Extended supercooled storage of red blood cells

Abstract

Red blood cell (RBC) transfusions facilitate many life-saving acute and chronic interventions. Transfusions are enabled through the gold-standard hypothermic storage of RBCs. Today, the demand for RBC units is unfulfilled, partially due to the limited storage time, 6 weeks, in hypothermic storage. This time limit stems from high metabolism-driven storage lesions at +1-6 °C. A recent and promising alternative to hypothermic storage is the supercooled storage of RBCs at subzero temperatures, pioneered by our group. Here, we report on long-term supercooled storage of human RBCs at physiological hematocrit levels for up to 23 weeks. Specifically, we assess hypothermic RBC additive solutions for their ability to sustain supercooled storage. We find that a commercially formulated next-generation solution (Erythro-Sol 5) enables the best storage performance and can form the basis for further improvements to supercooled storage. Our analyses indicate that oxidative stress is a prominent time- and temperature-dependent injury during supercooled storage. Thus, we report on improved supercooled storage of RBCs at -5 °C by supplementing Erythro-Sol 5 with the exogenous antioxidants, resveratrol, serotonin, melatonin, and Trolox. Overall, this study shows the long-term preservation potential of supercooled storage of RBCs and establishes a foundation for further improvement toward clinical translation.

Fig. 1. Schematic of experimental steps for the supercooled storage of red blood cells (RBCs).

a Additive solutions were prepared fresh in-house in sterile conditions. b Additive solutions were characterized for pH and osmolarity. c A single experiment was performed from a pool of packed RBCs from 3 donors. d Pools were washed and resuspended in their respective additive solutions. e Samples were aliquoted as 1 mL units at ∼ 50% hematocrit levels and sealed with a 0.5 mL mineral oil layer to prevent stochastic freezing at subzero temperatures. Samples were stored in coolers (Engel MHD-13, Engel, FL, USA) set at designated subzero temperatures. The figure was prepared in Biorender.com.

Fig. 2. Comparison of E-Sol 5, AS-7, CPDA-1, and SAGM for hemolysis, hemolysis-corrected lactate, TBARS, MCHC, and MCH at +4 °C and −5 °C after 6 and 10 weeks of storage.

a The samples were stored for 6 weeks, and hemolysis was measured. b The samples were stored for 10 weeks, and hemolysis was measured. c Data shows lactate level corrected for hemolysis after 6 weeks. Measured total lactate level was normalized by fraction of unlysed RBCs. d Data shows TBARS levels after 6 weeks. e Data shows MCHC levels at both temperatures after 6 weeks. f Data shows MCH levels at both temperatures after 6 weeks. Colored and dashed lines represent the mean day 1 levels following 3x washing, overnight storage at +4 °C, and final 1x washing in the respective solutions. Data represent mean ± standard deviation from 3 biological replicates (N = 3) and three technical replicates (n = 3, n = 1-3 for lactate, n = 2 for TBARS) for each biological replicate. Each biological replicate was from a pool of 3 donor samples. A two-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was performed to evaluate significant differences between conditions: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Comparisons were shown across the two temperatures for each solution and for E-Sol 5 against the other solutions. TBARS Thiobarbituric Acid Reactive Substances. MCHC mean cell hemoglobin concentration. MCH mean corpuscular hemoglobin. See Supplementary Fig. 3 for comparisons across the solutions at each temperature.

Fig. 3. Comparison of storage performance in E-Sol 5 at 3 different temperatures after 3, 7, and 10 weeks of storage.

a Hemolysis increased with increasing time of storage and decreasing temperature. b Data shows lactate levels normalized by fraction of unlysed red blood cells after 3, 7, and 10 weeks. Measured total lactate level was normalized by percent of unlysed RBCs. c Data shows TBARS levels after 3, 7, and 10 weeks. d Data shows TAS levels after 3, 7, and 10 weeks. Dashed lines represent the mean day 1 levels following 3x washing, overnight storage at +4 °C, and final 1x washing in E-Sol 5. Data represent mean ± standard deviation from 3 biological replicates (N = 3) and 1-3 technical replicates for each biological replicate (n = 3 for hemolysis, n = 1-3 for lactate, n = 2 for TBARS and TAS). Each biological replicate was from a pool of 3 donor samples. A two-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was performed to evaluate significant differences between conditions: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Comparisons were shown across the three temperatures at each time point. See Supplementary Fig. 6 for comparisons across the time points at each temperature. TBARS Thiobarbituric Acid Reactive Substances. TAC Total Antioxidant Capacity.

Fig. 4. Long-term comparison of storage performance in E-Sol 5 at +4 °C and −5 °C after 3, 21, 22, and 23 weeks of storage.

Samples were assessed for their supernatant colors where redness is proportional to hemolysis. At week 21 and later time points, the hemolysis for the samples stored at +4 °C was significantly higher than the hemolysis for the samples stored at −5 °C. Black dashed line represents the mean day 1 level following 3x washing, overnight storage at +4 °C, and final 1x washing in E-Sol 5. Data represent mean ± standard deviation from 2 biological replicates (N = 2) and 3 technical replicates (n = 3) for each biological replicate. Each biological replicate was from a pool of 3 donor samples. A two-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was performed to evaluate significant differences between conditions: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Comparisons were shown across the three temperatures at each time point. See Supplementary Fig. 7 for comparisons across the time points at each temperature.

Fig. 5. Comparison of antihemolytic effects of 4 antioxidants during supercooled (−5 °C) storage after 6 and 10 weeks of storage.

a The samples were stored for 6 weeks at −5 °C. Serotonin, melatonin, and Trolox decreased the hemolysis below 1%. b The samples were stored for 10 weeks at −5 °C. All antioxidants decreased the hemolysis relative to the antioxidant-unsupplemented case at −5 °C (control, N/A). Data represent mean ± standard deviation from 3 biological replicates (N = 3) and 2 or 3 technical replicates (n = 2-3) for each biological replicate. Each biological replicate was from a pool of 3 donor samples. A one-way analysis of variance (ANOVA) followed by Dunnett’s post hoc test was performed to evaluate significant differences between conditions: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. For clarity, comparisons were made for the control (N/A) against the antioxidant cases at −5  °C.