Clot Waveform Analysis Parameters Research Articles

The Role of Clot Waveform Analysis and Related Parameters in the Diagnosis and Treatment of Hemophilia A

Background Hemophilia A (HA) is an inherited bleeding disorder caused by a deficiency or defect in factor VIII (FVIII). Methods We investigated the role of clot waveform analysis (CWA) of activated partial thromboplastin time in the diagnosis and therapeutic monitoring of HA. The changes in CWA parameters the maximum clotting velocity (|Min1|), maximum clotting acceleration (|Min2|), and maximum clotting deceleration (|Max2|) were detected among mild, moderate, and severe HA groups. Results As the severity of HA subtypes increased, the levels of |Min1|, |Min2|, and |Max2| progressively decreased (p < 0.05). Receiver operating characteristic curve analysis showed that |Max2| and |Min2| were more effective than |Min1| in distinguishing different types of HA patients, with higher diagnostic efficacy. The standard curves based on Actin FSL reagent for normal and low levels of FVIII:C-|Max2| were established, with R2 values of 0.98 and 0.99, respectively. These curves can be utilized for monitoring during replacement therapies involving full-length recombinant FVIII and B-domain-deleted FVIII. Thirty cases of HA patients utilized the FVIII-|Max2| standard curve to obtain individual pharmacokinetics characteristic parameters. The clearance, half-life (t1/2), time to FVIII:C of 1% above baseline (tt1%), and predicted dosage showed no statistically significant differences compared with one-stage assay (p > 0.05). Conclusion CWA is an economical and practical tool, and its related parameters are associated with the severity of HA. It has promising clinical prospects in predicting FVIII:C levels and individualized treatment when HA patients undergo replacement therapy.

Reagent Effects on the Activated Partial Thromboplastin Time Clot Waveform Analysis: A Multi-Centre Study.

(1) Background: The activated partial thromboplastin time (APTT)- based clot waveform analysis (CWA) quantitatively extends information obtained from the APTT waveform through its derivatives. However, pre-analytical variables including reagent effects on the CWA parameters are poorly understood and must be standardized as a potential diagnostic assay. (2) Methods: CWA was first analysed with patient samples to understand reagent lot variation in three common APTT reagents: Pathromtin SL, Actin FS, and Actin FSL. A total of 1055 healthy volunteers were also recruited from seven institutions across the Asia-Pacific region and CWA data were collected with the Sysmex CS analysers. (3) Results: CWA parameters varied less than 10% between lots and the linear mixed model analysis showed few site-specific effects within the same reagent group. However, the CWA parameters were significantly different amongst all reagent groups and thus reagent-specific 95% reference intervals could be calculated using the nonparametric method. Post-hoc analysis showed some degree of influence by age and gender with weak correlation to the CWA (r < 0.3). (4) Conclusions: Reagent type significantly affects APTT-based CWA with minimal inter-laboratory variations with the same coagulometer series that allow for data pooling across laboratories with more evidence required for age- and gender-partitioning.

Effects of Hemodilution on Clot Waveform Analysis Parameters, Clotting Factors, and Thrombin Generation Assays in a Dilutional Model Based on Analysis of 11 Healthy Male Blood Donors.

BackgroundClot wave analysis (CWA) during activated partial thromboplastin time (aPTT) measures the speed and extent of fibrin polymerization in the plasma. This study aimed to evaluate the effects of hemodilution on CWA parameter, clotting factors, and thrombin generation assays in a dilutional model.Material/MethodsPlatelet-poor plasma obtained from 11 healthy male volunteers was diluted with 0.9% sodium chloride by 10–80% to analyze coagulation profiles, CWA, clotting factors, and thrombin generation assays. CWA includes 5 parameters: the time-dependent variable (aPTT), rate/acceleration (min1, min2, and max2), and magnitude of signal change (delta).ResultsCritically low activities of 30% for clotting factors and 100 mg/dl of fibrinogen were determined at dilutions of 70% and 60%, respectively. Peak thrombin and endogenous thrombin potential were significantly lower compared with baseline after 50% and 80% dilution, respectively. aPTT did not correlate with the decrease in the clotting factors up to dilutions of 50% and subsequently became abnormal values. As the change in rate/acceleration parameters parallels the time course of clotting factor activity in a dilution-dependent linear manner, these parameters indicate an intervention threshold at critically low activities of clotting factors. The strongest correlations were observed between clotting factors and aPTT, clotting factors and min2, delta and peak thrombin (r=0.95), and delta and fibrinogen (r=0.98).ConclusionsaPTT was significantly correlated with clotting factors, while the rate/acceleration parameters and delta changed with variation in thrombin and fibrinogen generation. These findings may help in evaluating coagulability.

Activated partial thromboplastin time-based clot waveform analysis enables measurement of very low levels of factor IX activity in patients with severe hemophilia B.

The precise measurement of very low levels of factor IX activity (FIX:C < 1IU/dL) is essential for understanding clinical severity and risk of inhibitor development in patients with severe hemophilia B (Pw-SHB). However, such measurement sensitivity has not yet been achieved. We aimed to establish a measurement method using clot waveform analysis (CWA). Residual FIX:C by adding anti-FIX monoclonal antibody, FIX:C by adding recombinant (r)FIX to the commercial Pw-SHB plasmas, and FIX:C in our Pw-SHB were determined by CS-2000i™/CS-2400™, followed by analysis of CWA parameters. The presence of anti-FIX antibody in the commercial Pw-SHB plasmas significantly decreased coagulation potential compared to its absence. The addition of rFIX to these innate plasma samples produced significant changes in three parameters upon adding FIX:C at 0.1-1IU/dL, supporting the presence of trace FIX:C in Pw-SHB. Therefore, appropriate FIX-depleted plasma containing minimum residual FIX:C was chosen from reference curves of FIX:C (0.01-1IU/dL). Among patients with untreated Pw-SHB, two had FIX:C 0.6-0.7IU/dL and two had lower than detectable levels using FIX-depleted plasma. One of the latter had detectable trough levels post-rFIX administration. In conclusion, CWA enabled measurement of very low levels of FIX:C using appropriate FIX-deficient plasma.

A correlation of thrombin generation assay and clot waveform analysis in patients on warfarin

ABSTRACT Objectives Thrombin generation assays and activated partial thromboplastin time (aPTT)-based clot waveform analysis (CWA), are some examples of global coagulation assays. Each modality evaluates different aspects of the clot forming process to globally define haemostasis with exclusive measurement parameters. Data on CWA are emerging, but its performance against other haemostatic assays is yet to be ascertained. This study evaluates the correlation between aPTT-based CWA and CAT parameters across a range of INR in warfarin-treated patients. Patients/Methods A prospective study consisting of patients on warfarin anticoagulation with varying INR levels. CWA and CAT were performed for the study subjects. Results 54 samples were included covering an INR range from 1.33–6.89, with a mean of 4.31 +/- 1.13. For CAT parameters, endogenous thrombin potential (ETP) and peak thrombin were assessed. Both unadjusted and adjusted (adjusted for final plateau transmittance) aPTT-based CWA were evaluated for parameters min1 (maximum velocity), min2 (maximum acceleration) and max2 (maximum deceleration). Peak thrombin showed significant correlation with all CWA parameters (min1: r = 0.435, P<0.001; min2: r = 0.485, P<0.001; max2: r = 0.578, P<0.001; adjusted min1: r = 0.734, P<0.001, adjusted min2: r = 0.693, P<0.001; adjusted max2: r = 0.751, P<0.001). ETP correlated significantly with all CWA parameters except unadjusted min1 (min1: r = 0.235, P = 0.087; min2: r = 0.326, P = 0.016; max2: r = 0.437, P<0.001; adjusted min1: r = 0.610, P<0.001, adjusted min2: r = 0.563, P<0.001; adjusted max2: r = 0.642, P<0.001). Conclusion We demonstrated a modest correlation between CAT and CWA parameters. Adjusted CWA improved this correlation. These findings provide additional understanding of CWA and it’s role in the evaluation of global haemostatic function.

Clot characterization by multidisciplinary approach: biochemical and imaging parameters in a hypocoagulative setting. A pilot study.

BackgroundClot characterization is, to the present days, a multimodal approach: scanning the clot by electron microscopy (SEM) is helpful for the visualization of fibrin structure along with laboratory parameters such as the clot waveform analysis (CWA) and thrombin generation in different settings of clot abnormalities. This study aimed to assess whether the coagulative parameters were consistent with the clot images texture acquired by SEM, and therefore to propose a more generalist and integrative approach to clots classification.Design and methodsIn this pilot study, the examined population consists of eight healthy subjects, seven patients affected by Acquired Hemophilia A (AHA) and seven patients treated with Vitamin K Antagonists (VKAs), similar for age and gender. We studied the velocity and acceleration (1st and 2nd derivative of the aPTT) of clot formation (CWA), the thrombin generation, and the clots’ scanning by SEM. Images acquired with SEM were then analyzed with the MATLAB software with the “Texture Analysis” methods to perform classification. Among the various texture parameters, we reported Contrast and Energy.ResultsSignificant differences among healthy subjects, patients with AHA and those treated with VKAs were detected for the coagulative parameters. We found no differences between VKAs and AHA patients. Contrast and energy highlighted a significant difference among the three groups in agreement with the laboratory's parameters. We found no significant differences between VKAs and AHA patients.ConclusionsThe use of SEM, CWA and thrombin generation parameters may be a starting point for studies aimed to demonstrate the general characteristics of clot formation in different clinical conditions with a multiparametric approach.

Evaluation of coagulation status using clot waveform analysis in general ward patients with COVID-19.

Coronavirus disease (COVID-19)-related systemic cytokine response induces the production of procoagulant factors, which predisposes patients to a prothrombotic state. Viscoelastic testing can identify the degree of hypercoagulability, which is related to outcomes. We aimed to study the changes in clot waveform analysis (CWA) parameters in COVID-19 patients on hospital admission compared to those in a group of healthy individuals. We conducted a retrospective study of COVID-19 patients admitted to general wards and evaluated demographic and clinical parameters as well as laboratory parameters, including coagulation parameters. CWA data from patients (n = 62) with COVID-19 prior to the initiation of anticoagulation therapy were compared with those from healthy controls (n = 67). The measured CWA parameters were min1, min2, max2, and delta change. CWA, fibrinogen, and D-dimer values were higher in COVID-19 patients than in healthy controls (p < 0.001). CWA profiles were consistent with hypercoagulability and characterized by an increase in density, velocity, and acceleration of clot formation. Activated partial thromboplastin time, fibrinogen, D-dimer, and C-reactive protein (CRP) values were higher in patients in whom all CWA parameters were raised than in patients with just a few elevated CWA parameters, while Sequential Organ Failure Assessment scores, prothrombin time, fibrin degradation product levels and platelet counts did not differ between the two groups. CWA variables showed hypercoagulopathy on admission in COVID-19 patients who were hospitalized in the general ward, and this pattern was more pronounced in critically ill patients with elevated fibrinogen, D-dimer, and CRP levels. Our results may help identify patients at high risk of thromboembolism.

Clinical and laboratory features of hypercoagulability in COVID-19 and other respiratory viral infections amongst predominantly younger adults with few comorbidities

COVID-19 caused by Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) and other respiratory viral (non-CoV-2-RV) infections are associated with thrombotic complications. The differences in prothrombotic potential between SARS-CoV-2 and non-CoV-2-RV have not been well characterised. We compared the thrombotic rates between these two groups of patients directly and further delved into their coagulation profiles. In this single-center, retrospective cohort study, all consecutive COVID-19 and non-CoV-2-RV patients admitted between January 15th and April 10th 2020 were included. Coagulation parameters studied were prothrombin time and activated partial thromboplastin time and its associated clot waveform analysis (CWA) parameter, min1, min2 and max2. In the COVID-19 (n = 181) group there were two (1.0 event/1000-hospital-days) myocardial infarction events while one (1.8 event/1000-hospital-day) was reported in the non-CoV-2-RV (n = 165) group. These events occurred in patients who were severely ill. There were no venous thrombotic events. Coagulation parameters did not differ throughout the course of mild COVID-19. However, CWA parameters were significantly higher in severe COVID-19 compared with mild disease, suggesting hypercoagulability (min1: 6.48%/s vs 5.05%/s, P < 0.001; min2: 0.92%/s2 vs 0.74%/s2, P = 0.033). In conclusion, the thrombotic rates were low and did not differ between COVID-19 and non-CoV-2-RV patients. The hypercoagulability in COVID-19 is a highly dynamic process with the highest risk occurring when patients were most severely ill. Such changes in haemostasis could be detected by CWA. In our population, a more individualized thromboprophylaxis approach, considering clinical and laboratory factors, is preferred over universal pharmacological thromboprophylaxis for all hospitalized COVID-19 patients and such personalized approach warrants further research.

Inhibitor Index in the Clot Waveform Analysis-Based Mixing Test Differentiates among Hemophilia A without and with Inhibitors, and Lupus Anticoagulant.

The mixing test is used to identify the pathway to follow-up testing and is also useful for the investigation of lupus anticoagulant (LA) positivity. "To completely correct" indicates clotting factor deficiency, while "to not correct" indicates the presence of a clotting factor inhibitor including LA. "Index of circulation anticoagulant" and/or "percent correction" is used to interpret the results of mixing studies, but it does not accurately differentiate factor inhibitors from LA. To precisely differentiate hemophilia A (HA), HA with inhibitor (HA-inh), and LA using the clot waveform analysis (CWA)-based mixing test. Plasma samples from HA, LA, and HA-inh including acquired HA were incubated with normal plasma in 9:1, 1:1, and 1:9 mix ratios. From activated partial thromboplastin time CWA at 0-minute (immediately) and 12-minute incubation, the ratios of CWA parameters at 12 minutes/0 minute (inhibitor index) were assessed. The inhibitor index values of CWA parameters obtained using the mixing test in a 1:1 ratio demonstrated a significant difference between HA-inh and LA but could not differentiate LA from HA-inh completely. Plasmas used for the mixing tests in 9:1 and 1:9 ratios were able to fully distinguish between HA-inh (>0.5 BU/mL) and LA. These indices significantly correlated with inhibitor titer below 40 BU/mL (r > 0.90), possibly estimating FVIII inhibitor titer from the inhibitor index. Plasmas in HA and LA could be distinguished by mixing in a 1:1 ratio at 0 minute (immediately). The inhibitor index from CWA-based mixing tests with a 12-minute incubation could differentiate among HA, HA-inh, and LA quickly.

Thrombotic Events in COVID-19 Patients and Its Comparison with Other Non-Sars-Cov-2 Respiratory Viruses

IntroductionAn increasing number of evidence have reported the association of COVID-19 with increased incidence of thrombotic events. High incidences were initially reported in critically ill COVID-19 patients, but subsequently an increased incidence was also noticed in non-critically ill general ward patients. This has led to a universal recommendation of thromboprophylaxis for all COVID-19 patients by ASH and ISTH. As the data on COVID-19 and thrombosis continue to develop and evolve, we examined the data in two aspects. Firstly, other non-SARS-CoV-2 viral respiratory infections have also been reported to be associated with thrombotic events, be it arterial or venous. Thus, we aimed to compare the thrombotic rates between these two groups of patients directly to hopefully ascertain the actual thrombotic tendency in COVID-19 infections. Secondly, global hemostatic assays such as thromboelastogram and clot waveform analysis (CWA) have been used to demonstrate hypercoagulability in COVID-19 patients, albeit in a small group of patients and only in the critically ill. Incorporating these laboratory results into the management of thromboprophylaxis in COVID-19 is an attractive notion but more data and studies are definitely needed. Here, we evaluate the dynamic changes of hemostatic assays in patients with COVID-19 to better understand the overall coagulation profiles of COVID-19 infection.MethodsWe performed a single center, retrospective cohort study. All consecutive patients admitted to our hospital between 15 January and 10 April 2020 that were tested positive for COVID-19 or other non-SARS-CoV-2 respiratory viruses were included in our study. The main coagulation assays studied were prothrombin time and activated partial thromboplastin time and its associated CWA, min1, min2 and max2.FindingsWe included a total of 181 COVID-19 patients and 165 patients with non-SARS-CoV-2 respiratory viral infections. The respiratory viruses were rhinovirus (n=65), influenza A and B (n=46), adenovirus (n=13), human coronavirus 229E/NL63/OC43 (n=15), human enterovirus (n=3), metapneumovirus (n=6), parainfluenza virus 1 to 4 (n=11), respiratory syncytial virus (n=6) and human bocavirus 1 to 4 (n=0). The median age of COVID-19 patients was 37 (interquartile range [IQR], 30.5-51 years) versus 35 (IQR, 29-51.5) in the non-SAR-CoV-2 respiratory viruses group (P=0.12). Comorbidities, assessed by Charlson score, was also not statistically different between both groups (median score 0 (IQR, 0-1) in both groups, P=0.39). Majority of our patients had relatively mild infection as reflected by the low proportions of them requiring oxygen supplementation (11.0% in COVID-19 vs 4.8% in non-SARS-COV-2, P=0.035). COVID-19 patients had longer hospital stay (7 days (IQR, 5.5-13) vs 3 days (IQR, 2-3), P<0.001) and more required ICU support (5.0% vs 1.2%, P=0.04). Mortality rate was low in both groups. We reported two (1.0 event/1000-hospital-days) and one (1.8 event/1000-hospital-day) thrombotic events amongst COVID-19 group and non-SARS-COV-2 group respectively (P=0.63). All were myocardial infarction and occurred in intensive care unit. No venous thrombotic event was noted. There was no significant difference in all the coagulation parameters throughout the course of mild COVID-19 infection (Table 1). However, CWA parameters were significantly higher in severe COVID-19 infection compared with mild disease (min1: 6.48%/s vs 5.05%/s, P<0.001; min2: 0.92%/s2 vs 0.74%/s2, P=0.033), suggesting hypercoagulability in severe COVID-19 infection (Table 2 and Figure 1). We also observed that critically ill COVID-19 patients had higher absolute CWA parameters as compared to non-SARS-CoV-2 patients, albeit in small number of patients (Table 3).ConclusionThe thrombotic rates were low in both groups and did not differ significantly between COVID-19 and Non-SARS-CoV-2 patients. Nonetheless, our analysis of hemostatic parameters demonstrated hypercoagulability in COVID-19 as a dynamic process with the risk highest when the patients are critically ill. These changes in hemostasis could be detected by CWA. With our findings, we suggest that a more individualized thromboprophylaxis approach, considering clinical and laboratory factors, is probably preferred over universal pharmacological thromboprophylaxis for all hospitalized COVID-19 patients and warrants further research.DisclosuresLee:Sanofi: Honoraria, Other: travel grants; Pfizer: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees, Other: travel grants ; Medtronics: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.

Screening method for congenital dysfibrinogenemia using clot waveform analysis with the Clauss method.

Congenital fibrinogen disorders (CFDs) are classified as afibrinogenemia or hypofibrinogenemia (Hypo), dysfibrinogenemia (Dys), or hypodysfibrinogenemia (Hypodys), according to functional and antigenic fibrinogen concentrations. However, in routine laboratory tests, plasma fibrinogen levels are mostly measured using the functional Clauss method and not as an antigenic level. Therefore, it is difficult to discriminate CFD from acquired hypofibrinogenemia (aHypo). To establish a screening method for CFD, we investigated the parameters of clot waveform analysis (CWA) from the Clauss method. We compared fibrinogen concentrations determined using Clauss and prothrombin time (PT)-derived methods for 67 aHypo and CFD cases (19 Dys, 4 Hypodys, and 1 Hypo determined using antigen levels and DNA sequence analysis) with a CS-2400 instrument, and the CWA parameters, dH and Min1, were analyzed automatically with an on-board algorithm. dH and Min1 are the maximum change in transmittance at the end of coagulation and the maximum velocity of transmittance change during coagulation, respectively. Clauss/PT-derived ratios detected 18 cases of Dys and Hypodys but no Hypo cases, whereas Clauss/dH plus Clauss/Min1 ratios were calculated from fibrinogen concentration using the Clauss method and CWA parameters detected 21 cases of Dys and Hypodys and one Hypo case. Moreover, the Clauss/PT-derived ratio and Clauss/dH plus Clauss/Min1 ratio detected 22 cases of Dys and Hypodys cases and one Hypo case. This report demonstrates that CWA parameters of the Clauss method, Clauss/dH plus Clauss/Min1 ratio, screened Dys patients with a higher rate, whereas Clauss/PT-derived ratios did not.

Assessment of aPTT-based clot waveform analysis for the detection of haemostatic changes in different types of infections

Infections cause varying degrees of haemostatic dysfunction which can be detected by clot waveform analysis (CWA), a global haemostatic marker. CWA has been shown to predict poor outcomes in severe infections with disseminated intravascular coagulopathy. The effect of less severe bacterial and viral infections on CWA has not been established. We hypothesized that different infections influence CWA distinctively. Patients admitted with bacterial infections, dengue and upper respiratory tract viral infections were recruited if they had an activated partial thromboplastin time (aPTT) measured on admission. APTT-based CWA was performed on Sysmex CS2100i automated analyser using Dade Actin FSL reagent. CWA parameters [(maximum velocity (min1), maximum acceleration (min2) and maximum deceleration (max2)] were compared against control patients. Infected patients (n = 101) had longer aPTT than controls (n = 112) (34.37 ± 7.72 s vs 27.80 ± 1.59 s, p < 0.001), with the mean (± SD) aPTT longest in dengue infection (n = 36) (37.99 ± 7.93 s), followed by bacterial infection (n = 52) (33.96 ± 7.33 s) and respiratory viral infection (n = 13) (29.98 ± 3.92 s). Compared to controls (min1; min2; max2) (5.53 ± 1.16%/s; 0.89 ± 0.19%/s2; 0.74 ± 0.16%/s2), bacterial infection has higher CWA results (6.92 ± 1.60%/s; 1.04 ± 0.28%/s2; 0.82 ± 0.24%/s2, all p < 0.05); dengue infection has significantly lower CWA values (3.93 ± 1.32%/s; 0.57 ± 0.17%/s2; 0.43 ± 0.14%/s2, all p < 0.001) whilst respiratory virus infection has similar results (6.19 ± 1.32%/s; 0.95 ± 0.21%/s2; 0.73 ± 0.18%/s2, all p > 0.05). CWA parameters demonstrated positive correlation with C-reactive protein levels (min1: r = 0.54, min2: r = 0.44, max2: r = 0.34; all p < 0.01). Different infections affect CWA distinctively. CWA could provide information on the haemostatic milieu triggered by infection and further studies are needed to better define its application in this area.

Plasma-derived factors VIIa and X mixtures (Byclot®) significantly improve impairment of coagulant potential ex vivo in plasmas from acquired hemophilia A patients

A combined product of plasma-derived factor (F)VIIa and FX (pd-FVIIa/FX; Byclot®) is currently available for the hemostatic treatment of hemophilia A and B patients with inhibitors in Japan. Limited information is available, however, on its coagulant effect in acquired hemophilia A (AHA). In the present study, we assessed the coagulant effect of pd-FVIIa/FX on impairment of coagulation potentials in AHA. The bypassing agents, pd-FVIIa/FX, recombinant FVIIa (rFVIIa), and activated prothrombin complex concentrates (aPCC) were spiked with normal plasma preincubated with anti-FVIII monoclonal antibody (AHA-model plasma), and added to plasmas from AHA patients. Clot waveform analysis (CWA) triggered by the mixture of tissue factor and ellagic acid was subsequently performed. In the AHA-model, pd-FVIIa/FX improved all of the CWA parameters in a dose-dependent manner, irrespective of epitope specificity, with significant improvements relative to rFVIIa and aPCC. The coagulant effect of pd-FVIIa/FX at 1.6µg/mL (corresponding to 120µg/kg infusion) at the maximum therapeutic dose was outside the normal range. Moreover, the addition of pd-FVIIa/FX led to a greater improvement in the coagulant potentials in AHA plasmas than those of rFVIIa and/or aPCC. These data suggest that pd-FVIIa/FX significantly improves the impaired coagulant potentials in AHA and is potentially therapeutic.

A Comparison of Clinical and Laboratory Manifestations of Thrombotic Events in Patients with COVID-19 and Other Respiratory Viral Infections

Background: COVID-19 caused by Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) and other respiratory viral (non-CoV-2-RV) infections are associated with thrombotic complications. The differences in prothrombotic potential between SARS-CoV-2 and non-CoV-2-RV have not been well characterised. We compared the thrombotic rates between these two groups of patients directly and further delved into their coagulation profiles. Methods: In this single-center, retrospective cohort study, all consecutive COVID-19 and non-CoV-2-RV patients admitted between January 15th and April 10th 2020 were included. Coagulation parameters studied were prothrombin time and activated partial thromboplastin time and its associated clot waveform analysis (CWA) parameter, min1, min2 and max2.Findings: In the COVID-19 (n=181) group there were two (1.0 event/1000-hospital-days) thrombotic events while one (1.8 event/1000-hospital-day) was reported in the non-CoV-2-RV (n=165) group. All of these events were myocardial infarction occurring in the intensive care unit. Coagulation parameters did not differ throughout the course of mild COVID-19. However, CWA parameters were significantly higher in severe COVID-19 compared with mild disease, suggesting hypercoagulability (min1: 6.48%/s vs 5.05%/s, P 2 vs 0.74%/s2, P =0.033).Interpretation: The thrombotic rates were low and did not differ between COVID-19 and non-CoV-2-RV patients. The hypercoagulability in COVID-19 is a highly dynamic process with the highest risk occurring when patients were most severely ill. Such changes in haemostasis could be detected by CWA. In our population, a more individualized thromboprophylaxis approach, considering clinical and laboratory factors, is preferred over universal pharmacological thromboprophylaxis for all hospitalized COVID-19 patients and such personalized approach warrants further research. Funding: This research was funded by the SingHealth Duke-NUS Academic Medicine COVID-19 Research Grant.Declaration of Interests: The authors declare no competing interests.Ethics Approval Statement: This database is part of a prospective study to characterize emerging infectious diseases and was approved by our institutional review board (CIRB no. 2018/3045).

Elevated activated partial thromboplastin time-based clot waveform analysis markers have strong positive association with acute venous thromboembolism.

IntroductionA hypercoagulable state is a predisposition for venous thromboembolism (VTE). The activated partial thromboplastin time (aPTT)-based clot waveform analysis (CWA) is a global haemostatic measure but its role in assessment of hypercoagulability and thrombotic disorders is uncertain. We aimed to study the changes of CWA parameters in acute VTE. We hypothesized that patients with acute VTE would demonstrate higher CWA values than control patients without VTE and having elevated CWA parameters is associated with acute VTE.Materials and methodsClot waveform analysis data from patients (N = 45) with objectively proven acute VTE who had an aPTT performed prior to initiation of anticoagulation were compared with controls (N = 111). The CWA parameters measured were min1, min2, max2 and delta change.ResultsWhile the mean aPTT between VTE patients and controls did not differ (P = 0.830), the mean CWA parameters were significantly higher among VTE patients than controls (min1, P < 0.001; min2, P = 0.001; max2, P = 0.002; delta change, P < 0.001). There were significantly more cases within the VTE group exhibiting CWA values above their reference intervals than the control group (all P < 0.001), with the odds ratios for VTE of 8.0, 5.2, 4.8 and 18.6 for min1, min2, max2 and delta change, respectively (all P < 0.001).ConclusionsPatients with acute VTE had elevated aPTT-based CWA parameters than controls. Higher CWA parameters were significantly associated with acute VTE.

Clot waveform analysis in Clauss fibrinogen assay contributes to classification of fibrinogen disorders

BackgroundClauss fibrinogen assay (CFA) is widely used as a screening test to detect fibrinogen disorders. However, CFA alone cannot distinguish quantitative and qualitative defects because it depends on functional fibrinogen activity (Ac), and fibrinogen antigen (Ag) determination is required to classify fibrinogen disorders. ObjectivesTo establish a novel approach to classify fibrinogen disorders, we investigated the potential of clot waveform analysis (CWA) of CFA and searched for a surrogate marker for fibrinogen Ag. Materials and methodsWe analyzed CWA parameters obtained from CFA using plasma from normal patients (n = 91) and those with fibrinogen disorders (n = 27, including 15 hypofibrinogenemia, 6 dysfibrinogenemia and 6 hypodysfibrinogenemia) with a CS-5100 autoanalyzer. ResultsWe found that maximum coagulation velocity (Min1) levels were most strongly correlated with fibrinogen Ag in both normal and fibrinogen disorders. Hence, Min1 appeared to function as a surrogate for fibrinogen Ag. Although the Ac/Min1 ratio did not simply reflect the measured Ac/Ag ratio, we found that the Ac/Min1 ratio was significantly higher than normal in hypofibrinogenemia and hypodysfibrinogenemia, but not in dysfibrinogenemia. On the other hand, we could distinguish type II deficiency from type I using estimated fibrinogen Ag (eAg) predicted from Min1. The Ac/eAg ratios of dysfibrinogenemia and hypodysfibrinogenemia were significantly lower than those of normal and hypofibrinogenemia. ConclusionThe CWA of CFA could distinguish fibrinogen disorders using a combination of Ac/Min1 and Ac/eAg values. This analysis allows the qualitative detection of fibrinogen disorder easily and represents a novel screening test for fibrinogen disorders.

A Novel Clot Waveform Analysis to Measure the Plasma Coagulation Potency in the Presence of Anti-Fixa/Fx Bispecific Antibody, Emicizumab

Emicizumab (also termed ACE910) is a humanized anti-factor (F)IXa/FX bispecific antibody with FVIIIa cofactor function. A clinical phase 3 study was initiated in 2015 for hemophilia A patients (HA-pts) with FVIII inhibitors. Since emicizumab, unlike FVIII, does not require activation by thrombin, its APTT-shortening effect is much greater than that of FVIII. Thus, APTT, a conventional assay to assess whole coagulation potency, would have limited utility in emicizumab-administered HA-pts, because emicizumab would mask the effect of residual FVIII or a FVIII agent on APTT. Clot waveform analysis (CWA) can provide multidimensional coagulation potencies by monitoring the process of plasma clot formation with an automated coagulation analyzer. We considered the possibility, therefore, that this assay system would overcome the above issue on APTT. In this study, we aimed to optimize concentrations of tissue factor (TF) and ellagic acid (Elg) in a trigger reagent for CWA as well as CWA parameters to provide precise evaluation of coagulation potency even in the presence of emicizumab with neither masking nor being masked by FVIII or bypassing agents. First, we determined an optimal concentration of TF/Elg trigger reagent. Various concentrations (10, 30, 100, and 300 μg/mL) of emicizumab were spiked into commercially available FVIII-deficient plasmas (George King) for testing. Recombinant (r)FVIII (Kogenate FS; Bayer)-spiked samples were also tested as a reference. PT reagent (under development; Sysmex) and APTT reagent (Thrombocheck APTT-SLA; Sysmex), used as a source of TF and Elg, respectively, were mixed in various ratios. The optimized mixture ratio (PT:APTT:buffer=1:15:135) was chosen to ensure that the maximum coagulation velocity (|min1|) in the presence of emicizumab would be in agreement with the animal study-based estimated conversion rate "0.2-0.4 IU/dL of equivalent FVIII per 1 μg/mL of emicizumab" (Muto. J Thromb Haemost. 2014). When evaluating several lots of FVIII-deficient plasmas, however, we observed large variations in transmittance depending on fibrinogen concentration of each plasma, which resulted in large variations of |min1| between donor plasmas. To decrease the bias due to fibrinogen concentration, % transmittance of clot waveform (CW) was adjusted to 100% and 0% at the pre- and post-coagulation phase, respectively. By using |min1| from the adjusted CW (adjusted-|min1|), we successfully reduced the inter-donor variations and chose it as a main parameter. Next, we evaluated adjusted-|min1| using plasmas from HA-pts without inhibitors (severe; n=2, moderate; n=2) and HA-pts with inhibitors (<10 BU/mL; n=2, >10 BU/mL; n=2) by adding emicizumab (30, 100, and 300 μg/mL) in vitro. After the addition of emicizumab, concentration-dependent increases in adjusted-|min1| were observed in all plasmas with rather small individual variations. Finally, we examined whether adjusted-|min1| reflected the effects of FVIII or bypassing agent that was added to plasmas containing emicizumab. Additive effects of a plasma-derived FVIII agent (CROSS EIGHT M; Japan Blood Products Organization), a rFVIII agent (ADVATE; Baxalta) and activated prothrombin complex concentrate (FEIBA; Baxalta) were confirmed by the increase of adjusted-|min1|. As for rFVIIa agent (NovoSeven; Novo Nordisk), its additive effect on adjusted-|min1| was not clear enough in this assay condition, but its additive effects were confirmed by the clot time. In conclusion, we established the Elg/TF-triggered CWA assay condition and parameters for measuring coagulation potency in plasmas from HA-pts even in the presence of emicizumab and a FVIII/bypassing agent without masking each other. DisclosuresNogami:Sysmex Corporation: Patents & Royalties, Research Funding; Chugai Pharmaceutical Co., Ltd.: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties, Research Funding; F. Hoffmann-La Roche Ltd.: Honoraria, Membership on an entity’s Board of Directors or advisory committees. Matsumoto:Sysmex Corporation: Patents & Royalties, Research Funding; Chugai Pharmaceutical Co., Ltd.: Patents & Royalties, Research Funding. Tabuchi:Sysmex Corporation: Employment, Patents & Royalties; Chugai Pharmaceutical Co., Ltd.: Patents & Royalties. Soeda:Sysmex Corporation: Patents & Royalties; Chugai Pharmaceutical Co., Ltd.: Employment, Patents & Royalties. Arai:Sysmex Corporation: Employment. Kitazawa:Chugai Pharmaceutical Co.: Employment, Equity Ownership, Patents & Royalties; Sysmex Corporation: Patents & Royalties. Takaoka:Sysmex Corporation: Employment. Hattori:Chugai Pharmaceutical Co.: Employment, Equity Ownership, Patents & Royalties. Shima:F. Hoffmann-La Roche Ltd.: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Chugai Pharmaceutical Co., Ltd.: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties, Research Funding; Sysmex Corporation: Patents & Royalties, Research Funding.

Anti-Factor IXa/Factor X Antibody (ACE910) Improves the Coagulation Function in Acquired Hemophilia A ex vivo

<Introduction> Acquired hemophilia A (AHA) is a rare bleeding disorder in which autoantibodies (autoAbs) against coagulation factor (F)VIII impair the coagulation system. Anti-FVIII autoAbs developed in AHA are polyclonal and the majority of these autoAbs bind to the A2, A3, and/or C2 domains in FVIII. Different inhibitory actions of anti-FVIII autoAbs, depending on their epitopes, are well-known. We have recently developed a humanized anti-FIXa/X bispecific antibody, ACE910, which mimics the cofactor function of FVIIIa even in the presence of anti-FVIII alloantibodies.<Aim> In this study, we examined whether ACE910 improved the lower coagulation function in AHA patients using the comprehensive coagulation assays.<Methods> Two representative experiments were performed as follows; 1) In vitro effects of ACE910 on normal pooled plasmas (PNPs) mixed with various anti-FVIII monoclonal Abs (mAbs) as the reconstituted AHA-models. For this experiments, several anti-FVIII mAbs; VIII-9222 (epitope: anti-a1), VIII-2236 (anti-A2), VIII-3776 (anti-A3C1), ESH4 (anti-C2) were prepared. 2) Ex vivo effects of ACE910 on plasmas obtained from nine patients with AHA, whose regions of Abs were A1, A2 and light chain for four patients, A2 and light chain for three patients, A2 for one patient and light chain for one patient. The coagulation functions in obtained samples were evaluated using the comprehensive coagulation assays, clot waveform analysis (CWA) and thrombin generation assays (TGA). In CWA, the minimum value of the first derivative (min1) and the second derivative (min2) were calculated as an index of the maximum acceleration of the reaction and the maximum velocity of coagulation, respectively. In TGA, calibrated automated thrombin generation assays derived the standard parameters; peak thrombin, lagtime, and time to peak. Total thrombin generation at intervals from the beginning to peak level was quantified. These values (nmol/l) were divided by the sample times (min, time to peak - lagtime), and represented the mean velocity of thrombin generation until the reach to peak level, expressed as mean velocity to peak thrombin (MV-peak thrombin). We defined the attenuation of inhibitory effect (% of AIE) represented that ACE910 attenuated the parameters inhibited by mAbs in artificial AHA or by allo-Abs in AHA patients, respectively. Figure 1 represents the % of AIE on thrombin peak in TGA.<Results> PNPs were incubated with various anti-FVIII mAbs (100-500 mcg/ml) at 37 degrees Celsius for 2 hrs, and FVIII activities in these samples were decreased to 2.0-43%. These reactant mixtures were reacted with ACE910 (0-20 mcg/ml) for a further 2 hr-incubation. ACE910 improved the parameters of CWA (|min1|; 40-62%, and |min2|; 49-74% of AIE) and of TGA (thrombin peak; 46-72%, and MV-peak thrombin; 51-70% of AIE, respectively) in all PNPs with mAbs. These data indicated that ACE910 improved the coagulant activity in AHA patients. Therefore, we determined the efficacy of ACE910 for nine AHA patients ex vivo. FVIII activities and FVIII inhibitors were <1.0-7.5 % and 2.2-134 BU/ml, respectively. ACE910 improved the parameters for eight AHA patients in CWA (|min1|; 36-100 %, and |min2|; 52-100 % of AIE, respectively). For one AHA patient whose epitope was A2 and light chain, ACE910 showed the slight improvement in |min1| (12 % of AIE) and in |min2| (31 % of AIE). The reason was that the range of concentration of ACE910 was only from 0 to 5 mcg/dl. On the other hand, ACE910 improved the parameters for all AHA patients in TGA. ACE910 showed effect on thrombin peak (27-77 % of AIE). Furthermore, ACE910 improved MV-peak thrombin (17-73 % of AIE). Figure 2 shows representative TGA curve for the effect of ACE910 on one AHA patient whose allo-Ab was anti-light chain.<Conclusion> Our results indicated that ACE910 could improve the coagulant activities in AHA patients independent of the regions of antibodies and might be useful for the treatment of AHA patients. [Display omitted] [Display omitted] DisclosuresTakeyama:Chugai Pharmaceutical Co., Ltd.: Research Funding. Nogami:Bayer, NovoNordisk, Baxalta, Chugai, Kaketsuken, Pfizer, Biogen: Honoraria; Chugai: Membership on an entity's Board of Directors or advisory committees; Bayer, Novo Nordisk, Baxalta. Biogen: Research Funding. Matsumoto:Chugai Pharmaceutical Co., Ltd: Research Funding. Kitazawa:Chugai Pharmaceutical Co., Ltd: Employment, Equity Ownership, Patents & Royalties. Hattori:Chugai Pharmaceutical Co., Ltd: Employment, Equity Ownership, Patents & Royalties. Shima:Chugai Pharmaceutical Co., Ltd. and F. Hoffmann-La Roche Ltd.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.