Loss of CD34 + hematopoietic progenitor cells due to washing can be reduced by the use of fixative-free erythrocyte lysing reagents

Abstract

Current protocols for sample preparation before flow cytometric enumeration of CD34 + hematopoietic progenitor cells (HPC) include both lyse-non-wash and lyse and wash methods. Erythrocyte lysis without washing is the method of choice when absolute cell counts are to be assessed, whilst a washing step is recommended for immunological subtyping of CD34 + cells in order to reduce background fluorescence. Here, we analyzed the effect of the interaction between type of erythrocyte lysis reagent and washing on the outcomes of (i) CD34 + cell enumeration and (ii) expression of CD38 by CD34 + cells in a single-platform, whole-blood staining assay [Gratama, J.W., Keeney, M., Sutherland, D.R., 1999. Enumeration of CD34 + hematopoietic stem cell and progenitor cells. Curr. Protocols Cytometry 6(4), 1–22.]. We studied seven commercially available lysing reagents (five containing fixative and two fixative-free) using 12 samples from cord blood ( n=4), mobilized peripheral blood ( n=4) and apheresis products ( n=4). Using the lyse and wash technique, significant reductions of absolute and relative numbers of CD34 + cells, as well as in the numbers of lymphocytes and leukocytes, were observed on samples that had been lysed using fixative-containing buffers as compared to the lyse-no-wash technique. Cell losses due to washing could be significantly reduced when samples were lysed using fixative-free buffers. ‘Postfixation’ using PBS+1% paraformaldehyde of samples that had been lysed using fixative-free buffers and then washed did not result in additional loss of CD34 + cells or other cell types. Finally, washing unfixed samples led to a slight decrease of CD38 monoclonal antibody bound to CD34 + cells as compared to samples that had been fixed during erythrocyte lysis. These results indicate that fixation renders (CD34 +) cells sticky and leads to their loss from the cell suspension upon centrifugation and resuspension. We conclude that all seven lysing reagents can be used with confidence in a lyse-no-wash technique, but that only fixative-free lysing reagents should be used when a washing step is considered necessary.