Comparison of Russell Viper Venom Based and Activated Partial Thromboplastin Time Based Screening Assays for Resistance to Activated Protein C

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1 Coagulation and Transfusion Medicine / RVV and aptt Screening Assays for APCR Comparison of Russell Viper Venom Based and Activated Partial Thromboplastin Time Based Screening Assays for Resistance to Activated Protein C Adrianna Z. Herskovits, MD, PhD, Susan J. Lemire, MT(ASCP), Janina Longtine, MD, and David M. Dorfman, MD, PhD Key Words: Factor V Leiden; Hereditary thrombophilia; Screening assays; Russell viper venom; Activated partial thromboplastin time; Activated protein C resistance; Economic evaluation DOI: /AJCP7YBJ6URTVCWP Abstract Thrombotic disease is a significant cause of mortality and morbidity, with an estimated lifetime risk of greater than 10% in Western populations. One of the most common hereditary thrombophilias is the factor V Leiden mutation, which is identified with a screening assay for activated protein C (APC) resistance and confirmed by DNA analysis. In this study, we compared the commercially available Pefakit (Pentapharm, Basel, Switzerland) and Cryocheck (Precision BioLogic, Dartmouth, Canada) assays, 2 recently developed Russell viper venom (RVV)-based screening tests, with the activated partial thromboplastin time (aptt)- based screening test currently used in our hospital s clinical laboratory. We found that the aptt-based assay for resistance to APC had a sensitivity of 100%, a specificity of 70%, and a positive predictive value (PPV) of 70%, whereas both of the RVV-based assays exhibited high sensitivity, specificity, and PPV at 100%. In addition, we found that these new functional assays are more cost-effective relative to the screening algorithm previously used in our clinical laboratory and could potentially eliminate the need for DNA analysis, although further study is required. Thrombosis is a pathogenic process in which clots are formed within arterial or venous systems that disrupt the normal flow of blood. Thrombotic disease is a significant cause of mortality and morbidity, with an estimated lifetime risk of greater than 10% in Western populations. 1,2 Venous thrombosis is more commonly associated with acquired risk factors such as surgery, hospitalization, malignancy, immobilization, pregnancy, autoimmune disease, and oral contraceptive use or can be caused by a number of genetic mutations that affect different clotting cascade 1 and fibrinolytic proteins. One of the most common hereditary thrombophilias is the factor V Leiden mutation, a single nucleotide substitution that causes an amino acid change from arginine to glutamine at position 506 in the factor V protein. This single residue change occurs at one of the sites where activated protein C (APC) cleaves factor V. This proteolytic step is important for maintaining normal hemostasis, and resistance to APC caused by the R506Q mutation disrupts the inactivation of factor V. Patients with a mutation at this site in factor V have an increased risk of pulmonary emboli, deep vein thrombosis, recurrent pregnancy loss, stroke, and other thrombotic complications. 3-5 The prevalence of heterozygotes for the factor V Leiden mutation in the United States differs in ethnic groups and has been estimated at 5.27% in Caucasians, 2.21% in Hispanic Americans, 1.25% in Native Americans, 1.23% in African Americans, and 0.45% in Asian Americans. 6 Heterozygotes are 7 times more likely to experience a thrombotic event relative to a person without the factor V Leiden mutation, and the risk is elevated 80-fold in patients who carry 2 copies of this allele. 3,7 Although functional clotting assays are useful screening tests to identify carriers of this genotype, the gold standard to verify whether a individual person carries the factor V 796 Am J Clin Pathol 2008;130: DOI: /AJCP7YBJ6URTVCWP

2 Coagulation and Transfusion Medicine / Original Article Leiden mutation is DNA sequence-based analysis. Although DNA analysis is more costly, the alternative functional assays have less sensitivity and specificity for detecting homozygote or heterozygote carriers of the mutation. Moreover the more commonly used functional biologic tests are activated partial thromboplastin time (aptt)-based assays that can give misleading results in patients undergoing anticoagulant therapy or who have an elevated aptt for other reasons, such as the presence of a lupus anticoagulant. 8 APC resistance in the absence of a factor V Leiden mutation has also been observed in patients who are pregnant, taking oral contraceptives, or have high factor VIII levels or autoimmune conditions such as lupus anticoagulants that may predispose them to venous thromboembolism. 9 In addition, several less common changes in the coding region of the factor V gene such as the Liverpool mutation, I359T, and the R2 haplotype marked by the H1299R polymorphism have also been demonstrated to affect APC resistance. Lupus anticoagulants are acquired autoantibodies directed against phospholipid-protein complexes, which can interfere with activated partial thromboplastin based APC resistance assays. This autoimmune phenomenon is associated with thrombosis, thrombocytopenias, and recurrent fetal loss. Antibodies in the patient s plasma can affect the phospholipid-dependent reactions in the coagulation cascade, increasing the measured clotting time in the absence of physiologic APC resistance. 3 Several strategies have been recently devised to eliminate these interferences by measuring clotting time on a more focused portion of the clotting cascade that involves factor V and protein C. Some of the advantages of these systems are that these tests are less likely to be affected by anticoagulant therapy or lupus anticoagulants. Many patients begin anticoagulant therapy before samples for their hypercoagulable workup are drawn and sent to the laboratory; therefore, it would be extremely useful to have a screening assay that is not affected by these interferences. Two recently developed functional assays for APC resistance are the Pefakit Activated Protein C Resistance Factor V Leiden (Pentapharm, Basel, Switzerland) and the Cryocheck Clot APCR (Precision BioLogic, Dartmouth, Canada). Both of these screens incorporate the use of activating reagents and Russell viper venom (RVV) in a clotting assay. The Pefakit system works by using venom from the Russell viper (Daboia russelli russelli), which contains an activator of factor V. It is used in conjunction with a prothrombin activator extracted from the Australian tiger snake (Notechis scutatus scutatus) to increase the velocity of thrombin activation. The time required for clotting is measured in the presence and absence of APC. The addition of APC will substantially increase the time to clot for people with a wild-type genotype, whereas patients with a factor V Leiden mutation have APC resistance and their clotting times will not be greatly affected by the addition of this reagent. 10 However, this is not the only approach to increase the sensitivity and specificity of functional APC resistance screens. The Cryocheck Clot APCR combines the activating effect on factor V provided by RVV with a direct protein C activator derived from the venom of the southern copperhead snake (Agkistrodon contortrix contortrix) to increase the sensitivity of the functional APC resistance assay. Healthy people have a clotting time that is prolonged 2- to 3-fold relative to people with the factor V Leiden mutation in the presence of APC with RVV, making it possible to distinguish people with the factor V Leiden mutation with greater ease from a functional assay. 11 Although a recent study favorably compared the Pefakit APCR test with the Coattest APC Resistance V (Chromogenix, Milan, Italy), 8 we sought to independently verify the usefulness of this assay. Because the initial study was sponsored by the manufacturer of one of the reagents, it was unclear whether the exclusion criteria used in the study would affect the practical usefulness of these assays in a hospital setting, and we were interested in assessing whether it would be economically feasible to switch to another method of functional protein C resistance screening owing to the large volume of samples received at our clinical facility during a year. The design of this study was to compare the Pefakit and Cryocheck RVV functional tests with the aptt-based APC resistance assay screening algorithm currently used in our hospital s clinical laboratory in terms of accuracy and cost efficiency as a screening test. Materials and Methods Sample Collection Venous blood was collected in blue-top collection tubes containing citrate, and the samples were centrifuged at 1,300g for 15 minutes at room temperature. The plasma supernatants were removed and placed on ice for immediate use or maintained in aliquots at 70 C for longer-term storage. aptt-based APC Resistance Assay and Testing Algorithm The APC resistance assay was performed according to the method previously described. The aptt was measured directly in the patient s plasma and also after mixing the sample with a sufficient quantity of APC (Hematologic Technologies, Essex Junction, VT) to prolong the baseline aptt to 100 to 160 seconds. Samples with baseline elevation in prothrombin time (PT) or aptt were not analyzed using this APC resistance assay because the testing was previously shown to be inaccurate with these plasma samples, and samples were sent directly for molecular testing. 12,13 Am J Clin Pathol 2008;130: DOI: /AJCP7YBJ6URTVCWP 797

3 Herskovits et al / RVV and aptt Screening Assays f o r APCR The APC sensitivity ratio (APC-SR) was calculated by dividing the aptt with added APC by the aptt from the same person s plasma in the absence of exogenous APC. The same APC-SR measurement was also performed on a pool of plasma from healthy subjects (George King Pooled Normal Plasma, George King Biomedical, Overland Park, KS) each day. Reported values for the aptt-based assay were normalized to maintain consistency with prior descriptions of this method. 5,13 The normalized APC value for the patient was determined by dividing the APC-SR of the patient s sample by the APC-SR of the normalized control samples. All measurements were performed on an MDA-II instrument (Biomerieux, Durham, NC) using the Actin FS activated PTT reagent (Dade Behring, Miami, FL), and samples with a normalized ratio less than 0.80 were considered outside the standard range and diagnostic of APC resistance. Pefakit APC Resistance Assay Patient samples were diluted by mixing 30 µl of sample plasma into 20 µl of normal human plasma according to the instructions of the manufacturer (Pentapharm). The RVV reagent was added to each sample in the absence or presence of APC. After incubation for 3 minutes, both samples were incubated in a prothrombin activator reagent. The clotting time in the presence and absence of APC was determined. All measurements were performed on a STA Compact (Diagnostica Stago, Parsippany, NJ). The initial cutoff values were set at more than 2.9 for wild-type, as suggested by the manufacturer s protocol; however, after running the initial batch of tests with the Actin FS aptt reagent and comparing the results with DNA analysis (see DNA Analysis Using the Invader Assay ), we established that samples with a ratio of less than 1.9 should be considered diagnostic for APC resistance. Cryocheck Clot APC Resistance Assay Reaction cuvettes were set up containing 50 µl of patient s plasma according to the instructions of the manufacturer (Precision BioLogic). The RVV reagent was warmed to 37 C and incubated with 50 µl of the activator reagent in 1 cuvette or 50 µl of 0.15 mol/l of saline in a second cuvette, and the clotting time was measured after 5 minutes. The APCR ratio was calculated by dividing the clotting time recorded from the first cuvette with the activator reagent by the second cuvette with the normal saline. All measurements were performed on a STA Compact (Diagnostica Stago), and samples with a ratio less than 1.57 were considered outside the standard range and diagnostic for APC resistance. DNA Analysis Using the Invader Assay DNA sequence-based analysis for the factor V Leiden mutation was performed using the Invader assay (Third Wave Technologies, Madison, WI) as previously described. 14,15 Oligonucleotide probes were designed to target the G1691 or A1691 single nucleotide polymorphism of the factor V Leiden gene forming a 3-dimensional structure recognized by a flap endonuclease (FEN) enzyme. The FEN cleaves the flap sequences, which then become invader probes for a secondary reaction, binding to a distinct fluorescence resonance energy transfer (FRET) cassette containing a fluorophore and an internal quencher. The FEN enzyme now recognizes a 3-dimensional structure formed by the FRET cassette partially annealed to itself and the 59 flap released from the first reaction. Cleaving this structure separates the fluorophore and quencher molecules, and each allele generates a different fluorescent signal enabling distinction of wild types, heterozygotes, and homozygotes for the factor V Leiden mutation. 14,15 Data Collection and Analysis Samples were from inpatients at Brigham and Women s Hospital (Boston, MA) and its affiliated clinics sent for testing between September 2007 and February While there were no specific inclusion or exclusion criteria used in this study, we attempted to include many samples with abnormalities in the initial screening PT and PTT tests to maximize the number of study samples with other coagulation abnormalities that might affect functional screening. Of 115 patient plasma samples, 10 did not have enough sample material available to perform all of the functional screens. In those cases, the plasma sample was run using 1 or 2 tests, and results were compared with DNA analysis. Information about risk factors for hypercoagulability and other clinical parameters was collected by chart review. Statistical analysis was performed using KyPlot, version 2.0 (KyensLab, Tokyo, Japan). Results A total of 115 samples from patients ranging in age from 18 to 93 years were studied using the functional and molecular assays to investigate APC resistance. All patient samples were subject to DNA sequence-based testing, which is the gold standard for determining APC resistance due to the factor V Leiden mutation. A total of 19 samples were genotyped as heterozygous and 96 as wild type by DNA analysis using the Invader assay. There were no homozygotes present in this study because this genotype is rare and our center did not receive any samples with the R506Q factor V mutation at both alleles between September 2007 and February 2008, when this analysis was performed. The characteristics of the patients whose samples were studied are summarized in ztable 1z. Of the patients, 29.6% were men and 70.4% were women. There were no exclusion criteria precluding the use of patient plasma samples in this study, and the majority of the samples were from patients 798 Am J Clin Pathol 2008;130: DOI: /AJCP7YBJ6URTVCWP

4 Coagulation and Transfusion Medicine / Original Article with clinical risk factors for thromboembolic disease such as recent surgery, prior thromboembolic events, malignancy, or pregnancy. More than 90% of the patients had at least 1 clinical risk factor for hypercoagulability. Of the patients, 58.3% were known to be taking anticoagulant medications such as heparin, enoxaparin, or warfarin at the time samples were drawn. This pharmacologic therapy is significant because these samples would ordinarily be automatically sent for molecular analysis under the hospital s current testing algorithm, because prior studies have shown that this assay is not accurate in patients with elevated PT or PTT values before the addition of APC. 12,13 In this study, these samples were analyzed using the Pefakit and Cryocheck assays to see whether these tests could accurately predict the patient s genotype and, therefore, increase the proportion of samples that could be screened by functional assay. Low protein S levels were found in 21 patients, and 10 patients had protein C levels below 50% of the normal range; however, these results must be taken in context: 8 of the patients with low protein S levels and 3 of the patients with low protein C levels had started warfarin therapy around the time of testing, which affects γ carboxylation of vitamin K dependent clotting factors produced by the liver, and 11 patients in the study had a current or recent pregnancy. Moreover, these tests were not ordered for every patient within the study. Four patients in the study group had low antithrombin III (ATIII) levels. Protein C, protein S, and ATIII levels were important factors to assess because the snake venom activators and factor V Leiden mutation impact many of the same points of the clotting cascade zfigure 1z. ztable 1z Overview of Patient Plasma Samples Included in This Study Variable Result Age (y) Average 50.1 Range Sex Men 34 Women 81 Hypercoagulability testing Confirmed factor V Leiden mutation 19/115 (16.5) Prothrombin G20210A mutation 3/89 (2.6) Positive for lupus anticoagulant 4/81 (3.5) Low protein S (<50%) 21/76 (18.3) Low protein C (<50%) 10/75 (8.7) Low ATIII 4/85 (3.5) Factor VIII >150% 4/9 (3.5) PT/PTT abnormalities 75/115 (65.2) Known anticoagulation therapy at time of testing 67 (58.3) Predisposing clinical risk factors Clinical risk factors for hypercoagulability 104 (90.4) Family history of thrombophilia 11 (9.6) Immobility 14 (12.2) Obesity 8 (7.0) Recurrent pregnancy loss 21 (18.3) Second- or third-trimester pregnancy 11 (9.6) Current or prior thrombotic event 68 (59.1) Malignancy 15 (13.0) Current or recent oral contraceptive use 4 (3.5) Atrial fibrillation 5 (4.3) ATIII, antithrombin III; PT, prothrombin time; PTT, partial thromboplastin time. Data were obtained by chart review. Patients with confirmed testing for low protein S, low protein C, low ATIII, presence of lupus anticoagulant, elevated factor VIII, and prothrombin G20210A or clinical risk factors for hypercoagulability were recorded. Complete hypercoagulability testing and patient records were not available for all subjects. For hypercoagulability testing, data are given as number/ total tested (percentage) because not all patients had all types of hypercoagulability testing. For predisposing risk factors, data are given as number with risk factor (percentage of total). A Intrinsic pathway Extrinsic pathway B Intrinsic pathway Extrinsic pathway Xa C S Xa C S Va V Va V Prothrombin Thrombin VIII Prothrombin Thrombin VIII Fibrinogen Fibrin Fibrinogen Fibrin VIIIa VIIIa Clot Clot zfigure 1z Schematic diagram illustrating the differences between the 2 Russell viper venom based screening methods used in this study. A, The Pefakit APC Resistance Assay utilizes Russell viper venom, which contains an activator of factor V, along with a prothrombin activator from the Australian tiger snake. B, The Cryocheck Clot APC Resistance Assay utilizes Russell viper venom, along with a direct protein C activator from the southern copperhead snake. V, VIII, VIIIa, and Xa are clotting factors; C, protein C; S, protein S. Am J Clin Pathol 2008;130: DOI: /AJCP7YBJ6URTVCWP 799

5 Herskovits et al / RVV and aptt Screening Assays f o r APCR During pregnancy, the blood concentration of most clotting factors increases, while fibrinolytic activity and some anticoagulant levels decrease, creating an overall shift in the direction toward hypercoagulation. These changes may be physiologically advantageous to protect against excessive bleeding during delivery; however, they increase a woman s chances of developing a thromboembolic event during pregnancy or the postpartum period. 16 Although we had samples from 11 patients with current (second- or third-trimester) or recent (within 8 weeks postpartum) pregnancies at the time of testing, we did not find that pregnancy-associated hemostatic changes had any effect on the RVV-based functional assays. Four patients had positive results for the lupus anticoagulant on at least 1 occasion; however, heparin and lowmolecular-weight heparin can interfere with testing for the lupus anticoagulant and many APC functional assays. Elevated factor VIII levels may increase thrombotic risk, 17 and levels of this factor were tested in 9 patients as part of their hypercoagulability workup. Of the 9 patients, 4 had a documented elevation in their factor VIII levels. Of the 4 patients with factor VIII levels above the reference interval, 3 had a wild-type genotype, and the fourth patient was a heterozygote for the factor V Leiden mutation. Both of the RVV-based screening assays correctly identified all 4 of these patients, whereas the PTT-based assay correctly identified only the heterozygote. Three patients had a documented prothrombin G20210A mutation, and 2 of these 3 patients were also found to be heterozygous for the factor V Leiden mutation. Clinical risk factors, including current or prior thrombotic events, immobility, obesity, malignancy, current or recent oral contraceptive use, and atrial fibrillation, were also evaluated (Table 1). The mean values for APC resistance are summarized in ztable 2z, and all 3 methods enabled the discrimination of wild-type and mutant alleles. The Cryocheck assay not only exhibited clear separation between the mean values for these 2 populations but also had low variance relative to the Pefakit assay (Table 2). All of the assays achieved statistical significance at a P value less than.001 in distinguishing the wild-type and mutant alleles using the Mann-Whitney U test; however, it is clear that the Pefakit and Cryocheck assays demonstrate a greater difference between the mean values for the R506Q and R506 alleles relative to the aptt screen zfigure 2z. A clear separation between APC ratios for wild-type and heterozygous samples using the Pefakit and Cryocheck assays is shown in zfigure 3z. This finding suggests that the RVVbased assays should be valuable as screening tests because a APC Ratio aptt Pefakit Cryocheck zfigure 2z Comparison of genotyped plasma using activated partial thromboplastin time (aptt)- and Russell viper venom based assays using plasma samples with prothrombin time/ aptt values within the normal range. Genotype analysis was confirmed by using molecular DNA testing. Samples from wild type R506 are represented with triangles and samples with the factor V Leiden R506Q allele by circles. ztable 2z Descriptive Statistics for Activated Protein C Sensitivity Ratios and Functional Screening Assays aptt napc-sr Pefakit APC-SR Cryocheck APC-SR R506Q Mutation R506 Wild Type R506Q Mutation R506 Wild Type R506Q Mutation R506 Wild Type Mean SE of mean SD Variance Coefficient of variation Minimum Maximum No. of samples aptt, activated partial thromboplastin time; napc-sr, normalized activated protein C sensitivity ratio. 800 Am J Clin Pathol 2008;130: DOI: /AJCP7YBJ6URTVCWP

6 Coagulation and Transfusion Medicine / Original Article number of samples characterized as wild type by DNA analysis were miscategorized as heterozygous in the aptt-based functional screen owing to the clustering of values around the cutoff value of 0.8, which affected the specificity of the aptt-based assay. Although resistance to APC using the aptt-based assay had a sensitivity of 100% with a specificity of 70% and a positive predictive value of 70%, both of the RVV-based assays showed improved sensitivity, specificity, and PPV at 100%, correctly identifying all wild-type and heterozygous plasma Mean APC-SR (± SD) aptt Method Pefakit Cryocheck zfigure 3z Comparison of mean activated protein C (APC) sensitivity ratios (SRs) using Russell viper venom and activated partial thromboplastin time (aptt)-based screening assays. White bars represent heterozygotes for the factor V Leiden mutation, and black bars represent the wild-type genotype as determined by molecular DNA testing with error bars showing the SD. Statistical analysis was performed using KyPlot 2.0, and comparisons marked with an asterisk () were statistically significant at P <.001 using the Mann- Whitney U test. ztable 3z Comparison of Sensitivity and Specificity Using Russell Viper Venom and aptt-based Functional Assays for Resistance to Activated Protein C Result/Assay aptt Pefakit Cryocheck Positive assay R506Q genotype 15 (TP) 18 (TP) 18 (TP) Wild-type (R506) genotype 8 (FP) 0 (FP) 0 (FP) Negative assay R506Q genotype 0 (FN) 0 (FN) 0 (FN) Wild-type (R506) genotype 15 (TN) 96 (TN) 87 (TN) Indeterminate assay R506Q genotype Wild-type (R506) genotype Prevalence Sensitivity Specificity Positive predictive value aptt, activated partial thromboplastin time; FN, false negative; FP, false positive; TN, true negative; TP, true positive. samples that were assayed. Moreover, the RVV-based assays did not have any indeterminate samples and yielded informative results on all samples that were run ztable 3z. Although the Pefakit assay enabled clear distinction between the heterozygous and wild-type plasma samples, it was necessary to readjust the certified ranges given for our instrumentation in the manufacturer s protocol from a cutoff value of greater than or equal to 2.9 to greater than or equal to 1.9 for normal genotype samples. The Cryocheck assay also clearly distinguished the mutant and wild-type alleles. It worked perfectly in accordance with the cutoff ranges in the manufacturer s protocol and did not require any adjustments. One of the other factors that can be more difficult to quantify in evaluating different testing methods is preparation time and ease of changing to an alternative screening method. In this initial validation study, we found that the Pefakit and Cryocheck assays were easily adaptable to automation using equipment already on hand in our clinical laboratory and decreased technician time for preparing reagents and stock solutions relative to the assay previously used in our laboratory. The aptt-based assay has been long used in our clinical laboratory because its components are nonproprietary, so the assay can be inexpensively run on the large volume of samples that we receive. Therefore, cost analysis was performed to compare the different methods of testing, factoring in the purchase of reagent kits, cuvettes, control plasma samples, and other reagents used to perform these tests. Of approximately 1,014 samples received by the clinical laboratories in our hospital during the previous year, 384 were sent directly for DNA analysis, owing to PT or PTT abnormalities at baseline, at a cost of $43.03 per assay. The remaining 630 were screened directly using the aptt APC test, and from these samples, approximately 5% were positive for the factor V Leiden mutation. The Pefakit and Cryocheck assays do not yield indeterminate results in patients with abnormal baseline PT or PTT results, necessitating fewer DNA tests. All positive functional screens (approximately 51 heterozygous factor V Leiden plasma samples received per year) are sent for confirmatory molecular testing. Although the aptt test seems to be the least expensive screening assay because its components are inexpensive, nonproprietary reagents, and the Pefakit and Cryocheck assays have a higher cost per test, these savings are offset by the relative expense of molecular testing on a larger number of indeterminate samples. We estimate that a savings of approximately $10,173 or $11,139 per year ztable 4z can be realized by adopting one of the newer functional assays, which could reduce our budget for APC/factor V Leiden testing by approximately 50%. These figures are only approximate because the costs may vary depending on the source of control plasma samples for the functional RVV assays and are also likely to change in the future as DNA assay technology advances. Am J Clin Pathol 2008;130: DOI: /AJCP7YBJ6URTVCWP 801

7 Herskovits et al / RVV and aptt Screening Assays f o r APCR ztable 4z Cost Analysis Comparing Estimated Annual Expenses for Performing RVV- and aptt-based Functional Assays for Resistance to Activated Protein C Pefakit Cryocheck BWH aptt Estimated annual cost (eg, reagents, controls, buffers, cleaners) $6,870 (n = 1,014) $7,836 (n = 1,014) $2,312 (n = 630) Molecular testing for indeterminate samples $0 (n = 0) $0 (n = 0) $16,524 (n = 384) Molecular testing to confirm positive functional screen $2,182 (n = 50.7) $2,182 (n = 50.7) $1,355 (n = 31.5) Total cost $9,052 $10,018 $20,191 Estimated annual savings for changing from aptt method to RVV-based assay $11,139 $10,173 aptt, activated partial thromboplastin time; BWH, Brigham and Women s Hospital, Boston, MA; RVV, Russell viper venom. Molecular testing was performed on the 384 samples with abnormal prothrombin time or partial thromboplastin time results at a cost of $43.03 per assay; however, the Pefakit and Cryocheck functional screens do not yield indeterminate results under these conditions. The cost of confirmatory molecular testing is lower in the BWH aptt category relative to the Pefakit and Cryocheck assays because an estimated 19 plasma samples would have been tested with samples yielding indeterminate results for the functional screen and 32 samples tested as a result of an abnormal aptt based functional screen. Approximately 51 plasma samples from patients with the factor V Leiden mutation are received per annum, and all of these samples are sent for confirmatory molecular testing at a cost of $43.03 per assay. Discussion In this study, we compared 2 recently developed methods of performing functional APC resistance assays and found that the snake venom based APC assays correlate perfectly with patient genotype as determined by DNA testing. These assays were not subject to the same kinds of interferences that affect the aptt-based testing used at our clinical center. The newer functional APC resistance strategies not only provide improved sensitivity but also offer a significant cost savings by decreasing the number of samples sent for molecular analysis. One of the difficulties in performing functional protein C resistance assays is that by the time the samples are sent to the clinical laboratories, many patients have already started anticoagulant therapy. Because many of the risk factors that predispose patients to venous thromboembolic events are common in hospitalized patients, such as immobilization, surgery, and pregnancy, patients are often given heparin prophylactically. The origins of oral anticoagulation therapy can be traced to the 1920s when Schofield observed that cattle foraging on spoiled sweet clover developed an unusual clotting disorder. During the next several decades, the compound causing this hemorrhagic disease was identified as dicoumarol, and its more potent derivative warfarin was used as an agent to kill rodents. Warfarin works by irreversibly antagonizing vitamin K reductases, preventing the γ carboxylation necessary to produce active coagulation factors II, VII, IX, and X, protein C, and protein S by the liver. After toxicity and therapeutic ranges were established, warfarin has become one of the most frequently prescribed anticoagulant agents. 18,19 One of the concerns at the outset of the study was whether patients taking warfarin would have accurate functional APC resistance testing, because one of the components in the Cryocheck assay is a direct protein C activator derived from copperhead snake venom, and warfarin decreases levels of protein C and protein S, which act in concert to cleave activated factor V at the R506Q site. Of the 58.3% of patients who were receiving anticoagulation therapy with heparin or warfarin at the time of testing in this study, no discrepancies between the functional screening and genotype were found by the Cryocheck or Pefakit assay. Heparin is another commonly used anticoagulant that we wanted to assess when evaluating the new functional APC resistance assays. Heparin was initially discovered by McLean in 1915, who began working in a research laboratory while awaiting admission to medical school. While McLean was actually searching for procoagulants, he stumbled on a compound that exerted the opposite effect when extracted from dog liver. It was several decades before the compound could be purified sufficiently for clinical use, and the first human studies using heparin for prevention of venous thromboembolism were initiated in At low plasma concentrations, heparin binds ATIII to inactivate thrombin, factor IXa, and factor Xa. Low-molecular-weight heparin cannot bind ATIII and thrombin simultaneously, and these anticoagulants primarily work by helping ATIII inactivate factor Xa. 18 These mechanisms of action are important to consider because one of the active components in the Pefakit assay is Noscarin, an extract from the Australian tiger snake, which functions as a prothrombin activator. In our analysis, anticoagulation with heparin did not interfere with the Pefakit or Cryocheck assay; however, further studies should be performed on a larger cohort of patients because patients with a blood concentration of greater than 2 U/mL of heparin were excluded from one of the prior studies on Pefakit 8 and it is suggested in the Cryocheck product literature that higher doses of heparin might cause interference. 11 This problem of interference from anticoagulant therapy has practical implications because anticoagulant medications have been shown to interfere with APC, so identifying APC resistance assays that are not subject to these limitations is greatly advantageous for clinicians who want to initiate 802 Am J Clin Pathol 2008;130: DOI: /AJCP7YBJ6URTVCWP

8 Coagulation and Transfusion Medicine / Original Article treatment before completing a hypercoagulability workup. Although the 2 RVV-based assays have different mechanisms of activity that have the potential to interact with multiple factors involved in thrombosis (Figure 1), we have found that both tests offer excellent sensitivity and specificity for routine APC screening. Conclusions Factor V is an exceedingly complex protein that has an important role in modulating procoagulant and anticoagulant pathways. Proteolysis at the Arg506 site by APC and protein S influences factor V to become a natural anticoagulant, acting as a secondary cofactor for APC (in addition to protein S) synergistically effecting the proteolytic inactivation of factor VIIIa. These proteins regulate the tenase complex, composed of activated factors IX and VIIIa, which activate factor X as part of a membrane-associated group of factors. 20 Alternatively, factor V can behave as a procoagulant molecule if it is cleaved at Arg709, Arg1018, and Arg1545. In this capacity, factor V becomes a cofactor for prothrombin activation, binding with factor Xa to form the prothrombinase complex, which converts prothrombin to thrombin to promote clot formation. 1 As an anticoagulant or a procoagulant, activated factor V can be relieved of its duties by subsequent cleavage at the R306 site by APC. 20 APC can down-regulate factor Va at 3 proteolytic sites Arg306, Arg 506, and Arg679; however, they are not of equal importance for the general functioning of the protein. The Arg 506 residue is the only APC-mediated cleavage site critical for mediating the anticoagulant function of factor V, and in vitro studies suggest that the increased risk of thrombosis in patients who have the factor V Leiden mutation is due to this altered anticoagulant potency of inactive factor V. 1 Although the majority of people who have inherited APC resistance have the Arg506Glu mutation, the factor V Leiden allele is not the only determinant of APC resistance. A common haplotype of linked polymorphisms that affect amino acids in the A2, B, A3, and C2 domains of factor V, known as the HR2 haplotype, is associated with decreased levels of circulating factor V and mild APC resistance. Resistance to APC can also be due to the I359T mutation, which causes a missense mutation in the protein and leads to increased venous thromboembolic events clinically. 21 A recent study investigating the genetic determinants of hypercoagulability in a cohort of Asian-Indian patients with deep venous thrombosis showed that only 16 of 32 patients with APC resistance carried the factor V Leiden mutation. Although they did not identify other mutations associated with venous thromboembolic risk, their results suggested a possible association between the factor V HR2 haplotype and APC resistance using an aptt-based assay. 22 Although it seems likely that the HR2 haplotype would yield APC resistance using an RVV-based assay, the influence of these mutations should be studied experimentally. Some people have substitutions at Arg306, a site that is also subject to APC cleavage. The factor V Cambridge mutation is an Arg306Thr mutation that causes APC resistance, whereas the factor V Hong Kong mutation results in an Arg306Gly amino acid change. It is interesting that these mutations have not been shown to cause APC resistance in patient samples but may have a mild effect with in vitro recombinant protein based assays. Because these are rare mutations, further studies of the physiologic importance and compensatory mechanisms through alternative APC cleavage sites may further elucidate the role of additional factor V mutations. 1 Although factor V in all of its biologic functions is complex, the thrombotic effect of the factor V Leiden mutation has been relatively well characterized. In this study, we have shown that several new snake venom derived functional protein C assays offer improved sensitivity and specificity over the aptt-based assay currently used in our clinical laboratory. We have also found that these tests offer substantial cost savings owing to the decreased need for more expensive DNA analysis. In the several months since this study was completed, our laboratory has received 1 sample from a patient homozygous for the factor V Leiden mutation, and the APC resistance ratio using the Pefakit assay was Although this value is within the expected range for a homozygote, more samples will need to be tested to establish whether this assay is effective for identifying homozygotes before deciding whether confirmatory DNA analysis can be eliminated. At this point, there are advantages of combining functional APC resistance assays with confirmatory DNA sequencebased analysis. Although functional assays may more accurately capture a range of biologically relevant phenotypes, DNA testing helps to confirm which patients have genetic mutations at the factor V Leiden mutation. People with discrepancies in their phenotypic and genotypic analysis results can undergo analysis for alternative or compound mutations, which will broaden our understanding of the interplay among protein C, factor V, and the numerous other players in the process of hemostasis. From the Department of Pathology, Brigham and Women s Hospital and Harvard Medical School, Boston, MA. Supported by Brigham and Women s Hospital Clinical Laboratories. Address reprint requests to Dr Dorfman: Dept of Pathology, Brigham and Women s Hospital, 75 Francis St, Boston MA Am J Clin Pathol 2008;130: DOI: /AJCP7YBJ6URTVCWP 803

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