Pitfalls in Argatroban Monitoring: Heparin Interference With Dilute Thrombin Time Assays

Direct thrombin inhibitors (DTI), such as argatroban, dabigatran, or bivalirudin, prolong clotting times due to their ability to bind and inhibit thrombin. Among these, argatroban requires particularly careful monitoring as it is commonly used to treat Heparin Induced Thrombocytopenia (HIT). This condition presents a dual risk: thrombosis caused by HIT and bleeding due to anticoagulation. For instance, Beyer et al. established that there was a 38% bleeding risk for supratherapeutic concentrations of argatroban, while subtherapeutic levels carried a 5% risk of thrombosis [1].

To monitor argatroban, several assays are available, including activated partial thromboplastin time (aPTT), ecarin chromogenic assay (ECA) and dilute thrombin time (dTT). Studies have established that aPTT is not ideal for monitoring argatroban because of a plateau effect and a risk of non-therapeutic variations (for instance, CRP may prolong aPTT [2, 3]), whereas ECA and dTT are more reliable and specific [1, 4-6].

ECA utilizes ecarin, which derives from a snake venom and converts prothrombin into meizothrombin rather than thrombin. Meizothrombin is inactivated by DTI but unaffected by heparin [7], making it useful for monitoring HIT patients on argatroban therapy.

In contrast, dTT consists of a thrombin time performed on a prediluted plasma, as standard thrombin time is too sensitive to DTI. While predilution can also reduce heparin interference, no study has yet evaluated the effect of heparin on dTT when used for argatroban measurement.

This issue is relevant because argatroban is typically administered immediately after stopping heparin for HIT treatment. As a result, both argatroban and heparin may be present in the bloodstream for a short period during this transition. Therefore, this study aims to assess whether heparin can interfere with argatroban measurement using a dTT assay.

In this study, approved by the institution's ethics board (CE-2024-103), samples collected during December 2024 from inpatients at University Hospital of Strasbourg (France) were included. Inclusion criteria were as follows: patients over 18 years old anticoagulated with unfractioned heparin (UFH) or enoxaparin. Underaged patients, patients undergoing a switch from another anticoagulant to heparin, or samples with insufficient plasma volume were excluded. Patient blood was drawn in Vacuette PET citrated tubes (Greiner Bio One, Kremsmünster, Austria) or Vacutest PET citrated tubes (Kima, Padua, Italy) with 0.109 M of trisodium citrate. Samples were centrifuged at 2500 g for 10 min at 20°C.

Anti-Xa activity (AXA) was measured using STA Liquid anti-Xa on a STA-R Max analyzer (both Diagnostica Stago, Asnières-sur-Seine, France) and anti-IIa activity was measured using a dTT assay, Hemoclot Thrombin Inhibitor (Hyphen Biomed, Neuville-sur-Oise, France), calibrated with an argatroban standard (Hyphen Biomed) on a STA-R Max analyzer (Diagnostica Stago).

First, to evaluate whether UFH and enoxaparin could interfere with anti-IIa argatroban measurement, artificial standards with increasing concentrations of UFH and enoxaparin were prepared using Héparine Choay (Cheplapharm France, Levallois-Perret, France) and Lovenox (Sanofi, Gentilly, France), respectively, diluted in Cryocheck normal pooled plasma (Cryopep, Montpellier, France). AXA and anti-IIa activity were then measured simultaneously.

Second, to assess whether such interference could occur in a clinical setting, samples of patients treated with heparin were analyzed for both AXA and anti-IIa activity simultaneously.

Pearson correlation tests were carried out to determine whether a correlation existed between AXA and anti-IIa activity. A p value ≤ 0.05 was considered statistically significant. Analyses were performed with Prism v6.05 (GraphPad Software).

Artificial standards AXA was 0.10–1.81 IU/L and 0.12–1.74 IU/L, while anti-IIa activity was 0.03–0.55 μg/mL and 0.00–0.14 μg/mL for UFH and enoxaparin, respectively. There was a significant correlation (p < 0.001) between AXA and anti-IIa for both UFH and enoxaparin (see Figure 1).

In conclusion, this study on 58 patients anticoagulated with UFH or enoxaparin highlighted that UFH could interfere with anti-IIa activity measurement using a dTT assay calibrated for argatroban. We first demonstrated that there was a correlation (p < 0.001) between AXA and anti-IIa activity on normal pooled plasmas spiked with increasing concentrations of UFH or enoxaparin. This interference was more pronounced for UFH, with anti-IIa activity reaching 0.55 μg/mL at an AXA of 1.74 IU/mL. Second, we showed that the same interference was present in samples from patients receiving UFH (p < 0.001), whereas no such effect was observed in samples from patients anticoagulated with enoxaparin, which is consistent with the generally low anti-IIa activity of LMWH.

This finding suggests that dTT results may be falsely elevated in the initial hours following UFH cessation. Indeed, it is usually recommended to monitor anti-IIa activity of argatroban 4 h after initiation. Considering that UFH half-life usually ranges from 1 to 2 h [8-10], argatroban anti-IIa activity using dTT assays may be overestimated in the first hours following heparin cessation, depending on residual UFH concentration. However, this overestimation is most likely slight because therapeutic UFH usually ranges between 0.3–0.7 IU/mL.

Finally, several reliable assays that are unaffected by UFH are commercially available: besides ECA, some manufacturers also provide dTT assays incorporating heparin neutralizers, which could be an alternative.

This study presents limitations: this experiment was conducted on a single type of analyzer (STA-R Max) using a single type of reagent (Hemoclot Thrombin Inbibitor). Therefore, depending on the methodology and reagent used for dTT assays, the interference of UFH may vary. Additionally, we did not conduct spiking experiments that included both UFH and argatroban, which prevents us from fully assessing the impact of UFH on argatroban activity.

Clinicians and pathologists should be aware that dTT-based argatroban monitoring may yield falsely elevated anti-IIa activity measurements shortly after UFH cessation, depending on the reagent used. This could cause an unnecessary change in the infusion regimen. However, this interference correlates with UFH levels, which should usually be below 0.2 IU/mL 4 h after discontinuing heparin, suggesting that the risk is limited in most cases.

To minimize misinterpretation, it may be useful to perform anti-Xa simultaneously to anti-IIa activity in order to determine whether UFH is likely to interfere.

A.H. designed the study, analyzed data, and wrote the manuscript. J.W., L.S., and L.M. revised intellectual content.

This study was approved by the institution's ethics board (CE-2024-103).

The authors have nothing to report.

The authors declare no conflicts of interest.