Association for Academic SurgeryIn vitro efficacy of RiaSTAP after rapid reconstitution
Introduction
Hemorrhage is the most common cause of early death in critically injured trauma patients. Loss or dilution of coagulation factors is thought to exacerbate bleeding and contribute to the development of hemorrhagic shock [1]. In particular, levels of the coagulation protein fibrinogen are the first to reach critical levels during hemorrhage, possibly because of early consumption or nonspecific and systemic polymerization distant from sights of injury [2]. Fibrinogen is necessary for achieving effective hemostasis, forming a strong and insoluble mesh that reinforces platelet plugs following its enzymatic conversion to fibrin by thrombin [3], [4]. Sufficient concentrations of fibrinogen are crucial for forming stable clots that are resistant to rapid fibrinolysis. As such, transfusable products (both old and new) designed to supplement fibrinogen are used to help facilitate hemostasis in bleeding trauma patients [5], [6], [7]. Unfortunately, little high-quality data are available to help guide fibrinogen-based therapy [8].
Currently, the three available treatments used to supplement fibrinogen are fresh frozen plasma (FFP), cryoprecipitate, and human fibrinogen concentrate, RiaSTAP. RiaSTAP is a lyophilized fibrinogen concentrate that was approved by the Food and Drug Administration in 2009 for treating acute bleeding episodes in those patients with hypofibrinogenemia [9]. However, it is widely used in many European countries to control bleeding in both the elective and the trauma surgical settings [10], [11], [12]. Ongoing clinical trials exploring the effectiveness of RiaSTAP in aortic reconstruction and heart valve surgery, respectively, present preliminary data showing that RiaSTAP can predictably and precisely increase fibrinogen levels, reduce need for platelet transfusions, and mitigate bleeding [13], [14]. Compared with FFP and cryoprecipitate, RiaSTAP provides the highest concentration of fibrinogen, lowest infusion volume, most consistent dosing, and lowest pathogen risk [10] (Table 1).
RiaSTAP's standard protocol reconstitution requires injecting 50 mL of distilled water followed by gently swirling for 15 min to prevent protein damage and allow all complete solubilization of the protein. RiaSTAP has shown promise in reestablishing normal fibrinogen levels in numerous surgical situations as varied as obstetric to orthopedic; however, these surgeries are elective and not restricted by RiaSTAP's 15-min protocol reconstitution time [10], [15], [16], [17]. In situations of time constraint such as in a trauma setting, physicians usually resort to a forceful and rapid reconstitution. Because of the unstable nature of some protein structures, only gentle agitation is recommended for reconstitution of protein solutions. This recommendation is provided in the preparation and reconstitution section of the RiaSTAP package insert [9]. Vigorous shaking can cause mechanical denaturation of proteins. When oxygen is introduced into the solution during shaking, rejoining of the hydrophobic and hydrophilic amino acids from the denatured proteins results in foaming bubble formation, providing an indication of protein damage [18], [19], [20]. RiaSTAP's rapid reconstitution requires injecting 50 mL of distilled water followed by vigorously shaking for up to 30 s to quickly force the protein into solution. This shaking causes excessive foaming of the solution, possible mechanical degradation of the protein, and loss of protein in the uninjectable foam, thus compromising the hemostatic potential of the product.
RiaSTAP is usually reconstituted slowly over 15 min, ensuring complete solubilization of the protein. However, in emergency situations, when patients are massively bleeding, clinicians may use a more rapid reconstitution process. This study aims to address the effectiveness of RiaSTAP in vitro after rapid reconstitution. In vitro hemostatic potential is measured by comparing the concentration of functional fibrinogen and fibrinogen efficacy in protocol-reconstituted RiaSTAP versus rapidly reconstituted RiaSTAP versus cryoprecipitate. Functional fibrinogen is that which is capable of being converted from its soluble zymogen form to insoluble fibrin following activation by thrombin and therefore able to participate in clot formation and/or hemostasis. We hypothesized that the rapidly reconstituted RiaSTAP is less effective than the protocol-reconstituted RiaSTAP in facilitating hemostasis. Rapidly reconstituted RiaSTAP and protocol-reconstituted RiaSTAP are compared against cryoprecipitate, which is the current standard of care for high fibrinogen concentration dosing.
Section snippets
RiaSTAP and cryoprecipitate preparation
RiaSTAP (A registered trademark of CSL Behring, Marburg, Germany) contains lyophilized fibrinogen protein concentrate pooled from human plasma [9]. Six vials of RiaSTAP were reconstituted: three using a protocol reconstitution and three using a rapid reconstitution. For both conditions, the metal cap coverings were removed, and a syringe was used to inject 50 mL of distilled water into each vial. For the protocol reconstitution, the vial was gently swirled on a shaker at 30 rpm for 15 min until
Gross findings—protocol-reconstitued RiaSTAP and rapidly reconstituted RiaSTAP
As can be seen in Figure 1, the protocol reconstitution of RiaSTAP results in a solution with minimal foaming. In comparison, the rapidly reconstituted RiaSTAP results in excessive foaming. It was thought that the rapid reconstitution of RiaSTAP results in a solution with a lower concentration of functional fibrinogen because of possible mechanical degradation of protein or loss of protein in the foam.
Differences in functional fibrinogen concentration and fibrinogen efficacy between protocol-reconstitued RiaSTAP and rapidly reconstituted RiaSTAP
The functional fibrinogen concentration values for each theoretical fibrinogen concentration
Discussion
RiaSTAP, when reconstituted with a 15-min protocol reconstitution, has been shown to be effective in controlling bleeding in elective surgery [10]. However, in trauma scenarios where time is of the essence, RiaSTAP is reconstituted with forceful shaking to quickly solubilize the protein. This forceful shaking could cause mechanical degradation of the fibrinogen protein and yield a less effective solution. Rapidly reconstituted RiaSTAP was compared with protocol-reconstituted RiaSTAP and
Acknowledgment
The authors thank the laboratory personnel at Center for Translational Injury Research for helping make this project possible, the University of Texas Medical School at Houston's Summer Research Program for providing funds, and Dr Pär I. Johansson's laboratory for supplying the RiaSTAP. All authors contributed to conception and design; J.V.K., J.C.C., C.E.W., and J.B.H. for analysis and interpretation; J.V.K. and J.C.C. for data collection; and J.V.K., J.C.C., and C.E.W. for writing the
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