F2c.*C20209T mutation in patients with a history of thrombosis: A case report, retrospective 2 site-results and review of the literature.

INTRODUCTION: G20210A (c.*97G>A) prothrombin gene variant, found in white population has been associated with an increased risk of venous thromboembolism (VTE). Other rare polymorphisms in F2 gene (C20209T) have been reported, more rare and touching black people, but its potential association with VTE remain uncertain. METHODS: About a 69 years-old Caucasian woman presenting an unprovoked deep venous thrombosis of the leg, we analyzed retrospectively 25.000 thrombophilia tests on a 11-year period of time (2007-2018), at Nice and Marseille University Hospitals, and performed extensive review of the literature. RESULTS: Genetic determination included a similar PCR protocol and sequencing. Twenty-one heterozygous cases out of 25.585 determinations (0.08%) was found. The C20209T mutation detected in our Caucasian patient is rare, with a frequency that differed from what was reported in the previous literature, mainly in non-Caucasian patients (Africans, Africans-Americans, and Caribbeans). One hundred and thirteen patients with this mutation have been described in the literature, of which only one homozygous. CONCLUSION: This study is the most important on C20209T mutation performed at present, allowing to precise its frequency and its potential role in venous thromboembolism.

APTT therapeutic range for monitoring unfractionated heparin therapy. Significant impact of the anti-Xa reagent used for correlation.

INTRODUCTION: Unfractionated heparin (UFH) therapy is monitored by using the anti-activated factor X (anti-Xa) activity, or the activated partial thromboplastin time (APTT), which remains the most widely used assay. One of the main advantages of anti-Xa relies on its hypothesized standardization, with a unique therapeutic range (0.30-0.70 IU/ml) for all reagents, whereas APTT is influenced by numerous preanalytical and analytical parameters not related to the anticoagulant activity of UFH. METHODS: The aim of this study was to compare the anti-Xa-correlated APTT therapeutic ranges calculated using different combinations of APTT (n = 4) and anti-Xa reagents (n = 4) in frozen citrated plasmas from 87 inpatients on UFH. RESULTS: The median APTT ratio ranged from 2.19 for the less sensitive to 3.23 for the most sensitive reagent, whereas the median anti-Xa activity was between 0.37 IU/ml and 0.57 IU/ml. The APTT therapeutic ranges calculated to correlate with anti-Xa activities between 0.30 and 0.70 IU/ml were found to be highly different from one combination of APTT reagent and analyzer to another. The same applied to the therapeutic range of a single APTT reagent calculated using different anti-Xa assays performed on the same analyzer, leading to a lack of agreement as to whether a sample was classified as subtherapeutic, therapeutic or supratherapeutic in 8.0% to 23.0% of the patients, with kappa coefficients between 0.908 and 0.753. CONCLUSIONS: These results suggest that the APTT therapeutic range calculated to correlate with anti-Xa activities between 0.30 and 0.70 IU/ml is influenced not only by the APTT reagent, but also by the anti-Xa reagent used for calculation.

Age-adjusted D-dimer cut-off levels to rule out venous thromboembolism in patients with non-high pre-test probability: Clinical performance and cost-effectiveness analysis.

BACKGROUND: As aging was found to be associated with increased D-dimer levels, the question arose whether D-dimer measurement was useful in the diagnostic strategy of venous thromboembolism (VTE) in elderly patients. AIM OF THE STUDY: To compare retrospectively the performance of six diagnostic strategies based on the three-level Wells scores and various cut-off levels for D-dimer, evaluated using the HemosIL D-Dimer HS 500 assay, in a derivation cohort of 644 outpatients with non-high pretest probability (PTP) of VTE. The clinical usefulness of the best-performing strategy was then confirmed in a multicenter validation study involving 1255 consecutive outpatients with non-high PTP. RESULTS: The diagnostic strategy based on the age-adjusted cut-off level calculated by multiplying the patient's age by 10 above 50 years was found to perform the best in the derivation study with a better sensitivity-to-specificity ratio than the conventional strategy based on the fixed cut-off level (500 ng/ml), a higher specificity and a negative predictive value (NPV) above 99%. Such an increase in test specificity was confirmed in the validation cohort, with the NPV remaining above 99%. Taking into account the local reimbursement rates of diagnostic tests, using this strategy led to a 6.9% reduction of diagnostic costs for pulmonary embolism and a 5.1% reduction for deep vein thrombosis, as imaging tests would be avoided in a higher percentage of patients. CONCLUSION: The diagnostic strategy of VTE based on the age-adjusted cut-off level for D-dimer in patients over 50 years was found to be safe, with NPV above 99%, and cost-effective.

Monitoring unfractionated heparin therapy: Lack of standardization of anti-Xa activity reagents.

INTRODUCTION: One of the main advantages of using anti-Xa instead of activated partial thromboplastin time in monitoring of unfractionated heparin (UFH) therapy relies on its hypothesized standardization, with a unique therapeutic range defined to be 0.30 to 0.70 IU/mL. The aim of the present study was to compare the inter-reagent agreement of anti-Xa activity. METHODS: Citrate tubes were obtained from 104 inpatients on UFH. Plasma samples were stored frozen in aliquots at -70°C before being shipped to three accredited coagulation laboratories to be evaluated for anti-Xa activity using their routine assay(s). Pooled normal plasmas spiked with dilutions of the 6th International Standard of UFH to achieve anti-Xa activities up to 1.0 IU/mL were evaluated using the same techniques. RESULTS: In the plasmas from patients on UFH, the median anti-Xa activity ranged from 0.37 IU/mL with one reagent to 0.57 IU/mL with another; results were in between (0.45 IU/mL) using two other reagents. Comparisons of results obtained using the different reagents demonstrated unacceptable bias up to 0.24 IU/mL between some reagents (41% difference). The concordance as whether anti-Xa activities measured using different reagents were within or outside the therapeutic range was between 0.411 and 0.939 (kappa). Similar discrepancy was demonstrated for anti-Xa activities when evaluating normal plasma spiked with the International Standard. A discrepancy of the same order of magnitude was demonstrated in the 2017 External Quality Assessment Program provided by the External Quality Control in Assays and Tests exercises. CONCLUSIONS: The reported discrepancy between test results obtained using different anti-Xa assays clearly suggests a lack of standardization of that assay with potentially significant impact on the patients' anticoagulation.

Age dependency for coagulation parameters in paediatric populations. Results of a multicentre study aimed at defining the age-specific reference ranges.

Understanding of developmental haemostasis is critical to ensure optimal prevention, diagnosis, and treatment of haemorrhagic and thrombotic diseases in children. As coagulation test results are known to be dependent on the reagents/analysers used, it is recommended for each laboratory to define the age-dependent reference ranges by using its own technical condition. That study was carried out in seven centers to establish age-specific reference ranges using the same reagents and analyser. Plasma samples were obtained from 1437 paediatric patients from the following age groups: 15 days-4 weeks (n=36), 1-5 months (n=320), 6-12 months (n=176), 1-5 years (n=507), 6-10 years (n=132) and 11-17 years (n=262). Indication of coagulation testing was pre-operative screening for non-acute diseases in most cases. PT values were similar in the different age groups to those in adults, whereas longer aPTTs were demonstrated in the younger children. Plasma levels of all clotting factors, except for FV, were significantly decreased (p<0.0001) in the youngest children, adult values being usually reached before the end of the first year. The same applied to antithrombin, protein C/S, and plasminogen. In contrast, FVIII and VWF levels were elevated in the youngest children and returned to adult values within six months. The same applied to D-dimer levels, which were found elevated, particularly until six months of life, until puberty. These data suggest that most coagulation test results are highly dependent on age, mainly during the first year of life, and that age-specific reference ranges must be used to ensure proper evaluation of coagulation in children.