Hi Doug, Both Procount and StemKit are designed for the clinical evaluation of CD34+ cells in peripheral blood and apheresis samples. They are both fluorescent bead-based single platform methods that allow the direct determination of CD34+ cells from a flow cytometer, without the need for a hematology analyser. Procount uses a vital nuclear dye in FL1, CD34PE in FL2 and CD45PerCp in FL3. Cells are defined as dye positive, CD34+/CD45dim. The nucleic acid dye is not a viability dye. It is used to delineate nucleated cells from unlysed red cells and other debris present after lyse-no-wash sample processing. Since the lysing agent contains a fixative, Procount is incompatible with viability dyes such as 7-AAD. The Procount Kit requires three channels of a cytometer to measure the absolute CD34+ cell number precluding the use (on three channel instruments at least) of other antibody conjugates in a more sophisticated qualitative analysis of CD34+ cell subsets. A useful feature for the clinical laboratory is that Procount comes with automated setup and analysis software. The Stem-Kit from Coulter Immunotech is based on the single platform version of the ISHAGE Protocol using CD45FITC and CD34PE (see J Hematotherapy 3: 213-226, 1996 and Cytometry [Communications in Clinical Cytometry]) 34: 61-7, 1998). Fixative-free ammonium chloride based lyse-no-wash sample processing is utilised and as such is entirely compatible with viability dyes. While Procount (that was designed to measure CD34 cells in fresh peripheral blood and apheresis products) and Stem-Kit can be expected to give very similar results on fresh blood and apheresis samples in good condition, approximately 10% of apheresis samples contain platelet aggregates and/or other debris that can be problematic for the automated Procount software. BD have issued a 'circular' indicating that such samples must be analysed manually by 'experienced laboratory personnel'. A potential problem with both assays is the need for extremely accurate pipetting (reverse pipetting is recommended). The Procount method requires pipetting of the sample into a Trucount tube pre-loaded with lyophilized beads, whereas the Stemkit requires pipetting of both beads and sample. Note however that for many apheresis samples, accurate dilution will also be required for both protocols. The basic ISHAGE method was originally developed to be capable of enumerating CD34+ cells on a wide variety of samples currently used in both the research and clinical laboratories, including cord blood, fetal, normal and abnormal marrow, ex vivo manipulated (purged) samples and when properly set-up, can do so with great accuracy and sensitivity. Isotype controls are not required and the method can be performed on all modern cytometers tested to date including Coulter XL and Elite, BD FACScan, Calibur and Vantage instruments. Incorporation of counting beads and the viability dye 7-AAD allows the determination of absolute viable CD34+ cells with this methodology. This capability has great utility on samples that may have been 'held-overnight' prior to analysis,or otherwise 'mishandled', or have been shipped in less than pristine condition to the flow laboratory (eg cord blood samples shipped to the local 'bank'). Dual platform absolute counting (flow cytometry plus automated hematology analyser) using so-called simple methods like the Milan protocol or its derivatives can generate highly inaccurate data on such samples due to the loss of neutrophils, increased platelet and other debris, and dead cells that can masquerade as CD34+ cells. Similarly, dual platform methods can generate inaccurate data on cord blood samples due to the presence of significant numbers of nucleated red cells that alter the denominator in the calculation of '%CD34+ cells'. More sophisticated analysis is possible starting from the basic ISHAGE protocol, and we routinely perform three and four colour subset analysis/sorting on CD34+ cells in a variety of clinical and research settings. Thus, for example, the utility of new growth factor/chemotherapy combinations that mobilise candidate stem cells within the total CD34+ cell compartment can be assessed. Alternatively, if needed, one can also simultaneously perform CD34 cell enumeration alongside residual T cell quantitation in the allogeneic setting. Many of these methods are detailed in an upcoming article (Enumeration of CD34 Hematopoietic Stem and Progenitor Cells by Gratama et al) to be published in Current Protocols in Cytometry. With specific reference to 'which kit to use in the clinical laboratory', except for the above caveats re platelet aggregates and debris in a monority of samples, either kit should do an adequate job on enumerating CD34+ cells in fresh blood and apheresis samples. We hope this is helpful. Rob Sutherland Rakash Nayar The Toronto Hospital Mike Keeney Ian Chin-Yee London Health Sciences Center Ontario.
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