Coombs' Activity

Introduction

Antibody-mediated agglutination is a technique developed by Dr. Robin Coombs, a professor of Pathology at Cambridge University. Although originally developed as a test for autoimmune hemolytic anemia, the test has since been applied to both blood and tissue typing. When applied to the prevention of Hemolytic Disease of the Newborn (HDN), the procedure can either be used directly to test the infant, or indirectly to test the mother.

HDN occurs in infants with an Rh blood group antigen (D antigen) whose Rh-negative mothers have been exposed to D antigen, usually from a previous childbirth. Exposure occurs during birth, when fetal cells enter the mother's blood stream. In response to such an exposure, the mother produces IgG antibodies which will pass through the placenta and attack the red blood cells of a future Rh+ child.

Once diagnosed as having been exposed to the D antigen using an Indirect Coombs' Test, the mother can be given passive anti-Rh gamma globulin should she become pregnant again, to prevent her from generating antibodies capable of destroying her baby's red blood cells. If passively immunized at seven months and on delivery, the mother's immune response is suppressed and successive pregnancies should be uncomplicated by HDN.

The Coombs' Test is a technique that detects anti-red blood cell antibodies (anti-D) using a second antibody which binds to the first anti-Rh antibody. Visualization of a positive result is made possible by this second antibody, which cross-links antibodies bound to individual red blood cells.

The Direct Coombs' Test is used to test infants' red blood cells for the presence of bound maternal anti-D antibodies. Coombs' reagent (anti-human IgG) is then added to infant RBCs which, if positive, will already contain pre-bound anti-D antibody from the mother. Cross-linking occurs when anti-human IgG binds to multiple infant RBCs. A negative result indicates that the mother does not have anti-D antibodies or that the infant RBCs do not have the D antigen on their surfaces.

The Indirect Coombs' Test involves addition of Rh positive red blood cells to the mother's serum in vitro. If an Rh-negative mother has been exposed to the Rh protein from a previous childbirth, her anti-D IgG will bind to Rh+ (D antigen) red blood cells and indicate the need for precautionary measures during future pregnancies. Conversely, serum from a mother that has not been exposed will test negative.

Immunological Techniques
University of Arizona
douglas.lake@asu.edu