An optimized microplate-based method to evaluate complement-dependent hemolysis mediated by intravenous immunoglobulins (IVIG).

Hemolytic reactions can cause serious complications after administration of Intravenous Immunoglobulin (IVIG), due to passive transfer of anti-A and anti-B IgG antibodies (isoagglutinins). A maximum allowable amount of isoagglutinins is established in the US and EU for licensed IVIG, as measured by a specified direct hemagglutination test (DHAT). Despite this limit, reports of hemolysis have increased over time, raising the question of how well the DHAT predicts clinically significant hemolysis. This study was undertaken to develop a microplate-based complement-dependent hemolysis assay (CDHA) that reproducibly measures functional hemolytic activity of IVIG, for assessment of IVIG products. An IVIG working reference reagent (NIBSC 14/160) was qualified as an assay control and for quantitation purposes. Hemolytic activities of 36 IVIG product lots encompassing seven brands and including 6 clinically hemolytic lots were measured. Hemolytic activity varied among IVIG product brands, and to a lesser extent, from lot-to-lot for individual brands. Correlation between the CDHA and DHAT was not robust which may reflect imprecision of the DHAT method or additional variables that influence complement-dependent hemolysis after opsonization. In conclusion, the CDHA provides a simple, specific, and sensitive tool for IVIG product characterization and investigation of hemolytic events by manufacturers, researchers, and regulatory authorities.