Flow cytometric determination of the absolute number of CD34 positive hematopoitetic stem cells using the CD34Count Kit (DAKO)

DOI 7 References
  • Hayashida Masahiko
    Tenri Institute of Medical Research Department of Biomedical Informatics, Division of Health Science, Osaka University Graduate School of Medicine
  • Kitada Kayo
    Tenri Institute of Medical Research
  • Iwatani Yoshinori
    Department of Biomedical Informatics, Division of Health Science, Osaka University Graduate School of Medicine

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  • CD34陽性造血幹細胞数の絶対数測定-CD34Count Kit（DAKO社）の使用について

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Abstract

<p>The determination of the number of CD34 positive cells is essential for evaluating the availability of hematopoietic stem cells in autologous and homologous peripheral stem cell transplantations and for optimizing the timing of collecting a sufficient number of peripheral stem cells. Recently the CD34 Count Kit has been marketed by DAKO to flow-cytometrically determine the absolute number of CD34 positive cells by the single platform method, following the International Society for Cellular Therapy (ISCT) guidelines for stem cell analysis in human peripheral blood or its fractions. We assessed the performance of this kit using peripheral blood and simultaneously compared it with the Stem-kit, which is made by Beckman-Coulter Inc. on the same principle. </p><p></p><p>There was no essential difference between the two kits in their performance including cellular staining as well as time required for the whole procedure, which was completed within 30 minutes. The absolute number of CD34 cells collected from peripheral blood ranged from 4 to 161/μl, and the number obtained by the CD34Count Kit (Y) agreed well with that obtained by the Stem Kit (X) with a regression of y = 1.019 + 1.5 and a coefficient of determination (R2) of 0.9984. Similarly, the white cell counts obtained by the two kits agreed well with each other in the range of 3,900 to 70,000 /μl with a regression of y=0.966 x -240.6 and an R2 of 0.9984. However, when the two kits were compared using bone mallow cells, forward scatter versus side scatter cytograms of the CD34 Count Kit allowed many non-specifically stained cells such as debris and platelets to enter the stem cell region, occasionally giving falsely high counts of CD34 positive cells and leukocytes. This difference is due to the effect of the solution used to suspend internal standard particles in the Stem Kit, enhancing forward scatter of CD34 positive cells and thus discriminating them from non-specifically stained debris.</p>

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