Cryopreserved Erythrocytes Processed for Blood Transfusion

Preservation of blood for transfusion purposes has represented a lifesaving advance in clinical practice over many years, but although cryostorage has attracted a great deal of attention clinically, little is known about the biochemistry and physiology of cryostored erythrocyte concentrates.

Specialists at the University of Tuscia (Viterbo, Italy) investigated cryostorage of RBCs through monitoring of cell processing steps, from fresh blood, to glycerolization, thawing, and deglycerolization, followed by washing. Whole blood was collected from 10 healthy donor volunteers into 63 mL of citrate phosphate dextrose (CPD) anticoagulant, and leukodepleted. All glycerolized RBC units were frozen and stored at −80 ± 10 °C in a mechanical freezer for at least 12 months.

The erythrocytes were monitored through repeated assays of standard parameters of mean cell volume (MCV), red cell distribution width-standard deviation (RDW-SD), the mean cell hemoglobin concentration (MCHC), and the hematocrit (Hct). These were determined with a CA 530-Oden hematology analyzer. Scanning electron microscopy was also performed. Other standard biological indicators, such as hemoglobin content, pH values both internal and in the supernatant, lactate levels, osmotic fragility and hemolysis, were also calculated.

Cell processing for cryostorage resulted in increased RBC volumes. Mean cell volume (MCV) increased significantly upon glycerolization, from 89.4 ± 4.5 fL of fresh RBCs to 126.04 ± 2.3 fL of glycerolized RBCs, while it remained constant at 129.6 ± 3.3 fL upon cryostorage even after thawing. Deglycerolization and washing restored lower MCV values 93.7 ± 5.9 fL, though they were still higher than controls. Shape alterations caused an increase in osmotic fragility and permeability to ions. A significant pH drop was observed which could not to be attributed to a higher metabolic rate, since the levels of lactate did not show substantial fluctuation during the cell processing steps tested in this study. The membrane anomalies reported are likely related to the hemolysis observed which preferentially affected the densest and oldest cell sub-populations, as confirmed by means of discontinuous density gradients. The CA 530-Oden hematology analyzer used in the study is manufactured by Medonic (Stockholm, Sweden).

The authors concluded that their results indicate that cryostorage itself in presence of glycerol does not significantly affect RBCs. Most of the alterations observed were related to cell processing and, in particular, to the increase of cytosolic glycerol as a consequence of the glycerolization step. Further studies might profitably investigate replacing glycerol with nonpenetrating cryoprotectants such as polyvinylpyrrolidone or polyethylene glycol, or the less toxic sucrose and trehalose.The study was published online on March 14, 2012, in the journal Blood Cells, Molecules, and Diseases.

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University of Tuscia
Medonic