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.

Relative Efficiency of Leucocyte Removal Procedures for the Production of Leucocyte-Poor Red Cell Concentrates Assessed by Flow Cytometry

“…However, below 20 fluorescence events, we observed a have shown that leucocyte-depleting platelet filters differ ing in their overall efficacy also differed in their relative efficacy in absorbing different lymphocyte subsets, so that the less-efficient filter removed more dendritic cells, thought to be of central importance in inducing alloimmu nization [27]. Al et al [22] characterised the different leucocyte classes on the basis of light scatter. Although they confirmed the identity of the cells using fluoresceinlabelled monoclonal antibodies, we feel that immunophenotyping as used by us and others [20,21] is a more reliable method, as changes to light-scattering properties, e.g.…”

“…Previous studies [20][21][22] have described the use of flow cytometry for this purpose. Freedman et al [20] The change in transfusion policy described in Methods is summarised in figure 2.…”

“…A number of techniques have been recently described which address the problem of counting leucocytes in leucocyte-depleted blood com ponents [13][14][15][16], but access to a flow cytometer has al lowed us to use this methodology for this purpose, as described by other groups [17][18][19]. We chose this approach as flow cytometry allows counting of very low numbers of leucocytes, as well as permitting characterisation of the residual cells [20][21][22]. This results in increased insight into the mechanism by which leucocyte depletion procedures achieve the desired effects in the reduction of post-trans fusion reactions.…”

Preliminary assessment of whole‐blood, red‐cell and platelet‐ leucodepleting filters for possible induction of prion release by leucocyte fragmentation during room temperature processing

“…Leucocyte counting was achieved by flow cytometry on a Becton Dickinson FACSort using the Becton Dickinson LeucoCOUNT kit, and in some cases by visual microscopy using a Nageotte chamber. The method of flow cytometric evaluation of residual leucocytes involved staining with propridium iodide (Al et al , 1991; Deneys et al , 1994; Dzik, 1997). This approach was validated for sensitivity and linearity by spiking of double‐filtered units with fresh whole blood, and allows enumeration to a level of 2 × 10 5 per unit (data not shown).…”

Original Paper

“…Counting leukocytes in RCC becomes more difficult as their number decreases. Although counting methods with the flow cytometer [22] or a radioimmunoassay [23] can be applied, we showed in this study that dilution of the sam ple in Turk's solution and the use of a Bürker hemocytometer suffice for routine checks. The use of the Fuchs-Rosenthal hemocytometer or fluorescent microscopy did not give clearer results.…”