ECA, ecarin chromogenic assay; ECT, ecarin clotting time; LMWH, low molecular weight heparin; UFH, unfractionated heparin.

Conventional: Approved clinical laboratory tests not specific for DOACs. Results useful as a positive or negative indication of the presence of DAOC activity. For dabigatran, if the TT is normal, there is either no drug on board or the concentration is not high enough to impair coagulation; if the result is elevated, the dabigatran concentration is high enough to affect coagulation. Similarly, for the Xai, if the anti-Xa for LMWH/UFH is normal, the Xai concentration is 0 or not high enough to impair coagulation. If the anti-Xa LMWH/UFH is elevated, the Xai concentration is high enough to impair coagulation. However, elevated results cannot be interpreted as they would be for LMWH/UFH regarding degree of anticoagulation.

Specific: Designed to quantitate DOAC concentration. LC-MS/MS directly quantitates drug concentration. Other types of assays require construction of a standard curve using known concentration of DOACs added to the assay to create a calibration curve. Results obtained from the patient sample are then compared with the curve to determine drug concentration and are reported as nanograms per milliliter. Threshold concentration values for administering reversal agents have been determined,²⁸  and ranges of DOAC therapeutic windows have been identified.^56,57  Reference curves must be run for each QC of the machine. The development of packaged sets of standards to construct reference curves has increased the appeal of these assays in the clinical laboratory because of decreased labor and decreased turnaround time. Commercial assays for these tests have been developed and are available but not yet approved by regulatory agencies.

LC-MS/MS: LC-MS/MS is a sophisticated method for detecting plasma concentrations of all DOACs by analyzing the actual drug molecules in the sample. It is considered the gold-standard method but requires large expensive equipment and well-trained staff and has limited throughput, so it is not well suited for routine clinical laboratories.⁶⁵ 

dTT: Plasma-based assay. The TT measures the time it takes for thrombin to cleave fibrinogen and form a clot. The standard test is measured in seconds, with the normal range usually 15 to 20 seconds. Because direct thrombin inhibitors significantly interfere with thrombin activity, patient plasma is diluted 1:3 with normal plasma to dilute drug concentration but maintain normal levels of coagulation factors and fibrinogen. A small amount of bovine thrombin is added to initiate coagulation. The presence of dabigatran inhibits the thrombin activity proportional to drug concentration. The result of the test in seconds is compared with a reference curve constructed from known concentrations of dabigatran.^66,69 

Ecarin-based assay: Plasma-based assay that uses the snake venom ecarin to cleave thrombin to meizothrombin, which is insentitive to heparins. For the ECT, a clot-based assay is run similar to the TT, with the patient sample results compared with the calibration curve to determine dabigatran concentration. In the ECA, meizothrombin activity is determined by cleavage of a chromogenic substrate. The cleavage is inhibited by dabigatran in a concentration-dependent manner; results are compared with known calibrators to determine dabigatran concentration.⁶⁷ 

Calibrated anti-Xa: Anti-Xa assays use a chromogenic substrate that is cleaved by factor Xa. Color production is proportional to Xa activity; exogenous Xa is added so that the patient’s Xa level is not rate limiting. The presence of DOACs that inhibit factor Xa (apixaban, edoxaban, rivaroxaban, betrixaban) will inhibit color generation. This assay is also used for heparins and fondaparinux. Drug concentration is inversely proportional to color generation and can be determined by comparing with the reference curve generated from calibration standards specific for each DOAC. When used for LMWH/UFH, the results from the heparin-specific reference curves are expressed in units per milliliter, and when used for factor Xai DOACs, the results from the reference curve are expressed as nanograms per milliliter.^57,68