Pathogen Reduced Cryoprecipitate and Fibrinogen Concentrate Have Delayed Resuscitation of Simulated Dilutional Coagulopathy in a Microfluidic Model of Bleeding When Compared to Cryoprecipitate
Authors: Thomas Kimberly A. , Spinella Philip C. , Shea Susan M. 
Published: November 2021

Abstract
Impaired fibrinogen function, one component of trauma-associated coagulopathy, is highly associated with increased mortality in patients with severe traumatic bleeding. Fibrinogen replacement is crucial for improving outcomes in bleeding patients. The two most common clinically-used hemostatic adjuncts for fibrinogen supplementation are fibrinogen concentrates (FibCon) and cryoprecipitate (Cryo), yet which of these products provides better hemostatic resuscitation remains controversial. Where FibCon is predominantly a source of fibrinogen, Cryo contains additional factors which may enhance hemostatic efficacy, such as FVIII and FXIII (inherent to clot strength and fibrin crosslinking) and von Willebrand factor (VWF, important for platelet adhesion and aggregation). Cryo poses more logistical challenges than FibCon, as Cryo is a frozen product that requires thawing (20 minutes) prior to use, and has a shelf life of 4-6 hours post thaw. FibCon is lyophilized, and can be reconstituted and used within 10 minutes, but with a cost roughly 3 times that of Cryo, use of FibCon can be cost-prohibitive. Ultimately, in the setting of hemostatic resuscitation, every minute matters and each minute delay in blood product transfusion is associated with a 5% increase in mortality. Moreover, Cryo is associated with an increased risk of transfusion transmitted infection (TTI).Thus, there is a need for an immediately available and safe fibrinogen source for use in hemostatic resuscitation.
Pathogen reduction (PR) of blood products renders any nucleic acid-containing source replication incompetent via crosslinking using psoralens and ultraviolet light. PR was recently adapted for use with cryoprecipitate, yielding a novel hemostatic adjunct - pathogen reduced cryoprecipitated fibrinogen complex, or PR-Cryo FC. We have previously shown PR-Cryo FC stored out to 10 days performs similarly to Cryo and FibCon in current standard assays used to assess hemostatic function. However, as primary hemostasis is dictated by physiologically relevant flow conditions, we wanted to determine if PR of cryoprecipitate altered its hemostatic function during resuscitation of dilutional coagulopathy using a microfluidic model of hemorrhage.
Healthy human whole blood (WB), Cryo, FibCon, and PR-Cryo FC were stained with fluorescent antibodies specific for VWF, CD41, fibrinogen, and FXIII. Stained WB was diluted 3:7 in 0.9% NaCl to induce dilutional coagulopathy (dWB). dWB was resuscitated 1:5 with stained adjuncts (Cryo:dWB, FibCon:dWB, or PR-Cryo FC:dWB) and perfused at three different shear rates (150, 500, 3500 1/s) through a microfluidic model of hemorrhage (a lumen that "bleeds" through an injury site into a collagen/tissue factor-coated extravascular space). Occlusion of the injury site was defined as the point at which clot formation sealed the injury site for &gt; 3 minutes. The time from initial perfusion to occlusion was defined as the bleeding time (BT, seconds). If no seal was formed, the assay was stopped at 20 minutes, and the assay given a BT of 1200 seconds. Real-time phase and fluorescent images of the injury site were acquired. Data was extracted from real-time phase and fluorescent images using MATLAB.
Both FibCon:dWB and PR-Cryo FC:dWB had significantly increased BT compared to Cryo:dWB at low shear (150 1/s). PR-Cryo FC:dWB had significantly increased BT compared to Cryo:dWB at medium shear (500 1/s), and at high shear (3500 1/s) there were no significant differences in BT between hemostatic adjuncts. However, kinetic analysis at high shear revealed there was a significant delay in clot formation and accumulation in the injury site, such that by 5 minutes, Cryo:dWB had filled 75% of the injury site and FibCon:dWB and PR-Cryo FC:dWB had only filled 50% of the injury site. Real-time fluorescent image analysis showed that both FibCon:dWB and PR-Cryo FC:dWB had reduced VWF deposition at the injury site compared to Cryo:dWB, and this led to a delays in platelet recruitment. FibCon has less VWF than Cryo, which would explain the delayed VWF deposition and platelet recruitment. In contrast, PR-Cryo FC and Cryo have similar amounts of VWF, suggesting that VWF from PR-Cryo FC has limited binding to the collagen-coated injury site, and as PR-Cryo FC:dWB phenocopies FibCon:dWB during clot formation at high shear, this suggests that pathogen reduction of Cryo may impair early VWF mediated capture of platelets at high shear.

Disclosures
Spinella: Secure Transfusion Services: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company; Cerus Corporation: Consultancy, Research Funding.