Hi Tracie, In general, the successful practise of single platform flow methods requires a high level of precision when it comes to such mundane issues as pipetting. This requirement takes on huge importance when applied to CD34+ cell enumeration because we are dealing with 'rare event' analysis and the consequences of inaccuracy could be disastrous for patients undergoing 'Armageddon' therapies such as myeloablation and stem cell transplantation. Single platform methods should utilise positive displacement/reverse pipetting techniques, such that accuracy is maintained when pipetting relatively viscous samples such as peripheral blood and apheresis products. Such accuracy requirements apply to both Stem-Kit and ProCOUNT methodologies since samples usually have to be pipetted for both dilution and aliquoting purposes. The Stem-Kit additionally requires accurate pipetting of the counting beads. We use an electronic pipettor (that can be set to perform reverse pipetting) for performing dilutions (when needed), for subsequent sample pipetting, and for pipetting counting beads (Stem-CountTM, part of the Stem-Kit). Regarding the two kits, BDB have developed specialised software for semi-automated data acquisition and analysis of ProCOUNT stained samples (on BDFacs instruments) and this product can accurately measure CD34+ cells in most fresh PB and apheresis samples. However, the presence of platelet aggregates, non-viable cells and other debris that stain non-specifically with CD34PE conjugates can cause the software to 'flag' even some fresh samples and BDB have issued a directive to ProCOUNT users to the effect that manual gating of such flagged samples must be performed by an experienced flow cytometrist. Unfortunately, manual analysis of such listmode files (sent to us by people who have experienced this problem) does not always generate reliable data. A recent study by Gutensohn et al of 90 fresh apheresis samples (Transfusion 39: 1220, 1999) found that 23% of them were 'flagged', and even after manual analysis, significant differences were noted between ProCOUNT and both the German Reference and ISHAGE protocols (Stem-Kit is closely modelled on the single platform ISHAGE protocol, as originally described by Keeney et al, Cytometry (Comm Clin Cytom) 34: 61, 1998). Other problems have been noted by others when analysing cord blood samples with this kit (Logdberg et al, Blood 96: (Suppl 1), 2000 (abstr 1645). If you are using the range of samples indicated (PB, apheresis, cord blood and marrow), I would not recommend ProCOUNT since strictly speaking, it is marketed for use on fresh peripheral blood and apheresis products only. In contrast, the ISHAGE protocol and its single platform derivative, can be used to enumerate CD34+ cells in all of the samples you listed. You indicated you were having problems with reproducibiliy using the Stem-Trol control cells, supplied in the Stem-Kit. If you are confident that these problems are not due to pipetting errors, the problems could lie elsewhere. Specifically, it is important to resuspend the Stem-Trol cells adequately prior to pipetting. This also applies to the Flow-Count beads. If methods are used that are too 'violent', you can end up pipetting air bubbles (particularly wrt the beads). Inadequate resuspension of either product will lead to erroneous results and warrant the discarding of the remainder of either product since its assayed value will no longer be correct. These and other issues pertaining to enumerating CD34+ cells have been detailed in Current Protocols in Cytometry Unit 6.4, 1999 (maybe Paul Robinson will give you a free copy!) and the ISHAGE Graft Evaluation web-site (www.ishage.org/committees/Committees/Graft_Evaluation/graft.htm). In some respects, a more valid method involves comparing the white blood cell count from a hematology analyser, with the absolute CD45+ cell count from the flow cytometer. This can be readily obtained since the Stem-Kit contains antibodies to CD45 and CD34 in addition to the counting beads). On PB samples, the numbers obtained with the two methods should be very similar. If not, you need to either go to pipetting school, or check that the assayed bead (or Stem-Trol) count has not been compromised by inadequate storage and improper handling procedures as described above. On appropriately diluted apheresis samples, the results should also be very similar. Larger differences between methods might be expected on CB and marrow samples due to the presence of nucleated red blood cells, that are counted as nucleated cells by the hematology analyser, but excluded by CD45 staining on the cytometer. Thus I would proceed as follows: on a fresh PB sample, compare the absolute CD45 count from flow, with the absolute leukocyte count from the hematology analyser. If these numbers look very similar, then the beads and pipetting issues discussed above are probably OK. If not, a fresh batch of beads should be acquired and used appropriately. If results are now similar, discard the old beads. Now run Stem-Trol. If you obtain the 'wrong' answer, a new batch of Stem-Trol should be assessed. HTH cheers, Rob Sutherland Casey Nayar University Health Network, Princess Maragaret Hospital, Toronto Tel:416 946 2218
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