Cellular volume and marker expression in human peripheral blood apheresis stem cells

Abstract

Coulter volume is far more accurate measure of cell volume than forward angle light scatter. In this report, we have used Coulter volume to determine the mean cell volume and diameter of normal human peripheral blood cells and hematopoietic progenitor cells obtained by apheresis (HPC-A) from patients with hematological malignancies. Fresh peripheral blood samples (treated with Beckman Coulter IMMUNOPrep erythrocyte lysis solution), HPC-A samples (treated with BD Biosciences FACSLysing solution), or processed by Ficoll Hypaque sedimentation method were stained with CD45-FITC and PE-labeled CD34, CD90, CD117, and CD133 antibodies and analyzed for electronic volume and two color fluorescence. The mean electronic volume and diameter of mononuclear cells from fresh peripheral blood samples prepared with IMMUNOPrep were lymphocytes (191 μm3, 7.16 μm), monocytes (370 μm3, 9.91 μm), and granulocytes (328 μm3, 8.56 μm). In mononuclear cells of HPC-A samples prepared by Histopaque-1077 sedimentation, the lymphocytes had volume and diameter of 311 μm3, 8.4 μm, monocytes were 486 μm 3, 9.76 μm, and granulocytes were 515 μm3, 9.95 μm. In contrast, HPC-A samples prepared after lysis with FACSLysing solution had mean electronic volume and diameter of lymphocytes (414 μm3, 9.25 μm), monocytes (797 μm3, 11.5 μm), and granulocytes (670 μm3, 10.85 μm). Cell volume of mononuclear cells in the HPC-A samples prepared by Histopaque-1077 sedimentation method was correlated with the expression of stem cell markers CD34, CD90, CD117, and CD133. CD90 positive cells had the smallest mean electronic volume of 299.93 μm 3 when compared with cells with positive expression of CD133 (322 μm3), CD117 (349 μm3), CD34 (407 μm3), and CD45 (453 μm3). Correlation of cell volume with stem cell marker expression may allow for the identification of small stem cells, which may not express the conventional markers used for the identification of stem cells in HPC-A samples. © 2008 International Society for Analytical Cytology.