Red blood cell phenotype fidelity following glycerol cryopreservation optimized for research purposes.
Journal Article (Journal Article)
Intact red blood cells (RBCs) are required for phenotypic analyses. In order to allow separation (time and location) between subject encounter and sample analysis, we developed a research-specific RBC cryopreservation protocol and assessed its impact on data fidelity for key biochemical and physiological assays. RBCs drawn from healthy volunteers were aliquotted for immediate analysis or following glycerol-based cryopreservation, thawing, and deglycerolization. RBC phenotype was assessed by (1) scanning electron microscopy (SEM) imaging and standard morphometric RBC indices, (2) osmotic fragility, (3) deformability, (4) endothelial adhesion, (5) oxygen (O2) affinity, (6) ability to regulate hypoxic vasodilation, (7) nitric oxide (NO) content, (8) metabolomic phenotyping (at steady state, tracing with [1,2,3-13C3]glucose ± oxidative challenge with superoxide thermal source; SOTS-1), as well as in vivo quantification (following human to mouse RBC xenotransfusion) of (9) blood oxygenation content mapping and flow dynamics (velocity and adhesion). Our revised glycerolization protocol (40% v/v final) resulted in >98.5% RBC recovery following freezing (-80°C) and thawing (37°C), with no difference compared to the standard reported method (40% w/v final). Full deglycerolization (>99.9% glycerol removal) of 40% v/v final samples resulted in total cumulative lysis of ~8%, compared to ~12-15% with the standard method. The post cryopreservation/deglycerolization RBC phenotype was indistinguishable from that for fresh RBCs with regard to physical RBC parameters (morphology, volume, and density), osmotic fragility, deformability, endothelial adhesivity, O2 affinity, vasoregulation, metabolomics, and flow dynamics. These results indicate that RBC cryopreservation/deglycerolization in 40% v/v glycerol final does not significantly impact RBC phenotype (compared to fresh cells).
Full Text
- Published version (via Digital Object Identifier)
- Pubmed Central version
- Open Access Copy from Duke
- Link to Item
Duke Authors
Cited Authors
- Rogers, SC; Dosier, LB; McMahon, TJ; Zhu, H; Timm, D; Zhang, H; Herbert, J; Atallah, J; Palmer, GM; Cook, A; Ernst, M; Prakash, J; Terng, M; Towfighi, P; Doctor, R; Said, A; Joens, MS; Fitzpatrick, JAJ; Hanna, G; Lin, X; Reisz, JA; Nemkov, T; D'Alessandro, A; Doctor, A
Published Date
- 2018
Published In
Volume / Issue
- 13 / 12
Start / End Page
- e0209201 -
PubMed ID
- 30576340
Pubmed Central ID
- PMC6303082
Electronic International Standard Serial Number (EISSN)
- 1932-6203
Digital Object Identifier (DOI)
- 10.1371/journal.pone.0209201
Language
- eng
Conference Location
- United States