Hello Emanuel, You have touched on a very tricky issue here. There have been several attempts to generate stabilised CD34+ cells for Quality Control and Laboratory Proficiency testing schemes. Additionally, it is desirable to have at hand, a product that contains a known concentration of CD34+ cells that can be used as a 'process control' for the single platform methodology you are using. We have evaluated many different products over the last few years and have yet to find the 'perfect solution' for these problems. Clearly, it is verydifficult to obtain large volumes of 'normal' mobilised blood that can be stabilised and stored for long periods of time for the sort of uses indicated above. Thus, Stem-Trol cells, based upon an engineered cell-line (KG1a) were developed for this purpose, and included in the Beckman-Coulter Stem-Kit. As you indicated, while the CD45 and CD34 expression levels of the KG1a cells have been engineered to mimic 'normal' CD34+ cells in mobilised PB and apheresis products, their light scatter characterstics are quite different, and the gating regions established for Stem-Trol must be carefully altered to include all the stabilised KG1a cells therein. You also indicated that you are practising the dark art of reverse pipetting etc. as recommended. I assume you used such techniques for pipetting the Stem-Trol cells also. Again, as with the beads, you need to be careful to fully suspend them prior to pipetting, without generating air bubbles etc. Note however, that Stem-Trol cells can withstand (and indeed require) much more vigorously vortexing than Flow Count beads. This process should be performed immediatley prior to use, as they are currently shipped in a conical vial and they 'pellet' quite rapidly . You may have received a 'bad' batch of Stem-Trol cells, or you or one of your colleagues may have inadvertently 'altered' the concentration of the Stem-Trol by a failing to re-suspend them properly on an earlier occasion. Of course, if an aliquot has been removed from an improperly suspended sample, the assayed cell concentration is permanently altered. I suggest you obtain another batch of Stem-Trol and try again. Although we regularly 'run' Stem-Trol as a control, our day-to-day samples are 'internally' controlled by assessing (as you also do) the WBC count on all blood and apheresis samples. In that way, we can compare the absolute CD45+ cell count with the absolute leukcoyte count from the hematolgy analyser. Analysis of our results over hundreds of samples shows that this is an extremely effective way of demonstrating that the flow assay is working well. This situation only pertains to fresh blood and apheresis samples of course. Samples held overnight prior to processing sometimes show differences between the absolute WBC and the absolute CD45+ cell counts. Presumably these differences are due to loss of neutrophils, platelet aggregate formation and other metaphysical side effects. However, the absolute CD34+ cell counts (derived by 'single platform' Stem-Kit protocols) on such samples change very little between 'fresh' and 'overnight' storage (assuming appropriate dilution and storage conditions are observed). This reinforces our view that single platform analysis represents the most accurate manner in which to enumerate CD34+ cells (regardless of sample condition). I hope this helps. Cheers, Rob Sutherland University Health Network, Toronto Emanuel Raniolo wrote: > Hi all > > Our standard method for CD34 enumeration is a dual platform ISHAGE gated > (wash) method. > In order to establish and validate a single platform (no wash) ISHAGE method > in the laboratory, it would be nice to be able to run an internal control. > > I have used Beckman-Coulter's commercial Stem-Trol control cells for this > purpose, but have been unable to recover the stated assayed concentration. > In fact I my determinations are consistently higher than the allowable +/- > 15% range. > > Apart from some difficulties in gating placement, as these cells display > increased forward and side light scatter characteristics to normal human > haematopoetic precursors, I cannot see why my results are too high (around > 40% above the stated concentration). I practice reverse pipetting, and mix > the fluorospheres adequately prior to dispensing. Single vs dual platform > comparisons for peripheral blood demonstrate a small positive bias in favour > of the single platform method, whereas results are identical for apheresis > product. In addition, the absolute CD45+ count is in general agreement with > the analyser WBC count. > > I would like to ask: > What is the group experience with the use of internal controls for CD34 > enumeration, and in particular with Stem-Trol cells? > Is the stated Stem-Trol assayed concentration and range reflective of common > experience? > Are internal CD34+ controls regularly utilised in routine practice? > What alternative sources are available? > > Thanks > > Emanuel Raniolo > IMVS Laboratories > The Queen Elizabeth Hospital > Department of Haematology > Cell Analysis and Cryopreservation Laboratory > Adelaide, South Australia.
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