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Article

Optimization of Blood Handling and Peripheral Blood Mononuclear Cell Cryopreservation of Low Cell Number Samples

1
Robinson Research Institute, Adelaide Medical School, University of Adelaide, Adelaide, SA 5005, Australia
2
Women’s and Children’s Hospital, Adelaide, SA 5006, Australia
3
Children’s Diabetes Centre, Telethon Kids Institute, The University of Western Australia, Perth, WA 6009, Australia
4
Child Health Research Unit, Barwon Health, Geelong, VIC 3220, Australia
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School of Women’s and Children’s Health, Faculty of Medicine and Health, University of New South Wales, Sydney, NSW 2052, Australia
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Walter and Eliza Hall Institute of Medical Research, Melbourne, VIC 3052, Australia
7
Virology Research Laboratory, Serology and Virology Division, NSW Health Pathology, Prince of Wales Hospital, Sydney, NSW 2031, Australia
*
Author to whom correspondence should be addressed.
Authors contributed equally.
Membership of the ENDIA Study Group is provided in the Acknowledgments.
Academic Editor: Vladimir Isachenko
Int. J. Mol. Sci. 2021, 22(17), 9129; https://doi.org/10.3390/ijms22179129
Received: 13 July 2021 / Revised: 14 August 2021 / Accepted: 19 August 2021 / Published: 24 August 2021
(This article belongs to the Section Molecular Biology)
Background: Rural/remote blood collection can cause delays in processing, reducing PBMC number, viability, cell composition and function. To mitigate these impacts, blood was stored at 4 °C prior to processing. Viable cell number, viability, immune phenotype, and Interferon-γ (IFN-γ) release were measured. Furthermore, the lowest protective volume of cryopreservation media and cell concentration was investigated. Methods: Blood from 10 individuals was stored for up to 10 days. Flow cytometry and IFN-γ ELISPOT were used to measure immune phenotype and function on thawed PBMC. Additionally, PBMC were cryopreserved in volumes ranging from 500 µL to 25 µL and concentration from 10 × 106 cells/mL to 1.67 × 106 cells/mL. Results: PBMC viability and viable cell number significantly reduced over time compared with samples processed immediately, except when stored for 24 h at RT. Monocytes and NK cells significantly reduced over time regardless of storage temperature. Samples with >24 h of RT storage had an increased proportion in Low-Density Neutrophils and T cells compared with samples stored at 4 °C. IFN-γ release was reduced after 24 h of storage, however not in samples stored at 4 °C for >24 h. The lowest protective volume identified was 150 µL with the lowest density of 6.67 × 106 cells/mL. Conclusion: A sample delay of 24 h at RT does not impact the viability and total viable cell numbers. When long-term delays exist (>4 d) total viable cell number and cell viability losses are reduced in samples stored at 4 °C. Immune phenotype and function are slightly altered after 24 h of storage, further impacts of storage are reduced in samples stored at 4 °C. View Full-Text
Keywords: PBMC; blood handling; delay in processing; cryopreservation; cell concentration PBMC; blood handling; delay in processing; cryopreservation; cell concentration
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MDPI and ACS Style

Hope, C.M.; Huynh, D.; Wong, Y.Y.; Oakey, H.; Perkins, G.B.; Nguyen, T.; Binkowski, S.; Bui, M.; Choo, A.Y.L.; Gibson, E.; Huang, D.; Kim, K.W.; Ngui, K.; Rawlinson, W.D.; Sadlon, T.; Couper, J.J.; Penno, M.A.S.; Barry, S.C.; on behalf of the ENDIA Study Group. Optimization of Blood Handling and Peripheral Blood Mononuclear Cell Cryopreservation of Low Cell Number Samples. Int. J. Mol. Sci. 2021, 22, 9129. https://doi.org/10.3390/ijms22179129

AMA Style

Hope CM, Huynh D, Wong YY, Oakey H, Perkins GB, Nguyen T, Binkowski S, Bui M, Choo AYL, Gibson E, Huang D, Kim KW, Ngui K, Rawlinson WD, Sadlon T, Couper JJ, Penno MAS, Barry SC, on behalf of the ENDIA Study Group. Optimization of Blood Handling and Peripheral Blood Mononuclear Cell Cryopreservation of Low Cell Number Samples. International Journal of Molecular Sciences. 2021; 22(17):9129. https://doi.org/10.3390/ijms22179129

Chicago/Turabian Style

Hope, Christopher M., Dao Huynh, Ying Ying Wong, Helena Oakey, Griffith Boord Perkins, Trung Nguyen, Sabrina Binkowski, Minh Bui, Ace Y. L. Choo, Emily Gibson, Dexing Huang, Ki Wook Kim, Katrina Ngui, William D. Rawlinson, Timothy Sadlon, Jennifer J. Couper, Megan A. S. Penno, Simon C. Barry, and on behalf of the ENDIA Study Group. 2021. "Optimization of Blood Handling and Peripheral Blood Mononuclear Cell Cryopreservation of Low Cell Number Samples" International Journal of Molecular Sciences 22, no. 17: 9129. https://doi.org/10.3390/ijms22179129

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