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

Stephen C Rogers,Joseph Herbert,Travis Nemkov,Parhom Towfighi,Melanie Ernst,Asa Cook,Reid Doctor,Gabi Hanna,Jacqueline Atallah,Xue Lin,Mark Terng,Hengtao Zhang,Ahmed Said,Hongmei Zhu,Angelo D’Alessandro,Gregory M Palmer,Jaya Prakash,Matthew S Joens,Julie A Reisz,Allan Doctor,Timothy J Mcmahon,James A.j Fitzpatrick ,David D Timm ,Laura Beth Mann Dosier

doi:10.1371/journal.pone.0209201

Abstract

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).

Highlights

Red blood cell (RBC) cryopreservation was described almost seventy years ago[1] and subsequently explored as means to prolong RBC storage prior to transfusion[2, 3]
The effect of temperature on this process was significant, with warmer glycerol resulting in less RBC lysis (Fig 1B)
We found the order of component addition to strongly influence hemolysis; adding buffer (PBS 300μl) and 300μl buffer/glycerol stock (60% v/v glycerol in starting buffer solution) to the tube first, followed by RBCs (300μl), resulted in lowest lysis (Fig 1C)

Summary

Introduction

Red blood cell (RBC) cryopreservation was described almost seventy years ago[1] and subsequently explored as means to prolong RBC storage prior to transfusion[2, 3]. Between -10 to -60 ̊C) to which cells are exposed twice–during cooling and warming[5]. This intermediate temperature zone may damage cells due to imbalanced transmembrane water transport, a temperature-dependent process[6]. If water exits too quickly, injury may result from cell shrinkage and related changes in intracellular solute concentrations (“solution effects” injury) [8, 9]. Between these two ends of the spectrum, optimal cooling and warming rates exist that minimize freezing injury[10, 11]

Methods

Results

Conclusion