Use of supernatant refractive index and supernatant hemoglobin concentration to assess residual glycerol concentration in cryopreserved red blood cells.

BACKGROUND: Red blood cells (RBCs) cryopreserved in glycerol must be deglycerolized prior to transfusion. The adequacy of glycerol removal is commonly assessed by measurement of the refractive index (RI) of the supernatant fluid. However, the presence of free hemoglobin in the supernatant falsely increases the RI and may lead to discard of units that have an acceptable residual glycerol concentration. METHODS: We performed an analysis of the diagnostic accuracy of 3 methods for residual glycerol measurement - refractometry, osmometry, and a glycerol assay kit. Residual glycerol measurement using these methods was performed on 12 deglycerolized, citrate-phosphate-dextrose (CPD)/saline-adenine-glucose-mannitol (SAGM) leukoreduced RBCs. A calculation that estimates the glycerol concentration based on the refractive index and supernatant hemoglobin concentration was developed and ensures that units with an elevated RI due to the presence of hemoglobin are not discarded if their residual glycerol concentration was <1.0% (w/v). RESULTS: Osmometry was an accurate method for estimating residual glycerol concentration. Refractometry overestimated the residual glycerol concentration due to the interference from hemoglobin. However, when supernatant hemoglobin values were measured and used in the calculation for glycerol concentration, refractometry accurately estimated the residual glycerol concentration. CONCLUSION: The residual glycerol concentration of cryopreserved, deglycerolized CPD/SAGM RBCs can be accurately estimated using the supernatant refractive index and an equation that accounts for the supernatant hemoglobin concentration.

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. STUDY DESIGN AND 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. CONCLUSION: 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.