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Improved cord blood thawing procedure enhances the reproducibility and correlation between flow cytometry CD34+ cell viability and clonogenicity assays

  • Author Footnotes
    * These authors contributed equally to this work.
    Cristian Camilo Galindo
    Footnotes
    * These authors contributed equally to this work.
    Affiliations
    Cord Blood Bank, Instituto Distrital de Ciencia, Biotecnología e Innovación en Salud, Bogotá DC, Colombia
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  • Author Footnotes
    * These authors contributed equally to this work.
    Diana María Vanegas Lozano
    Footnotes
    * These authors contributed equally to this work.
    Affiliations
    Cord Blood Bank, Instituto Distrital de Ciencia, Biotecnología e Innovación en Salud, Bogotá DC, Colombia
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  • Bernardo Camacho Rodríguez
    Affiliations
    Cord Blood Bank, Instituto Distrital de Ciencia, Biotecnología e Innovación en Salud, Bogotá DC, Colombia
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  • Ana-María Perdomo-Arciniegas
    Correspondence
    Correspondence: Ana-María Perdomo-Arciniegas, MD, MSc, PhD, Cord Blood Bank, Instituto Distrital de Ciencia, Biotecnología e Innovación en Salud, Carrera 32 12 – 81, Bogotá DC, Colombia.
    Affiliations
    Cord Blood Bank, Instituto Distrital de Ciencia, Biotecnología e Innovación en Salud, Bogotá DC, Colombia
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  • Author Footnotes
    * These authors contributed equally to this work.
Published:April 18, 2018DOI:https://doi.org/10.1016/j.jcyt.2018.03.033
      Umbilical cord blood (UCB) is cryopreserved and stored in cord blood banks (CBBs) for allogeneic transplantation in pediatric and adult patients [
      • Ballen K.
      Challenges in umbilical cord blood stem cell banking for stem cell reviews and reports.
      ]. It is known that cord blood unit (CBU) cell viability is deeply affected by both cryopreservation and thawing processes [
      • Fry L.J.
      • Querol S.
      • Gomez S.G.
      • McArdle S.
      • Rees R.
      • Madrigal J.A.
      Assessing the toxic effects of DMSO on cord blood to determine exposure time limits and the optimum concentration for cryopreservation.
      ,
      • Regan D.M.
      • Wofford J.D.
      • Wall D.A.
      Comparison of cord blood thawing methods on cell recovery, potency, and infusion.
      ]. Cell viability is determined by flow cytometry using 7-Aminoactinomycin D (7-AAD) before and after cryopreservation. However, some authors report that the obtained data may be inaccurate when performing post-thawing assays due to membrane instability and cellular unspecific uptake of 7-AAD [
      • Zhu F.
      • Heditke S.
      • Kurtzberg J.
      • Waters-Pick B.
      • Hari P.
      • Margolis D.A.
      • et al.
      Hydroxyethyl starch as a substitute for dextran 40 for thawing peripheral blood progenitor cell products.
      ]. It is possible that this phenomenon varies during the staining protocols and, consequently, affects the flow cytometry results. On the other hand, clonogenic efficiency (CLONE; percentage of effective colonies from a known number of seeded CD34+ cells) is a complementary assessment to flow cytometry and provides information about cell viability, lineage commitment and proliferation ability. Therefore, it may be a more suitable quality control of the unit. The correlation between CD34+ cell viability, as assessed using flow cytometry and CLONE, is a subject under research and may depend on several pre-freezing variables [
      • Pope B.
      • Hokin B.
      • Grant R.
      Effect of umbilical cord blood prefreeze variables on postthaw viability.
      ,
      • Yang H.
      • Pidgorna A.
      • Loutfy M.R.
      • Shuen P.
      Effects of interruptions of controlled-rate freezing on the viability of umbilical cord blood stem cells.
      ]. We hypothesize that if the thawing and staining procedure-associated biases are removed, a real correlation factor may be determined.
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