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- Title
Optimization of Blood Handling and Peripheral Blood Mononuclear Cell Cryopreservation of Low Cell Number Samples.
- Authors
Hope, Christopher M.; Huynh, Dao; Wong, Ying Ying; Oakey, Helena; Perkins, Griffith Boord; Nguyen, Trung; Binkowski, Sabrina; Bui, Minh; Choo, Ace Y. L.; Gibson, Emily; Huang, Dexing; Kim, Ki Wook; Ngui, Katrina; Rawlinson, William D.; Sadlon, Timothy; Couper, Jennifer J.; Penno, Megan A. S.; Barry, Simon C.
- Abstract
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.
- Subjects
MONONUCLEAR leukocytes; BLOOD collection; CELL physiology; KILLER cells; BLOOD banks; CRYOPRESERVATION of cells
- Publication
International Journal of Molecular Sciences, 2021, Vol 22, Issue 17, p9129
- ISSN
1661-6596
- Publication type
Article
- DOI
10.3390/ijms22179129