STANDARD OPERATING PROCEDURE (SOP) : INDIRECT ANTIGLOBULIN / COOMB’S TEST (IAT / ICT)

BLOOD CENTRE NAME

SCOPE AND APPLICATION

The IAT is performed to determine in vitro sensitization of RBCs and is used in the following situations:

 • Detection of incomplete (non-agglutinating) antibodies to potential donor RBCs (compatibility testing) or to screening cells (antibody screen) in serum

 • Determination of RBC phenotype using known antisera (e.g., weak D, any other antigen testing that requires IAT)

 • Titration of incomplete antibodies

RESPONSIBILITY

It is the responsibility of the resident doctor/Technician posted in Immunohaematology Lab to perform Indirect Antiglobulin Test and document the results

REFERENCES

• Modern Blood Banking and Transfusion Services, Denise M. Harmening 6^th edition
• Technical Manual of American Association of Blood Banks, 18^th edition

MATERIAL REQUIRED

Equipment:

• Tabletop centrifuge
• Microscope
• Incubator 

Specimen:

• Clotted blood sample of donors/patients.

Reagents:

• Group O pooled cells 
• Antihuman globulin reagent (anti-IgG+anti-C3d) 
• Coomb’s control check cells (IgG Coated cells) 
• 0.9% Normal Saline. 

Glassware:

• Pasteur pipettes.
• Glass slides
• Glass Test tubes 

Miscellaneous:

• Rubber teats. 
• Disposal box
• plastic beakers
• Test tube Stand

PROCEDURE:

• Label the test tube with Patient’s Name or MRD or CR number
• Add 2 drops of patient’s serum to the labelled test tube 
• Add 1 drop of 5% red cell suspension of reagent red cells (Pooled O) to the tube and mix well
• Centrifuge the tube at 1000 rpm for 1 minute
• Observe for haemolysis and agglutination. Grade and record the results
• Incubate at 37⁰ C for 45 to 60 minutes
• Wash the red cells at least 3 times using Normal Saline at 3000 rpm for 3 minutes
• Decant completely after last wash
• Add 1 drop of AHG reagent to the dry red cell button and mix well 
• Centrifuge the tube at 1000 rpm for 1 minute
• Observe for any agglutination
• If the test is negative, add 1 drop of IgG sensitised control cells 
• Centrifuge the tube at 1000 rpm for 1 minute and observe for agglutination
• If no agglutination, the test is invalid and needs to be repeated
• If there is agglutination, titration should be performed
• Label 10 test tubes according to the serum dilution (1:1, 1:2, 1:4 etc)
• Deliver 1 volume of saline to all the test tube except the first two test tube
• Using pipette, mix the contents of the 1:2 dilution several times and transfer one volume into the next tube (1:4 dilution)
• Continue the same process for all dilutions. Remove one volume of diluted serum from the final tube and save it for use if further dilutions are required
• Perform IAT to each dilution. Grade and record the results.

INTERPRETATION:

• The test is positive when agglutination is observed after centrifugation and the agglutination reaction is graded
• The test is negative when there is no agglutination after centrifugation, provided the check cells give a positive reaction
• The test is invalid when no agglutination is observed after adding Coomb’s check cells

DOCUMENTATION:

• Enter results in Immunohaematology register
• Report positive findings to the In-Charge

END OF DOCUMENT