Red Blood Cell Storage In Pediatric Transfer Bags Is Correlated With Increased Levels Of Hemolysis and Altered Osmotic Fragility

Red blood cells (RBCs) intended for pediatric transfusion are currently obtained from banked RBC units, from which selected volumes of RBC aliquots are transferred from to smaller pediatric bags. Certain conditions, such as repeated transfusions, limited inventory, and hospitals’ distance from blood banks, may require storage at 1-6 °C of pediatric bags for up to six weeks. In this study, we compared the recovery of RBCs stored in pediatric bags from a selected manufacturer (Pedi-Pak, Genesis BPS) to that of their parent unit. We demonstrated that temporary storage in pediatric bags can modify RBC quality, particularly in regard to end of storage hemolysis, and stress-induced oxidative hemolysis.

Packed leukoreduced RBCs from 10 healthy donors were stored under routine blood banking procedures (4 °C) for six weeks. At day 1, 20 mL of packed RBCs from each parent unit were transferred to a pediatric bag by sterile docking. RBC hemolytic propensity in each bag type was evaluated at selected time points (3, 7, 14, 21, 28, 35 and 42 days). Storage hemolysis was measured by supernatant cell-free hemoglobin using Drabkin’s assay. Osmotic stress was induced by incubating RBCs in hypotonic buffer containing glycerol. Oxidative hemolysis was induced by incubating RBCs with 2,2′-azobis (2-methylpropionamidine) dihydrochloride (AAPH). Mechanical stress was achieved by shaking RBC suspensions in the presence of one metal bead (3/32”) for 180 minutes on a plate shaker. Complete blood counts were obtained by a commercial blood counter (Hemavet). Statistical analysis was performed by repeated measures two-way ANOVA using a commercial software (GraphPad Prism 6).

Percent storage hemolysis was higher at all tested time points in the pediatric bags compared to their parent units. Significant differences were recorded on day 21 (0.24±0.09 % in Pedi-Pak versus 0.18±0.11 % in parent units, n=10, p=0.0484) and over. At the end of storage, hemolysis levels in pediatric bags were twice than that of their parent units (0.52±0.25 % in Pedi-Pak versus 0.25±0.14 % in parent units, n=10, p<0.0001). Similarly, RBCs stored in pediatric bags were more susceptible to AAPH-induced oxidative hemolysis (37.7±7.7 % in Pedi-Pak versus 31.7±7.4 % in parent units, Day 42, n=10, p<0.0001). Conversely, RBCs stored in pediatric bags demonstrated enhanced resistance to osmotic fragility at testing days 7-42 (21.2±6.9 % in Pedi-Pak versus 25.5±8.8 % in parent units, Day 42, n=10, p=0.0069). No significant differences between the two bag types were found in regard to mechanical fragility or RBC indices obtained by complete blood counts.

This study suggests that RBCs stored in pediatric bags from a selected manufacturer have altered in vitro recovery. It is not clear whether these changes affect transfusion outcomes, however, higher levels of free hemoglobin and increased susceptibility to oxidative stress may increase the risks of transfusion-related injuries, as free hemoglobin is redox active and capable of inducing hypertension via nitric oxide scavenging. The differences in hemolysis and osmotic fragility between the two RBC groups may stem from the type of plasticizers used in each bag (butyryl trihexyl citrate (BTHC) in Pedi-Pak versus bis(2-ethylhexyl) phthalate (DEHP) in parent units). Other factors may be related to differences in surface area to volume ratio of the different sized bags. Understanding the effects of the storage bag on RBC recovery can improve the quality and safety of RBC products used for pediatric transfusion.