Sensitivity and Specificity ofthe APC Resistance Assay in Detection of Individuals With Factor V Leiden

Transcription

1 COAGULATION AND TRANSFUSION MEDICINE Sensitivity and Specificity ofthe APC Resistance Assay in Detection of Individuals With Factor V Leiden JAMES L. ZEHNDER, MD, AND RENEE C. BENSON Resistance to activated protein C (APC) is the most common cause of familial thrombophilia. The partial thromboplastin time (PTT)-based test for resistance to APC has been widely employed as a screening test for this disorder. However, the utility of this test for screening is not well characterized. More than 90% of patients with resistance to APC have the G1691A mutation in factor V (factor V Leiden). The authors studied the ability of a commercial APC resistance assay to correctly identify the factor V Leiden genotype in 130 individuals. At the recommended assay cut-off value of 2, the sensitivity of the APC resistance assay was 50%, with a specificity of 98%. Increasing the cut-off value increased the sensitivity but decreased the specificity of the test. Re- Activated protein C (APC) limits the clotting process by proteolytic inactivation ofthe two clotting co-factors factor Va and factor Villa.'" 3 Resistance to activated protein C has been shown to be a common risk factor for venous thrombosis. 4,5 In >90% of cases, APC resistance is due to a mutation in the factor V gene, G1691A (factor V Leiden). 6 " 10 This mutation does not appear to alter the procoagulant activity of factor Va. However, the mutation occurs at one ofthe two APC cleavage sites, resulting in decreased inactivation of factor Va, and increases the risk of venous thrombosis in affected individuals. Horaozygotes for factor V Leiden have a relative risk of thrombosis of 50 to 100, whereas the risk in heterozygotes is 5- to 10-fold higher than healthy subjects." The factor V Leiden mutation appears to be relatively common with a prevalence of 5% to 10% in several populations studied, 46 although a recent report suggests that the incidence of this mutation in people of Japanese ancestry is much lower. 12 This defect is the most common cause of familial thrombophilia yet identified. From the Department of Pathology, Stanford University Medical Center, Stanford, California. Manuscript received October 10, 1995; revision accepted January 10, Address reprint requests to Dr. Zehnder: Department of Pathology, L235, Stanford University Medical Center, Stanford, CA ceiver operating characteristic (ROC) curve analysis indicated that the test was of intermediate utility. There was considerable overlap in APC ratios in the range of 2 to 3 between subjects with a normal factor V genotype and heterozygotes for factor V Leiden. The authors conclude that the APC resistance assay in its present form is not a useful screening test for factor V Leiden heterozygotes. Until the performance of this assay is improved, patients should have molecular diagnostic testing performed to determine their factor V Leiden status. (Key words: Resistance to activated protein C; Factor V Leiden; Thrombophilia; Receiver operating characteristics; Test utility; Test methodology) Am J Clin Pathol 1996; 106: Testing for this syndrome can be performed by an in vitro assay of resistance to activated protein C, or by isolating genomic DNA of patients, amplifying and analyzing the area ofthe mutation by restriction enzyme digestion or by oligonucleotide hybridization " 15 The most commonly used test for APC resistance is a partial thromboplastin time (PTT)-based test, reported as the ratio of a PTT performed in the presence of a standard quantity of APC divided by the PTT without APC added. It has been shown that most healthy individuals will have a ratio of >2, whereas those with APC resistance were defined as having a ratio < 2. 4 Because of its simplicity this test has been widely employed as a screening test for APC resistance. The definitive test for this syndrome is molecular diagnosis of factor V Leiden. 6 Determination of factor V genotype is performed by isolating genomic DNA from patient leukocytes. The DNA sequenceflanking the mutation site in the factor V gene is then amplified using the polymerase chain reaction (PCR), and the resultant product analyzed by restriction enzyme digestion. 6 In this way, patients can be classified as wild type (two normal factor V alleles), heterozygous for factor V Leiden or homozygous for factor V Leiden. There is limited information on the sensitivity and specificity and performance ofthe APC resistance test in identifying patients with factor V Leiden. We studied the 107

2 108 COAGULATION AND TRANSFUSION MEDICINE results of APC testing and factor V genotype in 130 patients referred for evaluation of thrombosis. Specimen Collection MATERIALS AND METHODS One hundred thirty sequential patients referred for APC and factor V Leiden testing were studied. Patients were excluded if they had a prolonged baseline PTT or were on heparin or Coumadin therapy. Activated protein C resistance and factor V Leiden determinations were made on all patients. For the APC resistance test, patient samples of venous blood were drawn into tubes containing 3.8% sodium citrate and kept on ice. Following centrifugation at 3,000 rpm for 10 minutes at 4 C twice, the platelet-poor plasma was aliquoted and stored at 20 C until use. At the time of testing, specimens were rapidly thawed at 37 C. For factor V Leiden, patient blood was drawn into tubes containing EDTA and refrigerated at 4 C before DNA extraction. APC Resistance Assay Activated protein C resistance was assessed using a commercially available kit (Coatest APC resistance, Chromogenix AB, Sweden) according to the manufacturer's instructions using a fibrometer (Dataclot, Helena Laboratories, Beaumont, TX). Normal and abnormal controls were performed before testing patients. All tests were performed in duplicate. This activated partial thromboplastin time (aptt)-based test is reported as an APC ratio: clotting time with added APC/clotting time without APC. Normal range was defined by the manufacturer as a PTT ratio > 2. Factor V Leiden Assay Genomic DNA was isolated from patient leukocytes (QIAamp Blood Kit, Qiagen) collected in EDTA tubes. A 267 base pair (bp) fragment of the factor V gene flanking the factor V Leiden mutation sit at position 1691 was amplified as described by Bertina and colleagues 6 with the following modifications: 50 /xl of a mixture containing 10 mm Tris-Hcl, ph 8.3, 50 mm KC1, 1.5 ram MgC12,0.001% gelatin, 1.25 U of Taq DNA polymerase, ng genomic DNA and 12.5 pm of each primer was heated to 80 C, then 0.8 mm final concentration of each nucleoside triphosphate was added. The mixture was heated to 95 C for 3 minutes and then subjected to 30 cycles of 94 C (1 minute), 55 C (1 minute) and 72 C (1 minute), followed by 72 C for 7 minutes using a microprocessor controlled thermal cycler (Perkin Elmer Cetus). The 267 bp amplification product was digested with Mnl I for 60 minutes at 37 C and the resulting fragments separated by electrophoresis in a 3% agarose gel. Under these conditions wild type factor V (1691G) yields a 167 bp fragment and factor V Leiden (1691 A) yields a 200 bp fragment. On this basis, patients can be classified as wild type factor V (only a 167 bp band is visualized), heterozygous for factor V Leiden (167 bp and 200 bp fragments are present), or homozygous for factor V Leiden (only a 200 bp band is present). Statistical Analysis Sensitivity, specificity and ROC curves were calculated using the LABROC1 program (Dr. Charles E. Metz, Department of Radiology, University of Chicago), which makes a maximum likelihood estimation of a binomial ROC curve from the continuous APC ratio data. Patients were divided into true positive and true negative groups on the basis of the factor V Leiden results, which constituted the gold standard in this analysis. Predictive value of a negative test was calculated as (true negatives)/ (true negatives + false negatives) X 100. Patient Characteristics RESULTS One hundred thirty consecutive patients were studied. All were referred for evaluation of thrombosis except for eight individuals who were asymptomatic family members of patients with factor V Leiden. The median age was 44 (range 5-87 years) 55% were female and 45% were male. Of the 130 patients studied, 26 (20%) were heterozygous for factor V Leiden and 4 (3.1%) were homozygotes. Three patients (2.3%) had an APC ratio < 2 and a normal factor V genotype. The mean APC ratio for healthy subjects was 3.00 (standard deviation [SD] 0.78); that of heterozygotes was 2.13 (SD 0.53) and homozygotes 1.19 (SD 0.11). The median age of the factor V Leiden-positive patients was 43. The median age of factor V Leiden-negative patients was 45. Of the eight asymptomatic family members tested, four were heterozygous for factor V Leiden. The mean APC ratio of these individuals was 2.1 (range ; mean of heterozygotes 2.05, range ; mean of healthy subjects 3.2, range ). Distribution of APC Ratios in Patients With and Without Factor V Leiden As shown in Figure 1, there was substantial overlap in APC ratios in patients with and without factor V Leiden. A.J.C.P. -July 19%

3 ZEHNDER AND BENSON 109 Utility ofapc Resistance Assay All Patients Excluding Factor V Leiden Homozygous FlG. 1. Results of the activated protein C resistance assay in all patients tested. Foreground: patients documented to have factor V Leiden (G1491 A). Background: patients with normal factor V genotype. Fifty-four percent of patients (14 of 26) who were heterozygous for factor V Leiden had APC ratios > 2. ROC Curves for the APC Ratio Test The ROC curves for all patients and patients not on anticoagulant therapy is shown in Figure 2. The area under the curve is with estimated SD for all patients and with estimated SD excluding the four patients who were homozygous for factor V Leiden. By this criterion, the test falls into a range of intermediate utility (>0.9 being highly useful, <0.7 being of little utility). 16 " 18 Analysis of Critical Cut-off Values Figure 3 demonstrates APC ratio test sensitivity and specificity as a function of test cut-off critical values for all 130 individuals studied. At the manufacturers recommended cut-off of 2.0, the test is highly specific (98%) but has only 50% sensitivity. Increasing the cutoff to 2.5 increases the sensitivity to 75%, but at a cost of decreased specificity (also 75%). The predictive value of a negative test result was 88% at a cut-off of 2; 92% at a cut-off of 2.5; 97% at a cut-off of 3.0; and 100% at a cut-off of A False Positive Fraction (1-Specificity) FIG. 2. Receiver operating characteristic (ROC) curves for the activated protein C resistance assay in identifying patients with factor V Leiden. Squares: all patients; diamonds: excluding factor V leiden homozygotes. The area under the curve is with estimated standard deviation (SD) for all patients and with estimated SD excluding the four patients who were homozygous for factor V Leiden. The ROC curve for the APC resistance assay is characteristic of an intermediate utility test (>0.9 being highly useful, <0.7 being of little utility) D Sensitivity -O Specificity DISCUSSION We examined the performance of a standard APC resistance assay in correctly identifying 130 patients referred for evaluation of thrombosis for the presence of the factor V Leiden mutation. This molecular defect is present in approximately 90% of patients with resistance to activated protein C." The incidence of heterozygosity APC Ratio Cutoff Value FIG. 3. Test cut-off value analysis. Sensitivity and specificity as a function of the cut-off value used in the APC resistance assay are plotted. At the manufacturer's recommended cut-off of 2.0, the test is highly specific (98%) but has only 50% sensitivity. Increasing the cut-off value increases the sensitivity at a cost of decreased specificity. Vol. 106-No. I

4 110 COAGULATION AND TRANSFUSION MEDICINE in our population was 20%, in close agreement with the incidence of this disorder in other unselected series of thrombosis patients.'' Although only four patients were homozygotes for factor V Leiden, the APC resistance test appears to be very sensitive and specific for detecting these individuals, with a mean ratio of However, most patients with factor V Leiden are heterozygotes, and in this group the APC ratios demonstrate a significant overlap with the normal range, resulting in a low sensitivity (50%) at the recommended cut-off value of 2 and the test ROC curve in the intermediate utility range. 16 Increasing the cut-off increases the sensitivity of the APC resistance test, but has the undesirable effect of increasing the number of false-positive tests as indicated by the decrease in specificity depicted in Figure 3. Inspection of Figures 1 and 3 reveals that at a ratio of >3.0, the diagnosis of factor V Leiden could be excluded with a high degree of certainty (predictive value of a negative test result = 97% for all patients). Thus, the standard APC resistance test is useful in identifying the small number of factor V Leiden homozygotes and in excluding the diagnosis of factor V Leiden in the 36% of healthy subjects with a ratio of >3.0. However, the test does not clearly discriminate between factor V Leiden heterozygotes and healthy subjects. This leaves the majority of patients (64% of healthy subjects and 54% of those with factor V Leiden; 60% overall) in a gray area between a ratio of , requiring a confirmatory test, the molecular diagnosis of factor V Leiden. As such, the APC resistance assay in its present form is not a useful screening test. It is possible that variants of the PTTbased assay using normalized ratios may more accurately separate heterozygotes from healthy subjects Recently, a tissue factor-dependent factor V assay has been developed and reported to reliably separate factor V Leiden heterozygotes from healthy subjects; 19 PTTbased assays diluting the patient plasma into factor V deficient plasma are reported to have less overlap between healthy subjects and factor V leiden heterozygotes as well as reliably test individuals on oral anticoagulants Until such a test is available and performance validated, given the overlap of APC ratios in factor V Leiden heterozygotes and healthy subjects using the first-generation Chromogenix APC resistance assay, it is important to document factor V Leiden status of patients with APC ratios in the gray area of with molecular diagnostic testing. An alternative strategy is to perform the factor V Leiden molecular test initially in patients suspected of inherited thrombophilia. In this study, 20% of patients were found to have the mutation. As the factor V Leiden test is by definition the standard, the sensitivity of the test is 100%, assuming that both chromosome 1 factor V alleles are amplified in all patients. This test has additional advantages over the APC resistance test of not being affected by Coumadin therapy or prolonged baseline PTTs in patients on heparin or those with lupus anticoagulants. A disadvantage of the molecular test in its current form is that it is labor intensive. However, automation of PCR-based analysis systems or single base mismatch detection hybridization systems on ELISA plates promise to streamline this type of analysis in the future and make these tests better suited for screening. 1 ' 2 " Another disadvantage of molecular testing for factor V Leiden would be the failure to identify the 10% to 20% of patients with resistance to APC who have a normal factor V genotype. The significance of APC resistance in the setting of a normal factor V genotype is unclear at present. To date, no mutations at the other APC cleavage site of factor Va or in factor Villa have been described to account for APC resistance in these patients. It is possible that some of these patients will have resistance to APC on the basis of an as yet undefined mechanism. As the risk of thrombosis and the mechanism(s) of thrombosis if present are unknown at in these patients, they remain an intriguing and important population to identify both for clinical and research purposes. Acknowledgments. The authors thank Dr. P. Joanne Cornbleet for her critical review of the manuscript. REFERENCES 1. Esmon CT. The roles of protein C and thrombomodulin in the regulation of blood coagulation. J Biol Cham 1989:264: Esmon CT. Molecular events which control the protein C anticoagulant pathway. Thromb Haemost 1993;70: Heijboer H, Brandjes D, Buller HR, Sturk A, ten Cate JW. 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