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Assessing pilot vial material as a surrogate for functional and phenotypic stem cell markers in cryopreserved haematopoietic stem cell product

Bone Marrow Transplantation volume 51, pages 1631–1632 (2016)Cite this article

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Cryopreservation is often used as a means of storing haematopoietic progenitor cell-apheresis (HPC-A) product when it is collected as part of high-dose therapy for the treatment of haematological malignancies. Processing and cryopreservation of HPC-A is known to reduce the viability and the absolute number of CD34+ cells available for reinfusion.1, 2 Therefore, a pilot vial (PV) containing a small aliquot of the same HPC-A material is usually cryopreserved and stored under the same conditions enabling enumeration and viability assessment on post-thaw HPC-A material without disturbing the primary infusion bag required for the transplantation. However, whether PV material can be used to assess other functional or phenotypic stem cell markers in HPC-A material following cryopreservation has not been fully investigated. We aimed to assess whether markers of viability, early apoptosis, DNA damage signalling and oxidative damage, together with CD34+ subset analysis performed similarly between PV and HPC-A material following cryopreservation.

Thirty matched pairs of HPC-A and PV were identified. Patient diagnoses included multiple myeloma (MM) (n=10), non-Hodgkin’s lymphoma (NHL) (n=11), Hodgkin’s lymphoma (n=2), AML (n=3), CML (n=1), 1 patient with POEMS (polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, and skin changes) and 2 with solid tumours. The nucleated cell concentration of the product for cryopreservation was 293±17 × 106/mL, expressed as mean±s.e. HPC-A was plasma reduced and cryopreserved in a controlled rate freezer that was validated for both HPC-A and PV material, freezing at a rate of ~1–3 °C/min till −50 °C. Frozen products were then transferred to a liquid nitrogen vapour-phase tank (Chart Inc., Biomedical, Sydney, NSW, Australia) for storage. In each case, the PV was stored in the same cryopreservation cassette with its corresponding infusion bag. Because of the availability of paired clinical samples, only 4 out of 30 samples could be duplicated using a second PV. HPC-A and PV were simultaneously thawed to 37 °C. Post-thaw CD45+ cell recovery and viable CD34+ cell number were assessed by 7-aminoactinomycin D (7-AAD) using the single-platform ISHAGE protocol,3 one tube for each sample; intracellular aldehyde dehydrogenase (ALDH) activity was assessed by the ALDEFLUOR Assay kit (Stemcell Technology, Melbourne, VIC, Australia)4, 5 and stem cell subsets were assessed by CD1336, 7 and CD38.8, 9 Mitochondrial membrane potential was assessed using 1,1′,3,3,3′-hexamethylindodicarbo-cyanine idodide (DilC5) staining as a marker of early apoptosis,10, 11 DNA damage was assessed using histone γH2AX as a marker of DNA double-stranded break formation12 and intracellular reactive oxygen species (ROS)13 was assessed using 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA). All assays were performed by flow cytometry. All markers, except CD45+ cell% recovery, are expressed as percentages of viable CD34+ cells.

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Acknowledgements

This work was supported by the Staff Specialist Trust Fund, Royal North Shore Hospital, Sydney, Australia.

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Authors and Affiliations

  1. Department of Haematology and Transfusion Medicine, Royal North Shore Hospital, Sydney, NSW, Australia

    L Bai, L Peters, W Xia, K Wong, C Ward & M Greenwood

  2. Department of Haematology, Cellular Therapeutic Laboratory, Northern Blood Research Centre, Kolling Research Institute, Sydney, NSW, Australia

    L Bai, L Peters, W Xia & G Best

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  1. L Bai
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  2. L Peters
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  3. W Xia
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  4. G Best
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Correspondence to L Bai.

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Bai, L., Peters, L., Xia, W. et al. Assessing pilot vial material as a surrogate for functional and phenotypic stem cell markers in cryopreserved haematopoietic stem cell product. Bone Marrow Transplant 51, 1631–1632 (2016). https://doi.org/10.1038/bmt.2016.236

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