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Assessment of cord blood hematopoietic cell parameters before and after cryopreservation

Authors

  • James Kurtz,

    1. From the American Red Cross, Jerome H. Holland Laboratory for the Biomedical Sciences, Rockville, Maryland; and the Department of Medicine, Division of Hematology/Oncology, Case Western Reserve University, Cleveland, Ohio.
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  • Shalini Seetharaman,

    1. From the American Red Cross, Jerome H. Holland Laboratory for the Biomedical Sciences, Rockville, Maryland; and the Department of Medicine, Division of Hematology/Oncology, Case Western Reserve University, Cleveland, Ohio.
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  • Nicholas Greco,

    1. From the American Red Cross, Jerome H. Holland Laboratory for the Biomedical Sciences, Rockville, Maryland; and the Department of Medicine, Division of Hematology/Oncology, Case Western Reserve University, Cleveland, Ohio.
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  • Gary Moroff

    1. From the American Red Cross, Jerome H. Holland Laboratory for the Biomedical Sciences, Rockville, Maryland; and the Department of Medicine, Division of Hematology/Oncology, Case Western Reserve University, Cleveland, Ohio.
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James W. Kurtz, American Red Cross, Jerome H. Holland Laboratory for the Biomedical Sciences, 15601 Crabbs Branch Way, Rockville, MD 20855; e-mail: kurtzj@usa.redcross.org.

Abstract

BACKGROUND: The testing of cord blood (CB) progenitor and stem cell units for transplantation suitability involves enumeration of total nucleated cells before freezing. CD34+ cell counts may also be a means of determining suitability. Studies have been conducted to evaluate how specific storage conditions influence cell counts.

STUDY DESIGN AND METHODS: CB units were processed by hydroxyethyl starch volume reduction. Cryopreserved-thawed samples were diluted 1:3 without washing. CD34+ cells were measured with three commercially available assay methods. In specific studies, apoptosis-indicating reagents were included. CB units were analyzed for nucleated cells, aldehyde dehydrogenase–containing cells, and progenitor colonies.

RESULTS: CD34+ cell levels and nucleated cells were retained during storage in test tubes at 1 to 6°C for 3 days. Cryopreserved-thawed samples showed a reduction in CD34+ cells relative to prefreeze levels with the largest decrease with the Stem-Kit (Beckman Coulter) restricted gating procedure. Prefreeze samples contained minimal numbers of presumed apoptotic cells detected with 7-aminoactinomycin D or SYTO16, but after cryopreservation-thawing there was an increase. Nucleated cell levels determined with a hematology analyzer or flow cytometry were reduced after thawing. Cryopreservation-thawing reduced the percentage of CD34+ cells positive for the presence of aldehyde dehydrogenase and the number of progenitor colonies. These differences were significant.

CONCLUSION: These studies indicate that CD34+ cell counts were maintained when CB samples were stored at 1 to 6°C in test tubes for 3 days. Cryopreservation-thawing resulted in changes in a number of parameters including the percentage of CD34+ cells that were aldehyde dehydrogenase+ and the number of 7-aminoactinomycin D+ cells and SYTO16low cells.

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