Comparison of two single-platform ISHAGE-based CD34 enumeration protocols on BD FACSCalibur and FACSCanto flow cytometers

Clinical Flow Cytometry Laboratory, University Health Network, Toronto, Canada.
Cytotherapy (Impact Factor: 3.29). 06/2009; 11(5):595-605. DOI: 10.1080/14653240902923161
Source: PubMed


Enumeration of viable CD34(+) cells provides critical information for the bone marrow (BM) transplant physician. The single-platform ISHAGE protocol is the most reliable method currently available to quantitate accurately this important subset of cells. Previous studies have shown that 5 CD34(+) cells/microL blood predicts the collection of at least 0.5x10(6) CD34(+) cells/kg patient weight. From the apheresis product, infusion of 2.5x10(6) viable CD34(+) cells (measured pre-cryopreservation)/kg patient weight will reliably permit engraftment of the hematopoietic system (as measured by the time to 20000 platelets/microL) by day 12-14 post-infusion.
We compared the CD34(+) cell numbers derived from Flow Count-based Stem-Kit; (Beckman Coulter) and Trucount tube-based stem cell enumeration (SCE) kit (BD Biosciences) ISHAGE templates on BD FACSCalibur and BD FACSCanto cytometers on 12 granulocyte-colony-stimulating factor (G-CSF)-mobilized peripheral blood (MPB) and 10 peripheral blood stem cell (PBSC) samples.
Comparison of results showed that there was no statistical difference between samples run with Stem-Kit on the FACSCalibur versus SCE kit-based assays on either the FACSCalibur or FACSCanto. Mean results for the Stem-Kit/Calibur combination were 137, for SCE kit/Calibur 140 and for SCE kit/Canto 137 cells/microL. Pair-wise comparison of data based on rank order showed no statistically significant difference and all correlation coefficients had an R(2)>0.98.
The two kits generated very similar data on a range of fresh samples regardless of instrument platform. These results confirm and extend the utility of the single-platform ISHAGE protocols with a variety of reagent kits and instrument platforms.

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    • "For those acquiring flow data with the use of automated software, it is essential that the cytometrist be aware of the potential presence of subsets of CD34 þ cells that play no documented role in engraftment. The auto-software itself cannot distinguish such subsets from candidate engrafting phenotypes, and, in such cases, manual analysis (performed after acquisition of data) is essential [9]. "

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    • "Blood count was analyzed before and after apheresis from peripheral blood using a hematology analyzer (Sysmex , Norderstedt, Germany). CD34+ cells were counted in pre-and post-apheresis peripheral blood samples as well as in a product sample by flow cytometry using a commercially available single-platform assay (BD stem cell enumeration kit, BectonDickinson, Heidelberg, Germany) [11] [12]. "
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    • "Using this approach a population of small Lin−CD45− cells with the lower possible number of haematopoietic contaminants could be isolated, with the main contaminant being platelets. These events are normally excluded using flow cytometry-based protocols for HSC [4], [19], [20]. Therefore, during our analysis a gating strategy based on that proposed by Zuba-Surma et al. [4] was employed. "
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