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 AJ

Hanna, Gabi

Lin, Xue

Reisz, Julie A

Nemkov, Travis

D'Alessandro, Angelo

Doctor, Allan

2019-07-12T20:17:32Z

2019-07-12T20:17:32Z

2018-01

2019-07-12T20:17:31Z

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).

PONE-D-18-25310

1932-6203

1932-6203

https://hdl.handle.net/10161/19098

eng

Public Library of Science (PLoS)

PloS one

10.1371/journal.pone.0209201

Erythrocytes

Animals

Humans

Mice

Mice, Nude

Glycerol

Hemoglobins

Cryoprotective Agents

Microscopy, Electron, Scanning

Erythrocyte Indices

Osmotic Fragility

Cryopreservation

Blood Preservation

Erythrocyte Transfusion

Transplantation, Heterologous

Cell Adhesion

Erythrocyte Deformability

Phenotype

Metabolome

Healthy Volunteers

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

Journal article

McMahon, Timothy J|0000-0002-3404-3223

Palmer, Gregory M|0000-0003-2955-8297

e0209201

12

School of Medicine

Duke

Medicine, Pulmonary, Allergy, and Critical Care Medicine

Medicine

Clinical Science Departments

Pediatrics, Pulmonary and Sleep Medicine

Pediatrics

Duke Cancer Institute

Institutes and Centers

Radiation Oncology

Published

13