(G-266) Effect of Lyophilization on Cryoprecipitated Plasma

Introduction:: The burden of traumatic injury on civilian and military mortality cannot be understated. In the United States (US), traumatic injury is the most common cause of life-years lost before 75 years of age. In the US, an estimated 30,000 deaths after injury from traumatic hemorrhage per year are preventable, meaning these patients could have been saved with more timely and more effective care. The vast majority (60-90%) of preventable deaths occur in the pre-hospital phase of hemostatic resuscitation, i.e., during emergency response and transport. Effective resuscitation therapies that can be rapidly administered are therefore a critical unmet need. Prehospital transfusion of hemostatic adjuncts may therefore improve outcomes for traumatic hemorrhage. Logistical barriers, including limited room temperature shelf-life, present challenges of implementation. Lyophilization of these therapies provides a potential solution, as these products can be rapidly reconstituted and administered, and due to their shelf stability have reduced maintenance burden on blood bank inventory, allowing more centers to stock them and potential availability for prehospital treatment. This study aims to investigate the effect of lyophilization on the hemostatic protein content and functional quality of cryoprecipitated plasma (cryo), a concentrated plasma hemostatic adjunct. We hypothesize that the biochemical and hemostatic properties among cryoprecipitate and lyophilized cryoprecipitate will be similar as measured by in vitro assays.

Materials and Methods:: Each research-grade, pooled Cryo unit was manufactured and provided, in kind, by Vascular Solutions LLC (wholly owned subsidiary of Teleflex, Inc.). Briefly, five individual Fresh Frozen Plasma (FFP) CPD units of the same ABO blood type were cold precipitated (4°C overnight) and the resulting cryoprecipitate from each unit was collected by centrifugation (4000 x g). The five individual cryoprecipitate were then pooled. The resulting pooled cryo units were then frozen and stored frozen (cryo, n=6 units) or lyophilized (lyo-cryo, n=8 units) into a dry powder form. Cryo was thawed and lyo-cryo was reconstituted with 110mL sterile water immediately prior to testing and assayed for biochemistry, plasma protein content, and coagulation function. Biochemical profiles of cryo and lyo-cryo included pH, sodium (Na+), potassium (K+), and glucose (Glu). Hemostatic panels assessed prothrombin time (PT)/activated partial thromboplastin time (aPTT), fibrinogen, Factor VIII, von Willebrand Factor (VWF), Protein C, Protein S, and Antithrombin III (ATIII). The biochemical assays were measured using an iSTAT Handheld Analyzer, and all other assays were run using a Stago Compact Max. All assays were performed per manufacturer instructions. Statistical analysis was performed via unpaired t-tests. Results are reported as mean +/- standard deviation.

Results, Conclusions, and Discussions:: There were more resulting biochemical differences between cryo and lyo-cryo than hemostatic differences. Lyo-cryo had increased concentrations of Na+, K+, and Glu. However, cryo had increased pH (Figure1). In our hemostatic comparison, lyo-cryo had a slightly increased PT, aPTT, and fibrinogen concentration. There were limited differences in ATIII, Protein S, Protein C, VWF, or Factor VIII activity (Figure2). Several units of cryo (n=4) yielded VWF results outside calculable range, and therefore are not included in this analysis.
Development of blood products capable of overcoming the logistical hurdles in prehospital resuscitation is an essential step towards improved clinical outcomes. The possibility of using lyophilized products opens the door to more accessible treatment options that are not commonly used in current prehospital protocols. Prior to integrating lyophilized products into a clinical setting, it is important to understand the resulting differential effects caused by this manufacturing method. Lyo-cryo demonstrated some slight differences in both biochemical and hemostatic characterization in comparison to cryo. While increases in PT and aPTT in lyo-cryo products were statistically significant, interestingly despite increased fibrinogen concentration, they’re slight and therefore may not be clinically meaningful. Given that there are comparable amounts of coagulation proteins between cryo and lyo-cryo, this slight reduction in function suggests possible changes in protein efficacy induced by lyophilization. However, the ability to increase the availability of hemostatic adjuncts for prehospital resuscitation and rapidly deliver lyophilized products likely outweighs slight changes (12% PT increase and 24% PTT increase) in function. In future, characterizing function of these products can be expanded to assessment via rotational thromboelastometry, as PT and aPTT are limited functional assays.
Lyophilized blood products are a critical innovation for the treatment of severe hemorrhage, as they enable rapid hemostatic resuscitation, can be implemented in austere environments, and leverage benefits of improved availability. Future work will characterize the in vivo performance of lyo-cryo in a simulated prehospital resuscitation animal model. We hypothesize the very slight changes in plasma protein function are outweighed considering lyo-cryo can be given more rapidly (estimated 30 minutes) than cryo, as the former can be reconstituted immediately, and the latter requires thawing.


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