The impact of erythrocyte lysing procedures on the recovery of hematopoietic progenitor cells in flow cytometric analysis.
Since preanalytic lysing of erythrocytes remains critical in flow cytometry, we investigated the influence of four lysing procedures on the quantification of leukocyte and CD34+ cells in hematopoietic cell transplants (HCTs). Samples were derived from stem cell-enriched mobilized whole blood collected by apheresis (unselected) and immunologically purified stem cell products (selected) and were measured using the dual-platform (2-PF) method with two flow cytometric systems. Additionally, cells were measured by a volume-based technique (single platform [1-PF]). Results were identical in the 2-PF mode (unselected HCTs, r = 0.998; selected HCTs, r = 0.999). In comparison with the 2-PF results, the single-platform (1-PF) measurements revealed a mean decrease of 59.5% for CD34+ cells (50.8% for CD45+ cells) in unselected HCTs and a mean decrease of 52% for CD34+ cells (49.8% for CD45+ cells) in selected HCTs. In order to check the accuracy of cell quantification using the 1-PF method, leukocyte reference values from hematology counter results were compared with flow cytometric (1-PF)-counted nucleated cells. That analysis revealed good congruency, with r = 0.998 for unselected HCTs and r = 0.999 for selected HCTs. In conclusion, all lysing procedures that we used induced substantial loss of leukocytes and CD34+ cells. As demonstrated by the high accuracy of the 1-PF technique, all erythrocyte lysing procedures caused significant cell loss, which led to inconsistent counting of CD34+ cells in nonvolumetric flow cytometric (2-PF) protocols.
Greve
Burkhard
B
Department of Radiobiology, University Hospital Münster, Robert Koch Strasse 43, D-48149 Münster, Germany. greveb@uni-muenster.de
Beller
Constanze
C
Cassens
Uwe
U
Sibrowski
Walter
W
Göhde
Wolfgang
W
D000293:Adolescent; D000328:Adult; D018952:Antigens, CD34; D002469:Cell Separation / Q000379:methods; D005260:Female; D005434:Flow Cytometry / Q000379:methods; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D006461:Hemolysis; D006801:Humans; D007958:Leukocyte Count / Q000379:methods; D008297:Male; D008875:Middle Aged; D009369:Neoplasms / Q000628:therapy; D036102:Peripheral Blood Stem Cell Transplantation / Q000379:methods; D015203:Reproducibility of Results; D014182:Transplantation, Autologous; D014184:Transplantation, Homologous
D018952:Antigens, CD34