title: The impact of erythrocyte lysing procedures on the recovery of hematopoietic progenitor cells in flow cytometric analysis.
abstract: 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.
authors.affiliation: Department of Radiobiology, University Hospital Münster, Robert Koch Strasse 43, D-48149 Münster, Germany. greveb@uni-muenster.de
authors.forename: Burkhard
authors.initials: B
authors.lastname: Greve
authors.forename: Constanze
authors.initials: C
authors.lastname: Beller
authors.forename: Uwe
authors.initials: U
authors.lastname: Cassens
authors.forename: Walter
authors.initials: W
authors.lastname: Sibrowski
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Göhde
mesh_terms: 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
chemical_list: D018952:Antigens, CD34