Analysis of standard Ficoll-Hypaque separation profiles of peripheral WBC from patients undergoing hemodialysis (HD) demonstrated that dialysis caused a marked alteration in the number of cells found at both the interface between the Ficoll-Hypaque and plasma which normally contains mononuclear cells and the cell pellet which normally contains granulocytes. By 30 min into dialysis, there was a 175% increase in white blood cells in the mononuclear band with a corresponding decrease in the number of cells obtained from the cell pellet. When peripheral blood samples from normal donors were pumped through various types of hemodialyzers, a shift in the cell separation profiles similar to that of patients undergoing HD was observed. Differential analysis of the cells obtained from both the interface between the Ficoll-Hypaque and plasma and the cell pellet showed that by 30 min into dialysis, the 'mononuclear' band contained 40-50% polymorphonuclear neutrophils (PMN). To ascertain whether the cell separation changes were possible due to C5a generation resulting from complement activation by the HD membrane, whole blood was incubated with the purified chemotatic factors C5a, C5a(des arg), and formyl-methionyl-leucyl-phenylalanine. This resulted in similar alterations in PMN densities. This study demonstrates that both in vivo and in vitro exposure of human peripheral blood to HD membranes as well as the chemotactic factors C5a, C5a(des arg), and formyl-methionyl-leucyl-phenylalanine results in density changes in PMN. These changes are reflected in altered Ficoll-Hypaque separation profiles of white blood cells and indicate that Ficoll-Hypaque centrifugation is not a suitable method for the isolation of PMN or mononuclear cell preparations from whole blood obtained from HD patients after the initiation of dialysis. Furthermore, these studies indicate that care must be taken when evaluating white blood cell function with cell preparations isolated in this manner, since PMN responding to C5a are removed from the cell pellet and separate with the mononuclear cells.
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