Evaluation of Hemolytic Complications of Intravenous Immunoglobulin Products Using Both Intravascular and Extravascular Hemolysis Assays

Intravenous immunoglobulin (IGIV), first licensed for the treatment of primary immunodeficiencies, is currently widely used in treatment of primary immunodeficiency and certain autoimmune diseases. Hemolysis is a potential adverse effect related to IGIV administration that occurs in approximately 1.6% of patients. Serious hemolysis complications are rare but may cause renal failure, severe anemia, and even death and are commonly associated with non-O blood group recipients receiving high doses (1-2 g/kg/day). It is known that antibodies to blood groups A and B may copurify with IGIV, depending on the manufacturing method. Limits on anti-A and anti-B in IG products are required, as measured by direct hemagglutination (DHAT), a European Pharmacopeial (EP) method, however hemolytic events continue to occur and trend higher in recent years due to widespread use of high-dose IGIV as well as certain manufacturing advances aimed at enhancing IgG yield but resulting in higher isoagglutinin content in the final products. In blood, both intra- and extravascular hemolysis can occur in various proportions dependent on the patients: via acute complement-dependent lysis of antibody-bound RBCs within the blood circulation or delayed-onset cell-mediated clearance of antibody- bound RBCs by activated monocytes in liver and spleen, respectively. We developed a complement-dependent hemolysis assay (CDHA), to measure hemolytic activity to model of intravascular hemolysis. Our current aims are to develop a hemolysis assay that reflects IGIV-mediated cell-based extravascular hemolysis, to employ both approaches to determine the hemolytic activities, characterize isoagglutinins in IGIV, and compare findings with DHAT.

In developing a functional assay to measure cell-mediated extravascular hemolysis, we adapted the monocyte monolayer assay (MMA) developed in Donald Branch's laboratory with certain modifications. Macrophage preparations included primary elutriated monocytes differentiated to macrophages, PBMC, and THP-1, a macrophage cell line. These cells were exposed to Type A RBC opsonized with IGIV or Rho(D) IG. Anti-Rho(D)-opsonized RBC were used as a positive control, and a known clinically hemolytic IGIV lot (DV-1) was used to evaluate hemolytic/hemophagocytic macrophage activity. These controls were used to test critical conditions for the assay. The results were observed by phase-contrast microscope and the phagocytic index (phagocytosed RBCs per 100 monocytes) were calculated. Elutriated monocytes and PBMC showed greater hemophagocytic activity compared to THP-1.

Macrophages can largely be categorized into two main types: M1 and M2 macrophages. The M1 type, referred to as classically activated macrophages, plays a large role in the immune response to foreign pathogens, and the M2 type, referred to as alternatively activated macrophages, play an anti-inflammatory role. To determine whether the M1 and M2 type of macrophages play different roles in MMA, we employed elutriated monocytes differentiated in vitro by various stimuli into unique subsets (M1, M2). Preliminary results indicated that M2 contains higher hemophagocytic activity than M1.

Finally, the CDHA and MMA were compared with the DHAT (EP) method. Seven IND products were tested in the FDA research laboratory using both DHAT and CDHA methods. All products met the HA industry standard of 1:64. Several IND products demonstrated hemolytic ability similar to that of clinically hemolytic lots. In such cases, FDA provides advice on potential manufacturing solutions to lower hemolytic activity. Using both CDHA and MMA, we have shown that a clinically hemolytic lot (DV-1) exhibited the highest hemolytic activities in both assays.

Taken together, we have developed two functional assays in evaluating the intravascular and extramacular hemolytic activities mediated by the IGIV products. Our findings reveal that the functional assays, i.e., CDHA and MMA, are more precise for measuring hemolytic activity than the currently applied EP method for lot release (DHAT). The CDHA and MMA may be useful for investigation of clinically hemolytic lots, and to increase understanding of product and patient risk factors. Further studies would be needed to determine predictive value of these test methodologies.

No relevant conflicts of interest to declare.