Flow cytometric quantitation of platelet phagocytosis by monocytes using a pH-sensitive dye, pHrodo-SE

Antibody-mediated phagocytosis of platelets using a flow cytometric monocyte-based phagocytosis assay (FMPA) has been shown to predict the outcome of platelet transfusion. The easy adherence between platelets and monocytes even in the absence of an antibody is regarded as one of limitations of the FMPA. To improve the FMPA for prediction of transfusion outcome, we used the pH-sensitive dye pHrodo succinimidyl ester (pHrodo-SE), which has weak fluorescence at neutral pH and has increased fluorescence intensity in low pH conditions such as in lysomes. Platelets stained with pHrodo-SE were sensitized with an HLA class I monoclonal antibody (w6/32 clone) or anti-HLA class I containing antisera. The platelets were incubated with monocyte-enriched mononuclear cells. Phagocytic activity was assessed by the percentage of monocytes that phagocytosed platelets. Sensitization of platelets with w6/32 significantly increased platelet phagocytosis by monocytes in dose- and time-dependent manners. Anti-HLA class I antibody-containing sera caused platelet phagocytosis in a cognate antigen-antibody-dependent manner. There was a significant correlation (r=0.69, p<0.01) between phagocytic index and titer of HLA class I antibody measured by lymphocyte immunofluorescence test-flow cytometry. In addition, the phagocytic index obtained by FMPA with pHrodo-SE was significantly higher than that obtained by FMPA with the previously used dye, carboxyfluorescein diacetate succinimidyl ester, when platelets were sensitized by w6/32 and anti-HLA class I antibody-containing sera. Because of the higher resolution and higher sensitivity than those of the previous method, the pHrodo-SE-based FMPA may be suitable for more precise quantitation of phagocytosis activity, which would enable qualitative evaluation of transfusion effectiveness.