyl phosphate (colorless)
ALP 4-Nitrophenol (yellow) The p-nitro phenol released is directly proportional to the
catalytic ALP activity. It is determined by measuring the increase in absorbance at 409
nm.

Requirement: -
Test tube, test tube stand, pipette, incubator, colorimeter and test kit.

Procedure: -
 Take 3 test tube and marks as test, standard and blank.
 Then add reagent 1 in all test tube 0.8 micro liter.
 Then add serum sample 0.02 micro liter in test marked tube
 Then add standard 0.02 ml in standard test tube.
 Then add distilled water 0.02 ml in blank test tube.
 Incubate the tube at room temperature for 05 minutes.
 Add reagent 2, 0.2 micro liters in all test tube.
 Mix well and read the observance.
Calculation: -
OD of test/OD of standard * concentration of standard.
 Experiment No. 4
Name of the experiment: - Estimation of serum Bilirubin
Name of the method: - Di-methyl Sulphoxide method
Principle of the test: -
The bilirubin is reacts with the diazonium salt of sulfonalic acid and produced
azobilirubin in an acidic medium. Di-methyl sulphoxide is act as an accelerator and help
in the production of azobilirubin and color compound.
Requirement: -
Test tubes, test tube stand, pipettes, colorimeter, sample and reagent.
Reagents: -
 Diazo reagent A
 Diazo reagent B
 Bilirubin standard
 Sulphonalic acid
Procedure: -
1) Take four clean and dry test tubes.
2) Mark them as total test, total blank, direct test and direct blank.
3) Pipette reagent A 2.8 micro liters in the total test and total blank.
4) Than pipette Sulphonalic acid 2.8 micro liters in the direct test and direct blank.
5) Add reagent B 1.0 micro liters in the total test and direct test.
6) Add serum sample in all the test tubes.
7) Mix all the content of the tubes.
8) Read the optical density of all the test tubes.

Normal value: -
 Total bilirubin: - up to 1.0 mg/dl.
 Direct bilirubin: - up to 0.2 mg/dl.
 Indirect bilirubin: - up to 0.8 mg/dl.
 Experiment No. 5
Name of the experiment: - ESTIMATION OF HDL
PRINCIPLE:
Phosphotungstic acid and magnesium ions selectively precipitating all lipoproteins
except the HDL fraction – cholesterol present in the supernatant can be determined by
the same method used for total cholesterol.

PROCEDURE:
Precipitation:
Sample 0.20 ml Precipitating reagent (R1) 0.02 ml Vortex, let stand 10 min., centrifuge
for 15 min. at 3000 rpm. Measure HDL –Cholesterol in the supernatant using the same
method for total Cholesterol. Determination of HDL-Cholesterol: Blank (ml) Standard
(ml) Sample (ml) Distilled water 0.05 - - Standard (R2) - 0.05 - Supernatant - - 0.05
Working reagent 1.0. Mix, incubate at 37ºC for 10 min. Read absorbance of sample
(Sample) and standard (A Standard) against the blank at 500 nm, (495 – 550 nm ).

CALCULATION:
Sample/standard * 55
SEROLOGY
 Experiment No. 1
Name of the experiment: - WIDAL TEST
PRINCIPLE:
Principle of Widal test is that if homologous antibody is present in patient’s serum, it will
react with respective antigen in the reagent and gives visible clumping on the test card
and agglutination in the tube. The antigens used in the test are “H” and “O” antigens
of Salmonella Typhii and “H” antigen of S. Paratyphii. The paratyphoid “O” antigen are
not employed as they cross react with typhoid “O” antigen due to the sharing of factor
12. “O” antigen is a somatic antigen and “H” antigen is flagellar antigen.

PROCEDURE:
1. Place one drop of positive control on one reaction circles of the slide.
2. Pipette one drop of isotonic saline on the next reaction circle. (-ve Control).
3. Pipette one drop of the patient serum tube tested onto the remaining four reaction
circles.
4. Add one drop of Widal test antigen suspension ‘H’ to the first two reaction circles.
(PC & NC).
5. Add one drop each of ‘O’, ‘H’, ‘AH’ and ‘BH’ antigens to the remaining four reaction
circles.
6. Mix contents of each circle uniformly over the entire circle with separate mixing
sticks.
7. Rock the slide, gently back and forth and observe for agglutination macroscopically
within one minute.
 Experiment No. 2
Name of the experiment: - DENGUE NS1
PRINCIPLE:
When a sample is added to the sample well dengue virus NS1 antigens will react with
colloidal gold conjugated with anti dengue NS1 monoclonal antibody and form a complex
of antibody- antigen as this complex migrate along the length of the test device by
capillary action, it will be capture by another anti dengue NS1 monoclonal antibody
immobilized in a test line across the test membrane and generate a colored line.

PROCEDURE:
 Bring all test device, reagent and sample to room temperature for 15 minutes prior
to performing the assay.
 Use a fresh test device for every sample. The device is not reusable.
 Just prior to use, remove the required number of dengue NS1 test device from
there wrapper and place them in a flat surface area.
 Take 100 micro liter of whole blood or serum, plasma with the including disposable
pipette and add into the sample well.
 Interpret test result at 15 to 20 minutes.
 Do not interpret the test result after 20 minutes, this can give false result.
 Experiment No. 3
Name of the experiment: -MALARIA PARASITE
PRINCIPLE:
If malaria antigen is present in the blood sample, Ag-Ab complex will be formed as it
combines with the labeled pan-specific antibody present in the mobile phase. This Ag-
Ab complex will migrate along the test strips, which will be captured by the specific
antibodies present in the immobile phase (here T1 contains monoclonal antibodies
specific to P.falciparum i.e. HRP2 antigen and T2 contains plasmodium pan specific
antibody i.e. pLDH) thus producing a visible colored line. Control line contains goat
anti-mouse antibody and ensures that system is controlled for migration.

PROCEDURE:

1.Check the expiry date on the test packet.

2.Put on the gloves. Use new gloves for each patient.
3.Open the test kit packet and remove, test pad, capillary tube and Desiccant sachet.
4.Write the patient’s name on the test.
5.Open the alcohol swab. Grasp the 4th finger on the patient’s left hand. Clean the
finger with the alcohol swab. Allow the finger to dry before pricking.
6.Open the lancet. Prick patient’s finger to get a drop of blood. Do not allow the tip
of the lancet to touch anything before pricking the patient’s finger.
7.Discard the lancet in the Sharps Box immediately after pricking finger. Do not set
the lancet down before discarding it.
8.Use the capillary tube to collect the drop of blood.
9.Use the capillary tube to put the drop of blood into the square hole marked “A”.
10. Discard the capillary tube in the Sharps Box.
11. Add buffer into the round hole marked “B”.
HAEMATOLOGY
 Experiment No. 1
Name of the experiment: - BLOOD GROUPING
PRINCIPLE:
The ABO and Rh blood grouping system is based on agglutination reaction. When
red blood cells carrying one or both the antigens are exposed to the corresponding
antibodies they interact with each other to form visible agglutination or clumping.

PROCEDURE:
 Inform the patient or individual about the procedure to be carry out
 Dangle the hand down to increase the flow of blood in the fingers.
 Clean the fingertip to be pierced with spirit or 70% alcohol (usually ring or middle finger)
and gently massage the finger to increase blood flow
 With the help of the sterile lancet or pricker, pierce the fingertip and place one drop of
blood in each of the four cavities
 Now add one drop of each antiserum into each cavity respectively
ABO and Rhesus Blood Grouping Tiles
 Mix each blood drop with the antiserum using a fresh mixing stick or applicator stick
 Now you can observe agglutination in the form of fine red granules within 30 seconds.
Anti RhD takes slightly longer time to agglutinate compared to Anti A and Anti B.
 Experiment No. 2
Name of the experiment: - BLEEDING TIME
PRINCIPLE:
In the Ivy method, a blood pressure cuff is placed on the upper arm and inflated to 40
mmHg. ... A stopwatch is started immediately and every 30 seconds filter paper is used
to draw off the blood. The time from when the incision is made until all bleeding has
stopped is called the bleeding time.

PROCEDURE:
A blood pressure cuff is first inflated on the upper arm to a standardized pressure, then a
small incision is made on the forearm (avoiding any veins) and the time until bleeding
stops completely (in seconds) is measured.

Bleeding time test results explained

A prolonged bleeding time may be due to:

 Low platelet count (thrombocytopenia)

 Decreased platelet function – for example due to kidney failure or aspirin therapy
 von Will brand Disease – a deficiency of von Will brand Factor
 Disseminated intravascular coagulation (DIC) – bleeding disorder in critically ill patients,
for example with severe infections
 Experiment No. 2
Name of the experiment: - CLOTTING TIME
PRINCIPLE:
Clotting time is the time required for a sample of blood to coagulate in vitro under
standard conditions. ... Normal value of clotting time is 8 to 15 minutes. For the
measurement of clotting time by test tube method, blood is placed in a glass test tube
and kept at 37° C.

PROCEDURE:
 Take a sterilized capillary tube.
 Collect the blood into the capillary tube by capillary action.
 Then start the stopwatch.
 Break the tube in small parts.
 When you see the coagulation into the blood then note the time.

Normal range: -
The normal range of the test is 7 to 14 minutes.