FLOW CYTOMETRIC ENUMERATION OF CD34(+) HEMATOPOIETIC STEM AND PROGENITOR CELLS

The need for a rapid and reliable marker for the engraftment potential of hematopoietic stem and progenitor cell (HPC) transplants has led t o the development of flow cytometric assays to quantitate such cells o n the basis of their expression of CD34, The variability associated wi th enumeration of low-frequency cells (i.e., as low as 0.1% or 5 cells /mu l) is exceedingly large, but recent developments have improved the accuracy and precision of the assay. Here, we review and compare the major techniques. Based on the current state of the art, we recommend i) bright fluorochrome conjugates of class II or III monoclonal antibo dies (mAbs) that detect all glycoforms of CD34, 2) use of a vital nucl eic acid dye to exclude platelets, unlysed red cells, and debris or us e of 7-amino actinomycin D to exclude dead cells during data acquisiti on, 3) counterstaining with CD45 mAb to be included in the definition of HPC, 4) during list mode data analysis, Boolean gating to resolve t he CD34(+) HPCs from irrelevant cell populations on the basis of the l ow levels of CD45 expression and low sideward light-scatter signals of HPCs, 5) inclusion of CD34(dim) and CD34(bright) populations in the C D34(+) cell count, 6) omission of the negative control staining, and 7 ) for apheresis products, enumeration of at least 100 CD34+ cells to e nsure a 10% precision. Unresolved technical questions are 1) the repla cement of conventional dual-platform by single-platform assay formats, i,e,, derivation of absolute CD34+ cell counts from a single flow cyt ometric assessment instead of from combined flow cytometer (percent CD 34(+)) and hematology analyzer (absolute leukocyte count) data, 2) the cross-calibration of the available single-platform assays, and 3) the optimal method for sample preparation. An important clinical question to be addressed is the definition of the precise phenotypes and requi red numbers of HPCs responsible for short- and long-term recovery to o ptimize HPC transplant strategies. 1998. (C) 1998 Wiley-Liss, Inc.