Mechanism of Fcγ Receptor-Mediated Trogocytosis-Based False-Positive Results in Flow Cytometry

The whole blood erythrocyte lysis method is the most common protocol of sample preparation for flow cytometry (FCM). Although this method has many virtues, our recent study has demonstrated false-positive results when surface markers of monocytes were examined by this method due to the phenomenon called Fc gamma receptor (Fc gamma R)-mediated trogocytosis. In the present study, similar Fc gamma R-mediated trogocytosis-based false-positive results have been demonstrated when granulocytes were focused on instead of monocytes. These findings indicated that not only monocytes but also granulocytes, the largest population with Fc gamma R expression in peripheral blood, could perform Fc gamma R-mediated trogocytosis. Since the capacity of Fc gamma R-mediated trogocytosis was different among blood samples, identification of factors that could regulate the occurrence of Fc gamma R-mediated trogocytosis should be important for the quality control of FCM. Our studies have suggested that such factors are present in the serum. In order to identify the serum factors, we employed the in vitro model of Fc gamma R-mediated trogocytosis using granulocytes. Investigation with this model determined the serum factors as heat-labile molecules with molecular weight of more than 100 kDa. Complements in the classical pathway were initially assumed as candidates; however, the C1 inhibitor did not yield an obvious influence on Fc gamma R-mediated trogocytosis. On the other hand, although immunoglobulin ought to be resistant to heat inactivation, the inhibitor of human anti-mouse antibodies (HAMA) effectively blocked Fc gamma R-mediated trogocytosis. Moreover, the inhibition rates were significantly higher in HAMA(high) serum than HAMA(low) serum. The collective findings suggested the involvement of heterophilic antibodies such as HAMA in the mechanism of false-positive results in FCM due to Fc gamma R-mediated trogocytosis.