Laboratory manual in AHG | Summaries Health sciences

ANTIHUMAN GLOBULIN TESTING (AHG)/

COOMBS TEST (DIRECT AND INDIRECT)

I. PRINCIPLE

This test is used to detect the

presence of human globulin molecules

either bound to the red cells or free

serum. It makes use of reagents

produced by rabbits which will react to

human antibodies. DIRECT

ANTIGLOBULIN TEST (DAT) is used

to detect antibody binding to antigen

on the surface of RBC’s that happens

in vivo. INDIRECT ANTIGLOBULIN

TEST (IAT) is used to detect for the in

vitro sensitization of red blood cells

with blood group antibodies.

II. MATERIALS

1. TEST TUBES

2. CENTRIFUGE

3. AHG REAGENT

4. NSS

5. MICROSCOPE

III. QUALITY CONTROL

Reagents must be stored at 2-8

degrees centigrade and must be at

room temperature prior to use. Make

sure that it is not beyond its expiry

date before using.

IV. SPECIMEN

No special preparation of the patient is

required.

Blood should be collected by approved

techniques.

V. PROCEDURE

DAT

1. Prepare 3-5% of RBC suspension

of the patient’s red cell.

2. In an appropriately labeled tube,

place 1-2 drops of cell suspension

and wash it with saline 3-4 times

3. Add 2 drops of the AHG reagent to

the cell button and resuspend the

cells.

4. Centrifuge at 3200 rpm for 15

seconds

5. Read and grade

6. Record

7. If negative, add 1 drop of check

cells to the negative tube. This

should give a positive result.

IAT

1. Prepare 3-5% of RBC suspension

of the patient’s cells.

2. Into a tube, place 2 drops of the

prepared red cell suspension.

3. Add 2 drops of patient’s serum into

the same tube.

4. Incubate the tube at 37 degrees

centigrade in a water bath for 15

minutes.

5. Remove the tube from the water

bath, centrifuge and resuspend and

observe for agglutination or

hemolysis.

6. If no agglutination, wash the

serum-cell mixture 3 times by NSS

7. After the 3rd washing. Add 2 drops

of AHG reagent, mix and centrifuge

at 3200 rpm for 15 seconds

8. Read and grade

9. Record

10. If negative, Add 1 drop of check

cells to the negative test tube.

This should give a positive result.

VI. REPORTING OF RESULTS

Agglutination in DAT – positive

result

oDue to in vivo sensitization of

antibodies coating to the

patient’s RBC’s.

Agglutination in IAT – positive result

oSuggests that there is in vitro

sensitization which tells that

there is an antibody which will

possibly react to the patient’s

RBC’s.

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ANTIHUMAN GLOBULIN TESTING (AHG)/

COOMBS TEST (DIRECT AND INDIRECT)

I. PRINCIPLE

 This test is used to detect the presence of human globulin molecules either bound to the red cells or free serum. It makes use of reagents produced by rabbits which will react to human antibodies. DIRECT ANTIGLOBULIN TEST (DAT) is used to detect antibody binding to antigen on the surface of RBC’s that happens in vivo. INDIRECT ANTIGLOBULIN TEST (IAT) is used to detect for the in vitro sensitization of red blood cells with blood group antibodies.

II. MATERIALS

1. TEST TUBES

2. CENTRIFUGE

3. AHG REAGENT

4. NSS

5. MICROSCOPE

III. QUALITY CONTROL

 Reagents must be stored at 2- degrees centigrade and must be at room temperature prior to use. Make sure that it is not beyond its expiry date before using.

IV. SPECIMEN

 No special preparation of the patient is required.  Blood should be collected by approved techniques.

V. PROCEDURE

 DAT

Prepare 3-5% of RBC suspension of the patient’s red cell.
In an appropriately labeled tube, place 1-2 drops of cell suspension and wash it with saline 3-4 times 3. Add 2 drops of the AHG reagent to the cell button and resuspend the cells. 4. Centrifuge at 3200 rpm for 15 seconds 5. Read and grade 6. Record 7. If negative, add 1 drop of check cells to the negative tube. This should give a positive result.  IAT
Prepare 3-5% of RBC suspension of the patient’s cells.
Into a tube, place 2 drops of the prepared red cell suspension.
Add 2 drops of patient’s serum into the same tube.
Incubate the tube at 37 degrees centigrade in a water bath for 15 minutes.
Remove the tube from the water bath, centrifuge and resuspend and observe for agglutination or hemolysis.
If no agglutination, wash the serum-cell mixture 3 times by NSS
After the 3rd^ washing. Add 2 drops of AHG reagent, mix and centrifuge at 3200 rpm for 15 seconds
Read and grade
Record
If negative, Add 1 drop of check cells to the negative test tube. This should give a positive result.

VI. REPORTING OF RESULTS

 Agglutination in DAT – positive result o Due to in vivo sensitization of antibodies coating to the patient’s RBC’s.  Agglutination in IAT – positive result o Suggests that there is in vitro sensitization which tells that there is an antibody which will possibly react to the patient’s RBC’s.

VII. PROCEDURE NOTES

 AHG is also known as – COOMB’S TEST.  1945 – Coomb’s, Maorant, and Race described a test for detecting nonagglutinating (coating) Rh antibodies in serum which they used to demonstrate in vivo coating of red cells with an antibody and a complement component.  DAT – is used to diagnose HDN, autoimmune hemolytic anemia, and hemolytic transfusion reaction.  IAT – used to demonstrate in vitto reactions between red cells and coating antibodies, as in: o Antibody detection o Antibody identification o Blood grouping o Compatibility testing  False positive results may arise in the following circumstances:  improper specimen (refrigerated or clotted) may cause in vitro complement attachment  autoagglutinable cells  bacterial contamination of cells or saline contaminated with heavy metals or colloidal silica  dirty glassware  overcentrifugation and over reading  polyagglutinable cells  preservative dependent antibody in LISS reagents  contaminating antibodies in the antihuman globulin reagent.  False negative results  Inadequate or improper washing of RBC’s  Deterioration of reagent or neutralization  AHG reagent not added  serum not added in the Indirect test  serum not reactive (complement inactivation)  inadequate incubation condition in the Indirect test  cell suspension too heavy or too weak

 undercentrifugation  poor reading technique  FACTORS AFFECTING THE SENSITIVITY OF IAT:  Temperature  Ionic strength  Ration of serum to cells  pH of the reaction mixture  incubation time

MAJOR CROSSMATCHING

I. PRINCIPLE

 Make use of patient’s serum mixed with donor’s RBC to detect antibodies in the patient’s serum that may be possibly damage or destroy the donor’s RBC’s.

II. MATERIALS

Test tube
Water bath
AHG reagent
Centrifuge
Microscope

III. QUALITY CONTROL

 No special preparation is required.  Blood should be collected by appropriate techniques  Clotted of anticoagulated blood drawn within 72 hours of testing may be used  Fresh serum must be used to assure the presence of adequate complement and calcium  If plasma is used, complement- dependent antibodies may not be detected.

IV. PROCEDURE

 Immediate spin/saline phase

Place 2 drops of Px serum into a 5 ml tube
Add 1 drop of 2-5% donor’s red cells
Mix and spin for 15 seconds at 3400 rpm then read for agglutination or hemolysis  Incubation at 37c/protein phase
Add 2 drops of 22% bovine albumin or LISS or PEG
Mix and incubate at 37c for 15- 30 minutes
Centrifuge for 15 seconds and observe for hemolysis or agglutination.  Antiglobulin phase
Wash 3-4 times with NSS
Add 2 drops of AHG reagent globulin reagent
Centrifuge for 15 sec at 3400 rpm
Read observe for agglutination or hemolysis 5. Rest all negative results with check cells.

V. REPORTING OF RESULTS

 Hemolysis/agglutination of a crossmatch indicates – presence of an antibody directed against the corresponding antigen which is present on donor’s cells. o INTERPRETED AS INCOMPATIBLE  NO agglutination/hemolysis is a negative test result indicates the ABSENCE of AB/AG present in the donor’s RBC’s and patient’s serum respectively. o INTERPRETED AS COMPATIBLE

VI. PROCEDURE NOTES

 The main aim of crossmatching is to ensure a safe transfusion. Upon the performance of crossmatching, it involves three procedures:  Pre-serologic procedures  Serological testing  Pos-serological testing

Laboratory manual in AHG, Summaries of Health sciences

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Download Laboratory manual in AHG and more Summaries Health sciences in PDF only on Docsity!

ANTIHUMAN GLOBULIN TESTING (AHG)/

COOMBS TEST (DIRECT AND INDIRECT)

I. PRINCIPLE

II. MATERIALS

1. TEST TUBES

2. CENTRIFUGE

3. AHG REAGENT

4. NSS

5. MICROSCOPE

III. QUALITY CONTROL

IV. SPECIMEN

V. PROCEDURE

 DAT

VI. REPORTING OF RESULTS

VII. PROCEDURE NOTES

MAJOR CROSSMATCHING

I. PRINCIPLE

II. MATERIALS

III. QUALITY CONTROL

IV. PROCEDURE

V. REPORTING OF RESULTS

VI. PROCEDURE NOTES