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Flow cytometric measurement of apoptosis and necrosis in cryopreserved PBPC concentrates from patients with malignant diseases

Bone Marrow Transplantation volume 29, pages 165–171 (2002)Cite this article

Abstract

The number of viable precursor cells actually reinfused into patients after high-dose chemotherapy is one of the most clinically important variables determining graft success or failure. A modified, previously described flow cytometric method based on annexin V staining was therefore applied to assess the degree of apoptosis and necrosis in cryopreserved PBPC concentrates from patients with malignant diseases. Twenty-two samples of unmanipulated cryopreserved PBPC concentrates were analyzed by flow cytometry. The samples were triple-stained with anti-CD34 PE, annexin V-FITC and actinomycin D, which enabled the separation of viable, early apoptotic and late apoptotic/necrotic CD34+ precursor cells. Apotosis and necrosis were also measured in the total cell population of the concentrates. Eighty-one percent (range 49–97) of the CD34+ cells were viable, while 7% (range 1–15) were early apoptotic and 12% (range 2–36) were late apoptotic/necrotic after freeze/thaw. There was no difference in apoptosis and necrosis in CD34+ cells harvested from mildly pretreated patients with multiple myeloma and heavily pre-treated patients with non-Hodgkin's lymphoma. Apoptosis and necrosis were higher in the total mature cell population of the concentrates. Thirty-two percent (range 7–69) of the cells were apoptotic and 33% (range 12–60) were necrotic. We conclude that flow cytometric analysis of annexinV/actinomycin D binding in PBPC concentrates is a simple technique that can give additional information of the viability status of the cells post thaw. The present study confirms the relative robustness of human CD34+ precursor cells concerning the freeze/thaw procedure, which are carried out in daily clinical practice.

Bone Marrow Transplantation (2002) 29, 165–171. doi:10.1038/sj.bmt.1703346

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Acknowledgements

This work was supported by grants from the Norwegian Cancer Society.

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Authors and Affiliations

  1. The Blood Bank, Haukeland University Hospital, Bergen, Norway

    JF Abrahamsen & AM Bakken

  2. Division of Hematology, Department of Medicine, Haukeland University Hospital and the University of Bergen, Bergen, Norway

    Ø Bruserud & BT Gjertsen

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  1. JF Abrahamsen
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  2. AM Bakken
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Abrahamsen, J., Bakken, A., Bruserud, Ø. et al. Flow cytometric measurement of apoptosis and necrosis in cryopreserved PBPC concentrates from patients with malignant diseases. Bone Marrow Transplant 29, 165–171 (2002). https://doi.org/10.1038/sj.bmt.1703346

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