P008 : AGGREGATED IgG CONTROL FOR PRONASE FC RECEPTOR (FcR) REMOVAL IN FLOW CROSSMATCH

Abstract

Aim This study evaluated heat aggregated (Agg) IgG in IVIG and normal human serum (NRS) as a control for pronase FcR removal in Flow crossmatch. Pronase treatment of lymphocytes is used to remove FC receptors on B cells to reduce false B cell positive Flow crossmatches due to Agg IgG in patient serum. The effectiveness of pronase treatment can be compromised if there are too many cells or serum proteins present, a positive control is needed to verify that FcRs have been removed from B cells. Methods 1. To aggregate IgG, 5 ml of NRS or IVIG (Gammaguard 100 mg/ml) is heated at 63 °C for 20 min. 2. The working dilution for Agg IVIG is found by titering it on unpronased and pronased cells in a crossmatch. 3. A working dilution for Agg NRS is also found by titering it in a crossmatch, but first the Agg IgG is concentrated at 130,000g, 60 min at 4 °C. 4. The NRS Agg IgG pellet is resuspended in 1 ml PBS, pH 8.1 for titering. Results See the Fig. 1 , Fig. 2 . Conclusion This study demonstrates the ability to use heat Agg IgG in IVIG or from NRS as a control for pronase B cell FcR removal in Flow crossmatch.