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Rapid potency assessment of autologous peripheral blood stem cells by intracellular flow cytometry: the PBSC-IL-3-pSTAT5 assay

Published:April 07, 2022DOI:https://doi.org/10.1016/j.jcyt.2022.03.003

      Abstract

      Background aims

      The current gold standard for stem cell product potency assessment, the colony-forming unit (CFU) assay, delivers results that are difficult to standardize and requires a substantial amount of time (up to 14 days) for cellular growth. Recently, the authors developed a rapid (<24 h) flow cytometry assay based on the measurement of intracellular phosphorylated STAT5 (pSTAT5) in CD34+ cord blood stem and progenitor cells in response to IL-3 stimulation. The present work presents a novel adaptation of the protocol for use with autologous peripheral blood stem cells (PBSCs) and a performance comparison with the CFU assay.

      Methods

      The flow cytometry intracellular staining assay was optimized for PBSCs, and patient samples were analyzed using the PBSC-IL-3-pSTAT5 and CFU assays. Warming events were also simulated to emulate impaired potency products.

      Results

      Optimization led to minor protocol adjustments, such as removal of the red blood cell lysis step, the addition of a formaldehyde fixation step and an increase in anticoagulant concentration. The PBSC-IL-3-pSTAT5 assay discriminated between normal and impaired samples and identified 100% (18 of 18) of the impaired samples, thus showing better specificity than the CFU assay.

      Conclusions

      The updated IL-3-pSTAT5 potency assay has several important advantages, such as accelerating the release of autologous stem cell products and enabling the detection of potentially impaired products. The assay could also be used to rapidly assess the potency of any cryopreserved allogeneic stem cell product, such as those processed during the coronavirus disease 2019 pandemic.

      Keywords

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      References

        • Majhail N.S.
        • Farnia S.H.
        • Carpenter P.A.
        • Champlin R.E.
        • Crawford S.
        • Marks D.I.
        • LeMaistre C.F.
        Indications for Autologous and Allogeneic Hematopoietic Cell Transplantation: Guidelines from the American Society for Blood and Marrow Transplantation.
        Biology of Blood and Marrow Transplantation. 2015; 21: 1863-1869
        • Hsu J.W.
        • Farhadfar N.
        • Murthy H.
        • Logan B.R.
        • Bo-Subait S.
        • Frey N.
        • Wingard J.R.
        The Effect of Donor Graft Cryopreservation on Allogeneic Hematopoietic Cell Transplantation Outcomes: A Center for International Blood and Marrow Transplant Research Analysis. Implications during the COVID-19 Pandemic.
        Transplantation and Cellular Therapy. 2021;
        • N Rich I.
        Potency, Proliferation and Engraftment Potential of Stem Cell Therapeutics: The Relationship between Potency and Clinical Outcome for Hematopoietic Stem Cell Products.
        Journal of Cell Science & Therapy. 2013; 4
        • Yang H.
        • Acker J.P.
        • Cabuhat M.
        • Letcher B.
        • Larratt L.
        • McGann L.E.
        Association of post-thaw viable CD34+ cells and CFU-GM with time to hematopoietic engraftment.
        Bone Marrow Transplantation. 2005; 35: 881-887
        • Pamphilon D.
        • Selogie E.
        • McKenna D.
        • Cancelas-Peres J.A.
        • Szczepiorkowski Z.M.
        • Sacher R.
        • Takanashi M.
        Current practices and prospects for standardization of the hematopoietic colony-forming unit assay: a report by the cellular therapy team of the Biomedical Excellence for Safer Transfusion (BEST) Collaborative.
        Cytotherapy. 2013; 15: 255-262
        • Shoulars K.
        • Noldner P.
        • Troy J.D.
        • Cheatham L.
        • Parrish A.
        • Page K.
        • Kurtzberg J.
        Development and validation of a rapid, aldehyde dehydrogenase bright–based cord blood potency assay.
        Blood. 2016; 127: 2346-2354
        • Patterson J.
        • Moore C.H.
        • Palser E.
        • Hearn J.C.
        • Dumitru D.
        • Harper H.A.
        • Rich I.N.
        Detecting primitive hematopoietic stem cells in total nucleated and mononuclear cell fractions from umbilical cord blood segments and units.
        Journal of Translational Medicine. 2015; 13: 94
        • Duggleby R.C.
        • Querol S.
        • Davy R.C.
        • Fry L.J.
        • Gibson D.A.
        • Horton R.B.V.
        • Madrigal J.A.
        Flow cytometry assessment of apoptotic CD34+ cells by annexin V labeling may improve prediction of cord blood potency for engraftment.
        Transfusion. 2012; 52: 549-559
        • Simard C.
        • Bonnaure G.
        • Fournier D.
        • Neron S.
        An objective flow cytometry method to rapidly determine cord blood potency in cryopreserved units.
        Transfusion. 2019; 59: 2074-2083
        • Yang Y.-C.
        • Clark S.C.
        Interleukin-3: Molecular Biology and Biologic Activities.
        Hematology/Oncology Clinics of North America. 1989; 3: 441-452
        • Bradley H.L.
        • Hawley T.S.
        • Bunting K.D.
        Cell intrinsic defects in cytokine responsiveness of STAT5-deficient hematopoietic stem cells.
        Blood. 2002; 100: 3983-3989
      1. The ISHAGE Guidelines for CD34+ Cell Determination by Flow Cytometry. Journal of Hematotherapy.

        • Reich-Slotky R.
        • Patel N.
        • Dael S.
        • Semidei-Pomales M.
        • Stephens H.
        • Reich M.
        • Schwartz J.
        Postthaw clotting of peripheral blood stem cell products due to insufficient anticoagulant.
        Journal of Clinical Apheresis. 2009; 24: 265-268
        • Mandrekar J.N.
        Receiver Operating Characteristic Curve in Diagnostic Test Assessment.
        Journal of Thoracic Oncology. 2010; 5: 1315-1316
        • Hamadani M.
        • Zhang M.-J.
        • Tang X.-Y.
        • Fei M.
        • Brunstein C.
        • Chhabra S.
        • Horowitz M.M.
        Graft Cryopreservation Does Not Impact Overall Survival after Allogeneic Hematopoietic Cell Transplantation Using Post-Transplantation Cyclophosphamide for Graft-versus-Host Disease Prophylaxis.
        Biology of Blood and Marrow Transplantation. 2020; 26: 1312-1317
        • Maurer K.
        • Kim H.T.
        • Kuczmarski T.M.
        • Garrity H.M.
        • Weber A.
        • Reynolds C.G.
        • Gooptu M.
        Impact of Cryopreservation and Transit Times of Allogeneic Grafts on Hematopoietic and Immune Reconstitution.
        Blood Advances. 2021;
        • Kim D.H.
        • Jamal N.
        • Saragosa R.
        • Loach D.
        • Wright J.
        • Gupta V.
        • Messner H.A.
        Similar Outcomes of Cryopreserved Allogeneic Peripheral Stem Cell Transplants (PBSCT) Compared to Fresh Allografts.
        Biology of Blood and Marrow Transplantation. 2007; 13: 1233-1243
        • Fernandez-Sojo J.
        • Azqueta C.
        • Valdivia E.
        • Martorell L.
        • Medina-Boronat L.
        • Martínez-Llonch N.
        • et al.
        Cryopreservation of unrelated donor hematopoietic stem cells: the right answer for transplantations during the COVID-19 pandemic?.
        Bone Marrow Transplantation. 2021; 56(10): 2489-2496
        • Bankova A.K.
        • Caveney J.
        • Yao B.
        • Ramos T.L.
        • Bögeholz J.
        • Heydari K.
        • et al.
        Real-World Experience of Cryopreserved Allogeneic Hematopoietic Grafts during the COVID-19 Pandemic: A Single-Center Report.
        Transplantation and Cellular Therapy. 2022; 28(4): 215.e1-215.e10
        • Morgenstern D.A.
        • Ahsan G.
        • Brocklesby M.
        • Ings S.
        • Balsa C.
        • Veys P.
        • Watts M.J.
        Post-thaw viability of cryopreserved peripheral blood stem cells (PBSC) does not guarantee functional activity: important implications for quality assurance of stem cell transplant programmes.
        British Journal of Haematology. 2016; 174: 942-951
        • Wiercinska E.
        • Schlipfenbacher V.
        • Bug G.
        • Bader P.
        • Verbeek M.
        • Seifried E.
        • Bonig H.
        Allogeneic transplant procurement in the times of COVID-19: Quality report from the central European cryopreservation site.
        Journal of Translational Medicine. 2021; 19: 145
        • Horwitz M.E.
        • Stiff P.J.
        • Cutler C.
        • Brunstein C.
        • Hanna R.
        • Maziarz R.T.
        • Sanz G.
        Omidubicel vs standard myeloablative umbilical cord blood transplantation: results of a phase 3 randomized study.
        Blood. 2021; 138: 1429-1440
        • Claveau J.
        • Cohen S.
        • Ahmad I.
        • Delisle J.
        • Kiss T.
        • Lachance S.
        • Roy J.
        Single UM171-expanded cord blood transplant can cure severe idiopathic aplastic anemia in absence of suitable donors.
        European Journal of Haematology. 2020;
        • Spellman S.
        • Hurley C.K.
        • Brady C.
        • Phillips-Johnson L.
        • Chow R.
        • Laughlin M.
        • Kurtzberg J.
        Guidelines for the development and validation of new potency assays for the evaluation of umbilical cord blood.
        Cytotherapy. 2011; 13: 848-855