Diagnostic high-throughput sequencing of 2396 patients with bleeding, thrombotic, and platelet disorders.

A targeted high-throughput sequencing (HTS) panel test for clinical diagnostics requires careful consideration of the inclusion of appropriate diagnostic-grade genes, the ability to detect multiple types of genomic variation with high levels of analytic sensitivity and reproducibility, and variant interpretation by a multidisciplinary team (MDT) in the context of the clinical phenotype. We have sequenced 2396 index patients using the ThromboGenomics HTS panel test of diagnostic-grade genes known to harbor variants associated with rare bleeding, thrombotic, or platelet disorders (BTPDs). The molecular diagnostic rate was determined by the clinical phenotype, with an overall rate of 49.2% for all thrombotic, coagulation, platelet count, and function disorder patients and a rate of 3.2% for patients with unexplained bleeding disorders characterized by normal hemostasis test results. The MDT classified 745 unique variants, including copy number variants (CNVs) and intronic variants, as pathogenic, likely pathogenic, or variants of uncertain significance. Half of these variants (50.9%) are novel and 41 unique variants were identified in 7 genes recently found to be implicated in BTPDs. Inspection of canonical hemostasis pathways identified 29 patients with evidence of oligogenic inheritance. A molecular diagnosis has been reported for 894 index patients providing evidence that introducing an HTS genetic test is a valuable addition to laboratory diagnostics in patients with a high likelihood of having an inherited BTPD.

The Influence of Prostaglandin E1 and Use of Inhibitor Percentage on the Correlation between the Multiplate and VerifyNow in Patients on Dual Antiplatelet Therapy.

Platelet function tests (PFT), such as the Multiple Electrode Analyzer (Multiplate) and VerifyNow, show little concordance in patients using antiplatelet drugs. A major difference between these tests is the use of prostaglandin E1 (PGE1) to inhibit P2Y1-platelet-receptor activation in VerifyNow and is proposed to be of influence in the discrepancy between these tests. We aimed to investigate whether the presence of PGE1 could provide an explanation for the moderate correlation and concordance between Multiplate and VerifyNow by adding PGE1 to the Multiplate ADP assay, also known as the ADP-high sensitivity (ADP-HS) assay. We also aimed to investigate whether the difference in baseline platelet function as measured by the VerifyNow and Multiplate could (partly) explain the moderate correlation between the tests, by plotting ADP assay results against baseline function as measured by the corresponding device, which is expressed as the 'inhibitor percentage.' Fifty-one patients who underwent percutaneous coronary intervention (PCI) received dual antiplatelet therapy and were considered to have a high risk of ischemic or bleeding complications were included. The addition of 20 µl PGE1 in the Multiplate resulted in a significant reduction in Arbitrary Aggregation Units, but did not improve correlation with the VerifyNow. The correlation between VerifyNow and Multiplate inhibitor percentage was moderate. Based on these results, we concluded that neither PGE1 nor the calculation of the inhibitor percentage greatly influenced the correlation between PFTs.

Screening for platelet function disorders with Multiplate and platelet function analyzer.

Light transmission aggregation (LTA) is the gold standard for the diagnosis of platelet function disorders (PFDs), but it is time-consuming and limited to specialized laboratories. Whole-blood impedance aggregometry (Multiplate) and platelet function analyzer (PFA) may be used as rapid screening tools to exclude PFDs. The aim of this study is to assess the diagnostic performance of Multiplate and PFA for PFDs, as detected by LTA.Data from preoperative patients, patients referred to the hematologist for bleeding evaluation, and patients with a diagnosed bleeding disorder were used. PFDs were defined as ≥2 abnormal LTA curves. Diagnostic performance of Multiplate and PFA for detecting PFDs was expressed as sensitivity and specificity. The ability of Multiplate agonists and PFA kits to detect corresponding LTA curve abnormalities was expressed as area under the receiver operating characteristic curve. Prevalence of PFDs was 16/335 (4.8%) in preoperative patients, 10/54 (18.5%) in referred patients, and 3/25 (12%) in patients with a diagnosed bleeding disorder. In preoperative and referred patients, the sensitivity of Multiplate and PFA for detecting mild PFDs varied between 0% and 40% and AUCs for detecting corresponding LTA curve abnormalities were close to 0.50. In patients with a diagnosed bleeding disorder, both assays could detect Glanzmann thrombasthenia (GT) with sensitivity of 100% and AUCs of 0.70-1.00. Multiplate and PFA cannot discriminate between preoperative and referred patients with and without mild PFDs, meaning that they cannot be used as screening tests to rule out mild PFDs in these populations. Both Multiplate and PFA can detect GT in previously diagnosed patients.

A high-throughput sequencing test for diagnosing inherited bleeding, thrombotic, and platelet disorders.

Inherited bleeding, thrombotic, and platelet disorders (BPDs) are diseases that affect ∼300 individuals per million births. With the exception of hemophilia and von Willebrand disease patients, a molecular analysis for patients with a BPD is often unavailable. Many specialized tests are usually required to reach a putative diagnosis and they are typically performed in a step-wise manner to control costs. This approach causes delays and a conclusive molecular diagnosis is often never reached, which can compromise treatment and impede rapid identification of affected relatives. To address this unmet diagnostic need, we designed a high-throughput sequencing platform targeting 63 genes relevant for BPDs. The platform can call single nucleotide variants, short insertions/deletions, and large copy number variants (though not inversions) which are subjected to automated filtering for diagnostic prioritization, resulting in an average of 5.34 candidate variants per individual. We sequenced 159 and 137 samples, respectively, from cases with and without previously known causal variants. Among the latter group, 61 cases had clinical and laboratory phenotypes indicative of a particular molecular etiology, whereas the remainder had an a priori highly uncertain etiology. All previously detected variants were recapitulated and, when the etiology was suspected but unknown or uncertain, a molecular diagnosis was reached in 56 of 61 and only 8 of 76 cases, respectively. The latter category highlights the need for further research into novel causes of BPDs. The ThromboGenomics platform thus provides an affordable DNA-based test to diagnose patients suspected of having a known inherited BPD.

[Side effects of medicinal products: assessment and registration of causality]. / Bijwerkingen van geneesmiddelen.

The Farmacotherapeutisch Kompas (FK) and the KNMP Kennisbank report on side effects of medicinal products, in order of frequency. However, data regarding causality and its assessment are lacking, while this information is crucial when the discontinuation of a drug due to putative side effects is considered. In this article, we describe the role of pharmaceutical companies, the agencies in charge of the evaluation and supervision of medicinal products and Lareb. We also describe how this information is included in the FK and the Kennisbank. Only side effects with a probable causal relation to the drug are registered. However, to err on the side of caution, side effects with a questionable causal relation to the drug are sometimes also registered. It would be helpful for the physician if the FK and the Kennisbank also reported the degree of assessed causality, next to the frequency.

Rotational Thromboelastometry in High-Risk Patients on Dual Antithrombotic Therapy After Percutaneous Coronary Intervention.

Aims: Patients using antithrombotic drugs after percutaneous coronary intervention (PCI) are at risk for bleeding and recurrent ischemia. We aimed to explore routine and tissue plasminogen activated (tPA) ROTEM results in a post-PCI population on dual antithrombotic treatment. Methods and Results: In this prospective cohort, 440 patients treated with double antithrombotic therapy after recent PCI and with ≥3 risk factors for either ischemic or bleeding complications were included and compared with a control group (n = 95) consisting of perioperative patients not using antithrombotic medication. Laboratory assessment, including (tPA) ROTEM, was performed one month post-PCI and bleeding/ischemic complications were collected over a five-month follow-up. Patients were stratified by antithrombotic regimen consisting of a P2Y12 inhibitor with either aspirin (dual antiplatelet therapy; DAPT, n = 323), a vitamin K antagonist (VKA, n = 69) or a direct oral anticoagulant (DOAC, n = 48). All post-PCI patients had elevated ROTEM clot stiffness values, but only the DAPT group additionally presented with a decreased fibrinolytic potential as measured with tPA ROTEM. Patients receiving anticoagulants had prolonged clotting times (CT) when compared to the control and DAPT group; EXTEM and FIBTEM CT could best discriminate between patients (not) using anticoagulants (AUC > 0.97). Furthermore, EXTEM CT was significantly prolonged in DAPT patients with bleeding complications during follow-up (68 [62-70] vs. 62 [57-68], p = 0.030). Conclusion: ROTEM CT has high potential for identifying anticoagulants and tPA ROTEM could detect a diminished fibrinolytic potential in patients using DAPT. Furthermore, the ability of EXTEM CT to identify patients at risk for bleeding may be promising and warrants further research.

Differential Impact of Cytochrome 2C19 Allelic Variants on Three Different Platelet Function Tests in Clopidogrel-Treated Patients.

On-treatment platelet reactivity in clopidogrel-treated patients can be measured with several platelet function tests (PFTs). However, the agreement between different PFTs is only slight to moderate. Polymorphisms of the CYP2C19 gene have an impact on the metabolization of clopidogrel and, thereby, have an impact on on-treatment platelet reactivity. The aim of the current study is to evaluate the differential effects of the CYP2C19 genotype on three different PFTs. METHODS: From a prospective cohort study, we included patients treated with clopidogrel following percutaneous coronary intervention (PCI). One month after PCI, we simultaneously performed three different PFTs; light transmission aggregometry (LTA), VerifyNow P2Y12, and Multiplate. In whole EDTA blood, genotyping of the CYP2C19 polymorphisms was performed. RESULTS: We included 308 patients treated with clopidogrel in combination with aspirin (69.5%) and/or anticoagulants (33.8%) and, based on CYP2C19 genotyping, classified them as either extensive (36.4%), rapid (34.7%), intermediate (26.0%), or poor metabolizers (2.9%). On-treatment platelet reactivity as measured by LTA and VerifyNow is significantly affected by CYP2C19 metabolizer status (p < 0.01); as metabolizer status changes from rapid, via extensive and intermediate, to poor, the mean platelet reactivity increases accordingly (p < 0.01). On the contrary, for Multiplate, no such ordering of metabolizer groups was found (p = 0.10). CONCLUSIONS: For VerifyNow and LTA, the on-treatment platelet reactivity in clopidogrel-treated patients correlates well with the underlying CYP2C19 polymorphism. For Multiplate, no major effect of genetic background could be shown, and effects of other (patient-related) variables prevail. Thus, besides differences in test principles and the influence of patient-related factors, the disagreement between PFTs is partly explained by differential effects of the CYP2C19 genotype.

Assessment and determinants of whole blood and plasma fibrinolysis in patients with mild bleeding symptoms.

Enhanced clot lysis is associated with bleeding, but assessment of lysis capacity remains difficult. The plasma turbidity lysis and whole blood tissue Plasminogen Activator-Rotational Thromboelastometry (tPA-ROTEM) assays estimate fibrinolysis under more physiological conditions than clinically used assays. We hypothesized that these assays could find signs of enhanced lysis capacity in patients who report bleeding symptoms, but are not diagnosed with bleeding disorders. We also aimed to gain insight in determinants of the results of these lysis assays. Data from 240 patients with and 95 patients without self-reported bleeding symptoms were obtained, who were included in a study that primarily aimed to assess prevalence of haemostatic abnormalities in preoperative patients. ROTEM and turbidity assays were performed with rtPA. Blood counts, fibrinolysis and coagulation factor activities were determined. Data were analysed using multivariable linear regression models. Remarkably, patients reporting bleeding symptoms showed signs of significantly impaired lysis capacity in the tPA-ROTEM, but not in the turbidity lysis assay. In these patients, the tPA-ROTEM results depended on FII, FXII, plasminogen, α2-antiplasmin, PAI-1 and TAFI levels. The turbidity lysis results were significantly influenced by fibrinogen, α2-antiplasmin, PAI-1 and TAFI. In conclusion, the tPA-ROTEM and the turbidity lysis assay could not detect enhanced fibrinolytic capacity in patients with bleeding symptoms. This suggests that these symptoms are not caused by enhanced fibrinolytic activity. As both assays were sensitive to important determinants of fibrinolysis they may be able to detect a fibrinolytic imbalance, but this needs to be validated in patients with known hypo- or hyperfibrinolytic disorders.

Preoperative screening for bleeding disorders: A comprehensive laboratory assessment of clinical practice.

BACKGROUND: Patients with mild bleeding disorders are at risk of perioperative bleeding, but screening for these disorders remains challenging. OBJECTIVES: We aimed to assess the prevalence of hemostatic abnormalities in patients with and without reported bleeding symptoms on a preoperative questionnaire, consisting of guideline-proposed questions, and appraised the diagnostic value of several screening modalities for the identification of patients with hemostatic abnormalities. METHODS: In this observational study, 240 patients with and 95 patients without bleeding symptoms on the preoperative questionnaire were included. Patients with known bleeding disorders, antithrombotic drugs, thrombocytopenia, and anemia were excluded. Preoperatively, all patients underwent elaborate hemostatic testing. Hemostatic abnormalities were defined as coagulation, vWF, or fibrinolysis factor levels below reference range and platelet function defects. Screening modalities included the ISTH Bleeding Assessment Tool (ISTH-BAT), PT, aPTT, TT, Euglobulin Lysis Time (ELT), and Platelet Function Analyser (PFA). RESULTS: In 21 of 240 (8.8%) patients reporting bleeding symptoms, hemostatic abnormalities were found, including 7 reduced coagulation factor levels, 10 platelet function abnormalities, and 4 reduced vWF levels. In comparison, 10 of 95 (10.5%) patients not reporting bleeding symptoms had abnormalities. The ISTH-BAT could not identify patients with abnormalities, while PT, aPTT, TT, ELT, and PFA had high specificity but low sensitivity to detect abnormalities. CONCLUSIONS: The prevalence of hemostatic abnormalities in both patients with and without reported bleeding symptoms was 9%-10%. This suggests that the guideline-based questionnaire cannot differentiate between patients with and without abnormalities, while the discriminative power of the screening modalities is also limited.

Determinants of agreement between proposed therapeutic windows of platelet function tests in vulnerable patients.

AIMS: Therapeutic windows for residual platelet reactivity in patients with coronary artery disease on P2Y12 inhibitors were proposed in a consensus document. We aimed to explore the level of agreement between windows for different platelet function tests (PFTs) used to classify patients in low, optimal, and high on-treatment platelet reactivity categories, and to identify variables contributing to the level of agreement. METHODS AND RESULTS: In this explorative clinical study, the VerifyNow P2Y12, Multiplate adenosine diphosphate (ADP), and light transmission aggregometry (LTA) 20 µmol/L ADP were performed simultaneously in 145 consecutive vulnerable patients. Measurements were performed within 6 months of percutaneous intervention. Patients were considered vulnerable if they had ≥2 risk factors for bleeding or ischaemic events. Window-agreement between PFT pairs was slight to moderate. Multiplate-VerifyNow agreed in 72 patients (50%), κ = 0.41; VerifyNow-LTA agreed in 76 patients (52%), κ = 0.36; and LTA-Multiplate agreed in 64 patients (44%), κ = 0.20. Several variables including the type of P2Y12 inhibitor, aspirin, haemoglobin level, platelet count, age, and previous stroke significantly influenced agreement between PFTs. CONCLUSIONS: Our results suggest that the PFTs, with accompanying therapeutic windows, are not interchangeable when determining the response to antiplatelet therapy in vulnerable coronary artery disease patients on P2Y12 inhibitors. Hence, the type of PFT can directly affect the treatment strategy, which may be especially relevant for patients with multiple factors influencing individual PFTs and thereby test agreement.

Assessment of bleeding risk in patients with coronary artery disease on dual antiplatelet therapy. A systematic review.

Patients with coronary artery disease are usually treated with dual antiplatelet therapy (DAPT) after percutaneous coronary intervention. Patients on DAPT are at risk of both ischaemic and bleeding events. Although side-lined for a long time, real-life studies have shown that both the incidence and the associated morbidity and mortality of out-of-hospital bleeding are high. This indicates that prevention of (post-interventional) bleeding is as important as prevention of ischaemia. For this purpose it is crucial to reliably identify patients with a high bleeding risk. In order to postulate an algorithm, which could help identifying these patients, we performed a systematic review to determine the value of previously proposed prognostic modalities for bleeding. We searched and appraised the following tools: platelet function tests, genetic tests, bleeding scores and questionnaires and haemostatic tests. Most studies indicated that low on-treatment platelet reactivity (LTPR), as measured by several platelet function tests, and the carriage of CYP2C19*17 allele were independent risk factors for bleeding. A bleeding score also proved to be helpful in identifying patients at risk. No studies on haemostatic tests were retrieved. Several patient characteristics were also identified as independent predictors of bleeding, such as older age, female sex and renal failure. Combining these risk factors we propose an algorithm that would hypothetically facilitate identification of those patients at highest risk, warranting prevention measures for bleeding. This could be a starting point for further research concerning the topic.