Original Article

Restoration of Normal Prothrombin Time/ International Normalized Ratio With Fresh Frozen Plasma in Hypocoagulable Patients

Clinical and Applied Thrombosis/Hemostasis 1-7 ª The Author(s) 2014 Reprints and permission: sagepub.com/journalsPermissions.nav DOI: 10.1177/1076029614550819 cat.sagepub.com

Arthur J. Only Jr, BS1, Phillip J. DeChristopher, MD, PhD1, Omer Iqal, MD1, and Jawed Fareed, PhD1

Abstract Fresh frozen plasma (FFP) is an effective reversal agent for hypocoagulable patients. Its proven efficacy continues to prompt its usage as both a prophylactic and a therapeutic therapy. Although published guidelines encouraging the appropriate administration of FFP exist, overutilization continues. The purpose of these ex vivo studies was to determine the effects of succeeding volumes of FFP supplementation on hypocoagulable plasma prothrombin time/international normalized ratio (PT/INR). By analyzing the decline in PT/INR with varying volumes of FFP, a minimal required volume of FFP could be identified representing the optimal volume to administer while still providing therapeutic effect. A total of 497 plasma samples were screened for elevated PT/INR values and 50 samples were selected for inclusion in this experiment. The initial PTs/INRs ranged from 12.5 to 43.4 seconds/1.42 to 4.91. Subsequent declines in PT/INR values were analyzed following addition of 50, 100, and 150 mL of FFP to a fixed volume of 250 mL of plasma (26.4 + 5.318 seconds/2.99 + 0.603, 13.3 + 1.077 seconds/1.51 + 0.122, 11.2 + 0.712 seconds/1.27 + 0.081, and 10.3 + 0.533 seconds/1.16 + 0.06, respectively). A nonlinear relationship between decline in INR values and percentage of FFP supplementation was demonstrated. The greatest effect on INR was obtained after supplementation with 50 mL (49%). Doubling and tripling the volume of FFP lead to significantly lower declines in INR (16% and 8%, respectively). Analysis of variance indicated a statistical significance with subsequent volume supplementation of FFP, but marginal clinical benefits exist between the PTs/INRs obtainable with increased FFP volume administration. Keywords fresh frozen plasma, hypocoagulable, prothrombin time, international normalized ratio

Introduction Hemostasis could be defined as a physiologic equilibrium between coagulation and fibrinolysis. Blood remains in a hemodynamically fluid and inert state unless the coagulation cascade is activated. Virchow first described a triad of factors responsible for disruption of hemostasis leading to thrombosis.1 Virchow triad describes the 3 primary factors that influence hemodynamics, stasis, endothelial damage, and hypocoagulable states.1 Primary hemostasis consists of platelet adhesion, aggregation, and activation on the exposed subendothelial collagen following a vascular injury resulting in the formation of a primary hemostatic plug following vascular damage.1 Secondary hemostasis involves interaction of coagulation factors related to extrinsic and intrinsic coagulation pathways culminating in the deposition of insoluble fibrin, which is further stabilized by crosslinking of the initial fibrin clot.1 Coagulopathy follows an imbalance between activation of coagulation and compromised inhibition of coagulation and fibrinolysis.2 Anticoagulant drugs are used to prevent and manage thrombotic disorders and their complications such as stroke.3,4

Warfarin is the most commonly prescribed oral anticoagulant in the United States for patients with hypercoagulable conditions5 although a number of other non-Vitamin K antagonists medications are licensed in the United States. In the United States as reported by the National Collection and Utilization Survey, approximately 5,700,000 units of plasma were manufactured for transfusion in 2009.5 An estimated 3 million units of fresh frozen plasma (FFP) are administered annually in the United States6 often to critically ill patients with coagulopathies. Prophylactic FFP is often administered prior to invasive procedures to hemodynamically unstable patients with elevated prothrombin time/international normalized ratio (PT/INR), despite lack of adequate supportive

1

Department of Pathology, Loyola University Chicago, Maywood, IL, USA

Corresponding Author: Jawed Fareed, Loyola University Chicago, 2160 First Ave, Maywood, IL 60153, USA. Email: [email protected]

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Clinical and Applied Thrombosis/Hemostasis

INR Changes Post FFP Supplementation

PT Changes Post FFP Supplementation 50

6

45 5

40 35 PT(sec)

INR

4 3

30 25 20 15

2

10 1

5 0 Initial PT

0 Initial PT

50ul Saline

50ul FFP

100ul FFP

150ul FFP

50ul Saline

50ul FFP

100ul FFP

150ul FFP

Dosage

Dosage

Figure 1. Baseline international normalized ratio (INR) of hypocoagulable patient samples (N ¼ 50). Hypocoagulable patients and the effects of fresh frozen plasma supplementation on the restoration of clotting profile. 7

evidence. Most consensus guidelines mention only few clear indications for transfusion of FFP including treatment of bleeding due to multiple factor deficiency, emergency reversal of vitamin K antagonist, and treatment of thrombotic thrombocytopenic purpura (TTP), and other thrombotic microangiopathies.8 Several published audits of FFP transfusion very often suggested inappropriate excess usage.9 This excessive usage of FFP, besides incurring unnecessary health care expenditures, is actually etiologic in causing harm to patients due to complications such as transfusion-related acute lung injury (TRALI) and transfusion-associated circulatory overload (TACO).10 Reports on intensive care unit transfusion practices in Europe and United States indicate an estimated 33% of plasma is transfused to nonbleeding patients, despite lack of evidence of benefit.6,11,12 This unnecessary use of FFP not only incurs increased health care costs but also consumes considerable resources of blood components, which could either be appropriately used in patients who need them or whose manufacture could be avoided at the outset. Availability and implementation of clearly established transfusion guidelines, which are actually followed by physicians, are desperately needed to prevent such wastage. Based on this background it is hypothesized that the maximum effect on restoration of hemostasis is most apparent after administration of the equivalent of 2 units of FFP in patients with initial PT/INRs above 20 seconds/2.0. In order to evaluate this hypothesis, this ex vivo study was conducted to analyze the effective restoration of normal PT/INR times to hypocoagulable plasma samples after supplementation of 3 varying quantities of FFP. Fresh frozen plasma is a blood component obtained from the centrifugal separation of the fluid component of anticoagulated whole blood,13 then frozen within 8 hours to at least 18 C. A unit of FFP has a volume of 175 to 250 mL and an inherent INR of up to 1.3.13 This solution contains the essential

Figure 2. Baseline prothrombin time (PT) of hypocoagulable patient samples (N ¼ 50). Hypocoagulable patients and the effects of fresh frozen plasma supplementation on the restoration of clotting profile.

coagulation factors, which upon activation promote fibrin clot formation. The FFP consist of vitamin K-dependent factors II, VII, IX, and X, factors V and VIII, fibrinogen, and fibrinolytic/complement factors, which are among the soluble coagulation factors.13-15 The FFP is routinely used in the United States for therapeutic reversal of anticoagulation or to stop bleeding in actively hemorrhaging patients, despite scarce evidence from randomized controlled trials to support its use.16 The FFP is easily produced and relatively inexpensive. Its primary drawbacks include the potential risk of transfusion-transmitted infections, allergic reactions, TRALI, and TACO. In emergency situations, FFP may require significant time to obtain from a regional blood bank and more time to thaw in the Transfusion Service. Furthermore, it may require several hours after administration in order to establish full physiologic anticoagulation reversal.17,18 Current clinical practices promote excessive usage of FFP, which may result in complications that are better prevented.19 Ideally, such an excessive volume of FFP may not be necessary for effective anticoagulant reversal. In light of the lack of proper guidelines, this ex vivo study was designed to determine the minimal required volume of FFP necessary to restore normal clotting times (PT/INR) in patients with altered vitamin K-dependent coagulation factors due to warfarin therapy or altered hepatic function.

Materials and Methods Deidentified and discarded blood samples were collected from the Loyola University Health System/Trinity Core Clinical Laboratory under an approved IRB protocol. Samples were randomly selected based on observation of icteric plasma. Icteric plasma was taken as an indicator of plasma with strong likelihood of elevated PTs. A total of 497 samples were screened for elevated PT/INRs using Innovin (Dade) on the Automated Coagulation Laboratory 300 Plus (ACL300 Plus,

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Instrumentation Laboratory). A total of 50 samples were selected for this experiment from the original 497 cohort. Selection criteria for inclusion into this study were based on an initial PT/INR of 12/1.4. The 50 samples’ PT/INR ranged between 12.5 and 43.4 seconds/1.42 and 4.91. A laboratory reference PT time of 9 seconds represented the experimental control value. The FFP used in this experiment was manufactured in the laboratory by a method, which is equivalent to Blood Center manufacturing steps of licensed FFP. Blood draws from 10 males and 10 females (apparently healthy) were obtained. The blood was then centrifuged after the supplementation of sodium citrate (1part of citrate to 9 parts of blood) at 3000 rpm to obtain platelet poor plasma (PPP). Then, all of the plasma was pooled, mixed, and then aliquoted in 10-mL tubes. All aliquots were then stored at 70 C until used.

Sample Preparation The following protocol was executed for each of the 50 specimens. From each specimen, 250 mL of plasma was placed into 4 vials individually labeled A, B, C, and D. Vial A was supplemented with 50 mL of saline, vial B was supplemented with 50 mL of FFP, vial C was supplemented with 100 mL of FFP, and vial D was supplemented with 150 mL of FFP, respectively. Saline- and FFP-supplemented vials were analyzed using the ACL300 Plus instrument to determine PT values. In the design of this in vitro experiment, it is assumed that based on the 5-L blood volume of a patient averaging 70 kg in weight, the approximate plasma obtained based on the normal 45% hematocrit is 2.75 L. Considering administration of 2 bags (500 mL) to such a patient, the new circulating volume of plasma in that patient would be 3.25 L. We have designed this in ex vivo experiments, where we placed 50, 100, and 150 mL of FFP in a fixed volume of 250 mL of plasma and then determined the PT/INR values. The percentage of FFP administered was 16.6% when 50 mL of FFP supplemented; 28.6% when 100 mL of FFP was supplemented; and 37.5% when 150 mL of FFP was supplemented. The thought behind this approach was to identify the minimal volume of FFP necessary to correct PT/INR to avoid complications of circulatory overload while still adequately restoring hemostasis. All PT values for the discarded blood samples included in this study were obtained within 4 hours of their collection from the laboratory. The results are tabulated and graphed. Statistical analysis was performed using Sigmaplot 12.0 statistical programing software. Means and standard deviations of all parameters were calculated for all values. The INRs based on PT values were calculated using a laboratory reference PT of 9 seconds and the International Sensitivity Index (ISI) value of 1.02 for Innovin. Optimal ISI reagents are standardized in clinical laboratories in order not to influence the PT/INR values. After all samples have been analyzed, all data were incorporated into a composite to generate composite means for each supplement group. One-way repeated measure analysis of variance (ANOVA) statistical testing was then performed using Graph Pad Prism. All

Table 1. Comparison of the International Normalized Ratio (INR) Values of Native Hypocoagulable Patient Plasma Samples With Saline Supplementation and Fresh Frozen Plasma (FFP)-Corrected Groups.a INR

Mean

St Dev

Initial 50 mL saline 50 mL FFP 100 mL FFP 150 mL FFP

2.71 2.99 1.51 1.27 1.16

0.557 0.603 0.122 0.081 0.060

Abbreviations: PT, prothrombin time; St Dev, standard deviation. a Saline supplementation of the different samples resulted in a slight elevation in PT values. FFP supplementation at varying volumes resulted in marked reduction in the INR values.

Table 2. Comparison of the PT Values of Native Hypocoagulable Patient Plasma Samples With Saline Supplementation and Fresh Frozen Plasma (FFP)-Corrected Groups.a PT

Mean

St Dev

Initial 50 mL Saline 50 mL FFP 100 mL FFP 150 mL FFP

23.9 26.4 13.3 11.2 10.3

4.915 5.318 1.077 0.712 0.533

Abbreviations: PT, prothrombin time; St Dev, standard deviation. a Saline supplementation of the different samples resulted in a slight elevation in PT values. FFP supplementation at varying volumes resulted in marked reduction in the PT values.

pairwise multiple comparison procedures were conducted using Student-Newman-Keuls method. The level of statistical significance was established using a P value of 2. Our results support that administration of an initial bolus of 2 units possibly followed by initiation of a third unit of FFP drip with monitoring provides substantial reverses of PT/INRs above 17.8 seconds/2 and appears to provide appropriate treatment while minimizing the excessive administration of this blood component. Two clinical trials, one called The TOPIC trial (http:// www.clinical trials.gov website, NCT00953901)—aimed at demonstrating that omitting prophylactic FFP transfusion in critical ill patients with coagulopathy is safe in terms of bleeding complications—and another called EPICC Trial (http:// www.clinicaltrials.gov website, NCT00302965)—studying the effect of prophylactic FFP use in actively bleeding patients— have both been recently completed. The publication of the results of these trials are eagerly anticipated to establish an improved evidence base for use of FFP in hypocoagulable patients. Acknowledgments The authors gratefully acknowledge the encouragement and support of Dr Eva Woijck, chairperson department of pathology. We are also thankful to Dr Debra Hoppensteadt for her scientific advice and logistic input for these studies. The authors are thankful to Dr Gail Hecht, assistant dean for medical students research, and Ms Kate Peterson, program coordinator of the STAR program, for their support in the completion of these studies.

Authors’ Note This study was carried out as part of the STAR program at the Stritch school of medicine.

Declaration of Conflicting Interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding The author(s) received no financial support for the research, authorship, and/or publication of this article.

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