Establishing a Rabbit Blood Bank: An Assessment of Storage Lesion

Introduction:

In trauma settings, hemorrhage and its related complications are one of the major causes of mortality. Blood, blood products, and fluid replacement are the mainstay of treatment in such cases. However with storage, the viability and shelf life of stored products start to decline, reducing their quality when administered. This, in turn, necessitates a validated and flexible model that can be tailored to transfusion needs without affecting product quality. In this study, we established a leporine blood banking model that meets established human requirements to support translational resuscitation studies. Crossmatching was performed to assess risk of hemolytic transfusion reaction. To assess the storage lesion, we analyzed hematology and hemostatic function out to 28 days of cold storage.

Methods:

Juvenile (4-8 months old) male New Zealand White rabbits from Envigo (strain Hsd:Hra NZW (SPF)) (n=11) were anesthetized with their vitals monitored to ensure adequate plane anesthesia. Whole blood and plasma-designated units were collected via direct cardiac exsanguination. Units were collected (n=11) in 100 milliliter (mL) animal blood bags containing 24% CPDA and stored at 4°C upright. Initially, samples (first n=3) were analyzed on days 0 (collection day), 3, 6, 9, 12, 16, and 21. Sampling was then adjusted to days 0, 4, 7, 10, 14, and 21 for the following n=2, and day 28 was also added for the remaining n=6. Complete blood counts (CBC), rotational thromboelastometry (ROTEM), and collagen agonist impedance aggregometry (IA) were performed to assess changes over storage duration. Before ROTEM analysis, units were recalcified with 1 M CaCl2 1:100 Vol:Vol to account for the increased citrated concentration. Statistical analysis was performed using one-way ANOVA with Dunnett's test for multiple comparisons.

Results:

Red blood cell count showed no significant change from baseline mean 3.905 M/uL to 4.157 M/uL over 28 days (mean difference: 0.2523 M/uL, p=0.8379). Hematocrit and hemoglobin levels, initially at mean 26.34% and mean 8.755 g/dL respectively, also remained stable (hematocrit: mean difference: 1.01%, p=0.7257; hemoglobin: mean difference: 0.267 g/dL, p=0.9172) with means of 26.34% and 8.488 g/dl on day 28. Platelet count, starting at mean of 228.2 K/μL, finalized at 161.0 M/ μL did not significantly change (mean difference: 67.2 M/μL, p=0.3402). Hemolysis, however, increased significantly from 0.2191% to 1.008% (mean difference: 0.7889, p=0.0333), though all measures remained below 2%. ROTEM INTEM clotting time (CT) and clot formation time (CFT) showed no significant changes from baselines of 188.7 sec to 201.3 sec and 68.0 sec to 179.4 sec respectively (CT: mean difference: 12.57 sec, p=0.3171; CFT: mean difference: 111.4 sec, p=0.2118). INTEM maximum clot firmness (MCF) decreased significantly from 56.86 sec to 25.60 sec (mean difference: 31.26 sec, p=0.001). ROTEM EXTEM CT and CFT exhibited no significant changes from baselines of 76.73 sec and 88.32 sec to 125.5 sec and 586.2 sec respectively (CT: mean difference: 48.77 sec, p=0.2367; CFT: mean difference: 497.9 sec, p=0.3677). EXTEM MCF, like INTEM MCF, decreased significantly from 61.05 sec to 23.10 sec (mean difference: 37.95 sec, p <0.001). IA AUC showed no significant change from baseline 15.73 to 8.99 (mean difference: 6.74, p=0.6488).

Conclusion:

Our study provides a successful methodology for establishing stable leporine whole blood unit manufacturing and storage. This model can be customized to different transfusion requirements and could be utilized to manufacture components. We plan on expanding our sample size with continuous monitoring for all parameters. Our group will also investigate other available blood collection methods (such as vascular access ports for more frequent unit collections) to refine our model, enhance safe and sustainable production and optimize collection strategies.

Neal:Haima Therapeutics: Other: Chief Medical Officer . Spinella:Haima: Consultancy; Hemanext: Consultancy; CSL Behring: Consultancy; Cerus: Consultancy.