Poster B1 - Examining Metabolic Function, Lysis, and Morphology in Red Blood Cells Stored in Different Preservation Solutions Over One Month

Friday, October 25, 2024

10:00 AM – 11:00 AM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

HN

Heather Neisser

Undergraduate Research Assistant
University of Maryland, College Park
College Park, Maryland, United States

Co-Author(s)

Gurneet Sangha, PhD

Postdoctoral Researcher
University of Maryland
Hyattsville, Maryland, United States

Primary Investigator(s)

Alisa Clyne, PhD (she/her/hers)

Professor
University of Maryland, College Park
College Park, Maryland, United States

Introduction: Blood transfusions are a vital procedure in medicine. For patients to receive effective transfusions in an emergency, hospitals need to have a blood bank to store blood. However, a grand challenge in transfusion medicine is blood storage that preserves red blood cell (RBC) viability and functionality. As RBCs are stored in blood banks, they become metabolically dysfunctional, prone to hemolysis, and spiculated rather than biconcave in their shape. These issues decrease RBC quality, which can impair circulatory function. For example, longer RBC storage time can increase the risk of poor oxygen delivery [2] and pneumonia [3]. Furthermore, healthy RBCs circulate for up to 120 days, while up to 30% of transfused RBCs are removed from circulation within 24 hours after the transfusion and the remaining transfused RBCs only circulate for up to 60 days [1]. Therefore, the objective of this project was to understand how different RBC storage solutions impact RBC metabolic function, lysis, and morphology to improve RBC storage methods and preserve RBC function.

Materials and

Methods: Whole blood was freshly collected from three human subjects (IRB protocol: 1818450-4). RBCs were isolated by centrifugation and washing with Hanks Buffered Saline Solution (HBSS) three times. We then stored 6% hematocrit at 4°C with continuous shaking for 1 month in four different solutions: citrate-phosphate-dextrose-adenine (CPDA); Alsever’s solution (AS); saline-adenine-glucose-mannitol (SAGM), and Endothelial Growth Media-2 (EGM2). CPDA, AS, and SAGM were chosen as they are used for RBC storage. EGM2 was used because it is a standard media to grow endothelial cells, which are in constant proximity to RBCs in circulation.

We quantified RBC ATP concentration, hemolysis, and morphology at baseline, as well as week 1, 2, 3, and 4 to observe the effects of the different storage solutions. RBC metabolism was quantified via ATP concentration, which was measured using the Cell Titer Glo Cell Viability Assay. In this assay, the enzyme luciferase reacts with luciferin to produce light that is proportional to the RBC ATP concentration. Hemolysis was quantified by first separating the RBCs from the storage solutions using centrifugation. We then measured the storage solution absorbance at 541 nm in the Spark Multimode Microplate reader to quantify hemoglobin released by lysed RBCs. Finally, we imaged 1% hematocrit on a glass bottom dish using brightfield microscopy and quantified RBC shape by manually counting cells as either biconcave, spherical, or spiculated using ImageJ. Two-way ANOVA with a Tukey multiple comparison test was used to determine statistical significance among groups using Prism 10.

Results, Conclusions, and Discussions: We used ATP concentration to assess RBC metabolic function (Figure 1A). Between baseline and week 4, ATP decreased by about 50X in RBCs stored in CPDA, 5X in RBCs stored in AS, and 4X in RBCs stored in EGM2 (p < 0.05). ATP increased 1.1X from baseline in RBCs stored in SAGM (p < 0.05), suggesting that SAGM preserved metabolic function. RBCs stored in SAGM contained significantly more ATP than RBCs stored in CPDA, AS, or EGM2 (p < 0.05). SAGM may increase ATP since it contains glucose and mannitol, as both support RBC metabolism [4].

We used hemoglobin in the storage solution to assess RBC lysis (Figure 1B). From baseline to week 4, hemoglobin absorbance increased by ~20X for RBCs in CPDA , 10X for RBCs in AS, 30X for RBCs in SAGM, and 35X for RBCs in EGM2. For weeks 1 and 2, hemoglobin quantity increased linearly. After week 2, hemoglobin increased exponentially. Only RBCs stored in CPDA experienced statistically significant hemolysis between weeks 0 and weeks 3 and 4 (p < 0.05). RBCs in CPDA had a sharper exponential increase after week 2 than the other solutions, causing CPDA to have higher quantities of lysis.

We visualized the RBCs to assess their morphology (Figure 1C). Over 70% of the cells in each solution showed a spiculated morphology, characterized by thorny projections from the cell. 97% of RBCs displayed a spherical morphology at baseline, and 93% of RBCs in CPDA displayed a spherical morphology at week 4. CPDA also had fewer RBCs at week 4, likely due to excessive hemolysis. These results suggest that the storage solutions lacked the biochemical signals necessary to maintain viable RBC with biconcave disk morphology.

SAGM appeared to be the best storage solution for preserving metabolic function and minimizing hemolysis. However, all storage solutions failed to enable RBCs to maintain a biconcave disk morphology. These results suggest that current RBC storage solutions can be improved. Future work will focus on developing a solution to enhance RBC storage by preserving RBC structure and function. This will improve the quality of blood used in transfusions and improve patient outcomes.

Acknowledgements (Optional): Funding was provided to A.M.C by National Institutes of Health grant R01HL140239 and R01HL165193 and National Institutes of Health NIGMS MARC T34GM149472 to H.V.N .

References:
1. Tuo, Wei-Wei, et al. "How cell number and cellular properties of blood-banked red blood cells of different cell ages decline during storage." Plos one 9.8 (2014): e105692.
2. García-Roa, María, et al. "Red blood cell storage time and transfusion: current practice, concerns and future perspectives." Blood Transfusion 15.3 (2017): 222.
3. Spinella, Philip C., et al. "Properties of stored RBCs: Understanding immune and vascular reactivity." Transfusion 51.4 (2011): 894.
4. Tozzi, Marco, et al. "The P2X7 receptor and pannexin-1 are involved in glucose-induced autocrine regulation in β-cells." Scientific Reports 8.1 (2018): 8926.