High-grade loss of leukocytes and hematopoietic progenitor cells caused by erythrocyte-lysing procedures for flow cytometric analyses.
All current-flow cytometric techniques use erythrocyte-lysing procedures before leukocyte analysis. We investigated the impact of four lysing procedures with different flow cytometric techniques on the loss of leukocytes and hematopoietic progenitor cells in blood samples. A total of 280 determinations out of 10 samples were measured by two flow cytometers (FCMs), using a FACS-Calibur (Becton Dickinson) and a particle-analyzing system (PAS) with a "true volumetric unit" (Partec). All samples were prepared with four different commercially available erythrocyte-lysing reagents (n = 10, respectively). CD34(+) cells were determined in relation to counted leukocytes with both FCMs (dual platform determinations, 2-PF). In addition, further immunologic and nuclear staining determinations of cells with and without erythrocyte-lysing procedures were performed in the "true volumetric unit" (single platform mode 1-PF) using the PAS system (n = 10, respectively). In the 2-PF mode, both systems showed identical results for CD34(+) cells (r = 0.997). The comparison of 1-PF and 2-PF modes with immunologic stainings revealed a mean decrease of 34.5% for absolute amounts of CD45(+) cells [in detail: Becton-Dickinson (BD) lysis 40%; Ortho Diagnostics (OD) lysis 31%; Uti lyse (UL) 38%; Cylyse (CL) 29%] and of 41.3% for absolute concentration of CD34(+) cells [in detail: BD lysis 45%; OD lysis 40%; UL lysis 45%; CL lysis 34%] by the lysing procedures. In contrast, the nuclear stainings revealed a mean leukocyte loss of only 5% for the nonlysed samples and of 12% for lysed samples. All investigated lysing procedures induced a large loss of leukocytes and progenitor cells, obviously due to cell membrane destruction as demonstrated for identical samples in the 1-PF and 2-PF modes by immunologic and nuclear staining methods.
Greve
Burkhard
B
Department of Radiobiology, University Hospital Münster, 48149 Münster, Germany. greveb@uni-muenster.de
Beller
Constanze
C
Cassens
Uwe
U
Sibrowski
Walter
W
Severin
Erhard
E
Göhde
Wolfgang
W
D015703:Antigens, CD / Q000097:blood; D018952:Antigens, CD34 / Q000097:blood; D002452:Cell Count; D005434:Flow Cytometry / Q000379:methods*; D006405:Hematology / Q000379:methods; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D006461:Hemolysis; D006801:Humans; D007962:Leukocytes / Q000166:cytology*
D015703:Antigens, CD; D018952:Antigens, CD34