Abstract
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.
Highlights
Flow cytometry (FCM) is an indispensable analytical method to detect cell surface markers in the field of laboratory medicine.Identification of surface markers of peripheral blood leukocytes is important in the diagnosis of hematological malignancy
We demonstrated that similar FccRmediated trogocytosis-based false-positive results occurred when granulocytes were focused on instead of monocytes, using antiCD8 and anti-CD15 Abs
The cells in both region 1 (R1) and R2 gates were examined for the expression of CD8a. (B) Correlation of CD8+ granulocytes and CD8+ monocytes (n = 32, r = 0.92, p = 9.46610212 in Pearson correlation test)
Summary
Flow cytometry (FCM) is an indispensable analytical method to detect cell surface markers in the field of laboratory medicine.Identification of surface markers of peripheral blood leukocytes is important in the diagnosis of hematological malignancy. Whole blood erythrocyte lysis method is the most common protocol of sample preparation for FCM [1]. In this method, antibodies (Abs) for detection, usually mouse anti-human. Erythrocytes are removed chemically, and leukocytes are subjected to FCM This method is quick and easy, utilizes a small sample volume, and does not change the leukocyte fraction. Another merit of this method is no need to use Fcc receptor (FccR) blockers because considerable amount of human IgG (8–16 mg/ml), which can mask FccRs, is included in the sample itself. Our recent study has pointed out that false-positive results occurred when monocytes were examined by this method [2]
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