Multi-laboratory assay for harmonization of enumeration of viable CD34+ and CD45+ cells in frozen cord blood units

Fournier, Diane; Lewin, Antoine; Simard, Carl; Trépanier, Patrick; Néron, Sonia; Ballerini, Lara; Codinach, Margarita; Elmoazzen, Heidi; Halpenny, Mike; Kögler, Gesine; Liedtke, Stefanie; Louis, Isabelle; Molluna, Carmen Azqueta; Pineault, Nicolas; Prasath, Arun; Querol, Sergio; Saccardi, Riccardo; Sutherland, D. Robert; Thérien, Cynthia; Urbani, Serena

doi:10.1016/j.jcyt.2019.10.009

Cited by 12 publications

(30 citation statements)

References 16 publications

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“…Multicenter studies of vCD34 + HPC enumeration using dry shipper transport have been restricted to the setting of cord blood units [ 16 , 17 ]. Further investigation into this important area for harmonization and standardization has been hampered by the expense of LN dry shipper transport that arises from their limited availability, dangerous goods classification and weight.…”

Section: Discussionmentioning

confidence: 99%

Meeting the COVID challenge: Optimizing vCD34+ in cryopreserved HPC samples for implementation of an external QA Program

2022

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Background The COVID-19 pandemic has forced a fundamental change in the global procurement of allogeneic hematopoietic progenitor cells (HPCs) for transplantation. To better meet the emergent challenges of transporting cryopreserved allogeneic HPC during pandemics, there is an urgent need for External Quality Assurance (EQA) programs to evaluate reproducibility and harmonization of viable CD34 + cell (vCD34 + ) HPC enumeration, as the current EQA programs are unsuitable for analysis of vCD34 + . The cost-effective distribution of HPC cryopreserved reference samples (CRSs) with acceptable reproducibility and specificity is key to the success of a vCD34 + EQA program. Methods Cryopreserved HPC samples (n = 11) were either stored on dry ice for 1 to 4 days or for 1 day followed by liquid nitrogen (LN) storage for 1 to 3 days to assess optimal conditions for vCD34 + EQA. Flow cytometric enumeration of vCD34 + HPCs was performed using a single platform assay combined with 7-AAD viability dye exclusion. The optimum transportation condition was validated in pilot and multicenter national studies (n = 12). Results A combination of 1 day on dry ice followed by LN storage stabilized viability compared with continuous storage on dry ice. This study demonstrates that dispatch of CRSs on dry ice to recipient centers across a distance of ≤4000 km within 26 h, followed by LN storage, resulted in reproducible intercenter vCD34 + enumeration. The estimated cost of safer and more convenient dry ice delivery is >20-fold lower than that of LN. Conclusion This approach can form the basis for economically and scientifically acceptable distribution of CRSs for external vCD34 + EQA.

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Section: Discussionmentioning

confidence: 99%

Meeting the COVID challenge: Optimizing vCD34+ in cryopreserved HPC samples for implementation of an external QA Program

2022

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“…Given our understanding of the challenges associated with flow cytometry and our experience in inter-laboratory standardization of flow assays 10 , we ought to provide the community with a standardized baseline which could be adapted for further characterization of the SARS-CoV-2 humoral response. For example, laboratories who only have residual or low sample volume could adapt this assay to measure the IgG1/2/3/4, IgA and IgM distinct response all within one tube using multiple fluorescent antibodies.…”

Section: Discussionmentioning

confidence: 99%

Standardization of a flow cytometry SARS-CoV-2 serologic test

Simard

Richard

Bazin

et al. 2021

Preprint

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The SARS-CoV-2 virus is the causing agent of the coronavirus disease 2019 (COVID-19) pandemic which is responsible for millions of deaths worldwide. The development of the humoral response to the virus has been the subject of intensive research and development. A flow cytometry-based assay using native full-length SARS-CoV-2 Spike protein expressed in 293T cells was recently proposed as a complementary seropositivity determination assay.The aim of our study was to further develop the flow cytometry assay for potential use as a confirmatory test and to standardize its parameters and results for reliable inter-laboratory use. We have optimized the protocol, established the Receiving Operating Characteristic (ROC) curve and tested reproducibility using pre-COVID plasma samples and convalescent, SARS-CoV-2 individual plasma samples.The flow-based assay was simplified and standardized by cultivating the 293T cells in suspension and expressing results in Mean Equivalent Soluble Fluorochrome (MESF) using an internal antibody positive control. The ROC curve was determined with an area under the curve (AUC) of 0.996 and the assay specificity and sensitivity were established at 100% and 97.7% respectively. Reproducibility was good as determined on multiple cytometers, on different days, and with data acquisition as far as 72h post-staining. The optimized and standardized assay could be used as a high throughput confirmation confirmatory assay in flow cytometry laboratories involved in serological testing.

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“…However, allowing a post-thaw resting period before staining cells for flow cytometry analysis has been shown to improve the detection and functionality of PBMC subpopulations and T cell subsets. This resting period may allow for the repair of some aspects of cell injury, yet the nature and duration of such a recovery period has yet to be determined, and must be consistent between all samples, as does the time between sample staining and data acquisition ( 127 , 129 – 133 ). This recovery time has been considered as enabling both the removal of apoptotic cells generated by the cryopreservation process and the pre-activation of T cells ( 131 ).…”

Section: The Freezing Processmentioning

confidence: 99%

“…During freezing, even when frozen together, there may be a significant difference in rate of heat loss and degree of supercooling before ice nucleation between the two volumes. This can produce differences in their post-thaw survival ( 133 , 163 ). Recovery of the smaller, QC sample may therefore not accurately reflect the outcome when the bulk sample is thawed and it is essential to be aware of, and compensate for, such issues.…”

Section: Regulatory Issuesmentioning

confidence: 99%

Cryopreservation as a Key Element in the Successful Delivery of Cell-Based Therapies—A Review

2020

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Cryopreservation is a key enabling technology in regenerative medicine that provides stable and secure extended cell storage for primary tissue isolates and constructs and prepared cell preparations. The essential detail of the process as it can be applied to cell-based therapies is set out in this review, covering tissue and cell isolation, cryoprotection, cooling and freezing, frozen storage and transport, thawing, and recovery. The aim is to provide clinical scientists with an overview of the benefits and difficulties associated with cryopreservation to assist them with problem resolution in their routine work, or to enable them to consider future involvement in cryopreservative procedures. It is also intended to facilitate networking between clinicians and cryo-researchers to review difficulties and problems to advance protocol optimization and innovative design.

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Multi-laboratory assay for harmonization of enumeration of viable CD34+ and CD45+ cells in frozen cord blood units

Cited by 12 publications

References 16 publications

Meeting the COVID challenge: Optimizing vCD34+ in cryopreserved HPC samples for implementation of an external QA Program

Meeting the COVID challenge: Optimizing vCD34+ in cryopreserved HPC samples for implementation of an external QA Program

Standardization of a flow cytometry SARS-CoV-2 serologic test

Cryopreservation as a Key Element in the Successful Delivery of Cell-Based Therapies—A Review

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