A multi-laboratory assessment of congenital thrombophilia assays performed on the ACL TOP 50 family for harmonisation of thrombophilia testing in a large laboratory network.

OBJECTIVES: Thrombophilia testing is commonly performed within hemostasis laboratories, and the ACL TOP 50 family of instruments represent a new 'single platform' of hemostasis instrumentation. The study objective was to evaluate these instruments and manufacturer reagents for utility of congenital thrombophilia assays. METHODS: Comparative evaluations of various congenital thrombophilia assays (protein C [PC], protein S [PS], antithrombin [AT], activated protein C resistance [APCR]) using newly installed ACL TOPs 550 and 750 as well as comparative assessments with existing, predominantly STAGO, instrumentation and reagents. Verification of manufacturer assay normal reference ranges (NRRs). RESULTS: HemosIL PC and free PS assays showed good comparability with existing Stago methods (R>0.9) and could be considered as verified as fit for purpose. HemosIL AT showed high relative bias with samples from patients on direct anti-Xa agents, compromising utility. Manufacturer NRRs for PC, PS and AT were verified with minor variance. Given the interference with direct anti-Xa agents, an alternate assay (Hyphen) was evaluated for AT, and the NRR also verified. The HemosIL Factor V Leiden (APC Resistance V) evidenced relatively poor performance compared to existing assays, and could not be adopted for use in our network. CONCLUSIONS: This evaluation of HemosIL reagents on ACL TOP 50 family instruments identified overall acceptable performance of only two (PC, free PS) of four thrombophilia assays, requiring use of third-party reagents on ACL instruments for the other two assays (AT, APCR).

Simple Laboratory Test Utilization Interventions to Reduce Inappropriate Specialty Coagulation Testing.

OBJECTIVES: The naming convention in coagulation may cause confusion in electronic ordering systems, leading to inappropriate test orders. We implemented test utilization efforts and studied utilization before and after interventions for two specialty coagulation assays. METHODS: Two interventions were implemented: test names were changed from factor assay to activity, and residents reviewed all factor V and X requests. A retrospective review of factor V and X activity orders was performed for the period 1 year before and after interventions. RESULTS: After interventions, factor V and X activity orders decreased by approximately 40%. Resulted tests decreased by 53.8% and 47.8%, corresponding to reductions of $2,493.05 and $1,867.80 per year in laboratory charges for factor V and factor X activity, respectively. Abnormal factor V activity results increased from 45% to 59%. Factor V activity orders from outpatient clinics decreased by 21.6%. CONCLUSIONS: Simple interventions can reduce inappropriate specialty coagulation test orders and unnecessary costs.

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.

Risk assessment of recurrence in patients with unprovoked deep vein thrombosis or pulmonary embolism: the Vienna prediction model.

BACKGROUND: Predicting the risk of recurrent venous thromboembolism (VTE) in an individual patient is often not feasible. We aimed to develop a simple risk assessment model that improves prediction of the recurrence risk. METHODS AND RESULTS: In a prospective cohort study, 929 patients with a first unprovoked VTE were followed up for a median of 43.3 months after discontinuation of anticoagulation. We excluded patients with a strong thrombophilic defect such as a natural inhibitor deficiency, the lupus anticoagulant, and homozygous or combined defects. A total of 176 patients (18.9%) had recurrent VTE. Preselected clinical and laboratory variables (age, sex, location of VTE, body mass index, factor V Leiden, prothrombin G20210A mutation, D-dimer, and in vitro thrombin generation) were analyzed in a Cox proportional hazards model, and those variables that were significantly associated with recurrence were used to compute risk scores. Male sex (hazard ratio versus female sex 1.90, 95% confidence interval 1.31 to 2.75), proximal deep vein thrombosis (hazard ratio versus distal 2.08, 95% confidence interval 1.16 to 3.74), pulmonary embolism (hazard ratio versus distal thrombosis 2.60, 95% confidence interval 1.49 to 4.53), and elevated levels of D-dimer (hazard ratio per doubling 1.27, 95% confidence interval 1.08 to 1.51) were related to a higher recurrence risk. Using these variables, we developed a nomogram that can be used to calculate risk scores and to estimate the cumulative probability of recurrence in an individual patient. The model was cross validated, and patients were assigned to different risk categories based on their risk score. Recurrence rates corresponded well with the different risk categories. CONCLUSIONS: By use of a simple scoring system, the assessment of the recurrence risk in patients with a first unprovoked VTE and without strong thrombophilic defects can be improved.

Near real-time immuno-optical sensor for diagnosing single point mutation: a model system: sensor for factor V Leiden diagnosis.

Factor V leiden (FVL) is an abnormality of factor V (FV), a blood coagulation factor. It is a hereditary blood coagulation disorder with a high frequency (3-7% of general population). The most common type of FVL is caused by a single amino acid mutation and, therefore, its diagnosis is currently done only by DNA analysis, which takes a long time and is expensive. We have developed a rapid, accurate, and cost-effective, sandwich immuno-optical sensing method. To produce monoclonal antibodies against FV or FVL, having minimal cross-reactivity with the other molecule, a 20 amino acid sequence (20-mer) of FV or FVL at around the mutation site was utilized. The antibodies were screened first with the 20-mers and then the ones showing no cross-affinity were reacted with native FV or FVL molecules and they showed some cross-reactivity. Using two antibodies having strongest affinity to either FV or FVL molecule, a FV and a FVL preferred sensors, were produced. After verifying that the levels of the antibody affinity to the two different molecules remained constant with changes in analyte concentration, a two-sensor system is developed to quantify FV and FVL in plasma samples. The system quantified the levels of FV and FVL at the maximum error of 0.5 microg/ml-plasma, in their physiological concentration range of 0-12 microg/ml-plasma. The levels of both molecules may provide us whether the patient has FVL or not but also the seriousness level of the disease (homozygous and different level of heterozygous).

Biosensor for diagnosing Factor V Leiden, a single amino acid mutated abnormality of Factor V.

Factor V Leiden (FVL) is an abnormality with a single amino acid mutation of Factor V (FV) and is the most common, hereditary blood coagulation disorder. FVL is currently diagnosed by DNA analysis, which takes a long assay time, high cost, and a specially trained person. We are developing a rapid, accurate, and cost-effective biosensing system to quantify both FV and FVL in blood plasma, to diagnose FVL and also to evaluate the seriousness of the disease status. This system is based on a sandwich immuno-reaction on an optical fiber. To produce the monoclonal antibody against only FV or only FVL without cross-reacting with the other molecule and with a higher probability, a 20 amino acid sequence (20-mer) of FV or FVL around the mutation region was injected into mice and then hybridoma cell lines specific to each 20-mer were selected. When these antibodies were tested with native FV or FVL molecules, they were found to be cross-reacting with the other molecules, but some with higher affinity to FV (FV preferred) and some to FVL (FVL preferred). Using these antibodies, two different sensors were developed: FV preferred and FVL preferred sensors. These two sensors allowed us to quantify FV and FVL in plasma with a maximum error of 4%. The plasma levels of both molecules provide us not only FVL diagnosis but also the level of the seriousness. The same principles may be used for developing diagnostic tools for other diseases with a single point mutation.

Assessment of the role of thrombin activatable fibrinolysis inhibitor in retinal vein occlusion.

PURPOSE: To determine whether elevated levels of thrombin activatable fibrinolysis inhibitor (TAFI) may contribute to thrombotic risk for patients with retinal vein occlusion (RVO) and to investigate the possible correlations between TAFI activity level and other conventional risk factors. METHODS: Ninety patients with RVO (cases), except those receiving medication affecting the study parameters, those undergoing a surgical procedure within the last week, and those with kidney and/or liver failure, were enrolled in the study. The control group included similar patients matched for age and sex. After written informed consent was obtained, parameters including TAFI activity levels, conventional risk factors, results of routine hematological examination, and factor V Leiden and prothrombin G20210A mutations were evaluated by analysis of blood samples obtained after an 8-hour fast. RESULTS: Although TAFI activity levels were slightly elevated in cases (190.5 +/- 43.8) compared with controls (183.9 +/- 41.8), the difference was not statistically significant (P = 0.36). According to evaluation of TAFI activity in subgroups (>200%, 150-200%, and 0-150%), 36.7% with central RVO, 40.0% with branch RVO, and 30% of controls were found to have TAFI activity of >200% (P = 0.83). TAFI activity levels did not correlate with age, sex, demographics, clinical status, and hematological variables. Finally, in stepwise regression analysis, TAFIa (carboxypeptidase U) activity was not found to be an important risk factor for RVO. CONCLUSION: On the basis of these data, TAFI activity was not found to be a new risk factor for either type of RVO. However, further larger studies may better identify the exact role of TAFI in the pathogenesis of RVO.

Variability in factor V:C assays in UK National External Quality Assessment Scheme surveys: there is a need for an international standard.

Severe familial factor V:C deficiency is a rare, recessively inherited coagulation disorder but there is little information in respect of the accuracy and reliability of factor V:C assays that are required for diagnosis and treatment monitoring. We present here the results of three External Quality Assessment exercises in respect of factor V:C assays undertaken by 192--225 participating laboratories performed over a 2-year period. Consistent significant differences were observed between results obtained using different reference plasmas and different thromboplastins. The relationship between results obtained with different reference plasmas was not constant and varied between the surveys. In-house studies confirmed the observation derived from the External Quality Assessment surveys that the choice of commercial reference plasma significantly affects the results of factor V:C assays. These results clearly indicate the necessity for an international standard for factor V:C.

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.

Simultaneous detection of FV Q506 and prothrombin 20210 A variation by allele-specific PCR.

BACKGROUND AND OBJECTIVE: Factor V Leiden is the most important risk factor for hereditary thromboembolism, whereas the mutation in the 3'-untranslated region of the prothrombin gene seems to be only a mild risk factor for thrombotic events. On the other hand the factor V mutation (Arg 506) is frequently coinherited with the prothrombin 3'-untranslated region G20210A variant and there is increasing evidence that the co-segregated prothrombin variant is an additional risk factor for venous thromboembolism, contributing to thrombotic manifestations. A rapid, simple and cost-effective screening method is, therefore, required for the detection of both factor V Leiden and the prothrombin variant A20210G. DESIGN AND METHODS: Eighty-eight patients were enrolled in this study. Forty-four had a previously identified factor V and/or prothrombin mutation, the remaining 44 patients served as negative controls. A multiplex allele specific oligonucleotide PCR was established for the simultaneous detection of the two genetic risk factors for thrombophilia. To test the specificity of the simultaneous ASO PCR approach, the mutated and physiological factor V and prothrombin amplification products were sequenced. RESULTS: The factor V Leiden mutation and the prothrombin variant were correctly identified in all of 44 patients with known mutations. Furthermore the test was able to detect the mutated factor V and the II variant alone, as well as in the cosegregated pattern. Five patients with a homozygous pattern of factor V Leiden or prothrombin variant were also correctly identified. The sensitivity of the test is therefore 100%. In none of the 44 control cases were false positive results seen. INTERPRETATION AND CONCLUSIONS: The ASO PCR test is a rapid, simple and cost-effective screening test for thrombophilia.

Activated protein C resistance assay when applied in the general population.

We estimated the predictive values of activated protein C resistance in the diagnosis of women who are carriers of factor V Leiden. When the prevalence of factor V Leiden is 2%, the positive predictive value is 44% and the cost of preventing one thromboembolic death is $44,180,000. Thus the activated protein C resistance assay is not cost-effective in ruling out factor V Leiden in this population.

Assessment of coagulation factor activation during cardiopulmonary bypass with a new monoclonal antibody.

Antithrombin-III (AT) is a key inhibitor of blood coagulation that neutralizes activated serine esterases by forming covalent modified complexes (ATm). A new monoclonal antibody directed against short-lived AT-activated serine protease complexes provides a means of measuring subclinical coagulation activity during cardiopulmonary bypass (CPB). Twelve patients undergoing CPB for coronary artery bypass grafting were studied and AT, ATm, D-dimers (DD), and several other coagulation and fibrinolytic markers were measured during the surgical procedure. There were decreases in AT, factors V, II, X, IX, protein S (total and free), C4b-binding protein, thrombomodulin, and platelets counts, whereas heparin, ACT, thrombospondin, plasminogen activator inhibitor (PAI-1), and tissue plasminogen activator (tPA) increased. ATm and the percentage of ATm available (ATm/AT) showed a peak during CPB. These results demonstrate that during CPB, the use of heparin produces an equilibrium involving increased coagulation activation and consumption in association with increased fibrinolysis. The equilibrated consumption of both coagulation and fibrinolytic factors leads to low levels of all factors after cardiac surgery. The ATm assay allows assessment of the differential effects of CPB and surgical trauma on coagulation activation. It is speculated that ATm levels may be useful in monitoring the consumption of coagulation factors.