CD34 Count Kit - Dako

The shape of Excellentlaboratory performanceCD34 Count Kit...CD34 positive stem cell counts are important in monitoringstem cell transplantation. The Dako CD34 Count Kit is anISHAGE compatible protocol that provides highly accurateresults and reduced sample handling.Not for sale in the US

BackgroundHematopoietic cell transplantations are used in the treatment of blooddisorders, malignancies and genetic abnormalities.Bone marrow was initially the source of choice for transplantation, however,the use of peripheral blood stem cells (PBSC) to reconstitute hematopoiesishas increased tremendously over the last decade.The success of the transplant is crucial depended on the total number ofCD34 cells in the donor material. Conventionally, the CD34 cell count isapproximated by a two platform method, using a hemacytometer to determinethe total white blood cell count, and a flow analysis where the CD34cell count is determined relative to the white cell count (Described in moredetail in the package insert, and references within).The CD34Count kit offers a method to obtain the total number of CD34positive cells in human mobilized peripheral blood samples and leukapheresissamples, applying only a single flow cytometry test.Enumeration of CD34+ CellsAs flow cytometry provides a rapid and simultaneous analysis of bothquantitative and qualitative properties of CD34+ cells, this method has becomean important tool for the monitoring of “on-line” yield of CD34+ cellsand for the determination of the optimal time for harvesting.However, the variation in the enumeration of CD34+ cells by flow cytometryis significant due to different gating strategies and the use of differentmonoclonal antibodies, fluorochromes and lysing solutions. Thus,standardized guidelines are important for obtaining reliable and comparableresults.Over the years, several guidelines such as Milan, Nordic and ISHAGEhave been created in the endeavor to standardize the enumeration ofCD34+ cells. Today, the majority of users recognizes the ISHAGE singleplatform protocol as the “golden standard”.A Reliable Kit for Absolute Count of ViableCD34+ CellsIn order to obtain accurate, reliable and reproducible CD34+ counts, Dakohas developed the CD34Count Kit which complies with the ISHAGE singleplatform protocol.The kit applies the four-parameter strategy of the above protocol andincludes two antibodies, anti-human CD34 and anti-human CD45. Furthermore,it contains CytoCount, a suspension of fluorescent microspheres,which is used as a reference population for the calculation of the absoluteCD34+ value.In addition, the kit includes EasyLyse, a no-wash, fixative-free, ammoniumchloride red-blood-cell lysing reagent, and the viability dye, 7-aminoactinomycinD (7-AAD), to exclude dead cells from the analysis.High AccuracyTo minimize potential sources of inaccuracy, allthe results used in the CD34+ cell calculationderive from a single platform, the flow cytometer.To increase the accuracy of the enumeratedviable CD34+ cells in the sample even more, theCD34Count Kit employs the practice of:n Exclusion of washing and fixing procedures ofthe sample to avoid cell lossn Identification and quantitation of viable CD34+cells by use of the viability dye, 7-aminoactinomycinDOptimized AnalysisAs the CD34Count Kit utilizes the ISHAGE singleplatform protocol, the absolute number of CD34+cells is directly derived from a single flow cytometricmeasurement. This optimizes the analysisby:n Reducing the sample handlingn Minimizing the workloadn Reducing the cost of the test compared to conventionalplatform countingUser-FriendlyThe CD34Count Kit is simple to use and isprovided with detailed instructions. Vials arenumbered according to the order of use.The kit has been designed and optimized for theISHAGE guidelines for single platform.Main steps in procedure:1. Pipette 100 µL of the sample into a test tubeusing reverse pipetting.2. Add 10 µL antibody mixture (anti-CD45/FITC +anti-CD34/RPE) and incubate for 15 min.3. Add 2 mL EasyLyse (the lysing solution) andincubate for 10 min.4. Add 10 µL 7-AAD and incubate for 5 min.5. Add 100 µL CytoCount (the referencebeads) using reverse pipetting, applying thesame pipet as used to pipette the sample.6. Analyze on the flow cytometer and calculatethe absolute viable CD34+ cells using thesingle platform ISHAGE protocol.Dako has a comprehensive portfolio of more than240 reagents for flow cytometry, including CD1a,CD90, CD117 and TdT, commonly used for stemcell analysis.For details please see instructions for use forK2370.

Enumeration of CD34+ Cells (example)CD34Count Kit is applied on a leukapheresis sample, and the ISHAGE gating protocol is usedfor determination of the absolute number of viable CD34+ cells.SSC-H0 64 128 192 256SSC-H0 64 128 192 256SSC-H0 64 128 192 256SSC-H0 64 128 192 25610 0 10 1 10 2 10 310 410 0 10 1 10 2 10 310 410 0 10 1 10 2 10 310 40 64 128 192256CD45FITCCD34RPECD45 FITCFSC-HFigure 1. CD45/FITC vs. SSCshowing all events excluding deadcells and beads. R1 is set aroundthe CD45-positive events. R5 is setaround the lymphocytes.Figure 2. CD34/RPE vs. SSC.The plot is gated on a logical gateinclu ding R1 and not R8 (the deadcells and reference beads). R2 is setaround CD34+ cells.Figure 3. CD45/FITC vs. SSC gatedon a logical gate including R1, R2and not R8. True CD34 events forma discrete cluster, and a region is setaround this population (R3).Figure 4. FSC vs. SSC gated on alogical gate including R1, R2, R3and not R8. R4 illustrates the CD34+progenitor cells. The counted numberfrom R4 is used to calculate the absoluteCD34+ cell number.SSC-H0 64 128 192 256CD34RPE0 64 128 192 256FSC-H0 64 128 192 256SSC-H0 64 128 192 25610 0 10 1 10 2 10 310 410 0 10 1 10 2 10 310 40 64 128 1922560 64 128 1922567-AAD7-AADTime (204.80 sec.)FSC-HFigure 5. 7-AAD vs. SSC. R8 isset around the 7-AAD-positive cellevents.Figure 6. CD45/FITC vs. CD34/RPE.R6 is set around the CytoCountbeads.Figure 8. FSC vs. SSC ungated.Figure 7. Time vs. FS gated on theR6 events, the CytoCount beads(see also Figure 6). The counts fromR7 are used for calculating the absoluteCD34+ number.CalculationAbsolute concentration of CD34+ cells:Counted number of CD34+ cells x CytoCount concentration x Dilution factorCounted number of CytoCount beadsCD34+ concentration:216 x 1006 x 13276= 66 CD34+ cells/µL

Kit ContentsVial 1Vial 2Vial 3Vial 4Monoclonal Mouse Anti-Human CD45/FITC, Clone T29/33, andMonoclonal Mouse Anti-Human CD34/RPE, Clone BIRMA-K31 mL, ready-to-useEasyLyseErythrocyte-lysing reagent, ammonium-chloride based2 x 5 mL, 20 x concentrated7-Aminoactinomycin D (7-AAD)For viability staining1 mL (0.01% w/v), ready-to-useCytoCountCount control beads17 mL, ready-to-use after resuspensionProduct Package Size Code Regulatory status EUCD34Count Kit 50 duplicate tests K2370 CE – IVD** Complies with Directive 98/79/EC of the European Parliament and of the Council on in vitro diagnosticmedical devices.Dako´s kappa and lambda reagents are polyclonal.Many polyclonal antibodies conjugated with fluorescentcompounds render brighter signals thancommensurate monoclonal antibodies.Corporate HeadquarterDenmark+45 44 85 95 00Find your local distributor through:www.dako.comor contactReagent Partnership Division onreagent.partnerships@dako.comDistributors in more than 50 countries28997 30AUG11 Layout and print: Faust Dyrbye