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

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).

Quantitative comparative analysis of human erythrocyte surface proteins between individuals from two genetically distinct populations

“…To facilitate simultaneous immunostaining for temporal flow cytometry analysis in supplementary fig. 6, RBC were stored in a high glycerol solution using a method adapted from a previously published protocol 43 . Briefly, RBC were isolated from packed spun whole blood as described above.…”

The roles of reactive oxygen species and antioxidants in cryopreservation

“…During cryopreservation, NOS activation or NO • production was observed in cryopreserved heart valves [118] and sperm [30,116,119]. While NO • itself has not been found to be significantly increased by freeze-thaw stress in RBC, the product of nitric oxide nitrosylation, S-nitrosohemoglobin was found to be increased by freeze-thaw stress [120]. At high levels of NO • , sperm functions can be antagonized [116,121–123].…”

Red Blood Cell Deformability, Vasoactive Mediators, and Adhesion

“…The mechanistic study of RBC function in acute and subacute diseases is logistically and technically challenging. We recently described an improved technique for the cryopreservation of human RBCs, resulting in diminished RBC lysis after a cycle of controlled freezing in glycerol followed by thawing and deglycerolization (Rogers et al, 2018), as compared to standard (clinical) RBC cryopreservation. The RBC phenotype with this approach matched closely that of aliquots from the same RBC samples studied before freezing and thawing.…”

“…Additionally, we advise that in every experiment, the cell-free hemoglobin concentration be measured as an index of RBC lysis. We typically report the extent of hemolysis, and calculate to what extent the measured ATP EC values could theoretically be secondary to the degree of lysis measured (taking into account the initial intra-RBC (ATP) and the hematocrit (Hct)] (Zhu et al, 2011; Rogers et al, 2018). For example, if RBC [ATP] is 100 μM, experiment Hct is 1%, lysis of the RBCs is 5%, and the supernatant [ATP] is 100 nM, then ∼50% of the “ATP EC ” could be from lysis.…”