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Red blood cell phenotype fidelity following glycerol cryopreservation optimized for research purposes

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 physiolo...

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Autores principales: Rogers, Stephen C., Dosier, Laura B., McMahon, Timothy J., Zhu, Hongmei, Timm, David, Zhang, Hengtao, Herbert, Joseph, Atallah, Jacqueline, Palmer, Gregory M., Cook, Asa, Ernst, Melanie, Prakash, Jaya, Terng, Mark, Towfighi, Parhom, Doctor, Reid, Said, Ahmed, Joens, Matthew S., Fitzpatrick, James A. J., Hanna, Gabi, Lin, Xue, Reisz, Julie A., Nemkov, Travis, D’Alessandro, Angelo, Doctor, Allan
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6303082/
https://www.ncbi.nlm.nih.gov/pubmed/30576340
http://dx.doi.org/10.1371/journal.pone.0209201
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author Rogers, Stephen C.
Dosier, Laura B.
McMahon, Timothy J.
Zhu, Hongmei
Timm, David
Zhang, Hengtao
Herbert, Joseph
Atallah, Jacqueline
Palmer, Gregory M.
Cook, Asa
Ernst, Melanie
Prakash, Jaya
Terng, Mark
Towfighi, Parhom
Doctor, Reid
Said, Ahmed
Joens, Matthew S.
Fitzpatrick, James A. J.
Hanna, Gabi
Lin, Xue
Reisz, Julie A.
Nemkov, Travis
D’Alessandro, Angelo
Doctor, Allan
author_facet Rogers, Stephen C.
Dosier, Laura B.
McMahon, Timothy J.
Zhu, Hongmei
Timm, David
Zhang, Hengtao
Herbert, Joseph
Atallah, Jacqueline
Palmer, Gregory M.
Cook, Asa
Ernst, Melanie
Prakash, Jaya
Terng, Mark
Towfighi, Parhom
Doctor, Reid
Said, Ahmed
Joens, Matthew S.
Fitzpatrick, James A. J.
Hanna, Gabi
Lin, Xue
Reisz, Julie A.
Nemkov, Travis
D’Alessandro, Angelo
Doctor, Allan
author_sort Rogers, Stephen C.
collection PubMed
description 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 (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 (-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, 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).
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spelling pubmed-63030822019-01-08 Red blood cell phenotype fidelity following glycerol cryopreservation optimized for research purposes Rogers, Stephen C. Dosier, Laura B. McMahon, Timothy J. Zhu, Hongmei Timm, David Zhang, Hengtao Herbert, Joseph Atallah, Jacqueline Palmer, Gregory M. Cook, Asa Ernst, Melanie Prakash, Jaya Terng, Mark Towfighi, Parhom Doctor, Reid Said, Ahmed Joens, Matthew S. Fitzpatrick, James A. J. Hanna, Gabi Lin, Xue Reisz, Julie A. Nemkov, Travis D’Alessandro, Angelo Doctor, Allan PLoS One Research 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 (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 (-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, 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). Public Library of Science 2018-12-21 /pmc/articles/PMC6303082/ /pubmed/30576340 http://dx.doi.org/10.1371/journal.pone.0209201 Text en © 2018 Rogers et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Rogers, Stephen C.
Dosier, Laura B.
McMahon, Timothy J.
Zhu, Hongmei
Timm, David
Zhang, Hengtao
Herbert, Joseph
Atallah, Jacqueline
Palmer, Gregory M.
Cook, Asa
Ernst, Melanie
Prakash, Jaya
Terng, Mark
Towfighi, Parhom
Doctor, Reid
Said, Ahmed
Joens, Matthew S.
Fitzpatrick, James A. J.
Hanna, Gabi
Lin, Xue
Reisz, Julie A.
Nemkov, Travis
D’Alessandro, Angelo
Doctor, Allan
Red blood cell phenotype fidelity following glycerol cryopreservation optimized for research purposes
title Red blood cell phenotype fidelity following glycerol cryopreservation optimized for research purposes
title_full Red blood cell phenotype fidelity following glycerol cryopreservation optimized for research purposes
title_fullStr Red blood cell phenotype fidelity following glycerol cryopreservation optimized for research purposes
title_full_unstemmed Red blood cell phenotype fidelity following glycerol cryopreservation optimized for research purposes
title_short Red blood cell phenotype fidelity following glycerol cryopreservation optimized for research purposes
title_sort red blood cell phenotype fidelity following glycerol cryopreservation optimized for research purposes
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6303082/
https://www.ncbi.nlm.nih.gov/pubmed/30576340
http://dx.doi.org/10.1371/journal.pone.0209201
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