TY - JOUR
T1 - Red blood cell phenotype fidelity following glycerol cryopreservation optimized for research purposes
AU - Rogers, Stephen C.
AU - Dosier, Laura B.
AU - McMahon, Timothy J.
AU - Zhu, Hongmei
AU - Timm, David
AU - Zhang, Hengtao
AU - Herbert, Joseph
AU - Atallah, Jacqueline
AU - Palmer, Gregory M.
AU - Cook, Asa
AU - Ernst, Melanie
AU - Prakash, Jaya
AU - Terng, Mark
AU - Towfighi, Parhom
AU - Doctor, Reid
AU - Said, Ahmed
AU - Joens, Matthew S.
AU - Fitzpatrick, James A.J.
AU - Hanna, Gabi
AU - Lin, Xue
AU - Reisz, Julie A.
AU - Nemkov, Travis
AU - D’Alessandro, Angelo
AU - Doctor, Allan
N1 - Publisher Copyright:
© 2018 Rogers et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
PY - 2018/12
Y1 - 2018/12
N2 - 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 ali-quotted 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 (O 2 ) affinity, (6) ability to regulate hypoxic vasodilation, (7) nitric oxide (NO) content, (8) metabolomic phenotyping (at steady state, tracing with [1,2,3- 13 C 3 ]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 (-80C) and thawing (37C), 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, O 2 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).
AB - 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 ali-quotted 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 (O 2 ) affinity, (6) ability to regulate hypoxic vasodilation, (7) nitric oxide (NO) content, (8) metabolomic phenotyping (at steady state, tracing with [1,2,3- 13 C 3 ]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 (-80C) and thawing (37C), 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, O 2 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).
UR - http://www.scopus.com/inward/record.url?scp=85058922813&partnerID=8YFLogxK
U2 - 10.1371/journal.pone.0209201
DO - 10.1371/journal.pone.0209201
M3 - Article
C2 - 30576340
AN - SCOPUS:85058922813
SN - 1932-6203
VL - 13
JO - PloS one
JF - PloS one
IS - 12
M1 - e0209201
ER -