Antihuman Globulin & Red Cell Suspension

• Before the discovery of the anti-globulin test, only IgM antibodies had been detected.
• The utilization of the AHG test permitted the detection of non-agglutinating IgG antibodies and led to the discovery of many new blood group systems.
• The Coomb's procedure involved the injection of human serum into rabbits to produce antihuman serum.
• Antihuman Globulin is used to detect RBCs sensitized with IgG alloantibodies, IgG auto antibodies, or complement components.
• Antiglobulin test, also called the Coomb's test, is based on the principle that AHG obtained from immunized nonhuman species bind to human globulins such as IgG, or complement, either free in serum or attached to antigens on red blood cells.
• The addition of AHG containing anti-IgG to RBCs sensitized with IgG antibodies, allows the hemagglutination of these cells.
• Antibody molecules and complement are globulins.
• Injecting an animal with human globulin stimulates the animal to produce antibody to the foreign CHON.
• Antihuman globulin reacts with human globulin molecules, either bound to the RBCs or free in the serum
• Washed RBCs coated with human globulins are agglutinated by AHG reagent
• Polyspecific AHG reagent contains antibody to human IgG and to C3d component of the complement system. Other anti-complement antibodies such as anti-C3b, anti-C4b or anti-C4d may also be present.
• Commercially prepared polyspecific AHG contains little or no activity against IgA and IgM
• Monospecific AHG reagent contains only one antibody specificity, either anti-IgG or antibody to specific complement components such as C3b or C3d.
• Anti-IgG contains no anti-complement activity; AHG reagent must have antibody activity to non-agglutinating blood group anti-bodies, the presence of anti-light chain activity allows detection of all IgG classes
• Anti-complement involves anti-complement reagents such as antiC3b-C3d and are reactive against the designated complement components only and no activity against human immunoglobulin
• Classic method in preparation of AHG reagent involves the injecting human serum into laboratory animals. The human globulin as foreign antigen triggers the animal's immune response and an antibody is produced.
• Classic method in preparing AHG reagent produces the polyspecific AHG reagent.
• Hybridoma technology can be used to produce monospecific AHG reagent using clones of plasma cells that will produce either anti-IgG or anti-complement.
• Direct Coomb’s test is used to detect in vivo sensitization of red cells by a one stage procedure
• Direct Coomb's test is based on the principle of the detection in vivo sensitization of RBCs with IgG and/or complement components.
• In direct coomb's test, if no agglutination was observed, a drop of IgG-Sensitized red cells is placed, and result is read and recorded
• Indirect AHG Test is performed to determine in vitro sensitization of red cells and is used in detection of antibodies to potential donor cells, identification of antibody specificity, determination of red cell phenotype and titration of incomplete antibodies
• Indirect coomb's test is performed to identify the presence or absence of antibodies in blood directed against antigens found on red blood
• A minimum ratio of 40:1 should be aimed in AHG test
• Albumin allows antibody-coated cells to come into closer contact with each other.
• LISS enhances antibody uptake
• Polyethylene glycol removes water, thereby concentrating the antibody
• Incubation time of saline suspension is 30-120 mins
• Incubation time of LISS is 10-15 mins
• Washing of cells is done to remove unbound serum globulins.
• Saline for washing should be fresh and buffered at to a pH of 7.2-7.4
• Cells used for DAT must be collected in EDTA or citrate
• In direct and indirect coomb's test, a 5% RBC suspension is prepared using the patient’s blood sample
• Vast majority of IgG has an optimal reaction rate at 37 °C