Jan Schwonberg

Standardization of light transmittance aggregometry for monitoring antiplatelet therapy: an adjustment for platelet count is not necessary.

Summary.  Background: Light transmittance aggregometry (LTA) is considered to be the ‘gold standard’ of platelet function testing. As LTA has been poorly standardized, we analyzed the results of LTA in healthy subjects and patients with antiplatelet therapy using different concentrations of agonists and performing tests in non‐adjusted and platelet count‐adjusted platelet‐rich plasma (PRP). Methods: LTA was performed in 20 healthy subjects and in patients treated with aspirin (n = 30) or clopidogrel (n = 30) monotherapy, as well as in patients on combination therapy (n = 20), using arachidonic acid (ARA 0.25 and 0.5 mg mL−1) and adenosine diphosphate (ADP 2 and 5 μm) as agonists and performing platelet function tests in non‐adjusted and platelet count (250 nL−1 ± 10%)‐adjusted PRP. Results: The overall platelet aggregation response is decreased after adjusting the PRP for platelet count compared with measurements in unadjusted PRP. The variability of aggregation results is high in adjusted PRP in the subgroup of healthy subjects, ranging from 9.2–95.3% (5th–95th percentile) relative to 77.6–95.5% in non‐adjusted PRP when determining maximum aggregation to ARA 0.5 mg mL−1. Late aggregation using ADP 2 μm ranges from 3.8–89.9% in adjusted PRP compared with 42.9–92.5% in non‐adjusted PRP. Maximum aggregation using ARA 0.5 mg mL−1 in non‐adjusted PRP differentiates between aspirin‐treated patients and healthy controls well, whereas late aggregation using ADP 2 μm in non‐adjusted PRP offers the best discrimination between clopidogrel‐treated patients and healthy controls. Conclusion: Adjustment of PRP for platelet count does not provide any advantage and therefore the time‐consuming process of platelet count adjustment is not necessary.

Determination of clopidogrel main metabolite in plasma: a useful tool for monitoring therapy?

This study was performed to determine whether analysis of clopidogrel and its main carboxylic acid metabolite in plasma provides additional information about the wide variability of platelet aggregation inhibition in clopidogrel-treated patients with peripheral arterial occlusive disease. Consecutive outpatients (n = 56) with stable peripheral arterial occlusive disease treated with 75 mg clopidogrel daily, without co-administration of aspirin, were investigated. With use of a standardized questionnaire, the time of drug intake was documented. Blood sampling was performed within 24 hours after the most recent drug intake. Platelet function was measured by optical aggregometry using adenosine diphosphate (ADP) (2 μmol/L) as the agonist. Plasma concentrations of clopidogrel and its main metabolite, clopidogrel carboxylic acid, were quantitated using high-performance liquid chromatography analysis coupled to mass spectrometry. In 95% (53/56) of patients, clopidogrel carboxylic acid was detected. In 40% (22/56) of patients, the ADP-induced aggregation response was within the normal range despite clopidogrel treatment. In 14% (3/22) of these patients, neither clopidogrel nor its main metabolite could be detected. Two of these patients agreed to ingest 75 mg/d clopidogrel under observation and to undergo blood sampling after 2, 12, and 24 hours. Clopidogrel carboxylic acid and a significant inhibition of platelet aggregation were detected even after 24 hours in both patients, confirming noncompliance as the reason for the lack of inhibition of ADP-induced platelet aggregation observed in the initial measurements. In the subgroup of patients who had taken clopidogrel within 4 hours before blood sampling, a large range of carboxylic acid concentrations was detected, indicating a high variability of drug metabolism among patients. In conclusion, determining clopidogrel metabolite plasma concentrations could be a useful tool for identifying poor compliance and variable metabolism in clopidogrel-treated patients. Nevertheless, in the majority of clopidogrel-treated patients, the variability of platelet response is not caused by noncompliance.

Hereditary and acquired thrombophilia in patients with upper extremity deep-vein thrombosis. Results from the MAISTHRO registry.

The prevalence of coagulation disorders in patients with upper extremity deep-vein thrombosis (UE-DVT) is unknown due to only a few observational studies of limited size reporting varying results. Therefore, we aimed to evaluate the prevalence of thrombophilia in patients with UE-DVT compared to patients with lower extremity deep vein thrombosis (LE-DVT). One hundred fifty consecutive patients (15 to 91 years of age) with UE-DVT were recruited from the MAISTHRO (MAin-ISar-THROmbosis) registry. Three hundred LE-DVT patients matched for gender and age served as controls. Thrombophilia screening included tests for the factor V Leiden mutation, the prothrombin G20210A mutation, antiphospholipid antibodies and factor VIII (FVIII), protein C, protein S and antithrombin activities. At least one thrombophilia was present in 34.2% of UE-DVT and 39.2% in UE-DVT that was unrelated to venous catheters relative to 55.3% in LE-DVT patients (p<0.001). In particular, a persistently elevated FVIII is less likely to be found in UE-DVT patients than in those with LE-DVT and is the only thrombophilia that is differentially expressed after controlling for established VTE risk factors [OR 0.46, (95% CI 0.25-0.83)]. Although less prevalent than in LE-DVT patients, thrombophilia is a common finding in patients with UE-DVT, especially in those with thrombosis that is unrelated to venous catheters.

Prevalence of thrombophilia according to age at the first manifestation of venous thromboembolism: results from the MAISTHRO registry

Thrombophilia is a well‐established risk factor for a venous thromboembolic event (VTE), and it has been proposed that hereditary thrombophilia may substantially contribute to the development of VTE in young patients. We aimed to analyse the prevalence of thrombophilia with special regard to the age of VTE manifestation. The study cohort consisted of 1490 patients (58% females) with a median age 43 years at the time of their first VTE. At least one thrombophilic disorder was identified in 50·1% of patients. The probability of detecting a hereditary thrombophilia declined significantly with advancing age (from 49·3% in patients aged 20 years and younger to 21·9% in patients over the age of 70 years; P < 0·001). This may be primarily attributed to the decreasing frequencies of the F5 R506Q (factor V Leiden) mutation and deficiencies of protein C or protein S with older age at the time of the initial VTE event. Moreover, thrombophilia was more prevalent in unprovoked compared with risk‐associated VTE (57·7% vs. 47·7%; P = 0·001). The decline in the prevalence of hereditary thrombophilia with older ages supports the use of a selected thrombophilia screening strategy dependent on age and the presence or absence of additional VTE risk factors.

Impact of sex and traditional cardiovascular risk factors on the risk of recurrent venous thromboembolism: results from the German MAISTHRO Registry.

As arterial and venous thrombosis share common risk factors, a link between arterial and venous thrombosis has been suggested recently. Therefore, we aimed to investigate the impact of established cardiovascular risk factors on the risk of recurrent venous thromboembolism (VTE). With a cross-sectional study design, we analyzed the data of 1006 patients (582 F, 424 M) consecutively treated in our outpatient department for VTE (i.e. lower extremity deep vein thrombosis and/or pulmonary embolism) and registered in the MAISTHRO (MAin-ISar-THROmbosis) database. Of the total cohort, 324 (32.2%) patients suffered a recurrent VTE. Compared with the patients with a single thromboembolic event, patients with recurrent VTE were more frequently male (39.4 vs. 27.0%, P < 0.001). In univariate analysis, the relative risk of recurrent VTE was 1.9 [95% confidence interval (CI) 1.53–2.39] for male sex and 1.6 (1.25–1.95) for age over 50 years (PAOD). After adjustments for age, sex, thrombophilia and other common VTE risk factors, male sex [hazard ratio (HR) = 1.7 (1.38–21.9)] and arterial hypertension [HR = 1.4 (1.05–1.78)] were independent risk factors of recurrent VTE. The higher risk in men than in women persisted even after the exclusion of women with transient hormonal risk factors [HR = 1.57 (1.19–2.07)]. In contrast, no association between the presence of diabetes, obesity, hypercholesterolemia or smoking and the risk of VTE recurrence was observed. Male sex and arterial hypertension are independently associated with an increased risk of recurrent VTE after termination of anticoagulant therapy for the first VTE event.

Performance of five D-dimer assays for the exclusion of symptomatic distal leg vein thrombosis

The diagnostic value of D-dimer (DD) in the exclusion of proximal deep-vein thrombosis (DVT) is well-established but is less well-known in the exclusion of distal (infrapopliteal) DVT. Therefore, we evaluated the diagnostic abilities of five DD assays (Vidas-DD, Liatest-DD, HemosIL-DD, HemosIL-DDHS, Innovance-DD) for excluding symptomatic proximal and distal leg DVT. A total of 243 outpatients whose symptoms were suggestive of DVT received complete compression ultrasonography (cCUS) of the symptomatic leg(s). The clinical probability of DVT (PTP) was assessed by Wells score. Thirty-eight proximal and 31 distal DVTs (17 tibial/fibular DVTs, 14 muscle DVTs) were diagnosed by cCUS. Although all assays showed high sensitivity for proximal DVT (range 97-100%), the sensitivity was poor for distal DVT (range 78-93%). None of the assays were individually able to rule out all DVTs as a stand-alone test (negative predictive value [NPV] 91-96%). However, a negative DD test result combined with a low PTP exhibited a NPV of 100% for all DVTs (including proximal, tibial/fibular, and muscle DVTs) with the HemosIL-DDHS and Innovance-DD. All proximal and tibial/fibular DVTs, but not all muscle DVTs, could be ruled out with this strategy using the Liatest-DD and Vidas-DD. The HemosIL-DD could not exclude distal leg DVT, even in combination with a low PTP. The combination of a negative DD with a low PTP showed a specificity of 32-35% for all DVTs. In conclusion, our study shows that when used in conjunction with a low PTP some DD assays are useful tools for the exclusion of distal leg DVT.

Variability of residual platelet function despite clopidogrel treatment in patients with peripheral arterial occlusive disease

Residual platelet function despite treatment with clopidogrel may predict an unfavourable cardiovascular outcome. The majority of studies have investigated the effects of clopidogrel administration in conjunction with aspirin in patients undergoing percutaneous coronary intervention. The primary objective of the present study was to assess the platelet response to clopidogrel in the absence of aspirin in patients with peripheral arterial occlusive disease (PAOD) and to investigate whether non-responsiveness to clopidogrel is reproducible during long-term follow-up. Fifty-four clinically stable PAOD patients on a maintenance dose of 75 mg/d clopidogrel were enrolled in this study. Platelet function was assessed at baseline and after a median follow-up of 18 months using light transmittance aggregometry (LTA) with 2 microM ADP as an agonist. HPLC-coupled mass spectrometry was used to detect clopidogrel and clopidogrel carboxylic acid, the main metabolite of clopidogrel. Residual platelet function, as defined by late aggregation values within the reference range (i.e., >43%), was observed in 35.2% of patients at baseline and 17.6% during follow-up. During the observation period, 26.5% had switched from responder to non-responder status or vice versa. Among non-responders, either clopidogrel or its metabolite was detected in 89.5% and 83.3% of patients at baseline and at follow-up, respectively. We conclude that non-responsiveness to clopidogrel as determined by ADP-induced LTA is not stable over time. This phenomenon cannot be attributed to non-compliance alone.

Thrombophilia and risk of VTE recurrence according to the age at the time of first VTE manifestation.

BACKGROUND Whether screening for thrombophilia is useful for patients after a first episode of venous thromboembolism (VTE) is a controversial issue. However, the impact of thrombophilia on the risk of recurrence may vary depending on the patients age at the time of the first VTE. PATIENTS AND METHODS Of 1221 VTE patients (42 % males) registered in the MAISTHRO (MAin-ISar-THROmbosis) registry, 261 experienced VTE recurrence during a 5-year follow-up after the discontinuation of anticoagulant therapy. RESULTS Thrombophilia was more common among patients with VTE recurrence than those without (58.6 % vs. 50.3 %; p = 0.017). Stratifying patients by the age at the time of their initial VTE, Cox proportional hazards analyses adjusted for age, sex and the presence or absence of established risk factors revealed a heterozygous prothrombin (PT) G20210A mutation (hazard ratio (HR) 2.65; 95 %-confidence interval (CI) 1.71 - 4.12; p < 0.001), homozygosity/double heterozygosity for the factor V Leiden and/or PT mutation (HR 2.35; 95 %-CI 1.09 - 5.07, p = 0.030), and an antithrombin deficiency (HR 2.12; 95 %-CI 1.12 - 4.10; p = 0.021) to predict recurrent VTE in patients aged 40 years or older, whereas lupus anticoagulants (HR 3.05; 95%-CI 1.40 - 6.66; p = 0.005) increased the risk of recurrence in younger patients. Subgroup analyses revealed an increased risk of recurrence for a heterozygous factor V Leiden mutation only in young females without hormonal treatment whereas the predictive value of a heterozygous PT mutation was restricted to males over the age of 40 years. CONCLUSIONS Our data do not support a preference of younger patients for thrombophilia testing after a first venous thromboembolic event.