Measurement of isoagglutinins in immunoglobulins for intravenous application by flow cytometry.

Isoagglutinins present in intravenous immunoglobulin (IVIG) products have been linked to haemolysis. Therefore, accurately assessing isoagglutinin content in IVIG products is important. The standard European Pharmacopoeia (Ph.Eur.) direct assay is limited by low precision. Here, we describe the development of a fluorescence-activated cell sorting (FACS) method for assessing isoagglutinin levels. Serially diluted IVIG samples were incubated with red blood cells (RBCs), RBC-bound anti-A and anti-B antibodies were detected using a fluorescently-labelled antibody and the median fluorescence intensity of samples was assessed by FACS. Results were compared with the Ph.Eur. direct assay. The method was used to determine isoagglutinins in commercial products produced with and without isoagglutinin reduction steps. Assay precision, reported as the coefficient of variation, for the FACS method was 14% and 8% for anti-A and anti-B, respectively versus 33% and 20% with the Ph.Eur. direct assay. Application of the method on commercially available IVIGs revealed differences in isoagglutinin content between products produced with and without isoagglutinin reduction steps. This FACS assay allows for quantification of isoagglutinin concentrations in IVIGs with higher precision than the Ph.Eur. direct assay. Also the FACS assay confirms differences in isoagglutinin levels between IVIG products and the efficacy of isoagglutinin reduction measures.