Activation of factor VII-activating protease in human inflammation: a sensor for cell death

textabstractIntroduction: Cell death is a central event in the pathogenesis of sepsis and is reflected by circulating nucleosomes. Circulating nucleosomes were suggested to play an important role in inflammation and were demonstrated to correlate with severity and outcome in sepsis patients. We recently showed that plasma can release nucleosomes from late apoptotic cells. Factor VII-activating protease (FSAP) was identified to be the plasma serine protease responsible for nucleosome release. The aim of this study was to investigate FSAP activation in patients suffering from various inflammatory diseases of increasing severity.Methods: We developed ELISAs to measure FSAP-C1-inhibitor and FSAP-α2-antiplasmin complexes in plasma. FSAP-inhibitor complexes were measured in the plasma of 20 adult patients undergoing transhiatal esophagectomy, 32 adult patients suffering from severe sepsis and 8 from septic shock and 38 children suffering from meningococcal sepsis.Results: We demonstrate plasma FSAP to be activated upon contact with apoptotic and necrotic cells by an assay detecting complexes between FSAP and its target serpins α2-antiplasmin and C1-inhibitor, respectively. By means of that assay we demonstrate FSAP activation in post-surgery patients, patients suffering from severe sepsis, septic shock and meningococcal sepsis. Levels of FSAP-inhibitor complexes correlate with nucleosome levels and correlate with severity and mortality in these patients.Conclusions: These results suggest FSAP activation to be a sensor for cell death in the circulation and that FSAP activation in sepsis might be involved in nucleosome release, thereby contributing to lethality

Prothrombinase-Induced Clotting Time to Measure Drug Concentrations of Rivaroxaban, Apixaban, and Edoxaban in Clinical Practice: A Cross-Sectional Study.

Prothrombinase-induced clotting time (PiCT) is proposed as a rapid and inexpensive laboratory test to measure direct oral anticoagulant (DOAC) drug levels. In a prospective, multicenter cross-sectional study, including 851 patients, we aimed to study the accuracy of PiCT in determining rivaroxaban, apixaban, and edoxaban drug concentrations and assessed whether clinically relevant drug levels could be predicted correctly. Citrated plasma samples were collected, and the Pefakit® PiCT was utilized. Ultra-high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed to measure drug concentrations. Cut-off levels were established using receiver-operating characteristics curves. We calculated sensitivities and specificities with respect to clinically relevant drug concentrations. Spearman's correlation coefficient between PiCT and drug concentrations was 0.85 in the case of rivaroxaban (95% CI 0.82, 0.88), 0.66 for apixaban (95% CI 0.60, 0.71), and 0.78 for edoxaban (95% CI 0.65, 0.86). The sensitivity to detect clinically relevant drug concentrations was 85.1% in the case of 30 µg L-1 (95% CI 82.0, 87.7; specificity 77.9; 72.1, 82.7), 85.7% in the case of 50 µg L-1 (82.4, 88.4; specificity 77.3; 72.5, 81.5), and 85.1% in the case of 100 µg L-1 (80.9, 88.4; specificity 73.2%; 69.1, 76.9). In conclusion, the association of PiCT with DOAC concentrations was fair, and the majority of clinically relevant drug concentrations were correctly predicted

Determination of Anti-Xa Inhibitor Plasma Concentrations Using a Universal Edoxaban Calibrator

A universal calibrator for the determination of all anti-Xa inhibitors would support laboratory processes. We aimed to test the clinical performance of an anti-Xa assay utilizing a universal edoxaban calibrator to determine clinically relevant concentrations of all anti-Xa inhibitors. Following a pilot study, we enrolled 553 consecutive patients taking rivaroxaban, edoxaban, or apixaban from nine study centers in a prospective cross-sectional study. The Technochrom$^{®}$ anti-Xa assay was conducted using the Technoview$^{®}$ edoxaban calibrator. Using ultra-high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS), anti-Xa inhibitor drug concentrations were determined. Sensitivities and specificities to detect three clinically relevant drug concentrations (30 µgL$^{-1}$, 50 µgL$^{-1}$, 100 µgL$^{-1}$) were determined. Overall, 300 patients treated with rivaroxaban, 221 with apixaban, and 32 with edoxaban were included. The overall correlation coefficient (r$_{s}$) was 0.95 (95% CI 0.94, 0.96). An area under the receiver operating characteristic curve of 0.96 for 30 µgL$^{-1}$, 0.98 for 50 µgL$^{-1}$, and 0.99 for 100 µgL$^{-1}$ was found. The sensitivities were 92.3% (95% CI 89.2, 94.6), 92.7% (89.4, 95.1), and 94.8% (91.1, 97.0), respectively (specificities 82.2%, 93.7%, and 94.4%). In conclusion, the clinical performance of a universal, edoxaban-calibrated anti-Xa assay was solid and most drug concentrations were predicted correctly

Accuracy of a Single, Heparin-Calibrated Anti-Xa Assay for the Measurement of Rivaroxaban, Apixaban, and Edoxaban Drug Concentrations: A Prospective Cross-Sectional Study

Background: Applying a single anti-Xa assay, calibrated to unfractionated heparin to measure rivaroxaban, apixaban, and edoxaban would simplify laboratory procedures and save healthcare costs. Aim: We hypothesized that a heparin-calibrated anti-Xa assay would accurately measure rivaroxaban, apixaban, and edoxaban drug concentrations and correctly predict clinically relevant drug levels. Methods: This analysis is part of the Simple-Xa study, a prospective multicenter cross-sectional study conducted in clinical practice. Patients treated with rivaroxaban, apixaban, or edoxaban were included. Anti-Xa activity was measured using the Siemens INNOVANCE® Heparin assay. Drug concentrations were determined using ultra-high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). Cut-off levels were determined in a derivation dataset (50% of patients) and sensitivities and specificities were calculated in a verification dataset (50% of patients). Results: Overall, 845 patients were available for analysis. Correlation coefficients (r s ) between the heparin-calibrated anti-Xa assay and drug concentrations were 0.97 (95% CI 0.97, 0.98) for rivaroxaban, 0.96 (0.96, 0.97) for apixaban, and 0.96 (0.94, 0.99) for edoxaban. The area under the receiver operating characteristics curve (ROC) was 0.99 for all clinically relevant drug concentrations. In the verification dataset, the sensitivity was 94.2% (95% CI 90.8-96.6) for 30 μg L-1, 95.8% (92.4-98.0) for 50 μg L-1, and 98.7% (95.5-99.9) for 100 μg L-1. Specificities were 86.3% (79.2-91.7), 89.8% (84.5-93.7), and 88.7% (84.2-92.2), respectively. Conclusion: In a large prospective study in clinical practice, a strong correlation of heparin-calibrated anti-Xa measurements with LC-MS/MS results was observed and clinically relevant drug concentrations were predicted correctly. Keywords: anti-Xa assay; diagnostic accuracy; direct oral anticoagulants; laboratory monitoring; rivaroxaban

Variability between laboratories performing coagulation tests with identical platforms: a nationwide evaluation study.

BACKGROUND While the assessment of analytical precision within medical laboratories has received much attention in scientific enquiry, the degree of as well as the sources causing variation between them remains incompletely understood. In this study, we quantified the variance components when performing coagulation tests with identical analytical platforms in different laboratories and computed intraclass correlations coefficients (ICC) for each coagulation test. METHODS Data from eight laboratories measuring fibrinogen twice in twenty healthy subjects with one out of 3 different platforms and single measurements of prothrombin time (PT), and coagulation factors II, V, VII, VIII, IX, X, XI and XIII were analysed. By platform, the variance components of (i) the subjects, (ii) the laboratory and the technician and (iii) the total variance were obtained for fibrinogen as well as (i) and (iii) for the remaining factors using ANOVA. RESULTS The variability for fibrinogen measurements within a laboratory ranged from 0.02 to 0.04, the variability between laboratories ranged from 0.006 to 0.097. The ICC for fibrinogen ranged from 0.37 to 0.66 and from 0.19 to 0.80 for PT between the platforms. For the remaining factors the ICC's ranged from 0.04 (FII) to 0.93 (FVIII). CONCLUSIONS Variance components that could be attributed to technicians or laboratory procedures were substantial, led to disappointingly low intraclass correlation coefficients for several factors and were pronounced for some of the platforms. Our findings call for sustained efforts to raise the level of standardization of structures and procedures involved in the quantification of coagulation factors

Safety and Usage of C1-Inhibitor in Hereditary Angioedema: Berinert Registry Data

BackgroundThe plasma-derived, highly purified, nanofiltered C1-inhibitor concentrate (Berinert; “pnfC1-INH”) is approved in the United States for treating hereditary angioedema (HAE) attacks and in many European countries for attack treatment and short-term prophylaxis.ObjectiveThe objective of this study was to describe safety and usage patterns of pnfC1-INH.MethodsA multicenter, observational, registry was conducted between 2010 and 2014 at 30 United States and 7 European sites to obtain both prospective (occurring after enrollment) and retrospective (occurring before enrollment) safety and usage data on subjects receiving pnfC1-INH for any reason.ResultsOf 343 enrolled patients, 318 received 1 or more doses of pnfC1-INH for HAE attacks (11,848 infusions) or for prophylaxis (3142 infusions), comprising the safety population. Median dosages per infusion were 10.8 IU/kg (attack treatment) and 16.6 IU/kg (prophylaxis). Approximately 95% of infusions were administered outside of a health care setting. No adverse events (AEs) were reported in retrospective data. Among prospective data (n = 296 subjects; 9148 infusions), 252 AEs were reported in 85 (28.7%) subjects (rate of 0.03 events/infusion); 9 events were considered related to pnfC1-INH. Two thromboembolic events were reported in subjects with thrombotic risk factors. No patient was noted to have undergone viral testing for suspected blood-borne infection during registry participation.ConclusionsThe findings from this large, international patient registry documented widespread implementation of pnfC1-INH self-administration outside of a health care setting consistent with current HAE guidelines. These real-world data revealed pnfC1-INH usage for a variety of reasons in patients with HAE and showed a high level of safety regardless of administration setting or reason for use