Standardization of antihuman globulin reagents

All antiglobulin reagents available at present, monospecific or polyspecific, are standardized by the producer and issued pre-diluted for immediate use or within the gel or bead matrix of a microcolumn.

Antiglobulin reagents standardized for use in haemagglutina-tion tests should not agglutinate red cells, regardless of their ABO type, after incubation with compatible serum (i.e. they should be free of unwanted antibodies, especially anti-species). The antiglobulin reagent should not agglutinate washed red cells taken from units of blood intended for transfusion when they are nearing the end of their shelf-life. The final dilution of the anti-IgG component should be sufficient to allow the optimal detection of weak examples of incomplete non-complement-fixing IgG alloantibodies (e.g. anti-D, -c) coated on single antigen dose (heterozygous) target cells (e.g. D/d or C/c). The anti-C3d component should be optimized so that complement bound to the cells will be detected but not so strong that stored cells, with traces of bound C3d and C4d, react non-specifically. As plasma is now used by most laboratories for pretransfu-sion testing, alloantibodies that theoretically might only react with the anti-C3 in an AHG reagent will be missed. However, there is no evidence that significant antibodies are not being detected.

When new batches of an AHG reagent or IAT microcolumns are acquired by a laboratory, they should be subjected to a minimum verification with several IgG antibodies of different specificities to ensure that all are detectable. Laboratories should confirm that, on each day of use, the AHG reagent is reacting according to the manufacturer's specifications, with control red cells coated weakly with IgG. The control IgG-coated cells are the same cells as those used to check the validity of negative antiglobulin tests when a washing step is part of the process; the weakly coated cells are added to each tube giving a negative result, followed by repeat centrifugation and reading. Agglutination of these cells confirms that the AHG reagent in the tube had not been neutralized by residual unbound IgG that was not removed during washing of the original test cells.

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