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J Immunol Methods. 2000 May 26;239(1-2):13-23.

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

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Department of Clinical and Tumor Immunology, University Hospital and Daniel den Hoed Cancer Center, P.O. Box 5201, 3008 AE, Rotterdam, The Netherlands.


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.

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