Red blood cell phenotype fidelity following glycerol cryopreservation optimized for research purposes.
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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).
Microscopy, Electron, Scanning
Published Version (Please cite this version)10.1371/journal.pone.0209201
Publication InfoMcMahon, Timothy; Palmer, Gregory; Dosier, Laura; Rogers, Stephen C; Zhu, Hongmei; Timm, David; ... Doctor, Allan (2018). Red blood cell phenotype fidelity following glycerol cryopreservation optimized for research purposes. PloS one, 13(12). pp. e0209201. 10.1371/journal.pone.0209201. Retrieved from https://hdl.handle.net/10161/19098.
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Assistant Professor of Pediatrics
My research focuses on abnormal breathing in sleep including central sleep apnea, obstructive sleep apnea, snoring, and abnormal sleep in children with complex medical conditions.In my practice, I focus on pediatric diseases of the lungs and sleep. I think that it is important to collaborate with primary care providers and families to create individualized care plans for each of my patients.
Professor of Medicine
The McMahon Lab at Duke University and Durham VA Medical Center is investigating novel roles of the red blood cell (RBC) in the circulation. The regulated release of the vasodilator SNO (a form of NO, nitric oxide) by RBCs within the respiratory cycle in mammals optimizes nutrient delivery at multiple levels, especially in the lung (gas exchange) and the peripheral microcirculation (O2 transport to tisses). Deficiency of RBC SNO bioactivity (as in human RBCs banked for transfusion), f
Associate Professor of Radiation Oncology
Greg Palmer obtained his B.S. in Biomedical Engineering from Marquette University in 2000, after which he obtained his Ph.D. in BME from the University of Wisconsin, Madison. He is currently an Associate Professor in the Department of Radiation Oncology, Cancer Biology Division at Duke University Medical Center. His primary research focus has been identifying and exploiting the changes in absorption, scattering, and fluorescence properties of tissue associated with cancer progression and therape
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