Assessment of acquired activated protein C resistance with the FibWave and comparison with the ETP-based APC resistance.

INTRODUCTION: Activated protein C (APC) resistance is a major risk factor of venous thrombosis which may be acquired by hormonal therapy or other causes. The FibWave, a sensitive global clot-based assay design to analyze the coagulation kinetics in plasma, may be a good candidate to assess this prothrombotic state. This study aims to assess the suitability of the FibWave to differentiate the coagulation kinetics of women on oral contraceptives. MATERIALS AND METHODS: Fifty-four healthy volunteers were divided into 5 groups: men [n = 13], women not using hormonal contraception [n = 12], women using second [n = 12] or third generation [n = 12] combined oral contraceptives, and women using progestin only contraceptive [n = 5]. Patients with coagulation abnormalities were also assessed [n = 8]. The APC resistance was assessed on the FibWave using exogenous APC or Protac, and on the Calibrated Automated Thrombogram using the ETP-based APC resistance assay. RESULTS: Either in presence or in absence of APC or Protac, the FibWave was able to detect a hypercoagulable state in plasma samples. All combined oral contraceptives showed a lower FW-Max1 , FW-Max2, and FW-Min2 percentage of inhibition and a lower FW-Ttpeak ratio than the other groups. The sensitivity of the FibWave was similar to the one of the ETP-based APC resistance assay. CONCLUSION: The FibWave is able to differentiate APC resistance levels observed in women on combined oral contraceptive. The FW-Max1 , FW-Max2, and to a lesser degree FW-Min2 were identified as the most sensitive parameters with a similar performance to the ETP-based APC resistance assay.

Diagnostic Testing Approaches for Activated Protein C Resistance and Factor V Leiden: A Comparison of Institutional and National Provider Practices.

OBJECTIVES: To analyze the economic impact of testing for activated protein C resistance (APC-R) due to factor V Leiden (FVL) mutation with APC-R with reflexive FVL genotyping (algorithmic approach) or genotyping alone. METHODS: OptumLabs Data Warehouse (OLDW) data were used to assess testing approaches. Insurance claims for APC-R and FVL in 2013 were compared with the Mayo Clinic database. Centers for Medicare & Medicaid Services diagnostic fee schedules were used to assign costs. RESULTS: Of 19.3 million OLDW-covered individuals, 74,242 (0.385%) received 75,608 tests: APC-R, 2,265 (2.9%); FVL genotyping, 70,619 (90.1%); and both APC-R and FVL, 2,724 (7.0%). In total, 1,317 tests were performed at Mayo Clinic: APC-R with reflex FVL (1,256; 95.4%) and FVL alone (61; 4.6%). Costs per evaluated individual and per total population (person/year) in OLDW and algorithmic approach were $83.77 vs $36.38 and $0.32 vs $0.14, respectively. CONCLUSIONS: The cost-optimized algorithmic approach reduces health care costs.

Use of a systematic risk analysis method (FMECA) to improve quality in a clinical laboratory procedure.

BACKGROUND: Risk management is a set of actions to recognize or identify risks, errors and their consequences and to take the steps to counter it. The aim of our study was to apply FMECA (Failure Mode, Effects and Criticality Analysis) to the Activated Protein C resistance (APCR) test in order to detect and avoid mistakes in this process. METHODS: We created a team and the process was divided in phases and sub phases. For each phase we calculated the probability of occurrence (O) of an error, the detectability score (D) and the severity (S). The product of these three indexes yields the RPN (Risk Priority Number). Phases with a higher RPN need corrective actions with a higher priority. RESULTS: The calculation of RPN showed that more than 20 activities have a score higher than 150 and need important preventive actions; 8 have a score between 100 and 150. Only 23 actions obtained an acceptable score lower than 100. CONCLUSIONS: This was one of the first experience of application of FMECA analysis to a laboratory process, and the first one which applies this technique to the identification of the factor V Leiden, and our results confirm that FMECA could be a simple, powerful and useful tool in risk management and helps to identify quickly the criticality in a laboratory process.

Activated Protein C-Resistance Determination and Vascular Access Thrombosis in Populations with High Prevalence of Factor V Leiden.

BACKGROUND/AIMS: Factor V Leiden heterozygosity occurs in 3-8% of the general European and US populations. Activated protein C resistance (APC-R)--a non-molecular laboratory test--can efficiently demonstrate the presence of this mutation and can be performed on most coagulation analyzers. On the other hand, fistula or graft thrombosis is a common and costly complication in hemodialysis patients. Our aim was to establish the value of APC-R determination in hemodialysis patients by assessing the risk of access thrombosis in patients with increased APC-R. METHODS: A total of 133 patients (81 men, mean age 64.5 ± 14.9 years and 52 women, mean age 63.6 ± 15 years) were selected. Participants were divided into 2 groups: those with access thrombosis (54 patients, 40.6%) and those with no access thrombosis (79 patients, 59.4%), and they were tested for the most common congenital or acquired thrombophilia risk factors. RESULTS: Overall, 12 patients (9%) had an increased APC-R and 10 of them had at least 1 episode of access thrombosis (83.3%). Univariate analysis to estimate crude odds ratio (OR) showed an OR of 8.8 (95% CI 1.8-41.8) times higher risk for access thrombosis in these patients. No significant differences were found after adjusting for age, hypertension, diabetes mellitus, coronary artery disease, cerebrovascular disease, peripheral arterial disease and malignancy. Sex was also a factor influencing thrombosis, presenting a higher OR for women (OR 2.2, 95% CI 1.1-4.4). CONCLUSION: This study revealed a significant association between access thrombosis and increased APC-R in hemodialysis patients. This indicates that the determination of APC-R should be considered--especially, in populations with a high prevalence of Factor V Leiden--as proper anticoagulant therapy in these patients may reduce the risk of access thrombosis.

Activated protein C resistance assay and factor V Leiden.

The authors suggest that functional testing for activated protein C resistance is cheaper and more clinically relevant than genetic testing to detect a factor V Leiden mutation in identifying persons who are at risk for thromboembolism.

Estimating the cost of redundancy in molecular diagnostics: the case of activated protein C resistance and factor V Leiden.

BACKGROUND: The activated protein C resistance--sensitivity ratio in the presence of Factor V deficient plasma (APC-SR/Factor V) exhibits a high sensitivity for factor V Leiden mutation and has been proposed as the diagnostic approach of choice, as an alternative to genetic tests, to evaluate activated protein C resistance. A survey, including 4969 requests, was performed on the activity of a typical Molecular Diagnostics Laboratory in order to estimate the costs due to reagents, instrumentation and personnel. METHODS: The global costs of three hypothetical diagnostic approaches were compared: (A) exclusive molecular test for FV Leiden; (B) APC-SR alone; (C) APC-SR and the exclusive confirmation of positive results with molecular test. RESULTS: The global cost for each patient with the three approaches investigated were respectively 42.20 euros (A), 1.09 euros (B), and 433 euros (C). The cost for finding a patient with factor V Leiden mutation was 549.00 euros for A, 14.18 euros for B, and 56.32 euros for C. It was calculated that a decrease of 97.42% and 89.74% can be obtained using the approaches B and C, respectively. The difference in cost between B and C can be justified by the avoidance of false positive cases (6%) and by the impossibility of distinguishing homozygous from heterozygous patients using APC-SR exclusively (B). CONCLUSIONS: In the case of suspected phenotype APC resistance, we suggest a laboratory approach, which provides the combined and sequential use of ProCGlobal/FV analysis and a subsequent genetic test for positive patients.

Activated protein C resistance, factor V Leiden and assessment of thrombotic risk.

Venous thromboembolism comprises deep vein thrombosis (DVT) and pulmonary embolism (PE). Acute venous thromboembolism (VTE) is a serious and potentially fatal disorder which often complicates the course of hospitalized patients, but also affects ambulatory and otherwise healthy people. The annual incidence of venous thromboembolism is 1 to 2 cases per 1000 person and the risk of the disorder rises exponentially with age, from an annual rate of less than 5 per 100,000 children to greater than 400 per 100,000 adults older than 80 years.

Comparison of Russell viper venom-based and activated partial thromboplastin time-based screening assays for resistance to activated protein C.

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.

Screening with the activated protein C resistance assay yields significant savings in a patient population with low prevalence of factor V leiden.

Testing for factor V Leiden can be performed with a molecular assay or a test for activated protein C resistance. We noted that physicians in our institution tended to order the molecular test 80% of the time, but the prevalence of the mutation in our patient population was less than 10%. Consequently, we decided to introduce the activated protein C resistance assay in house and consistently use it for screening before the more expensive genetic test and to negotiate a discounted charge for the latter at a reference laboratory. After 6 years since these interventions began, the prevalence of an abnormal screening test result remained low (202/2,475 [8.2%]), even among white patients (10.9%). With this simple approach, the cost to test patients for factor V Leiden decreased by more than 90%, while the productivity of our laboratory increased by the introduction of a high-volume, fully automated assay.

Multilaboratory testing in thrombophilia through the United Kingdom National External Quality Assessment Scheme (Blood Coagulation) Quality Assurance Program.

We describe here results from the United Kingdom National External Quality Assessment Scheme (UK NEQAS) Thrombophilia Screening Program, in which an average of 21% of 280 centers reported an incorrect diagnosis for a series of plasma samples. Three case studies are described, showing causes of error in individual laboratories, related to the source of reference plasma or reagents. Methodological bias is also described. For protein C (PC) assays 18% of centers reported PC deficiency in a patient homozygous for factor V Leiden. Studies in the NEQAS laboratory confirmed the effect of activated protein C resistance (APCR) on clot-based PC activity assays. Differences in results obtained for PS-deficient subjects with different protein S (PS) activity kits are reported; several subjects would be misdiagnosed as normal with one kit if the manufacturer's reported reference range was adopted instead of a locally determined reference range. Antithrombin (AT) assays were shown to vary in their sensitivity to different molecular defects in the antithrombin gene; 77% of centers employing human thrombin-based activity assays reported a normal AT level in a patient with antithrombin Cambridge II. Sensitivity of the APC resistance test in the absence of factor V-deficient plasma was shown to be improved through normalization of results, and errors in the genetic diagnosis of factor V Leiden and the P20210A prothrombin gene mutation are described. Errors in the diagnosis of thrombophilic defects can therefore be identified through participation in EQA programs, and following dissemination of information, improvements in diagnosis can be demonstrated.

A 9-year retrospective assessment of laboratory testing for activated protein C resistance: evolution of a novel approach to thrombophilia investigations.

AIMS: To assess international and local trends in laboratory testing for activated protein C (APC) resistance (APCR) and factor V Leiden (FVL). Also, to compare local results of FVL testing with a variety of different clot-based APCR assays to assess utility for detection of APCR both related and unrelated to FVL. METHODS: Local test statistics and test result patterns were evaluated and international literature was reviewed over the past 9 years. Direct comparisons of FVL testing by DNA analysis against (a) the standard APTT-based APCR assay, with and without pre-dilution with factor V deficient (FVD) plasma, or with and without normalisation, and (b) three alternative RVVT-based procedures (most recently using a commercial RVVT-based procedure called GradiLeiden V; GLV). In total, data obtained over the past 7 years, using referred samples from over 1,000 patients, have been assessed. RESULTS: The 9-year retrospective assessment has seen many changes in test-based processes. Locally, test requests for both APCR and FVL have consistently increased. We suspect this has been fuelled in part by media reports of 'economy class syndrome' (ECS) and associated general public and clinical concern. Current request patterns number around 800 APCR and 1,600 FVL per year. Interestingly, most requests are for one or either test, with joint requests comprising less than 20% of those overall. Although test requests are increasing, detection of the FVL defect as a proportion of test requests is actually falling (from a high of over 25% in 1996 to around 14% currently). Whether this suggests an increasing tendency for clinical ordering in the absence of appropriate clinical histories is a matter of concern. Consistent with previous findings, the original and commonly used APTT-based procedure was found to show the least correlation with DNA findings, with a large overlap between FVL and non-FVL individuals. The alternate-RVVT-based procedures showed much better differentiation. Thus, for the APTT-based method, in order to ensure 100% sensitivity, an APC ratio cut-off value of 3.1 was required, and this yielded only 49.1% specificity. In contrast, for the GLV RVVT-based method, in order to ensure 100% sensitivity, an APC ratio cut-off value of 1.65 was required, and this yielded 96.6% specificity. CONCLUSIONS: It is important to recognise the limitation of APTT-based assays to discriminate FVL. However, a combination of RVVT- and APTT-based testing is still recommended, as this will provide excellent discriminatory power for the FVL defect, particularly negative prediction, in addition to detection of potential APCR unrelated to FVL, as well as detection of other potential haemostatic disturbances. Accordingly, we detail strategies, including a test algorithm that we are currently using to improve our detection of APCR and prediction of FVL, and use of clotting-based procedures as the first-line approach.

Screening for activated protein C resistance before oral contraceptive treatment: a pilot study.

The feasibility and cost-effectiveness of screening women for congenital thrombophilic alterations before oral contraceptive (OC) treatment was investigated. A total of 525 women (mean age 21.9 years, 73% aged < 25 years) were examined before their first OC course. At first screening, completely normal results were recorded in 485 (92.4%) women, the remaining showing single (n = 34) or multiple (n = 6) alterations. At second examination (possible in 37 of 40), activated protein C resistance (APCR) was confirmed in 21 cases (4.0%, 18 with factor V Leiden), protein C, or protein S reduction in 8 (1.5%) and 2 (0.4%) cases, respectively. No cases with antithrombin III deficiency were detected. The global estimated cost ($US) to detect one altered case was: $7795 for protein S, $2696 for antithrombin III (no case found), $1374 for protein C and $433 for APCR. The present study confirms that extensive thrombophilic screening before OC treatment is not currently advisable. APCR assessment, however, seems to have a favorable cost-effectiveness ratio: the alteration is frequent and has a synergistic effect with OC; sensibility and specificity of some methods are good; family history is unreliable to single out possible carriers; finally, carriers can be fully informed of their increased thrombotic risk if treated with OC and can receive thromboprophylaxis during life situations associated with high thrombotic risk (e.g., pregnancy and puerperium).

PIP: This article investigates the feasibility and cost effectiveness of screening women for congenital thrombophilic changes before oral contraceptive (OC) treatment. The study population included 525 women who were examined before their first OC course between September 1995 and May 1997 in Bologna, Italy. A completely normal result was seen in 92.4% women during the first screening, which was conducted before the first OC course. The second examination showed that activated protein C resistance (APCR) was confirmed in 21 cases (4.0%, 18 with factor V Leiden), and protein C and protein S reduction in 8 (1.5%) and 2 (0.4%) cases, respectively. Antithrombin III deficiency cases were not detected. The detection of one altered case is estimated to cost $7795 for protein S, $2696 for antithrombin III, $1374 for protein C, and $433 for APCR. The study confirmed that extensive thrombophilic screening before OC treatment was not advisable. However, APCR assessment was found to be cost-effective. The alteration was frequent and APCR had a synergistic effect with OC, and the sensibility and specificity of some methods for detection of APCR are good. Family history is not reliable for identifying possible carriers for the thrombophilic trait. Carriers can be fully informed of their high risk if treated with OC and can receive thromboprophylaxis in conditions where thrombotic risk is high.