In vitro efficacy of RiaSTAP after rapid reconstitution

Abstract

Background

Fibrinogen is the first coagulation factor to reach critical levels during hemorrhage. Consequently, reestablishing normal fibrinogen levels is necessary to achieve adequate hemostasis. Fibrinogen is supplemented through administration of fresh frozen plasma, cryoprecipitate, or human fibrinogen concentrate, RiaSTAP. RiaSTAP is potentially the most advantageous fibrinogen replacement product because it offers the highest fibrinogen concentration, lowest volume, and most consistent dose. Unfortunately, RiaSTAP is limited by a protocol reconstitution time of 15 min. Conversely, physicians in emergency settings frequently resort to a forceful and rapid reconstitution, which causes foaming and possible protein loss and/or damage. This study aims to address the in vitro effectiveness of protocol-reconstituted RiaSTAP versus rapidly reconstituted RiaSTAP versus cryoprecipitate.

Methods

Three fibrinogen treatments were prepared: protocol-reconstituted RiaSTAP, rapidly reconstituted RiaSTAP, and thawed cryoprecipitate. Each treatment was added in theoretical doses of 0–600 mg/dL to fibrinogen-depleted plasma (normal fibrinogen level is 150–450 mg/dL). Samples were generated in triplicate, and each sample was subjected to rapid thrombelastography and Clauss assays. The rapid thrombelastography assay measures the hemostatic potential of a blood and/or plasma sample. The maximum amplitude (MA) parameter indicates overall clot strength and is a reflection of fibrinogen efficacy. The Clauss assay measures the time to clot formation in response to a known concentration of thrombin, and the amount of functional fibrinogen is then determined from a standard curve.

Results

For all fibrinogen treatments, increasing fibrinogen dose resulted in an increase in MA. There was no significant difference in MA between both RiaSTAP reconstitutions (slope of RiaSTAP [protocol], 10.85 mm/[100 mg/dL] and slope of RiaSTAP [rapid], 10.54 mm/[100 mg/dL]). However, both protocol-reconstituted RiaSTAP and rapidly reconstituted RiaSTAP have higher MA values than cryoprecipitate in doses of ≥100 mg/dL. Moreover, each replicate of cryoprecipitate showed a higher variance in fibrinogen efficacy (coefficient of variance [CV] = 44.7%) at a fibrinogen dose of 300 mg/dL. RiaSTAP, however, showed a lower variance in fibrinogen efficacy for both reconstitutions (RiaSTAP [protocol], CV = 3.3% and RiaSTAP [rapid], CV = 2.7%), indicating a consistent fibrinogen dose.

Conclusions

RiaSTAP (either reconstitution method) has greater hemostatic potential and less variability in fibrinogen concentration compared with cryoprecipitate. Rapidly reconstituted RiaSTAP does not compromise hemostatic potential and can be used to potentially facilitate hemostasis in rapidly bleeding patients.

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.