The role of anticoagulation clinics needs to be reassessed to include follow up of patients on direct oral anticoagulants.

A survey was carried out to assess the state of organization of care (including clinical and laboratory) delivered to patients on vitamin K antagonists (VKA) or direct oral anticoagulants (DOAC) followed by clinics affiliated with the Italian Federation of Thrombosis Centers (FCSA), traditionally engaged to assist anticoagulated outpatients within the country. Participants were asked to answer questions concerning (i) proportion of patients on VKA-vs-DOAC and (ii) whether dedicated testing for DOAC is available. The proportion of patients on VKA-vs-DOAC was 60 % vs 40 %. This proportion is in sharp contrast with the real-life distribution where DOAC outweigh VKA prescriptions. Furthermore, the proportion of anticoagulation clinics that provide DOAC testing (even in special situations) is relatively small (i.e., 31 % of the respondents). Furthermore, 25 % of those that declared to follow DOAC patients do not provide any testing at all. The answers to the above questions cause concerns as (i) most patients on DOAC within the country are probably on self-management, or they are managed by general practitioners or specialists outside thrombosis centers. (ii) Most patients on DOAC have no access to testing even in special situations where it would be needed. We feel that there is a (false) perception that the care needed for DOAC treatment can be much less than that required for VKA, as DOAC require prescription and not regular follow-up. A call for action should be urgently made to reassess the role of anticoagulation clinics, which should pay the same attention to patients on DOAC as those on VKA.

Thromboplastin calibration revisited to look for possible revision of the World Health Organization recommendations.

BACKGROUND: Thromboplastin calibration is essential to determine the international sensitivity index required to calculate the international normalized ratio (INR). The procedure for calibration recommended by the World Health Organization (WHO) calls for the selection of patients on stable anticoagulation in the range of 1.5 to 4.5 INR. These patients are difficult to be recruited as the conventional therapeutic interval for warfarin is 2.0 to 3.0. A possible solution could be including patients with less intense anticoagulation in the calibration. OBJECTIVES: We sought to investigate the impact of this amended procedure on the parameters of calibration. METHODS: Eight data sets from previous calibrations of a rabbit thromboplastin that included patients on anticoagulation as required by WHO were used for this pilot study. Parameters of calibration as determined by the full data sets are identified as "full calibrations" and are considered reference. Each of the data sets were used to recalculate the calibration parameters after including patients with INRs of <4.0, <3.5, or <3.0, which were identified as "trimmed calibrations" and compared with those from the full calibrations. RESULTS: There was marginal variation of the international sensitivity index, CV, and INR that can be hardly of practical significance. CV was the most affected parameter, which increased from the full to the trimmed <3.0 calibration, but never exceeded the 3% cutoff value recommended by WHO. CONCLUSIONS: Should the results of this pilot study be confirmed for the calibration of other thromboplastins, revision of the WHO recommendations to include patients with INR from 1.5 to 4.0 is warranted.

Impact of a commercially available DOAC absorbent on two integrated procedures for lupus anticoagulant detection.

BACKGROUND: Lupus anticoagulant (LA)-detection in anticoagulated patients is an unmet need, which becomes even more cogent with the introduction of direct oral anticoagulants (DOAC) that may lead to false-positive results. AIMS: We aimed to investigate the effect of a commercially available DOAC absorbent on residual drug concentrations, and on integrated procedures for LA-detection. METHODS: Blood from patients treated for atrial fibrillation with either dabigatran (n = 39), rivaroxaban (n = 55), apixaban (n = 47) or edoxaban (n = 47) were collected at peak and trough, and centralized for testing with two LA integrated procedures [i.e., the silica clotting time (SCT) and dilute Russel viper venom (dRVVT)] before and after DOAC absorbent exposure. RESULTS: The commercially available DOAC absorbent investigated in this study proved effective in reducing the concentrations of all the investigated DOAC, although small residual drug was detected after absorption, especially in patients on edoxaban. Results mimicking LA were observed in patients on DOAC before absorbent exposure, especially for rivaroxaban when testing was performed with dRVVT (88% rate at peak and 20% at trough) and much less with SCT (12% at peak and 8% at trough). The correspondent rate of results mimicking LA in patients on rivaroxaban after exposure was reduced [dRVVT (peak 8%, trough 4%); SCT (peak and trough 8%)], but not abolished. CONCLUSIONS: Overall, in vitro DOAC absorbance by active charcoal compounds is a useful laboratory tool for LA-detection in patients on DOAC. Caution should however be exerted when used in daily practice.

Anti-TNF-α Treatment Reduces the Baseline Procoagulant Imbalance of Patients With Inflammatory Bowel Diseases.

BACKGROUND: Inflammatory bowel diseases (IBD) are characterized by an increased thrombosis risk of uncertain etiology. Coagulation derangement arising from inflammation may be a triggering factor. We hypothesized that strong inflammation inhibitors (eg, anti-tumor necrosis factor-α drugs) may affect coagulation. METHODS: Forty patients with IBD were compared with 57 control patients for coagulation factors and endogenous thrombin potential (ETP), the latter being the most sensitive marker of in vivo pro- and anticoagulation balance. We measured ETP in the presence and absence of thrombomodulin (the physiologic protein C [PC] activator). Coagulation at different timepoints was also assessed for 28 of these patients during infliximab treatment. RESULTS: The median ETP (nM thrombin × minutes) and range (minimum-maximum) were each higher in patients at baseline than in control patients in both the absence (2120 [1611-3041] vs 1865 [1270-2337]) and the presence (1453 [464-2522] vs 831 [104-1741]) of thrombomodulin. The ETP ratio (with/without thrombomodulin) was high at baseline (0.73 [0.21-0.90] vs 0.45 [0.07-0.85]). The ETP and ETP ratio declined during treatment and were significantly lower at the end than at baseline. Factor (F) VIII and fibrinogen, which were high at baseline, decreased during treatment and at the end were significantly lower than at baseline. The FVIII/PC ratio, which was high in patients at baseline, declined during treatment and at the end was lower than at baseline. C-reactive protein recorded at the end of treatment was lower than at baseline. CONCLUSIONS: Patients with IBD have a procoagulant imbalance as shown by increased ETP at baseline. The ETP decreases during treatment with infliximab, which is related to decreased FVIII and FVIII/PC ratio. This effect is also related to the improvement of inflammation as shown by decreased fibrinogen and C-reactive protein.

Emicizumab, the factor VIII mimetic bi-specific monoclonal antibody and its measurement in plasma.

Objectives: Emicizumab, a monoclonal antibody mimicking the function of factor (F) VIII in the activation of FX by FIXa, is widely used for prophylaxis in hemophilia patients with or without inhibitors to FVIII. Although it is administered at fixed dose, its measurement could be occasionally required. In principle, the emicizumab procoagulant effect could be assessed by the one-stage assay (OSA) currently used to measure FVIII. However, the OSA for FVIII presents with limitations. Furthermore, owing to its potent FVIII-like activity, emicizumab interferes with the measurement of the inhibitor to FVIII, which is often needed in patients on emicizumab. Methods: We prepared test samples by spiking a FVIII-deficient plasma with graded amounts of emicizumab. We modified the OSA for FVIII and tested plasma samples for emicizumab concentrations. Furthermore the chromogenic assay (CA) for FVIII with bovine reagents was used to assess for the FVIII inhibitor in patients on emicizumab. Results: Slight modification of the OSA for FVIII (i.e., higher test plasma dilution and longer coagulometer acquisition time) made the regular OSA as a reliable laboratory tool to measure emicizumab concentration as shown by the identity of the regression (observed vs. expected) lines. Furthermore, the inhibitors to FVIII in patients on emicizumab, which were negative when measured by the regular Bethesda assay, were reliably measured by the CA assay employing bovine reagents. Conclusions: The methods currently used to measure FVIII can be easily modified to make the general clinical laboratory able to assist clinicians when dealing with patients on emicizumab.

Paving the way for establishing a reference measurement system for standardization of plasma prothrombin time: Harmonizing the manual tilt tube method.

BACKGROUND: International normalized ratio (INR) is traceable to World Health Organization (WHO) International Standards for thromboplastins. International Standards must be used with a manual tilt tube technique (MTT) for prothrombin time (PT) determination. An important part of the total variability of INR is due to poor harmonization of MTT across WHO reference laboratories. OBJECTIVES: To determine the origins of PT differences between operators performing MTT and to develop a harmonized MTT. METHODS: Two workshops were held where WHO reference laboratory operators could compare their PTs using MTT and the same equipment. A harmonized MTT was used by seven operators in the second workshop. RESULTS: Differences have been observed in tilting frequency and in the height of pipetting plasma in the test tube. At the beginning of the first workshop, the tilting cycle time varied between 1.1 and 2.7 seconds. The mean PT of normal plasma obtained by pipetting plasma at the top of the tube was 14.3 seconds but was 12.9 seconds when plasma was pipetted at the bottom of the tube. When using the harmonized MTT for WHO International Standard rTF/16, the differences between operators were not greater than 1.1 seconds in normal plasma, and not greater than 1.3 seconds in patient plasma with average INR of 3.0. INR between-operator coefficient of variation was 2.3%. CONCLUSION: Application of a harmonized MTT in three reference laboratories resulted in substantial reduction of between-operator variation of PT and INR. The harmonized MTT is proposed as Candidate Reference Measurement Procedure.

Hypercoagulability of COVID-19 patients in intensive care unit: A report of thromboelastography findings and other parameters of hemostasis.

BACKGROUND: The severe inflammatory state secondary to COVID-19 leads to a severe derangement of hemostasis that has been recently described as a state of disseminated intravascular coagulation (DIC) and consumption coagulopathy, defined as decreased platelet count, increased fibrin(ogen) degradation products such as D-dimer, as well as low fibrinogen. AIMS: Whole blood from 24 patients admitted at the intensive care unit because of COVID-19 was collected and evaluated with thromboelastography by the TEG point-of-care device on a single occasion and six underwent repeated measurements on two consecutive days for a total of 30 observations. Plasma was evaluated for the other parameters of hemostasis. RESULTS: TEG parameters are consistent with a state of hypercoagulability as shown by decreased values, and increased values of K angle and MA. Platelet count was normal or increased, prothrombin time and activated partial thromboplastin time were near(normal). Fibrinogen was increased and D-dimer was dramatically increased. C-reactive protein was increased. Factor VIII and von Willebrand factor (n = 11) were increased. Antithrombin (n = 11) was marginally decreased and protein C (n = 11) was increased. CONCLUSION: The results of this cohort of patients with COVID-19 are not consistent with acute DIC, rather they support hypercoagulability together with a severe inflammatory state. These findings may explain the events of venous thromboembolism observed in some of these patients and support antithrombotic prophylaxis/treatment. Clinical trials are urgently needed to establish the type of drug, dosage, and optimal duration of prophylaxis.

Effect of different methods for outlier detection and rejection when calculating cut off values for diagnosis of lupus anticoagulants.

BACKGROUND: Lupus anticoagulant (LA)-detection is performed by testing plasma with activated partial thromboplastin time (APTT)-derived and dilute-Russell-viper-venom (dRVV)-derived tests. Results are interpreted by comparison to cut-off values determined by testing plasma from healthy-subjects. Several issues are concerned with the determination of LA cut-offs. Among them, the identification/rejection of outliers (i.e., aberrant values) prior statistical analysis is one of the most important and poorly defined. OBJECTIVES: The primary aim of this collaborative study was to evaluate whether outliers' identification/removal by using three algorithms prior cut-off calculation would have any effect on the between-laboratory variability of cut-offs. As a secondary aim, we evaluated whether and to what extent outliers' removal would affect LA-detection rate. METHODS: Data sets stemming from 120 plasmas from healthy-donors, collected and tested at each of 11 laboratories were evaluated by three algorithms for outliers' identification. Furthermore, a well characterized set of plasmas certified as being positive at increasing LA-potency, were concomitantly tested and their results used to assess any effect of outlier detection/rejection on LA-detection rate. RESULTS: Relatively few outliers were identified and their elimination (regardless of algorithms) showed no appreciable effects on the inter-laboratory variability of cut-offs nor on the LA-detection rate, indicating that outliers are not the main cause of the inter-laboratory variability of cut-offs for LA-detection. CONCLUSIONS: These results strengthen the recommendation that cut-offs should be determined locally after outlier removal (to avoid inclusion of gross, obvious outliers) and that they cannot be interchangeably used in other laboratories even when using the same platform.

Procoagulant imbalance in preterm neonates detected by thrombin generation procedures.

Preterm newborns are considered at risk of acquired coagulopathy and are often prophylactically infused with fresh frozen plasma (FFP) even in the absence of bleeding. To assess the coagulation asset of preterm neonates and the biological plausibility of such infusions, we investigated at birth 87 very low birth weight (≤1500 g) preterm (gestational age <35 weeks) newborns and 64 full-term newborns. Preterm neonates were also investigated at different time-points up to 30 days after birth. Plasma from preterm and full-term neonates were subjected to the measurement of prothrombin and activated partial thromboplastin time (PT, APTT), pro- and anticoagulant factors as well as to thrombin-generation procedures both with and without thrombomodulin. PT and APTT of preterm newborns were longer than those of full-term neonates [PT: 15.9 s (11.7-51.2)-vs-13.8 (11.0-25.4), p < 0.001. APTT: 59.0 (37.8-97.5)-vs- 47.3 (28.1-71.9), p < 0.001] and tended to shortening after 30 days from birth. Thrombin-generation defined as endogenous thrombin potential (ETP) was increased in preterm as compared to full-term neonates at birth [1322 nM·min (474-2384)-vs-1006 (697-1612), p < 0.001] and did not change appreciably over time up to 30 days from birth. In conclusion, plasma from preterm neonates displays a procoagulant imbalance at birth as shown by increasing ETP, despite the prolongation of PT and APTT. The results define preterm newborns as having hyper- rather than hypo-coagulability and argue against the infusion of FFP when given prophylactically and/or based solely on prolongation of PT or APTT.

Effect of emicizumab on global coagulation assays for plasma supplemented with apixaban or argatroban.

Emicizumab is a bi-specific humanized monoclonal antibody mimicking the factor (F) VIII cofactor activity in mediating the activation of FX by FIXa. Recent observations showed that emicizumab when added to pooled normal plasma (PNP), hemophilic plasma or PNP added with unfractionated heparin is able to interfere with coagulation assays. To further explore the mechanisms of assay interference we investigated the effect of emicizumab on global coagulation assays for the PNP added with two direct oral anticoagulants, apixaban or argatroban. Aliquots of PNP were added with purified apixaban or argatroban at a concentration of 500 ng/mL and emicizumab at concentrations ranging from 0 to 100 µg/mL. Plasma samples were then tested for the activated partial thromboplastin time (APTT) and for thrombin generation (the latter for the apixaban plasma only). Emicizumab at a 25-50 µg/mL shortened the APTT of the PNP with or without apixaban or argatroban. The extent of correction was greater for the apixaban or argatroban plasma and amounted to 35% or 42%, respectively. The parameters of thrombin generation (lag-time and time-to-peak) for the PNP supplemented with apixaban were shortened by 30% or 25%, respectively and the endogenous thrombin potential and the peak-thrombin were marginally affected. Emicizumab attenuates in vitro the anticoagulant activity of the PNP induced by apixaban or argatroban as documented by the correction of prolonged APTT and velocity of thrombin generation (i.e., lag-time and time-to-peak). Whether the above effects have any relevance in vivo is unknown.

Procoagulant imbalance influences cardiovascular and liver damage in chronic hepatitis C independently of steatosis.

BACKGROUND & AIMS: Patients with chronic HCV infection besides hepatitis often present cardiovascular damage, the pathogenesis of which is not defined. In chronic liver diseases, including NAFLD and cirrhosis, a procoagulant imbalance, potentially responsible for atherosclerosis has been reported. We aimed at evaluating whether a procoagulant imbalance is present also in non-cirrhotic patients with HCV infection and whether the procoagulant imbalance correlates with cardiovascular damage. The correlation between the procoagulant imbalance, coexisting steatosis, and liver fibrosis was analysed. METHODS: From 2014 to 2018, 393 subjects (205 patients with chronic HCV infection from two liver units and 188 controls) were enrolled. Metabolic, cardiovascular, liver assessment and coagulation parameters-procoagulants (FII and FVIII) and anticoagulants (antithrombin and protein C [PC]), endogenous thrombin potential (ETP), peak-thrombin and their ratios (with/without thrombomodulin)-were determined. RESULTS: The procoagulant imbalance (defined as high FVIII, FVIII/PC ratio, ETP-ratio and peak-thrombin-ratio (with/without thrombomodulin)) was significantly higher in patients with chronic HCV than controls. Steatosis was detected in 87 patients (42%). No difference in coagulation imbalance, carotid and cardiac parameters and severity of liver fibrosis was observed in patients with or without steatosis, despite the latter had less severe metabolic alterations. The FVIII/PC ratio was independently associated with carotid intima-media thickness (coefficient 0.04, 95% CI 0.002-0.07, P = .04) and liver fibrosis (coefficient 0.64, 95% CI 0.37-0.92, P < .0001). CONCLUSION: Patients with HCV infection, even in the absence of cirrhosis have a procoagulant-imbalance that possibly plays a role in increasing the risk of cardiovascular disease and progression of fibrosis.

Body mass index reduction improves the baseline procoagulant imbalance of obese subjects.

Obesity is a risk factor for cardiovascular diseases. The latter being dependent (at least in part) on plasma procoagulant imbalance (i.e., hypercoagulability). Information on hypercoagulability associated with obesity is scanty and mainly based on global traditional coagulation tests or on the measurement of individual components of coagulation (i.e., pro- and anticoagulants). Plasma from 33 obese subjects was investigated soon before endoscopic balloon placement and after removal (6 months later) by thrombin-generation procedures that are thought to represent much better than any other in vitro test the coagulation process occurring in vivo. We found that obese subjects possess a state of hypercoagulability as demonstrated by the modification of the main parameters of thrombin-generation. In particular, the median value (min-max) of the endogenous thrombin potential (ETP) of obese subjects at baseline was higher than that of controls [1968 (1335-2533) vs. 1710 (1010-2119), p < 0.001]. Endoscopic balloon placement achieved a BMI reduction from 38.9 (31.7-62.3) to 31.6 (21.9-53.3), p < 0.001 and a parallel reduction of thrombin-generation as demonstrated by the following findings. The ETP measured soon after balloon removal was significantly smaller than that measured at baseline [1783 (1224-2642) vs. 1968 (1335-2533), p < 0.01]. The other parameters of thrombin-generation, including lag-time, peak-thrombin, time-to-reach the peak and velocity index showed a pattern consistent with the ETP, both at baseline and soon after balloon removal. Endoscopic balloon placement achieves concomitant reduction of BMI and thrombin-generation in subjects with obesity.

Advances in the Treatment of Hemophilia: Implications for Laboratory Testing.

BACKGROUND: Until recently, clinical laboratories have monitored hemophilia treatment by measuring coagulation factors before/after infusion of human-derived or recombinant factors. Substantial changes are expected in the near future based on new therapeutic approaches that have been or are being developed. CONTENT: Hemophilia treatment includes replacement therapy with human-derived/recombinant factors or treatment with bypassing agents for patients without or with inhibitors, respectively. Accordingly, laboratory methods for monitoring include one-stage clotting or chromogenic assays meant to measure either factor VIII/IX or global coagulation tests to measure the effect of bypassing agents. Recently, modified long-acting coagulation factors have been introduced for which discrepant results may be expected when measurement is performed with one-stage clotting or chromogenic assays. Currently, novel drugs not based on coagulation factors are under development and are being tested in clinical studies. These drugs do require new methods and therefore laboratory evaluation of hemophilia will undergo dramatic changes in the near future. SUMMARY: From the analysis of the current practice and literature, we draw the following conclusions: (a) Thrombin generation or thromboelastometry are the logical candidate assays to monitor bypassing agents. (b) Considerable differences are expected when measuring modified long-acting coagulation factors, depending on whether one-stage or chromogenic assays are used. Although no definitive conclusions can presently be drawn, chromogenic assays are probably more suitable than one-stage clotting. (c) Novel drugs not based on coagulation factors such as emicizumab, fitusiran, or concizumab that are entering the market do require alternative methods that are not yet well established.

The intra-assay reproducibility of thromboelastography in very low birth weight infants.

BACKGROUND AND AIMS: Despite the potential benefits of thromboelastography (TEG) for bedside hemostatic assessment in critical care settings, its accuracy remains to be determined, especially in critically ill neonates. We determined the intra-assay reproducibility of TEG parameters: Reaction time (R), clot kinetics (K) and Maximum Amplitude (MA) in a cohort of very low birth weight (VLBW) infants. STUDY DESIGN: Observational study. SUBJECTS: One hundred VLBW newborns. OUTCOME MEASURES: We performed TEG duplicate measurements for blood samples from VLBW newborns. To assess for correlation, we calculated the coefficients of correlation by plotting the values of the first vs the second measurement. Paired samples were compared with t-test and the coefficient of variation (CV) on paired results was also calculated as a measure of variability. To evaluate the agreement between duplicates, Bland-Altman (BA) analysis was performed. RESULTS: We evaluated 228 TEG pairs. Both the coefficient of correlation and the BA analysis showed an acceptable level of agreement between duplicates. TEG variability (CV, mean ±â€¯SD) was highest for K (10.4%, ±12.9), lowest for MA (3.6%, ±8.0) and moderate for R (7.9%, ±9.0). The results from ANOVA one-way analysis describe different variability trends: K-CV increased at higher values, while MA-CV and R-CV increased at lower values. CONCLUSIONS: In VLBW newborns, the agreement between TEG duplicate measurements for R and MA parameters is adequate for clinical purposes. TEG is a promising tool to quickly assess hemostasis ensuring a significant blood sparing in critically ill neonates.

Lupus Anticoagulant Testing: Activated Partial Thromboplastin Time (APTT) and Silica Clotting Time (SCT).

Actvated partial thromboplastin time and its congener test Silica clotting time are used for the laboratory detection of lupus anticoagulants. Their performance consists of mixing test plasma with activators of the contact coagulation factors, phospholipids and calcium chloride, and recording the clotting time. Here we describe the principle, performance and extensive practical details of the two methods, the problems that may be encountered and how they can be identified and overcome.