A flow cytometric method for determination of white cell subpopulations in filtered red cells

Abstract

A flow cytometric method for the detection of low amounts of lymphocytes, monocytes, and granulocytes in filtered red cells (RBCs) was evaluated. In this procedure, the RBCs in the samples were lysed by ammonium chloride treatment and the white cells (WBCs) were detected by flow cytometry according to their specific light-scattering properties. The identity of the WBC subpopulations was confirmed by immunofluorescence with monoclonal antibodies specific for each cell type. Flow cytometric determination of WBCs in filtered RBCs correlated with numbers obtained by both a hemocytometer (r = 0.76) and a radioimmunoassay (r = 0.79). Total numbers of WBCs in RBCs measured by flow cytometry were 59 +/- 13 percent (n = 7) of those measured by electronic particle counting, 32 +/- 6 percent (n = 25) by hemocytometer, and 48 +/- 11 percent (n = 29) by radioimmunoassay. Lymphocytes added to filtered RBCs in a concentration of 1.37 cells per microL were detected at an average of 0.56 +/- 0.22 cells per microL (n = 3). Results with monoclonal antibodies indicated an altered expression of membrane markers on granulocytes after RBC filtration, as seen with cell activation. The inefficiency of the flow cytometric method to detect the total number of WBCs calculated by other methods may reflect filtration-induced changes in light-scattering properties of the WBCs. Although the method described does not accurately quantitate the total numbers of WBCs present in filtered RBCs, it may provide useful information on qualitative aspects of WBC subpopulations.