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 Fcγ receptor (FcγR)-mediated trogocytosis. In the present study, similar FcγR-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 FcγR expression in peripheral blood, could perform FcγR-mediated trogocytosis. Since the capacity of FcγR-mediated trogocytosis was different among blood samples, identification of factors that could regulate the occurrence of FcγR-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 FcγR-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 FcγR-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 FcγR-mediated trogocytosis. Moreover, the inhibition rates were significantly higher in HAMAhigh serum than HAMAlow serum. The collective findings suggested the involvement of heterophilic antibodies such as HAMA in the mechanism of false-positive results in FCM due to FcγR-mediated trogocytosis.

全血红细胞裂解法是流式细胞术（flow cytometry，FCM）中最为常用的样本制备方案。尽管该方法具备诸多优势，但我们的前期研究证实，采用该方法检测单核细胞表面标志物时，会因Fcγ受体（Fcγ receptor，FcγR）介导的胞啃作用（trogocytosis）这一现象出现假阳性结果。本研究中，当研究焦点转向粒细胞而非单核细胞时，同样观测到了基于FcγR介导胞啃作用的假阳性结果。上述结果表明，不仅单核细胞，外周血中表达FcγR的最大细胞群体——粒细胞，同样可发生FcγR介导的胞啃作用。由于不同血液样本的FcγR介导胞啃作用能力存在差异，明确可调控该现象发生的影响因素，对于FCM的质量控制具有重要意义。我们的研究提示，此类调控因子存在于血清中。为鉴定血清中的调控因子，我们构建了以粒细胞为研究对象的FcγR介导胞啃作用体外模型。通过该模型开展的研究将血清中的调控因子鉴定为分子量大于100 kDa的热不稳定分子。补体经典途径组分最初被假定为候选调控因子，但C1抑制剂对FcγR介导的胞啃作用并未产生显著影响。另一方面，尽管免疫球蛋白本应耐热灭活，但人抗小鼠抗体（human anti-mouse antibodies，HAMA）抑制剂可有效阻断FcγR介导的胞啃作用。此外，HAMA高含量血清的抑制率显著高于HAMA低含量血清。综上，本研究结果提示，诸如HAMA这类嗜异性抗体，参与了FcγR介导胞啃作用所导致的FCM假阳性结果的发生机制。