The effects of cryopreservation on red blood cell microvesiculation, phosphatidylserine externalization, and CD47 expression.
BACKGROUND
Since the 1950s, cryopreservation has been used in transfusion medicine for long-term storage of phenotypically rare red blood cells (RBCs). Recent reports have identified phosphatidylserine (PS) exposure, loss of CD47 expression, and membrane microvesiculation as important indicators of RBC storage lesion and in vivo survival. The purpose of this study was to assess the effects of RBC cryopreservation and prefreeze storage length on these novel markers of membrane injury and to correlate them to traditional RBC quality indicators.


METHODS
Leukoreduced RBC units were collected in citrate-phosphate-dextrose (CPD)-saline-adenine-glucose-mannitol (SAGM), hypothermically stored (1-6 degrees C) for 2 to 3 days or 13 to 14 days after collection, and then cryopreserved in 40 percent (wt/vol) glycerol. In vitro RBC quality was assessed before freeze, after thaw, and 24 hours after thaw by evaluating RBC recovery, hemolysis, sterility, residual glycerol, adenosine triphosphate, extracellular potassium, RBC indices, and morphology. RBC membrane microvesiculation, PS externalization, and CD47 expression changes were evaluated using flow cytometry.


RESULTS
Leukoreduced CPD-SAGM RBCs showed acceptable in vitro quality after deglycerolization, according to conventional assays. Cryopreservation alone did not induce significant changes in PS exposure, CD47 expression, and membrane microvesiculation. Prolonged prefreeze storage, however, resulted in a significant increase in RBC PS exposure and microvesiculation after 24 hours of postthaw hypothermic storage (1-6 degrees C). No significant changes in CD47 expression were detected.


CONCLUSIONS
High-glycerol cryopreservation does not induce microvesiculation, PS exposure, and loss of CD47 expression in RBC membranes. Since prolonged prefreeze storage can result in RBC membrane injury during the postdeglycerolization storage period, more defined criteria for this variable should be adopted.
Holovati
Jelena L
JL
Research and Development, Canadian Blood Services, Edmonton, Alberta, Canada.
Wong
Kenneth A
KA
Webster
Jarret M
JM
Acker
Jason P
JP
D015415:Biomarkers / Q000378:metabolism; D001793:Blood Preservation / Q000379:methods*; D051928:CD47 Antigen / Q000378:metabolism*; D015925:Cryopreservation; D003451:Cryoprotective Agents; D022162:Cytoplasmic Vesicles / Q000378:metabolism; D004912:Erythrocytes / Q000166:cytology* / Q000378:metabolism; D005434:Flow Cytometry; D005990:Glycerol; D006461:Hemolysis; D006801:Humans; D010718:Phosphatidylserines / Q000378:metabolism*
D015415:Biomarkers; D051928:CD47 Antigen; C497894:CD47 protein, human; D003451:Cryoprotective Agents; D010718:Phosphatidylserines; D005990:Glycerol