1. COOMB’S TECHNIQUES
MLS 522

2. Introduction
With Landsteiner’s discovery in 1901 of the ABO blood group system, the first step towards a rational approach to blood transfusion became possible.
In 1945, Coombs and colleagues described the use of antihuman globulin(AHG) test for the detection of weak and non-agglutinating Rh antibodies in serum.
They described the use of AHG to detect invivo sensitization of the red cells of babies suffering from Hemolytic disease of the newborn.

3. Coombs Techniques
Apart from Rh HDN, Coomb’s test can also be used to detect the presence of other IgG antibodies e.g Kell blood group.
Coombs procedure involve the injection of human serum into rabbits to produce antihuman serum.
Heterospecific antibodies were removed by absorption and the antiserum was diluted in such a way that it will still retain its antibody activity.

4. Coombs Techniques
And this will permit crosslinking of RBCs sensitized with IgG antibodies.
This crosslinking of RBCs by the AHG will produce agglutination.
The agglutination reaction indicate that the RBCs had been sensitized by antibody which has reacted with the an antigen present on the cell surface.

5. Coombs Techniques
The Coombs test can be; Indirect Coombs test(ICT) ,also known as Indirect Antihuman globulin Test(IAT)
This is used to detect invitro sensitization of red cells.
It can also be Direct Coombs test(DCT) ,also known as Direct Antihuman globulin Test(DAT)
DAT can be used to detect invivo sensitization of red cells.

6. Direct Coomb’s Test
The DCT is used to detect in vivo sensitization of red cells.
The clinical conditions that can result into in vivo sensitization of these red cells include HDN, hemolytic transfusion reaction, autoimmune and drug induced hemolytic anaemia.
In performing DCT , polyspecific followed by monospecific reagents are to be used in order to determine the type of protein sensitizing the cell.

7. Direct Coomb’s Test
The DAT procedure involve washing of cells in normal saline to remove free globulin molecules.
Addition of AHG to bridge the gap between corresponding IgG molecules.
Centrifugation to accelerate the agglutination process.
Agglutination check both macroscopically and microscopically.

8. Significance of a Positive DAT
A positive DAT can be seen in;
Recipient alloantibody and donor antigens
Donor antibody and recipient antigen
Drug absorption
Immune complex absorption
Membrane modification
Warm AIHA IgG and /or C3
Maternal alloantibody crosses placenta
Administration of high dose IV gammaglobulin.

9. Indirect Coomb’s Test
This test which determines in vitro sensitization of red cells is used to;
Detect incomplete antibodies to donor red cells in compatibility testing or screening cells in antibody screen.
Identification of antibody specificity using panel of cells with known antigen profiles.
Determination of red cell phenotypes using known antisera e.g. Kell typing, Du testing.

10. Indirect Coomb’s Test The IAT procedure involve;
Incubation of red cells reagent with antisera to allow molecule attachment to red cells
Washing of cells in normal saline to remove free globulin molecules.
Addition of AHG to bridge the gap between corresponding IgG molecules.

11. Indirect Coomb’s Test
Centrifugation to accelerate the agglutination process.
Agglutination check both macroscopically and microscopically.
Grading of agglutination reaction to determine the strength of the reaction.
Addition of antibody coated cells (Coombs control cells) to those with negative reactions.

12. Factors Affecting the Coomb’s Tests
Cell to Serum ratio
Temperature
Incubation time
Reaction Medium
Washing of cells
Saline for washing
Addition of AHG
Centrifugation speed

13. Sources of Error in Coomb’s Techniques
False positive results can be obtained in;
Auto agglutination
bacterial contamination
dirty glassware
use of control cells as patient’s sample
poly agglutination
overcentrifugation.

14. Sources of Error in Coomb’s Techniques
False negative results can be obtained in ;
inadequate or improper washing
deterioration of AHG reagent
omission of serum
too weak or too heavy cell suspension
under or over centrifugation
poor reading techniques.