Confounding effects of platelets on flow cytometric analysis and cell-sorting experiments using blood-derived cells

Background: Flow cytometric analysis and cell-sorting of peripheral blood leukocytes is commonplace; however, platelet contamination is typically ignored during immunophenotypic analysis and sorting of blood-derived cells. Methods: Red blood cells, platelets, T & B lymphocytes, monocytes, and granulocytes were sorted from rat blood preparations. Presort enrichment was performed by differential centrifugation for all cell types. Additionally, leukocyte samples were prepared by ammonium chloride lysis of red blood cells. Results: Unless proper precautions were taken, significant numbers of platelets were sorted along with (nonplatelet) cells of interest. The amount of platelet contamination varied greatly from experiment to experiment with the highest level of leukocyte-platelet association observed in the neutrophil/granulocyte population in samples prepared using ammonium chloride-based red blood cell-lysing solution. Conclusions: Addition of an immunophenotypic marker for platelet identification is a simple, vet prudent, measure to help evaluate the impact of platelets on immunophenotypic staining when performing flow cytometric analysis or sorting of blood-derived cells and should become a routine practice. Platelet presence in postsort fractions can be due to free platelets as well as target cell-associated platelets and both sources of contamination must be addressed. (C) 2006 International Society for Analytical Cytology.