Loss of CD34(+) hematopoietic progenitor cells due to washing can be reduced by the use of fixative-free erythrocyte lysing reagents.
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.
Gratama
J W
JW
Department of Clinical and Tumor Immunology, University Hospital and Daniel den Hoed Cancer Center, P.O. Box 5201, 3008 AE, Rotterdam, The Netherlands. gratama@immh.azr.nl
Menéndez
P
P
Kraan
J
J
Orfao
A
A
D018952:Antigens, CD34; D002452:Cell Count / Q000379:methods; D002469:Cell Separation / Q000379:methods; D004912:Erythrocytes; D005404:Fixatives; D006412:Hematopoietic Stem Cells; D007202:Indicators and Reagents
D018952:Antigens, CD34; D005404:Fixatives; D007202:Indicators and Reagents