LABORATORY DETERMINATION OF BLOOD GROUPS
Traditional methods for determining blood groups rely on the agglutina-tion of erythrocytes by antibodies, usually referred to as hemagglutination. Hemagglutination can be carried out on glass microscopy slides or in micro-titer plates in which agglutination patterns are easily distinguished from the settling of erythrocytes. Recent years have seen increasing use of the Diamed typing system to detect hemagglutination. This is a system which uses mono-clonal typing antibodies, distributed in a gel, contained in individual tubes set in plastic ‘cards’. Cells are added to the antibodies and the cards are cen-trifuged. Where agglutination has occurred, the agglutinates remain on top of the gel, whereas nonagglutinated cells settle through the gel to the bottom (Figure 6.10). Most transfusion laboratories now use gel technology for blood grouping and compatibility testing.
Whichever technique is used, a blood group, such as the ABO grouping, is determined by incubating the individual’s erythrocytes with antibodies to known antigens (anti-A and anti-B in this case) and also mixing the indi-vidual’s plasma with erythrocytes of known A, B, AB or O blood groups. The pattern of hemagglutination shown will enable the determination of the blood group.
Hemagglutination occurs when antibodies to an erythrocyte antigen cross-link the cells, forming visible aggregates. The extent of hemagglutination depends on the temperature, pH and the ionic strength of the medium. Agglutination is favored in low ionic strength saline (LISS). Erythrocytes have a net electronegative charge and repulsive forces normally keep them about 20 nm apart. When antibodies bind to the erythrocyte, the reduced surface charge allows the cells to agglutinate. This is most effectively achieved with IgM antibodies, which can cause direct agglutination of erythrocytes. To obtain a direct agglutination with IgG antibodies, it is usually necessary to include bovine serum albumin in the medium, which masks the charges on the erythrocytes and allows them to come closer together. Another method of reducing the negative charge is to use proteolytic enzymes to remove sur-face proteins that carry the charge. The enzyme can be added to the eryth-rocytes prior to the addition of the antibody, or all the components can be added together. Polycationic polymers such as polybrene will also reduce the negative charge on erythrocytes.
The antiglobulin test uses the ability of antihuman globulin (AHG) to agglutinate erythrocytes coated with nonagglutinating erythrocyte-specific IgG. This can be used to detect erythrocytes already coated with anti-erythrocyte IgG in the direct antiglobulin test (DAT), or can be used on cells which have been incubated with antibody in vitro (see Figure 6.8).