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

Abstract

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. ReActivated 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). "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. 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. - 613" 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 sequence flanking 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 ...

Whole Blood Screening Test for Factor V Leiden Using a Russell Viper Venom Time–Based Assay

“…Increasing the threshold value to 2.5 increases the sensitivity to 0.75 but at the expense of a decrease in specificity (0.75). 19 The predictive value of a negative test result was 0.88 at a cutoff of 2.0; 0.92 at a cutoff of 2.5; 0.97 at cutoff of 3.0; and 1.0 at a cutoff of 3.5. 19 Essentially similar data were obtained in another study.…”

“…19 The predictive value of a negative test result was 0.88 at a cutoff of 2.0; 0.92 at a cutoff of 2.5; 0.97 at cutoff of 3.0; and 1.0 at a cutoff of 3.5. 19 Essentially similar data were obtained in another study. 26 The low sensitivity of the APTT ratio might be explained in part by the confounding effect of high levels of factor VIIFC, which could antagonize the anticoagulant action of APC, 26 resulting in a lesser degree of prolongation of the APTT and, thus, a ratio near or below the lower threshold in a non-FV Leiden sample.…”

“…However, the sensitivity of this test in identifying the patients with FV Leiden has been disappointing. Zehnder and Benson, 19 using a threshold ratio of 2.0, reported a sensitivity of only 0.5. Therefore, other screening tests based on the prothrombin time, 20 Russell viper venom (RVV) time, 21 modified APTT using FV-deficient plasma, 2 2 or a chromogenic assay 2 3 have been developed and shown to have superior sensitivity (Fig l).…”

Comparison of two methods for factor five Leiden detection in patients with thromboembolism

“…But the heterozygotes had a sensitivity of 50% and specificity of 98% because of overlap with normal individuals and due to this overlap, APCR rates of 2 to 3 need other confirming tests [6]. Nevertheless, this overlap is entirely eliminated by the means of dilution with FV-deficient plasma [13].…”

“…Since the mutation of Factor V Leiden is one of the most common factors for Thrombophilia especially in young children and pregnant women, we should consider factor V Leiden detecting tests in DVT patients. There are two common tests in laboratories for detection of factor V Leiden mutation consisting of Activated Protein C Resistance (APCR) and molecular analysis and detection of the mutation by the use of restrictive enzymes, RFLP-PCR in particular [6]. Although PCR-RFLP is the Gold Standard test for detection of Factor V Leiden mutation, it cannot be performed in any regular laboratory and its more expensive and time consuming than APCR test, we performed …”