COOMB’S TECHNIQUES MLS 522

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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

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.

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.