key: cord-0879306-fakm0lic
authors: Helin, Tuukka A.; Lemponen, Marja; Lassila, Riitta; Joutsi‐Korhonen, Lotta
title: Anticoagulated patients exhibit intact endogenous thrombin potential using ST Genesia unlike the Calibrated Automated Thrombogram
date: 2021-03-11
journal: Res Pract Thromb Haemost
DOI: 10.1002/rth2.12497
sha: cb5cb347aee81f85ee07add66adcc860b415767f
doc_id: 879306
cord_uid: fakm0lic

BACKGROUND: The thrombin generation (TG) assay is a feasible but labor‐intensive method for detecting global coagulation. It enables comprehensive assessment of anticoagulation, while drug‐specific assays assess only exposure. Traditionally, the Calibrated Automated Thrombogram (CAT) has been used, however the ST Genesia (Diagnostica Stago) allows automated evaluation. OBJECTIVE: We aimed to observe coagulation using the ST Genesia and compare the data with those of CAT in anticoagulated patients. PATIENTS AND METHODS: In total, 43 frozen‐thawed samples were studied using DrugScreen to assess direct oral anticoagulants (DOACs), warfarin, and low‐molecular‐weight heparin. Twenty samples (nine rivaroxaban, five apixaban, three warfarin, and three heparin) were also compared using CAT (5 pM tissue factor). RESULTS: TG reduction in DrugScreen depended on the specific drug and modestly correlated with DOAC levels (lag time R(2) = 0.36; peak R(2) = 0.50). The best correlation was observed with peak thrombin and rivaroxaban‐specified anti–activated factor X (anti‐Xa) activity (R(2) = 0.60). When comparing ST Genesia with CAT, only the results for apixaban concorded (R(2) = 0.97). Unlike CAT, ST Genesia yielded a normal endogenous thrombin potential (ETP) in 77% (24/31) activated factor X inhibitor cases, and it failed to give readouts at international normalized ratio (INR) ≥4.5 and at anti‐Xa ≥1.0 IU/mL. CONCLUSION: The ST Genesia data did not correlate with CAT, but it was independently associated with INR, anti‐Xa, and DOAC concentrations. The lag time and peak responses were similar; the major differences were that ST Genesia showed no ETP effect of DOACs and failed to give readout at high INR or anti‐Xa activity.

The thrombin generation (TG) assay is often used to assess coagulation in research settings. It can be used to monitor anticoagulant response, as well as to identify coagulation deficiencies and prothrombotic tendencies. 1 The semi-automated Calibrated Automated Thrombogram (CAT; Thrombinoscope, Diagnostica Stago, Asnières, France) method has been available to evaluate the TG for more than 2 decades. 2, 3 Although the CAT has proven useful in several clinical settings, such as when using hemophilia bypassing agents and emicizumab, the method is labor intensive and poorly standardized. For this reason, it has mainly been restricted to research use. 4, 5 The ex vivo capacity of citrated recalcified plasma to generate thrombin is measured by adding phospholipids and tissue factor (TF).

A new device, ST Genesia (Diagnostica Stago), enables automated TG measurement, which would make the assay more useful in clinical settings. However, the method is still being validated in patients, particularly those using anticoagulants. Currently, ST Genesia has three available test reagents: BleedScreen, ThromboScreen, and DrugScreen, which have low, intermediate, and high TF content, respectively. The exact TF concentrations are not publicly available.

DrugScreen is recommended for measuring anticoagulant effects.

The TF component is associated with the sensitivity of the assay to anticoagulants; the TG curve is negligible if TF is used at too low a concentration. Recently, the validation results and reference intervals of ST Genesia were reported in healthy volunteers. 6 The ST Genesia and CAT assays have both practical and methodological differences (Table 1) . Both are fluorogenic methods that use a substrate split by thrombin after initiation, with supplementation of varying amounts of TF and phospholipids into the plasma. In ST Genesia, TG is measured in single cuvettes that require a higher plasma sample volume, while microtiter plates are used in CAT. ST Genesia is calibrated using buffered human thrombin without patient plasma, unlike CAT, which uses alpha-2-macroglobulin. ST Genesia is calibrated daily. To this end, the individual plasma properties are compared using the FluoSet reagent. In CAT, each patient sample is run in parallel with an alpha-2-macroglobulin calibrant. Due to this difference, dabigatran likely has less influence on ST Genesia than on CAT. [6] [7] [8] Few studies have compared CAT with ST Genesia. 9 Recently, the plasma levels of direct oral anticoagulants (DOACs) were shown to correlate with the lag time and peak height of the DrugScreen test. 7 However, no studies have compared the DrugScreen test with the CAT assay in anticoagulated patient samples. In the present study, we aimed to verify the ST Genesia DrugScreen test in patients using different anticoagulants at different plasma concentrations. We also compared ST Genesia with the more established CAT method. DOACs, low-molecular-weight heparin (LMWH), and warfarin plasma samples were included in this comparison.

Blood samples were collected and assays performed at the Department of Clinical Chemistry of the Helsinki University Hospital (HUSLAB Laboratory Services, Finland). The samples comprised surplus plasma from clinical thrombophilia panels or DOAC concentration assessments. Citrated plasma (3.2% sodium citrate) was collected, centrifuged at 2500 g for 10 minutes, aliquoted within 2 hours of collection, and frozen at −80°C. All requested routine coagulation test analyses were freshly performed using ACL TOP coagulation analyzers (Instrumentation Laboratory, Werfen, Columbia, MD, USA), including international normalized ratio (INR), anti-activated factor Xa (anti-Xa), diluted thrombin time calibrated with dabigatran, and the anti-Xa method calibrated using either rivaroxaban or apixaban. For all the anti-Xa assays, we used the HemosIL Liquid anti-Xa (Instrumentation Laboratory, Werfen), which contains dextran sulfate (may affect heparin measurement) but no added exogenous antithrombin. 10 The TG analyses were performed within 1-2 weeks of sample collection. No corn trypsin inhibitors were added. All samples were anonymously analyzed, and thus the specific indications for anticoagulation were not available. The study received institutional approval from Helsinki University Hospital (HUS/211/2020; § 34, 30.9.2020). We compared the response of the CAT 5 pM TF reagent (Thrombinoscope, Diagnostica Stago) with those of the ST Genesia ThromboScreen and DrugScreen reagents (Diagnostica Stago).

We collected 31 samples from patients using DOACs: 4 using dabigatran, 17 using rivaroxaban, and 10 using apixaban. Six patient samples were used in the pilot study: three using rivaroxaban, two using apixaban, and one using edoxaban.

• The new ST Genesia DrugScreen method has been suggested for monitoring anticoagulation.

• We compared ST Genesia with the established Calibrated Automated Thrombogram (CAT) method.

• DrugScreen showed different sensitivities to direct oral anticoagulants than CAT.

• DrugScreen failed to give a readout at high international normalized ratio or anti-Xa activity.

We also prepared a series of pooled plasmas from samples in which treatment with warfarin or the LMWH effect was measured. There 

The following TG parameters were recorded: lag time (minutes), time to peak (minutes), peak height (nM), and endogenous thrombin potential (ETP) (nM × min). The methods were compared with consideration for the different TF concentrations of the reagents. For comparison, the same pooled samples or patient samples (different frozen aliquots) were used in parallel to assay DrugScreen and CAT.

The TG assays were analyzed without adding thrombomodulin.

CAT with 5 pM TF Heparin sample NA

Abbreviations: CAT, Calibrated Automated Thrombogram; ETP, endogenous thrombin potential; NA, not applicable; TF , tissue factor. a No readout of thrombin generation (the curve was too flat).

b CAT analysis not done due to limited sample volume.

c Genesia DrugScreen and CAT results obtained from two different pools. Table 2 ). Peak height was lowered in all warfarin and LMWH samples and in most DOAC samples (Figure 1 and 2). The peak was normal in only three anti-Xa inhibitor samples in which drug concentrations were <100 ng/mL ( Figure 1C , Table 2 ).

When the anticoagulation levels were within the expected treatment ranges, the peak values correlated best with the DOAC concentration in the rivaroxaban samples (R 2 = 0.60), while they correlated somewhat in the apixaban samples (R 2 = 0.46 after excluding the highest apixaban sample of 795 ng/mL as an outlier). Lag time and time to peak doubled when the DOAC concentrations increased from <100 ng/mL to >200 ng/mL ( Figure 1A-B) , while the peak halved from 200-300 nM to around 100 nM ( Figure 1C ). In the warfarin and LMWH samples, lag time and time to peak were 1.5-to 2-fold higher at treatment levels than at baseline. The peak at an INR level of 3.0 was around 30% of that at an INR of 1.0 ( Table 2) . The ETP was also reduced in the warfarin and LMWH samples, being 50%-60% lower than normal (Table 2 ). In sharp contrast, the ETP remained within the reference interval in 24 of 31 DOAC samples (77%) across the DOAC treatment concentration range ( Figure 1D ). The lag time and time to peak were prolonged at high DOAC levels, while the peak values were lower. However, the ETP remained essentially unchanged, even at anticoagulant levels high above the target range; that is, DOAC concentrations >400 ng/mL or apixaban concentrations of 428 ng/mL and 795 ng/mL, respectively.

To compare the CAT assay with ST Genesia, we studied 24 samples using the CAT 5 pM TF assay. ST Genesia failed to provide results in three samples with high anticoagulant concentration (Table 2) The differences in responses were calculated by plotting each result variation against the mean of the results (Bland-Altman). Based on lag time change, CAT seemed more sensitive to all the studied anticoagulants, especially rivaroxaban, which was the DOAC with the highest observed concentrations. In all, ST Genesia yielded more prolonged lag time and time to peak than CAT, while peak height and ETP were higher, indicating a slower initiation phase but more capacity to generate thrombin.

To our knowledge, this was the first study to compare the new ST Genesia DrugScreen thrombin generation method with the established CAT assay in plasma samples of anticoagulated patients. Since the DrugScreen is the preferred assay for assessing anticoagulant activity according to Stago, we chose this reagent to compare with CAT 5 pM TF concentration, which is commonly used to assess anticoagulated samples. 11, 12 When 5 pM TF is used, also factors VIII and IX contribute to TG. 13 In addition, our small pilot study tested the Stago ThromboScreen reagent, which is close in TF concentration to the CAT 5 pM assay. The results showed very modest responses in some strongly anticoagulated patients. Overall, the results differ between the assays. CAT seemed more robust and provided results even when anticoagulation levels exceeded their clinical or assumed targets.

The ST Genesia Drugscreen results correlated with the concentrations of DOACs, especially when the FXa inhibitor apixaban was used, the peak was decreased, or the lag time was prolonged. This is in concordance with a recent study. 7 The level of anticoagulation impacted the findings; the difference between the methods grew at anticoagulant levels that were higher than the target ( Table 2) Because the curves are flat, the DrugScreen reagent cannot be used to diagnose high warfarin, LMWH, or dabigatran levels, which is a clinical problem. Bleeding risks with warfarin increase by 8-to 10fold at INRs >4.5 and at anti-Xa levels >0.7 IU/mL, which are considered supratherapeutic in patients with trough levels of LMWH. 15, 16 These differences are highly clinically relevant after incidents such as a severe bleeding episode, impaired renal function, or thrombolysis, as well as during unfractionated heparin therapy and warfarin overdose assessment.

ST Genesia has been proposed as a tool to exclude the presence of DOACs, because it can detect levels <30-50 ng/mL. 7 We were unable to confirm this finding because only two samples had low DOAC levels (<50 ng/mL). However, we did find that lag time was prolonged in all DOAC samples using DrugScreen. The peak value was also diminished in >95% of the samples studied. In accordance with a previ- 

The authors declare that nonfinancial support was received from Diagnostica Stago (use of the ST Genesia device). The authors have no other disclosures to declare.

Tuukka A. Helin https://orcid.org/0000-0002-5273-8088

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