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Related Experiment Videos

Quantification of CD34+ cells: comparison of methods

G Fritsch1, D Printz, M Stimpfl

  • 1Children's Cancer Research Institute, St. Anna Kinderspital, Vienna, Austria.

Transfusion
|August 1, 1997
PubMed
Summary

Quantifying CD34+ cells using density separation or whole-blood lysis with washing leads to inaccurate cell counts. These methods significantly alter nucleated cell numbers, impacting CD34+ cell enumeration results.

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Area of Science:

  • Hematology
  • Flow Cytometry
  • Cellular Analysis

Background:

  • CD34+ stem and progenitor cell quantification is crucial in clinical settings.
  • Current methods often involve whole blood staining and red blood cell lysis, potentially causing nucleated cell loss during washing.
  • This study investigates cell preparation techniques to address potential biases in CD34+ cell enumeration.

Purpose of the Study:

  • To compare three different cell preparation techniques for accurate CD34+ cell quantification.
  • To evaluate the impact of cell washing and density separation on nucleated cell counts and CD34+ cell enumeration.

Main Methods:

  • Examined blood, bone marrow, and leukapheresis components (n=303 total).
  • Compared density separation of mononuclear cells (MNCs) with two red cell lysis procedures (wash and no-wash).

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  • Calculated absolute CD34+ cell counts using flow cytometry and white cell counts from original and prepared samples.

    • Main Results:

    • Cell washing significantly reduced nucleated cell numbers (12-89%), primarily affecting granulocytes.
    • Density-separated MNCs showed threefold higher relative CD34+ values compared to lyse-and-wash methods in blood and bone marrow.
    • The MNC procedure underestimated CD34+ cell content by 5-26%, while the lyse-and-wash method overestimated it by 1.13-1.33-fold.

    Conclusions:

    • Neither density separation nor the conventional whole-blood lysis with washing procedure is optimal for precise CD34+ cell quantification.
    • Washing steps in cell preparation protocols lead to significant nucleated cell loss, impacting enumeration accuracy.
    • Further optimization of cell preparation techniques is needed for reliable CD34+ cell quantification.