Background/Case Studies: P50 is the partial pressure of oxygen at which hemoglobin (Hgb) is 50% saturated with oxygen. It represents the affinity of Hgb for oxygen and is affected by factors such as pH, temperature, and the presence of 2,3 diphosphoglycerate (2,3DPG). 2,3-DPG is a molecule produced in red blood cells (RBCs) that binds to Hgb and decreases its affinity for oxygen. Both p50 and 2,3DPG are two important factors that influence the kinetics of oxygen unloading in the tissues. Storage of RBCs under the current normoxic storage condition can have significant effects on the relationship between p50 and 2,3DPG levels. One of the most significant changes is a decrease in the levels of 2,3-DPG over time. In the present study we measured the p50 and 2,3DPG in hypoxic/hypocapnic RBCs in phosphate -adenine-glucose-guanosine -saline-mannitol (PAGGSM) during refrigerated storage for 49 days.
Study
Design/Methods: Ten units of fresh whole blood were collected from healthy donors into CPD/PAGGSM blood collection set and processed into leukocyte reduced packed RBCs (LR-RBCs) in PAGGSM. The LR-RBCs were processed with a CE marked oxygen and carbon dioxide reduction system. The hypoxia and hypocapnic LR-RBCs were transferred into PVC DINCH hypoxic/hypocapnic storage bag for 49 days at 1-6˚C. The in vitro metrics of RBC quality, 2,3DPG, P50, ATP and hemolysis were measured before, after hypoxic/hypocapnic treatment and during storage. The concentration of 2,3DPG in the RBCs was measured with a microtiter plate enzymatic assay. The assay involved the stoichiometric conversion of 2,3DPG to glyceraldehyde-3-phosphate with measurement of the resultant change in the absorbance of the reduced nicotinamide adenine dinucleotide at 340nm in a spectrophotometer. The p50 was measured at 37˚C with the Hemox analyzer (TCS Scientific Corp, PA). Linear regression analysis was performed using the least square method.
Results/Findings: The data are presented as the mean±SD. Salient results showed a significant increase in 2,3DPG from 12.6±2.3µmol/gHb on Day 0 to 16.9±2.3 on Day 14, 13.9±3.7 on Day 21 and decreased to 0.8± 0.6 on Day 49 with corresponding changes in the p50 from 26.4±1.1mmHg on Day 0 to 31.3±1.0 on Day 14, 26.8±4.6 on Day 21 and 15.9±1.1 on Day 49 of storage. The Pearson correlation coefficient for the linear relationship between 2,3DPG and p50 was R=0.9623, p< 0.0001. The percent hemolysis levels in the RBCs on Day 42 and 49 of storage were 0.28±0.06 and 0.35±0.09%, respectively Conclusions: The present results show that when 2,3-DPG levels are high in hypoxic RBCs, the oxygen-hemoglobin dissociation curve shifts to the right, causing a higher p50 value, which will result in increased oxygen unloading. Improved oxygen delivery is important for medical conditions such as sickle cell disease that have high oxygen demands. These results also demonstrate that hypoxic RBCs in a non-DEHP (DINCH) bag system can be stored for 49 days.
Importance of research: The key purpose of blood transfusion is to improve oxygen delivery to tissues. The present study shows that hypoxic/hypocapnic treatment of RBCs in PAGGSM storage solution resulted in significant increase in 2,3DPG which correlated with the p50 an important metric for monitoring the efficacy of the RBCs in delivering oxygen to the tissues. In addition, the use of PVC DINCH hypoxic storage bag reduces the toxicities that may be associated with the use of PVC DEHP storage bag.
