Publications by authors named "Yvonne M C Henskens"

40 Publications

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

J Clin Med 2021 Sep 3;10(17). Epub 2021 Sep 3.

Laboratory for Clinical Thrombosis and Haemostasis, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, 6200 MD Maastricht, The Netherlands.

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 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 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 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 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 metabolizer status ( < 0.01); as metabolizer status changes from rapid, via extensive and intermediate, to poor, the mean platelet reactivity increases accordingly ( < 0.01). On the contrary, for Multiplate, no such ordering of metabolizer groups was found ( = 0.10).

Conclusions: For VerifyNow and LTA, the on-treatment platelet reactivity in clopidogrel-treated patients correlates well with the underlying 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 genotype.
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http://dx.doi.org/10.3390/jcm10173992DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8432532PMC
September 2021

Serial markers of coagulation and inflammation and the occurrence of clinical pulmonary thromboembolism in mechanically ventilated patients with SARS-CoV-2 infection; the prospective Maastricht intensive care COVID cohort.

Thromb J 2021 May 31;19(1):35. Epub 2021 May 31.

Department of Intensive Care Medicine, Maastricht University Medical Centre+, Maastricht, The Netherlands.

Background: The incidence of pulmonary thromboembolism is high in SARS-CoV-2 patients admitted to the Intensive Care. Elevated biomarkers of coagulation (fibrinogen and D-dimer) and inflammation (c-reactive protein (CRP) and ferritin) are associated with poor outcome in SARS-CoV-2. Whether the time-course of fibrinogen, D-dimer, CRP and ferritin is associated with the occurrence of pulmonary thromboembolism in SARS-CoV-2 patients is unknown. We hypothesise that patients on mechanical ventilation with SARS-CoV-2 infection and clinical pulmonary thromboembolism have lower concentrations of fibrinogen and higher D-dimer, CRP, and ferritin concentrations over time compared to patients without a clinical pulmonary thromboembolism.

Methods: In a prospective study, fibrinogen, D-dimer, CRP and ferritin were measured daily. Clinical suspected pulmonary thromboembolism was either confirmed or excluded based on computed tomography pulmonary angiography (CTPA) or by transthoracic ultrasound (TTU) (i.e., right-sided cardiac thrombus). In addition, patients who received therapy with recombinant tissue plasminogen activator were included when clinical instability in suspected pulmonary thromboembolism did not allow CTPA. Serial data were analysed using a mixed-effects linear regression model, and models were adjusted for known risk factors (age, sex, APACHE-II score, body mass index), biomarkers of coagulation and inflammation, and anticoagulants.

Results: Thirty-one patients were considered to suffer from pulmonary thromboembolism ((positive CTPA (n = 27), TTU positive (n = 1), therapy with recombinant tissue plasminogen activator (n = 3)), and eight patients with negative CTPA were included. After adjustment for known risk factors and anticoagulants, patients with, compared to those without, clinical pulmonary thromboembolism had lower average fibrinogen concentration of - 0.9 g/L (95% CI: - 1.6 - - 0.1) and lower average ferritin concentration of - 1045 μg/L (95% CI: - 1983 - - 106) over time. D-dimer and CRP average concentration did not significantly differ, 561 μg/L (- 6212-7334) and 27 mg/L (- 32-86) respectively. Ferritin lost statistical significance, both in sensitivity analysis and after adjustment for fibrinogen and D-dimer.

Conclusion: Lower average concentrations of fibrinogen over time were associated with the presence of clinical pulmonary thromboembolism in patients at the Intensive Care, whereas D-dimer, CRP and ferritin were not. Lower concentrations over time may indicate the consumption of fibrinogen related to thrombus formation in the pulmonary vessels.
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http://dx.doi.org/10.1186/s12959-021-00286-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8165953PMC
May 2021

Serial EXTEM, FIBTEM, and tPA Rotational Thromboelastometry Observations in the Maastricht Intensive Care COVID Cohort-Persistence of Hypercoagulability and Hypofibrinolysis Despite Anticoagulation.

Front Cardiovasc Med 2021 26;8:654174. Epub 2021 Apr 26.

Central Diagnostic Laboratory, Maastricht University Medical Centre+, Maastricht, Netherlands.

Coronavirus Disease 2019 (COVID-19) patients often present with thromboembolic events. In COVID-19 patients, routine hemostatic assays cannot correctly identify patients at risk for thromboembolic events. Viscoelastic testing with rotational thromboelastometry (ROTEM) might improve the characterization of COVID-19-associated coagulopathy. To unravel underlying coagulopathy and fibrinolysis over time as measured by serial assessment heparin-independent (FIBTEM and EXTEM) and fibrinolysis illustrating (tissue plasminogen activator; tPA) ROTEM assays. Between April 23 and June 12, consecutive adult patients enrolled within the Maastricht Intensive Care COVID (MaastrICCht) cohort were included, and a comprehensive set of clinical, physiological, pharmaceutical, and laboratory variables were collected daily. Twice per week, EXTEM, FIBTEM, and tPA ROTEM were performed. Clotting time (CT), clot formation time (CFT), maximum clot firmness (MCF), lysis onset time (LOT), and lysis time (LT) were determined to assess clot development and breakdown and were compared to routine hemostatic assays. In 36 patients, 96 EXTEM/FIBTEM and 87 tPA ROTEM tests were performed during a 6-week follow-up. CT prolongation was present in 54% of EXTEM measurements, which were not matched by prothrombin time (PT) in 37%. Respectively, 81 and 99% of all EXTEM and FIBTEM MCF values were above the reference range, and median MCF remained elevated during follow-up. The ROTEM fibrinolysis parameters remained prolonged with median LOT consequently >49 min and unmeasurable LT in 56% of measurements, suggesting a severe hypofibrinolytic phenotype. ROTEM tests in COVID-19 ICU patients show hypercoagulability and severe hypofibrinolysis persisting over at least 6 weeks.
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http://dx.doi.org/10.3389/fcvm.2021.654174DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8107372PMC
April 2021

Thrombin-Fibrin(ogen) Interactions, Host Defense and Risk of Thrombosis.

Int J Mol Sci 2021 Mar 4;22(5). Epub 2021 Mar 4.

Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, 6200 MD Maastricht, The Netherlands.

Fibrinogen is a well-known risk factor for arterial and venous thrombosis. Its function is not restricted to clot formation, however, as it partakes in a complex interplay between thrombin, soluble plasma fibrinogen, and deposited fibrin matrices. Fibrinogen, like thrombin, participates predominantly in hemostasis to maintain vascular integrity, but executes some important pleiotropic effects: firstly, as observed in thrombin generation experiments, fibrin removes thrombin from free solution by adsorption. The adsorbed thrombin is protected from antithrombins, notably α2-macroglobulin, and remains physiologically active as it can activate factors V, VIII, and platelets. Secondly, immobilized fibrinogen or fibrin matrices activate monocytes/macrophages and neutrophils via Mac-1 interactions. Immobilized fibrin(ogen) thereby elicits a pro-inflammatory response with a reciprocal stimulating effect of the immune system on coagulation. In contrast, soluble fibrinogen prohibits recruitment of these immune cells. Thus, while fibrin matrices elicit a procoagulant response, both directly by protecting thrombin and indirectly through the immune system, high soluble fibrinogen levels might protect patients due to its immune diminutive function. The in vivo influence of the 'protective' plasma fibrinogen versus the 'pro-thrombotic' fibrin matrices on thrombosis should be explored in future research.
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http://dx.doi.org/10.3390/ijms22052590DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7961882PMC
March 2021

Monitoring of Unfractionated Heparin in Severe COVID-19: An Observational Study of Patients on CRRT and ECMO.

TH Open 2020 Oct 19;4(4):e365-e375. Epub 2020 Nov 19.

Central Diagnostic Laboratory, Maastricht University Medical Centre, Maastricht, the Netherlands.

 Severe cases of coronavirus disease 2019 (COVID-19) can require continuous renal replacement therapy (CRRT) and/or extracorporeal membrane oxygenation (ECMO). Unfractionated heparin (UFH) to prevent circuit clotting is mandatory but monitoring is complicated by (pseudo)-heparin resistance. In this observational study, we compared two different activated partial thromboplastin time (aPTT) assays and a chromogenic anti-Xa assay in COVID-19 patients on CRRT or ECMO in relation to their UFH dosages and acute phase reactants.  The aPTT (optical [aPTT-CS] and/or mechanical [aPTT-STA] clot detection methods were used), anti-Xa, factor VIII (FVIII), antithrombin III (ATIII), and fibrinogen were measured in 342 samples from 7 COVID-19 patients on CRRT or ECMO during their UFH treatment. Dosage of UFH was primarily based on the aPTT-CS with a heparin therapeutic range (HTR) of 50-80s. Associations between different variables were made using linear regression and Bland-Altman analysis.  Dosage of UFH was above 35,000IU/24 hours in all patients. aPTT-CS and aPTT-STA were predominantly within the HTR. Anti-Xa was predominantly above the HTR (0.3-0.7 IU/mL) and ATIII concentration was >70% for all patients; mean FVIII and fibrinogen were 606% and 7.5 g/L, respectively. aPTT-CS correlated with aPTT-STA (  = 0.68) with a bias of 39.3%. Correlation between aPTT and anti-Xa was better for aPTT-CS (0.78 ≤   ≤ 0.94) than for aPTT-STA (0.34 ≤   ≤ 0.81). There was no general correlation between the aPTT-CS and ATIII, FVIII, fibrinogen, thrombocytes, C-reactive protein, or ferritin.  All included COVID-19 patients on CRRT or ECMO conformed to the definition of heparin resistance. A patient-specific association was found between aPTT and anti-Xa. This association could not be explained by FVIII or fibrinogen.
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http://dx.doi.org/10.1055/s-0040-1719083DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7676995PMC
October 2020

Thrombo-Inflammation in Cardiovascular Disease: An Expert Consensus Document from the Third Maastricht Consensus Conference on Thrombosis.

Thromb Haemost 2020 Apr 14;120(4):538-564. Epub 2020 Apr 14.

Department of Hematology and Central Hematology Laboratory, Inselspital, Bern University Hospital, University of Bern, Bern, Switzerland; Center for Thrombosis and Hemostasis, University Medical Center of the Johannes Gutenberg University, Mainz, Germany; Haemostasis Research Unit, University College London, London, United Kingdom.

Thrombo-inflammation describes the complex interplay between blood coagulation and inflammation that plays a critical role in cardiovascular diseases. The third Maastricht Consensus Conference on Thrombosis assembled basic, translational, and clinical scientists to discuss the origin and potential consequences of thrombo-inflammation in the etiology, diagnostics, and management of patients with cardiovascular disease, including myocardial infarction, stroke, and peripheral artery disease. This article presents a state-of-the-art reflection of expert opinions and consensus recommendations regarding the following topics: (1) challenges of the endothelial cell barrier; (2) circulating cells and thrombo-inflammation, focused on platelets, neutrophils, and neutrophil extracellular traps; (3) procoagulant mechanisms; (4) arterial vascular changes in atherogenesis; attenuating atherosclerosis and ischemia/reperfusion injury; (5) management of patients with arterial vascular disease; and (6) pathogenesis of venous thrombosis and late consequences of venous thromboembolism.
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http://dx.doi.org/10.1055/s-0040-1708035DOI Listing
April 2020

Role of Platelet Glycoprotein VI and Tyrosine Kinase Syk in Thrombus Formation on Collagen-Like Surfaces.

Int J Mol Sci 2019 Jun 7;20(11). Epub 2019 Jun 7.

Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, 6229 ER Maastricht, The Netherlands.

Platelet interaction with collagens, via von Willebrand factor, is a potent trigger of shear-dependent thrombus formation mediated by subsequent engagement of the signaling collagen receptor glycoprotein (GP)VI, enforced by integrin αβ. Protein tyrosine kinase Syk is central in the GPVI-induced signaling pathway, leading to elevated cytosolic Ca. We aimed to determine the Syk-mediated thrombogenic activity of several collagen peptides and (fibrillar) type I and III collagens. High-shear perfusion of blood over microspots of these substances resulted in thrombus formation, which was assessed by eight parameters and was indicative of platelet adhesion, activation, aggregation, and contraction, which were affected by the Syk inhibitor PRT-060318. In platelet suspensions, only collagen peptides containing the consensus GPVI-activating sequence (GPO) and Horm-type collagen evoked Syk-dependent Ca rises. In whole blood under flow, Syk inhibition suppressed platelet activation and aggregation parameters for the collagen peptides with or without a (GPO) sequence and for all of the collagens. Prediction models based on a regression analysis indicated a mixed role of GPVI in thrombus formation on fibrillar collagens, which was abolished by Syk inhibition. Together, these findings indicate that GPVI-dependent signaling through Syk supports platelet activation in thrombus formation on collagen-like structures regardless of the presence of a (GPO) sequence.
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http://dx.doi.org/10.3390/ijms20112788DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6600290PMC
June 2019

The daily practice of direct oral anticoagulant use in patients with atrial fibrillation; an observational cohort study.

PLoS One 2019 6;14(6):e0217302. Epub 2019 Jun 6.

Thrombosis Expert Center Maastricht and Laboratory for Clinical Thrombosis and Hemostasis, Maastricht, the Netherlands.

Background: Direct oral anticoagulants (DOACs) are administered in fixed doses without monitoring. There is still little published data on the impact of the absence of monitoring on adherence to medication and stability of DOAC plasma levels over time.

Objectives: To explore adherence and stability of DOAC plasma levels over time in patients with atrial fibrillation (NVAF) recently started on DOAC therapy.

Patients And Methods: A prospective observational cohort study with structured follow up including assessment of adherence to medication, plasma levels at baseline, 3,6 and 12 months and adverse events.

Results: We included 164 patients; 89% were previous users of a vitamin K antagonist (VKA). One-year adherence was reasonably good: Morisky adherence measurement scores of 6-8 in 92%. The majority of DOAC plasma levels were within reported on-therapy ranges; dabigatran (median 104.4 ng/ml, IQR 110.2), rivaroxaban (median 185.2 ng/ml, IQR 216.1) and on average levels were not different for full and adjusted doses. There was significant variation between patients, but no significant differences over time within individuals. A substantial proportion of patients starting in the upper-or lower 20th percentiles remained there during the entire follow up. Seventeen bleedings (16 minor, 1 major) were reported, no ischemic events and bleeding or thrombotic events were not associated with DOAC plasma levels.

Conclusions: Adherence was reasonably good in the majority of patients. Our data confirm the stability of DOAC plasma levels over time. Knowledge of such data may, in the individual patient, contribute to optimal drug and dose selection.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0217302PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6554016PMC
February 2020

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

Blood 2019 12;134(23):2082-2091

East Midlands and East of England Genomic Laboratory Hub, Cambridge University Hospitals NHS Foundation Trust, Cambridge Biomedical Campus, Cambridge, United Kingdom.

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.
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http://dx.doi.org/10.1182/blood.2018891192DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6993014PMC
December 2019

Fibrinolysis in patients with chemotherapy-induced thrombocytopenia and the effect of platelet transfusion.

J Thromb Haemost 2019 07 24;17(7):1073-1084. Epub 2019 May 24.

Department of Hematology, Internal Medicine, Maastricht University Medical Center+, Maastricht, the Netherlands.

Essentials Bleeding in chemotherapy induced thrombocytopenia (CIT) might be influenced by hyperfibrinolysis. t-PA-thromboelastography is a fast and reliable assay for hyperfibrinolysis in CIT patients. Clots of CIT patients are more susceptible to t-PA induced lysis compared to healthy individuals. Besides platelets, other factors are likely to influence clot lysis in CIT patients.

Background: Bleeding events in chemotherapy-induced thrombocytopenic (CIT) patients with similar platelet counts might be influenced by changes in clot lysis potential.

Objectives: To investigate, in an observational study, thromboelastographic lysis parameters, alterations in clot strength and susceptibility to clot lysis in CIT patients. To identify factors associated with fibrinolytic profiles, and to evaluate the effects of platelet transfusions.

Methods: Independent determinants of tissue-type plasminogen activator (t-PA)-ROTEM lysis parameters were identified with multivariable linear regression. Clot formation, strength and lysis parameters were compared with the results of healthy individuals. Characteristics of CIT patients with and without hyperfibrinolytic profiles were compared. t-PA-ROTEM results before, 1 hour after and 24 hours after platelet transfusion were compared.

Results: A total of 72 consecutive CIT patients were included. t-PA-ROTEM lysis parameters correlated with changes in fibrinolytic proteins. Clot formation time was longer, maximum clot firmness was weaker and lysis times were shorter than in healthy individuals. CIT patients had low plasminogen activator inhibitor-1 and thrombin-activatable fibrinolysis inhibitor levels, and 40% showed hyperfibrinolytic profiles. Platelet transfusions resulted in less hyperfibrinolytic profiles in many, but not all CIT patients. Patients without hyperfibrinolytic profiles had higher fibrinogen, factor VIII and α -antiplasmin levels.

Conclusions: t-PA-ROTEM can be used as a fast and reliable assay to detect hyperfibrinolytic profiles in CIT patients. CIT patients have weaker clots, which are more susceptible to clot lysis, than healthy individuals. Besides platelets, other factors are likely to influence clot susceptibility to fibrinolysis in CIT patients. The impact of a hyperfibrinolytic t-PA-ROTEM profile on bleeding remains to be investigated.
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http://dx.doi.org/10.1111/jth.14465DOI Listing
July 2019

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

Thromb Res 2019 02 4;174:88-94. Epub 2018 Dec 4.

Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, the Netherlands; Central Diagnostic Laboratory, Maastricht University Medical Centre (MUMC+), Maastricht, the Netherlands.

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.
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http://dx.doi.org/10.1016/j.thromres.2018.12.004DOI Listing
February 2019

Shifts of Transfusion Demand in Cardiac Surgery After Implementation of Rotational Thromboelastometry-Guided Transfusion Protocols: Analysis of the HEROES-CS (HEmostasis Registry of patiEntS in Cardiac Surgery) Observational, Prospective Open Cohort Database.

J Cardiothorac Vasc Anesth 2019 02 29;33(2):307-317. Epub 2018 Aug 29.

Department of Anaesthesiology and Pain Treatment, Maastricht University Medical Center, Maastricht, the Netherlands.

Objectives: Rotational thromboelastometry (ROTEM)-guided transfusion algorithms in cardiac surgery have been proven to be successful in reducing blood loss in randomized controlled trials. Using an institutional hemostasis registry of patients in cardiac surgery (HEROES-CS), the authors hypothesized that the use of ROTEM-guided transfusion algorithms would save blood products and overall costs in cardiac surgery in every day practice.

Design: Observational, prospective open cohort database.

Setting: Single-center academic hospital.

Participants: Cardiac surgery patients.

Interventions: Implementation of ROTEM-guided bleeding management.

Measurements And Main Results: A classical-guided algorithm and a ROTEM-guided algorithm were used for patient blood management in 2 cohorts. Primary outcome was the use and amount of blood products and hemostatic medication. Secondary outcomes were amount of rethoracotomies, length of stay, and 30-day mortality. Finally, costs and savings were calculated. The classical-guided cohort comprised 204 patients, and ROTEM-guided cohort comprised 151 patients. Baseline characteristics showed excellent similarities after propensity score matching of 202 patients. Blood loss was lower after ROTEM guidance (p < 0.001). Absolute risk reduction was 17% for red blood cells (p = 0.024), 12% for fresh frozen plasma (p = 0.019), and 4% for thrombocyte concentrates (p = 0.582). More tranexamic acid was given, but not more fibrinogen concentrate, while desmopressin was given less often. Hospital length of stay was reduced by an overall median of 2 and a mean of 4 days (p < 0.001). Mortality and rethoracotomy rates were not affected. Potential savings were about €4,800 ($5,630) per patient.

Conclusions: Implementation of a ROTEM-guided transfusion algorithm in cardiac surgery patients reduced the use of blood products and hemostatic medication, hereby saving costs. Reductions in mortality and rethoracotomy rates could not be found.
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http://dx.doi.org/10.1053/j.jvca.2018.08.203DOI Listing
February 2019

Platelet Aggregation in Healthy Participants is Not Affected by Smoking, Drinking Coffee, Consuming a High-Fat Meal, or Performing Physical Exercise.

Clin Appl Thromb Hemost 2019 Jan-Dec;25:1076029618782445. Epub 2018 Jun 19.

1 Central Diagnostic Laboratory, Cluster for Hemostasis and Transfusion, Maastricht University Medical Center (MUMC+), Maastricht, the Netherlands.

Platelet aggregation can be measured using optical aggregation (light transmission aggregometry, LTA) as well as by impedance (Multiplate analyzer). The LTA (the gold standard method) can be influenced by many preanalytical variables. Several guidelines differ in recommendations for the duration patients should refrain from smoking, coffee, fatty meals, and physical exercise prior to blood collection for performing platelet function tests. In this pilot study, the influence of smoking, coffee, high-fat meal, or physical exercise on platelet aggregation was investigated to improve patient friendliness and laboratory logistics in platelet function diagnostics. Standardized blood collection was performed when participants were fasting and after each parameter (n=5 per group). As a control for diurnal fluctuations, participants (n=6) were fasting during both blood collections. Platelet aggregation was executed using standardized methods for LTA and Multiplate analyzer. Statistical analysis of the results using Wilcoxon signed-rank test did not show any significant differences in platelet aggregation in healthy participants under different preanalytical variables. Therefore, these variables are not expected to adversely affect testing, which can avoid canceling tests for those patients who inevitably did.
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http://dx.doi.org/10.1177/1076029618782445DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6714925PMC
July 2019

Impaired mitochondrial activity explains platelet dysfunction in thrombocytopenic cancer patients undergoing chemotherapy.

Haematologica 2018 09 7;103(9):1557-1567. Epub 2018 Jun 7.

Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University Medical Centre, the Netherlands

Severe thrombocytopenia (≤50×10 platelets/L) due to hematological malignancy and intensive chemotherapy is associated with an increased risk of clinically significant bleeding. Since the bleeding risk is not linked to the platelet count only, other hemostatic factors must be involved. We studied platelet function in 77 patients with acute leukemia, multiple myeloma or malignant lymphoma, who experienced chemotherapy-induced thrombocytopenia. Platelets from all patients - independent of disease or treatment type - were to a variable extent compromised in Ca flux, integrin a β activation and P-selectin expression when stimulated with a panelof agonists. The patients' platelets were also impaired in spreading on fibrinogen. Whereas the Ca store content was unaffected, the patients' platelets showed ongoing phosphatidylserine exposure, which was not due to apoptotic caspase activity. Interestingly, mitochondrial function was markedly reduced in platelets from a representative subset of patients, as evidenced by a low mitochondrial membrane potential (<0.001) and low oxygen consumption (<0.05), while the mitochondrial content was normal. Moreover, the mitochondrial impairments coincided with elevated levels of reactive oxygen species (Spearman's rho=-0.459, =0.012). Markedly, the impairment of platelet function only appeared after two days of chemotherapy, suggesting origination in the megakaryocytes. In patients with bone marrow recovery, platelet function improved. In conclusion, our findings disclose defective receptor signaling related to impaired mitochondrial bioenergetics, independent of apoptosis, in platelets from cancer patients treated with chemotherapy, explaining the low hemostatic potential of these patients.
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http://dx.doi.org/10.3324/haematol.2017.185165DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6119160PMC
September 2018

Detecting clinically relevant rivaroxaban or dabigatran levels by routine coagulation tests or thromboelastography in a cohort of patients with atrial fibrillation.

Thromb J 2018 1;16. Epub 2018 Feb 1.

1Central Diagnostic Laboratory, Maastricht University Medical Centre (MUMC+), Maastricht, The Netherlands.

Background: Traditional coagulation tests are included in emergency guidelines for management of patients on direct oral anticoagulants (DOACs) who experience acute bleeding or require surgery. We determined the ability of traditional coagulation tests and fast whole blood thromboelastography (ROTEM®) to screen for anticoagulation activity of dabigatran and rivaroxaban as low as 30 ng/mL.

Methods: One hundred eighty-four citrated blood samples (75 dabigatran, 109 rivaroxaban) were collected from patients with non-valvular atrial fibrillation (NVAF), to perform screening tests from different manufacturers, (diluted, D) PT, aPTT, TT and ROTEM®. The activity of DOACs was quantitatively determined by clot detection assays: Hemoclot DTT and DiXaI test (Biophen), on CS2100 (Siemens). The clotting time (CT) of INTEM and EXTEM ROTEM® (Werfen) were used as test parameters.

Results: Dabigatran, ≥ 30 ng/mL, was accurately detected by five coagulation tests: APTT Actin FSL (93%), PT Neoplastin (93%), APTT Cephascreen, Thromboclotin, and Thrombin (all 100%), but not by PT Innovin (49%). CT-EXTEM (91%) was sufficiently sensitive, but not CT-INTEM (52%) APTT Cephascreen and Thrombin showed good linearity (R = 0.71,R = 0.72). For the other tests linearity was moderate to poor. Rivaroxaban was accurately detected by PT Neoplastin (98%) and less so by APTT Cephascreen (85%). In addition, rivaroxaban was also accurately detected by CT-INTEM (96%). PT Neoplastin showed good linearity (R = 0.81), all other tests had moderate to poor linearity.

Conclusion: In patients with NVAF, the ability of routine coagulation tests to detect the presence of significant levels of DOACs is test and reagent dependent. CT-INTEM and CT-EXTEM may be fast whole blood alternatives.

Trial Registration: The Institutional Review Board of the MUMC approved this study (December 2011, project number 114069).
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http://dx.doi.org/10.1186/s12959-017-0160-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5793444PMC
February 2018

The development of D antibodies after D-mismatched kidney transplantation in a setting of reduced immunosuppression.

Transfusion 2018 01 29;58(1):100-104. Epub 2017 Nov 29.

Central Diagnostic Laboratory, Cluster for Hemostasis and Transfusion.

Background: D antigens are not taken into account in the allocation of solid organs. Female transplant recipients with D antibodies as a consequence of D-mismatched kidney transplantation may develop hemolytic disease of the fetus and newborn in future pregnancies. We examined D antibody development in transplant recipients who received D-mismatched kidney transplantation in absence of D prophylaxis and in a setting of reduced immunosuppression.

Study Design And Methods: From 1993 until 2015, a total of 1355 kidney patients received transplantations in our center of whom 156 received a D-mismatched graft. A retrospective analysis was conducted; frozen stored sera obtained from transplant recipients 3 months after transplantation were tested for irregular red blood cell (RBC) antibodies using a three-cell screening and an identification panel. In the case of D antibody positivity, additional testing was performed 1 month before transplantation.

Results: In seven of 156 (4.5%) transplant recipients we found irregular RBC antibodies after transplantation, of which five (3.2%) were determined to be D antibodies. We observed only one (0.6%) recipient without D antibodies before transplantation.

Conclusion: Although the risk of D antibody development is considerably lower after D-mismatched kidney transplantation than D-mismatched pregnancy, anti-D prophylaxis may still be advisable for female transplant recipients of childbearing age.
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http://dx.doi.org/10.1111/trf.14405DOI Listing
January 2018

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

Platelets 2019 14;30(1):81-87. Epub 2017 Nov 14.

d Central Diagnostic Laboratory, Unit for Hemostasis and Transfusion, Maastricht University Medical Centre+ , Maastricht , The Netherlands.

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.
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http://dx.doi.org/10.1080/09537104.2017.1371290DOI Listing
March 2019

Routine haemostasis testing before transplanted kidney biopsy: a cohort study.

Transpl Int 2018 03 21;31(3):302-312. Epub 2017 Nov 21.

Central Diagnostic Laboratory, Cluster for Haemostasis and Transfusion, Maastricht University Medical Center (Maastricht UMC+), Maastricht, The Netherlands.

Kidney biopsy can result in bleeding complications. Prebiopsy testing using bleeding time (BT) is controversial. New whole blood haemostasis tests, such as platelet function analyser-100 (PFA-100) and multiple electrode aggregometry (MEA), might perform better. We postulated that PFA-100 would be suitable to replace BT prebiopsy. In 154 patients, transplanted kidney biopsies were performed after measurement of bleeding time, PFA-100, MEA and mean platelet volume (MPV). Bleeding outcome (haemoglobin (Hb) drop, haematuria (±bladder catheterization), ultrasound finding of a bleeding, need for (non)surgical intervention and/or transfusion) after the biopsy was correlated to each test. Male-female ratio was 2:1. 50% had a surveillance biopsy at either three or 12 months. Around 17% (had) used acetylsalicylic acid (ASA) prebiopsy. Of 17 bleeding events, one subject needed a transfusion. Most bleeding events were Hb reductions over 1 mmol/l and all resolved uneventful. BT, PFA-100, MEA and MPV did not predict a bleeding outcome; prior ASA use however could (odds ratio 3.19; 95%-CI 1.06 to 9.61). Diagnostic performance data and Bland-Altman analysis showed that BT could not be substituted by PFA-100. ASA use was the best determinant of bleeding after kidney biopsy. Routine haemostasis testing prebiopsy has no added value.
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http://dx.doi.org/10.1111/tri.13090DOI Listing
March 2018

Empirically Reduced Dosages of Tinzaparin in Patients with Moderate-to-Severe Renal Insufficiency Lead to Inadequate Anti-Xa Levels.

Nephron 2017 30;137(2):113-123. Epub 2017 Jun 30.

Department of Internal Medicine, Maastricht University Medical Center+ (MUMC+), Maastricht, The Netherlands.

Background: Due to the higher molecular weight of tinzaparin, the low molecular weight heparin (LMWH) is less dependent on renal excretion than other LMWH preparations. However, several international guidelines recommend the same preemptive dosage reduction for all therapeutic dose LMWHs prescribed in renal insufficient patients, to ensure that there is no accumulation of anticoagulant activity and increased risk of bleeding. This study is aimed at assessing whether a preemptive dosage reduction of tinzaparin in all renal insufficient patients (comprising 25% reduction in patients with Modification of Diet in Renal Disease - estimated glomerular filtration rate (MDRD-eGFR) 30-60 mL/min/1.73 m2 and 50% reduction in patients with MDRD-eGFR <30 mL/min/1.73 m2) leads to adequate anti-Xa levels.

Methods: We selected the anti-Xa levels of in-hospital patients (≥18 years) with moderate-to-severe renal insufficiency (MDRD-eGFR <60 mL/min/1.73 m2), on therapeutic dosages of tinzaparin. Anti-Xa levels were measured using a chromogenic assay.

Results: Preemptive dosage reduction resulted in a median anti-Xa activity of 0.50 IU/mL (interquartile range [IQR] 0.38-0.60). In 92.3% of patients the anti-Xa level was below the target anti-Xa level of >0.85 IU/mL for therapeutic indications. Unadjusted dosages led to a median anti-Xa activity of 0.74 IU/mL (IQR 0.56-0.92). The preemptive dosage reduction was significantly associated with anti-Xa activity below therapeutic range (p = 0.007). No difference in anti-Xa activity was observed between patients with moderate (0.71 IU/mL, IQR 0.61-0.95) versus severe (0.65 IU/mL, IQR 0.41-1.06) renal insufficiency in whom an unadjusted dose had been administered (p = 0.77). None of the anti-Xa levels were above the upper margin of the presumed therapeutic range of 2.0 IU/mL.

Conclusion: In renal insufficient patients, the preemptive dosage reduction of tinzaparin leads to inadequate anti-Xa levels.
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http://dx.doi.org/10.1159/000477474DOI Listing
June 2018

Application of an optimized flow cytometry-based quantification of Platelet Activation (PACT): Monitoring platelet activation in platelet concentrates.

PLoS One 2017 16;12(2):e0172265. Epub 2017 Feb 16.

Synapse Research Institute, Maastricht, the Netherlands.

Background: Previous studies have shown that flow cytometry is a reliable test to quantify platelet function in stored platelet concentrates (PC). It is thought that flow cytometry is laborious and hence expensive. We have optimized the flow cytometry-based quantification of agonist induced platelet activation (PACT) to a labor, time and more cost-efficient test. Currently the quality of PCs is only monitored by visual inspection, because available assays are unreliable or too laborious for use in a clinical transfusion laboratory. Therefore, the PACT was applied to monitor PC activation during storage.

Study Design And Methods: The optimized PACT was used to monitor 5 PCs during 10 days of storage. In brief, optimized PACT uses a ready-to-use reaction mix, which is stable at -20°C. When needed, a test strip is thawed and platelet activation is initiated by mixing PC with PACT. PACT was based on the following agonists: adenosine diphosphate (ADP), collagen-related peptide (CRP) and thrombin receptor-activating peptide (TRAP-6). Platelet activation was measured as P-selectin expression. Light transmission aggregometry (LTA) was performed as a reference.

Results: Both PACT and LTA showed platelet function decline during 10-day storage after stimulation with ADP and collagen/CRP; furthermore, PACT showed decreasing TRAP-induced activation. Major differences between the two tests are that PACT is able to measure the status of platelets in the absence of agonists, and it can differentiate between the number of activated platelets and the amount of activation, whereas LTA only measures aggregation in response to an agonist. Also, PACT is more time-efficient compared to LTA and allows high-throughput analysis.

Conclusion: PACT is an optimized platelet function test that can be used to monitor the activation of PCs. PACT has the same accuracy as LTA with regard to monitoring PCs, but it is superior to both LTA and conventional flow cytometry based tests with regard to labor-, time- and cost efficiency.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0172265PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5313179PMC
August 2017

The use of regression analysis in determining reference intervals for low hematocrit and thrombocyte count in multiple electrode aggregometry and platelet function analyzer 100 testing of platelet function.

Platelets 2017 Nov 9;28(7):668-675. Epub 2017 Jan 9.

a Department of Anaesthesiology and Pain Treatment , Maastricht University Medical Center (MUMC+) , Maastricht , The Netherlands.

Low platelet counts and hematocrit levels hinder whole blood point-of-care testing of platelet function. Thus far, no reference ranges for MEA (multiple electrode aggregometry) and PFA-100 (platelet function analyzer 100) devices exist for low ranges. Through dilution methods of volunteer whole blood, platelet function at low ranges of platelet count and hematocrit levels was assessed on MEA for four agonists and for PFA-100 in two cartridges. Using (multiple) regression analysis, 95% reference intervals were computed for these low ranges. Low platelet counts affected MEA in a positive correlation (all agonists showed r ≥ 0.75) and PFA-100 in an inverse correlation (closure times were prolonged with lower platelet counts). Lowered hematocrit did not affect MEA testing, except for arachidonic acid activation (ASPI), which showed a weak positive correlation (r = 0.14). Closure time on PFA-100 testing was inversely correlated with hematocrit for both cartridges. Regression analysis revealed different 95% reference intervals in comparison with originally established intervals for both MEA and PFA-100 in low platelet or hematocrit conditions. Multiple regression analysis of ASPI and both tests on the PFA-100 for combined low platelet and hematocrit conditions revealed that only PFA-100 testing should be adjusted for both thrombocytopenia and anemia. 95% reference intervals were calculated using multiple regression analysis. However, coefficients of determination of PFA-100 were poor, and some variance remained unexplained. Thus, in this pilot study using (multiple) regression analysis, we could establish reference intervals of platelet function in anemia and thrombocytopenia conditions on PFA-100 and in thrombocytopenia conditions on MEA.
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http://dx.doi.org/10.1080/09537104.2016.1257782DOI Listing
November 2017

Within-day biological variation and hour-to-hour reference change values for hematological parameters.

Clin Chem Lab Med 2017 Jun;55(7):1013-1024

.

Background: Middle- and long-term biological variation data for hematological parameters have been reported in the literature. Within-day 24-h variability profiles for hematological parameters are currently lacking. However, comprehensive hour-to-hour variability data are critical to detect diurnal cyclical rhythms, and to take into account the 'time of sample collection' as a possible determinant of natural fluctuation. In this study, we assessed 24-h variation profiles for 20 hematological parameters.

Methods: Blood samples were collected under standardized conditions from 24 subjects every hour for 24 h. At each measurement, 20 hematological parameters were determined in duplicate. Analytical variation (CVA), within-subject biological variation (CVI), between-subject biological variation (CVG), index of individuality (II), and reference change values (RCVs) were calculated. For the parameters with a diurnal rhythm, hour-to-hour RCVs were determined.

Results: All parameters showed higher CVG than CVI. Highest CVG was found for eosinophils (46.6%; 95% CI, 34.9%-70.1%) and the lowest value was mean corpuscular hemoglobin concentration (MCHC) (3.2%; 95% CI, 2.4%-4.8%). CVI varied from 0.4% (95% CI, 0.32%-0.42%) to 20.9% (95% CI, 19.4%-22.6%) for red cell distribution width (RDW) and eosinophils, respectively. Six hematological parameters showed a diurnal rhythm.

Conclusions: We present complete 24-h variability profiles for 20 hematological parameters. Hour-to-hour reference changes values may help to better discriminate between random fluctuations and true changes in parameters with rhythmic diurnal oscillations.
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http://dx.doi.org/10.1515/cclm-2016-0716DOI Listing
June 2017

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

Eur Heart J Cardiovasc Pharmacother 2017 01 7;3(1):11-17. Epub 2016 Sep 7.

Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, PO Box 616, 6200 MD Maastricht, The Netherlands.

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.
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http://dx.doi.org/10.1093/ehjcvp/pvw026DOI Listing
January 2017

Rapid and Correct Prediction of Thrombocytopenia and Hypofibrinogenemia with Rotational Thromboelastometry in Cardiac Surgery Reconsidered.

J Cardiothorac Vasc Anesth 2016 Dec 12;30(6):e55-e56. Epub 2016 Apr 12.

Central Diagnostic Laboratory, Cluster for Hemostasis and Transfusion, Maastricht University Medical Center, Maastricht, The Netherlands.

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http://dx.doi.org/10.1053/j.jvca.2016.04.009DOI Listing
December 2016

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

Blood 2016 06 15;127(23):2791-803. Epub 2016 Apr 15.

Department of Haematology, University of Cambridge, National Health Service Blood and Transplant, Cambridge Biomedical Campus, Cambridge, United Kingdom;

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.
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http://dx.doi.org/10.1182/blood-2015-12-688267DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5016734PMC
June 2016

Platelet Control of Fibrin Distribution and Microelasticity in Thrombus Formation Under Flow.

Arterioscler Thromb Vasc Biol 2016 Apr 4;36(4):692-9. Epub 2016 Feb 4.

From the Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, The Netherlands (F.S., C.C.F.M.J.B., R.V., T.G.M., J.M.E.M.C., J.W.M.H., P.E.J.v.d.M.); Research and Development, Optics11, Amsterdam, The Netherlands (N.R., K.O.v.d.L., E.J.B.); Arthur Bloom Haemophilia Centre, Cardiff Institute of Infection & Immunity, School of Medicine, Cardiff University, Cardiff, United Kingdom (P.W.C.); and Central Diagnostic Laboratory (Y.M.C.H.), Departments of Anaesthesiology (M.D.L.) and Internal Medicine (Y.M.C.H.), Maastricht University Medical Center, Maastricht, The Netherlands.

Objective: Platelet- and fibrin-dependent thrombus formation is regulated by blood flow and exposure of collagen and tissue factor. However, interactions between these blood-borne and vascular components are not well understood.

Approach And Results: Here, we developed a method to assess whole-blood thrombus formation on microspots with defined amounts of collagen and tissue factor, allowing determination of the mechanical properties and intrathrombus composition. Confining the collagen content resulted in diminished platelet deposition and fibrin formation at high shear flow conditions, but this effect was compensated by a larger thrombus size and increased accumulation of fibrin in the luminal regions of the thrombi at the expense of the base regions. These thrombi were more dependent on tissue factor-triggered thrombin generation. Microforce nanoindentation analysis revealed a significantly increased microelasticity of thrombi with luminal-oriented fibrin. At a low shear rate, fibrin fibers tended to luminally cover the thrombi, again resulting in a higher microelasticity. Studies with blood from patients with distinct hemostatic insufficiencies indicated an impairment in the formation of a platelet-fibrin thrombus in the cases of dilutional coagulopathy, thrombocytopenia, Scott syndrome, and hemophilia B.

Conclusions: Taken together, our data indicate that (1) thrombin increases the platelet thrombus volume; (2) tissue factor drives the formation of fibrin outside of the platelet thrombus; (3) limitation of platelet adhesion redirects fibrin from bottom to top of the thrombus; (4) a lower shear rate promotes thrombus coverage with fibrin; (5) the fibrin distribution pattern determines thrombus microelasticity; and (6) the thrombus-forming process is reduced in patients with diverse hemostatic defects.
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http://dx.doi.org/10.1161/ATVBAHA.115.306537DOI Listing
April 2016

Validation of a modified thromboelastometry approach to detect changes in fibrinolytic activity.

Thromb J 2016 14;14. Epub 2016 Jan 14.

Central Diagnostic Laboratory, Cluster for Hemostasis and transfusion, Maastricht University Medical Center (MUMC+), Maastricht, The Netherlands.

Background: Thus far, validated whole blood assays used in in vitro fibrinolysis experiments using thromboelastometry (ROTEM) are lacking or have yet to be tested in humans. The objective was first, to establish a standardized modified ROTEM approach to detect both hypo- and hyperfibrinolysis. And second, to perform a technical and clinical validation of the assay.

Methods: Blood was used of healthy volunteers, patients with sepsis, patients after cardiothoracic surgery, pregnant women, and cirrhotic liver disease patients. A whole blood tissue factor (TF) activated ROTEM assay with and without the addition of recombinant tissue plasminogen activator (rTPA) was developed. Plasma fibrinolysis determinants were measured in all volunteers and patients.

Results: Thirty five pM TF and additions of 125 and 175 ng/ml rTPA resulted in full lysis within 60 min in healthy volunteers. Coefficients of variation were below 10 % without and below 20 % with rTPA addition. In sepsis the hypofibrinolytic ROTEM profiles with 175 ng/ml rTPA were in line with the plasma determinants (high PAI-1, high fibrinogen, low tPA activity, and high d-dimers). After cardiothoracic surgery, reduced fibrinogen and platelet levels accounted for the reduced maximum clot firmness. The hypofibrinolytic profile is attributed to tranexamic acid use and elevated PAI-1 levels. The lowest rTPA concentration in cirrhosis resulted in hyperfibrinolysis in only few of the patients. In pregnancy normal profiles were found.

Discussion: Our high rTPA concentration demonstrates hypofibrinolytic profiles adequately in sepsis and after cardiothoracic surgery. Our low rTPA concentration of 125 ng/ml seems too high for demonstrating hyperfibrinolysis in cirrhotic liver disease.

Conclusions: We were able to present a validated whole blood ROTEM approach to fibrinolysis testing using added rTPA, which can be of added value next to classical plasma based fibrinolysis assays.
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http://dx.doi.org/10.1186/s12959-016-0076-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4712545PMC
January 2016

Coated platelets function in platelet-dependent fibrin formation via integrin αIIbβ3 and transglutaminase factor XIII.

Haematologica 2016 Apr 31;101(4):427-36. Epub 2015 Dec 31.

Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, The Netherlands

Coated platelets, formed by collagen and thrombin activation, have been characterized in different ways: i) by the formation of a protein coat of α-granular proteins; ii) by exposure of procoagulant phosphatidylserine; or iii) by high fibrinogen binding. Yet, their functional role has remained unclear. Here we used a novel transglutaminase probe, Rhod-A14, to identify a subpopulation of platelets with a cross-linked protein coat, and compared this with other platelet subpopulations using a panel of functional assays. Platelet stimulation with convulxin/thrombin resulted in initial integrin α(IIb)β3 activation, the appearance of a platelet population with high fibrinogen binding, (independently of active integrins, but dependent on the presence of thrombin) followed by phosphatidylserine exposure and binding of coagulation factors Va and Xa. A subpopulation of phosphatidylserine-exposing platelets bound Rhod-A14 both in suspension and in thrombi generated on a collagen surface. In suspension, high fibrinogen and Rhod-A14 binding were antagonized by combined inhibition of transglutaminase activity and integrin α(IIb)β3 Markedly, in thrombi from mice deficient in transglutaminase factor XIII, platelet-driven fibrin formation and Rhod-A14 binding were abolished by blockage of integrin α(IIb)β3. Vice versa, star-like fibrin formation from platelets of a patient with deficiency in α(IIb)β3(Glanzmann thrombasthenia) was abolished upon blockage of transglutaminase activity. We conclude that coated platelets, with initial α(IIb)β3 activation and high fibrinogen binding, form a subpopulation of phosphatidylserine-exposing platelets, and function in platelet-dependent star-like fibrin fiber formation via transglutaminase factor XIII and integrin α(IIb)β3.
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http://dx.doi.org/10.3324/haematol.2015.131441DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5004391PMC
April 2016

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

Thromb Haemost 2016 Jan 13;115(1):7-24. Epub 2015 Aug 13.

Minka Vries, CARIM, Universiteitssingel 50, 6229 ER Maastricht, P. O. Box 616, 6200 MD Maastricht, The Netherlands, Tel.: +31 433881542, Fax: +31 43884159, E-mail:

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.
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http://dx.doi.org/10.1160/TH15-04-0355DOI Listing
January 2016
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