2025+ADLM指南：使用直接口服抗凝剂患者的凝血试验

INTRODUCTION

The introduction of direct-acting oral anticoagu-lants (DOACs) has been a major advancement in the management of anticoagulation. Previously,outpatient oral anticoagulation was limited to vita-min K antagonists, namely warfarin. Vitamin K ago-nists have numerous drug-drug and dietary

interactions. They also require frequent laboratory monitoring for therapeutic ranges and require dosing changes chronically. In contrast, DOACs al-low for oral anticoagulation with fixed doses that have relatively predictable pharmacokinetic char-acteristics and do not generally require routine therapeutic monitoring. These drugs also have fewer drug-drug and drug-food interactions. In

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Table 1. Indications, mechanisms, and pharmacokinetic properties of DOACs.

|Anticoagulant characteristics|Anticoagulant|Anticoagulant|Anticoagulant|Anticoagulant| |---|---|---|---|---| |Anticoagulant characteristics|Dabigatran (Pradaxa®)|Rivaroxaban (Xarelto®)|Apixaban (Eliquis®)|Edoxaban (Savaysa®)| |FDA-approved uses|NVAF|+|+|+| ||VTE treatment|+|+|+| ||Primary VTE prevention|+|+|+| ||Recurrent VTE prevention|+|+|+| ||CAD|+||| ||PAD|+||| ||Pediatric patients|+|+|| |Mechanism of action|Direct thrombin inhibition|Selective FXa inhibition||| |Half-life(h)|12-17|Pediatric: 1.6-4.2|12|10-14| |||Adults: 5-9||| |Renal clearance/excretion (%)|80|36|27|50| |Abbreviations: CAD,coronary artery disease; NVAF, nonvalvular atrial fibrillation; PAD, peripheral artery disease; VTE, venous thromboembolism.|||||

a relatively healthy patient population, the dur-ation and efficacy of the DOACs are generally de-pendent on half-life, age, and renal excretion or clearance. Table 1 summarizes indications, me-chanisms of action, and the pharmacokinetic properties of the DOACs(1,2).

Commercially available DOACs in the United States currently include dibagtran(Pradaxa®),rivaroxaban(Xarelto®), apixaban(Eliquis®), and edoxaban(Savaysa®). Dabigatran is a competitive direct thrombin inhibitor, while rivaroxaban, apixaban, and edoxaban are competitive direct factor Xa(FXa) inhibitors.

Despite the significant improvement in anti-coagulant administration, DOACs have introduced challenges for both clinical laboratorians and clin-icians. Specifically, providers need to understand the impact DOACs have on diagnostic hemostasis testing. This comprehension is vital to accurately interpreting results, forming clinical impressions,and rendering sound management decisions.This document is an expert opinion and

serves to provide evidence-based guidance for co-agulation testing in patients who are taking DOACs. In preparing this guidance document,the focus was on the laboratory methods as the driving principle for our approach while also ex-tracting and highlighting the most practical infor-mation in the literature to answer the following questions:

1.What is the impact of DOACs on basic hemosta-sis testing?

2.Which coagulation tests can be performed while a patient is on a DOAC?

3. Which coagulation tests should be avoided while a patient is on a DOAC?

4.What is the role for point-of-care(POC) testing in the presence of DOACs?

5.What strategies can be used to mitigate testing interferences in patients taking DOACs?

6. Should DOAC concentrations be monitored?

7. What tests are available for measuring DOACs?

8. How should DOAC concentrations be interpreted?

Coagulation Testing and Direct Oral Anticoagulants

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![Intrinsic Pathway\nActivated Partial Thromboplastin Time\n[Prothrombin Time]\nFXa\nAnti-Xa DOACs\nProthrombin\nDTI DOACs\nFibrinogen\nFibrin\n[Thrombin Time]\n](2/aj0YEe4oUhYkBdfpda0fYP/figure35bb9ade02454e82818f9180c95d140a_0_2.png) Fig.1.A schematic illustrating the coagulation cascade in vitro. The intrinsic and extrinsic pathways intercept at FXa, which is the start of the common pathway.FXa is responsible for activating prothrom-bin to thrombin, and thrombin cleaves fibrinogen into fibrin, which arranges to form the fibrin clot.The anti-Xa DOACs inhibit the activity of FXa, while the thrombin inhibitor DOAC inactivates thrombin.Created in BioRender. Harris, N.(2024) https://BioRender.com/c54d428.

WHAT IS THE IMPACT OF DOACS ON BASIC HEMOSTASIS TESTING?

Basic hemostasis testing commonly consists of the prothrombin time(PT), activated partial thromboplastin time(aPTT),and fibrinogen activity assays. These assays rely on the principle that a clot can be formed due to appropriate thrombin generation. In vivo, DOACs affect the common co-agulation pathway that ultimately disrupts throm-bin generation(Fig. 1) and could hypothetically impact clot-based PT and aPTT testing. From a la-boratory perspective, however, this is much more complicated.

If the PT and/or aPTT are prolonged due to DOACs, the degree of their prolongation cannot be used to infer DOAC concentrations because of an unsuitable correlation(3-6). Further, there is variable sensitivity in testing to different DOACs

that adds another layer of complexity. For ex-ample, the PT is less sensitive to apixaban than it is to rivaroxaban, and most PT reagents cannot ad-equately detect peak and trough levels of apixa ban. On the other hand, whether DOACs will affect the PT or aPTT depends on the testing plat-form(i.e., the specific method, reagent lot-lot vari-ation), the specific DOAC, drug metabolism, and drug plasma concentration(3, 5, 7, 8). Fibrinogen activity testing using the Clauss fibrinogen method is generally considered to be insensitive to DOACs,though high dibagtran concentrations may cause a false decrease for some assay reagents.

Summary

●PT and aPTT may be affected by DOACs (plat-form/assay dependent) and should not be used to assess DOAC presence and/or concentration.

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●Fibrinogen activity measurements can generally be obtained without interference from DOACs.

WHICH COAGULATION TESTS CAN BE PERFORMED WITHOUT INTERFERENCE FROM DOACS?

The impact of DOACs varies based on the meth-odology used for the test. In general, methodolo-gies not affected by the presence of DOACs include polymerase chain reaction, immunoas-says, and platelet function testing. Polymerase chain reaction testing is commonly used for the detection of factor V Leiden and prothrombin gene mutations. Serology-based methods such as enzyme-linked immunosorbent assays, lateral flow immunoassays, agglutination methods, and chemiluminescent immunoassays do not rely on any type of clot formation and are unaffected by DOACs. Immunoassays are commonly used for measuring von Willebrand factor, antiphospholi-pid antibodies, D-dimer, protein S and C antigen,antithrombin antigen, and fibrinogen antigen.

Platelet function testing (PFT) availability varies widely between laboratories. Further, PFT can be performed on POC systems (e.g., PFA-100/200,VerifyNowTM etc.) or considered high-complexity testing (e.g., light transmission aggregometry) de-pending on the testing platform and method.Because PFTs do not generally rely on thrombin generation for their evaluation of platelets, they should not be affected by DOACs. The current evi-dence, albeit relatively limited, indicates that DOACs do not significantly affect PFT platforms(9-14). However, regarding light transmission ag-gregometry, specific reagent agonists used to in-duce platelet aggregation may be impacted by DOACs-specifically, thrombin and thrombin receptor-activating peptides (9, 10,14-19).

Summary

●Polymerase chain reaction and immunoassays are unaffected by DOACs.

●Based on the current evidence, platelet function testing is relatively unaffected by DOACs, but spe-cific agonists(thrombin and thrombin receptor-activating peptides) should be avoided for light transmission aggregometry.

WHICH COAGULATION TESTS SHOULD BE AVOIDED WHEN A PATIENT IS TAKING A DOAC?

In general, certain clot-based tests and some chromogenic assays should be avoided in patients taking a DOAC, as DOACs can lead to test result misinterpretation.

Should factor activity assays be avoided?

Coagulation factor activities are commonly mea-sured using clot-based or one-stage assays.Briefly, the patient plasma is diluted with factor-deficient plasma and assayed by the PT(for factors II,V,VII,and X) or aPTT(for factors VIII,IX,XI,and XII). The measured clotting time is inversely pro-portional to factor activity. As described earlier,DOACs variably affect the PT and aPTT, with the de-gree of prolongation dependent on the assay re-agent,type, and concentration of DOAC present.Therefore, factor activities determined via one-stage assays with DOAC-susceptible PT or aPTT reagents have the potential to underesti-mate factor activities in the presence of DOACs.

In vitro spiking studies have demonstrated that increasing concentrations of dibagtran lower PT- and especially aPTT-based factors in a concentration-dependent manner(20, 21). Of the PT-based factors, factor II may be the most af-fected and factor X the least affected. Misleading results can occur with dibagtran-containing sam-ples as they often display results suggestive of a nonspecific inhibitor effect.

Increasing concentrations of rivaroxaban and apixaban can also lead to factitiously underesti-mated one-stage factor activities (22-25).Rivaroxaban generally has more of an effect than

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Table 2. Summary of DOAC interference with clot-based assays.

|Test|Testing indication|FXa inhibitor interference?|DTI interference?| |---|---|---|---| |PT/aPTT and mixing study|Screening test|Yes|Yes| |Thrombin time|Screening test|No|Yes| |Fibrinogen activity|Screening test|No|No| |Factor activities|Hemophilia and other factor deficiencies|Yes|Yes| |Activated protein C resistance|Thrombophilia|Yes|Yes| |Protein S activity|Thrombophilia|Yes|Yes| |Protein C activity|Thrombophilia|Yes|Yes| |Dilute thrombin time|Anticoagulation|No|Yes| |Reptilase time|Hypofibrinogenemia or dysfibrinogenemia|No|No| |Lupus anticoagulant|Thrombophilia|Yes|Yes|

apixaban, which reflects the varying sensitivities of PT and aPTT reagents to these different FXa inhibitors. As with dibagtran, apixaban and rivaroxaban often induced a nonspecific inhibitor effect.

In addition to falsely lowering factor activities mea-sured by one-stage assays, DOACs may lead to the factitious presence of factor inhibitors(20,24).

Summary.

•Factor activities measured by one-stage or clot-based methods can be underestimated in the presence of DOACs.

•Factor inhibitors measured by one-stage or clot-based methods can be overestimated in the presence of DOACs.

Should Lupus anticoagulant assays be avoided?

Lupus anticoagulants(LA) are autoimmune het-erogeneous immunoglobulins directed against phospholipid-protein components of the cell membrane that may impact coagulation-related proteins and/or clotting factors in vivo. If these im-munoglobulins are present in a sample and bound to their targeted antigen, they may cause prolongation of phospholipid-dependent clotting times in laboratory assays. Currently, it is recom-mended that LA testing should employ 2

phospholipid-dependent clotting time assays based on different principles(26). Although LA testing is only 1 of the 3 laboratory criteria for the identification of antiphospholipid syndrome,it is the best-established risk factor of clinical man-ifestations(27-29). It is important for healthcare providers to understand the effect of DOACs in LA testing for appropriate testing utilization, diag-nosis, and management.

Similar to other clot-based assays, LA tests are vulnerable to interferences by anticoagulant ther-apy that usually leads to false positives but in rare scenarios may cause false negatives(26, 29).Generally,DOACs have been widely shown to pro-long conventional LA screening assays(3, 29, 30).This may potentially lead to misleading results and misinterpretation.

Summary.

•The presence of DOACs may lead to unreliable LA test results such as false positives and, in rare cases, false negatives.

Should other clot-based testing be avoided?

Many specialized coagulation assays also rely on clot formation as a measurement endpoint and are at higher risk for interference from DOACs. A

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summary of the clot-based assays, their asso-ciated conditions, and susceptibilities to DOAC interference is presented in Table 2.

Clot-based protein C activity. Protein C activity via clot-based assay is overestimated in the presence of rivaroxaban, edoxaban, or dibagtran but not apixaban(31, 32). Clot-based methods are gener-ally discouraged in favor of chromogenic assays unless there is a strong clinical suspicion for the rare type II protein C deficiency(33). False eleva-tion in protein C activity could mask true deficiency of this natural anticoagulant, delaying appropriate diagnosis and management. It is recommended that chromogenic protein C activity testing be used in the presence of DOACs.

Protein S activity. Functional testing for protein S ac-tivity can be performed via PT-,aPTT-, or Russell vi-per venom time (RVVT)-based clotting tests (34).aPTT- and RVTV-based protein S activity assays are exquisitely sensitive to dibagtran (20,35).Clot-based protein S activity may also be overesti-mated by FXa inhibitors and is particularly sensi-tive to rivaroxaban (31, 36-38). False elevation in protein S activity could mask true deficiency of this natural anticoagulant, delaying appropriate diagnosis and management. It is recommended that immunoassay-based free protein S antigen testing be used in the presence of DOACs.

Activated protein C resistance(APCR). APCR assays rely on clot formation with aPTT-, RVT-, or prothrombinase-based reagents where the patient plasma is prediluted in factor V-deficient plasma,analyzed with and without the addition of activated Protein C and a ratio between these 2 clotting times calculated. Increasing APCR ratios have been ob-served with increasing concentrations of dibaga-tran, apixaban, rivaroxaban, and edoxaban, which could lead to a false normal APCR ratio in an individ-ual who carries the factor V Leiden mutation(20,22,31, 35, 39). Different APCR reagents demonstrate variable susceptibility to DOAC interference, with

prothrombinase- and RWT-based assays more af-fected by dibagtran and aPTT-based assays less af-fected by apixaban(37, 39, 40). It is recommended that molecular testing be used to test for the factor V Leiden mutation in the presence of DOACs (41).

Thrombin time(TT)/dilute TT. The TT is unaffected by Fxa inhibitors but is too sensitive to dibagtran to give any indication of drug concentration (21,31,42). However, a modification of the thrombin time, the dilute TT, demonstrates a concentration-dependent prolongation of the clotting time and,thus, is more appropriate for dibagtran quantifi-cation (43).

Reptilase time. Reptilase time measures fibrin for-mation following fibrinogen cleavage and fibrino-peptide A release by the batroxobin snake venom. The reptilase test is unaffected by all DOACs(20,22,31,42).

Summary.

●Clot-based protein C and protein S activity may be overestimated in the presence of DOACs, po-tentially masking true deficiency of these natural anticoagulants.

●APCR testing is susceptible to interference from DOACs, but the interference is variable depend-ing on the reagent and anticoagulant.

●The TT and dilute TT are prolonged by dibagtran but unaffected by FXa inhibitors.

Are chromogenic assays impacted?

Relative to clot-based assays, chromogenic as-says are less sensitive to interferences from DOACs. Chromogenic assays comprise 2 stages that are both independent of clot formation:activa-tion of a serine protease capable of cleaving a spe-cific peptide nitroanilide substrate and cleavage of the substrate to release a chromophore whose concentration is measured spectrophotometrical-ly. Active forms of protein C, factor II, and factor X are commonly used in chromogenic reactions.

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Chromogenic protein C activity. Chromogenic protein C activity assays can be reliably performed in pa-tients taking any DOAC, since protein C is not the target of these anticoagulants and is activated dir-ectly via viper venom(21, 24, 32).

Antithrombin activity based on factor lla. It is import-ant to recognize that chromogenic antithrombin activity assays that rely on factor lla(i.e., thrombin)for chromophore generation can be performed in patients taking FXa inhibitors but will be falsely ele-vated in patients taking dabigatran(31). Dabigatran affects thrombin-based antithrombin assays in a dose-dependent manner(20, 21, 32, 35).

Antithrombin activity based on factor Xa. Chromogenic antithrombin activity assays commonly use factor X activity for chromophore generation and can be performed in patients taking dibagtran but will be unreliable in patients taking FXa inhibitors(20,31,32). Different FXa-based antithrombin re-agents may demonstrate varying sensitivities to the FXa inhibitors(31).

Anti-Xa activity. The use of chromogenic anti-Xa as-says for therapeutic monitoring of unfractionated heparin and low molecular weight heparin is now commonplace, but the measured activity reflects all anti-Xa activity in the specimen and will be ele-vated in the presence of FXa inhibitors.Therefore, anti-Xa activity assays are not reliable for heparin monitoring in patients with residual direct FXa inhibitor present(44, 45).

Chromogenic factor VIII, IX and X activities. FXa inhibi-tors will reduce factor VIII, IX, and X activities when measured by chromogenic principles since these assays rely on factor Xa activity for chromophore generation. Plasma apixaban and rivaroxaban concentrations well within the typical on-therapy ranges for these anticoagulants may be sufficient to yield a factitiously low chromogenic factor VIII activity(31).

Factor XIII. Dabigatran can lead to falsely low levels in the chromogenic FXIII assay(6).

A summary of the chromogenic assays, their clinical indications, and susceptibilities to DOAC interference is presented in Table 3.

Summary.

●Chromogenic protein C assays are unaffected by DOACs.

●Chromogenic assays utilizing factor Ila for chromophore generation are affected by dibaga-tran but unaffected by FXa inhibitors.

$\bullet$ Chromogenic assays utilizing factor Xa for chromophore generation are affected by FXa in-hibitors but unaffected by dabigatran.

WHAT IS THE ROLE FOR POC TESTING IN THE PRESENCE OF DOACS?

POC testing is available for several laboratory tests, such as the international normalized ratio,activated clotting times, and viscoelastic tests.POC international normalized ratios can be impre-cise, vary from their central laboratory counter-parts depending on the POC device, and are potentially subject to interferences(importantly,such as the presence of a lupus anticoagulant).Prolongation of international normalized ratios may be seen with a DOAC present, but a normal result also does not exclude the presence of a DOAC either. Currently, there is no role for POC testing for monitoring the therapeutic effects of DOACs.

Viscoelastic testing

The use of viscoelastic testing to measure DOAC effects is not recommended.The rationale for this is a lack of specificity of alterations to the coagula-tion cascade. Another consideration is the rela-tively wide reference range of the viscoelastic methods. The major effect of the DOACs is a pro-longation of the initial clotting reaction time, which reflects the coagulation cascade and the

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Table 3. Summary of chromogenic assays.

|Test|Testing indication|FXa inhibitor interference?|DTI interference?| |---|---|---|---| |Antithrombin activity (FXa-based)|Thrombophilia|Yes|No| |Antithrombin activity (Fla-based)|Thrombophilia|No|Yes| |Protein C activity|Thrombophilia|No|No| |Factor VIII|Hemophilia A|Yes|No| |Factor X|Anticoagulation|Yes|No| |Anti-Xa activity|Anticoagulation|Yes|No| |Factor IX|Hemophilia B|Yes|No| |Factor XIII|Bleeding disorder|No|Yes| |Abbreviations:DTI,direct thrombin inhibitor.||||

generation of thrombin(46-51).This interval is the R time(thromboelastography analyzer) or the CT(ROTEM and Quantra analyzers). In many reports,these reaction times, especially for the direct FXa inhibitors, are still within the population-based ref-erence range even though they are prolonged above an individual patient's baseline reaction time. Apixaban has the least discernable effect on the clot or reaction time, while edoxaban has the greatest effect. It appears that dibagtran is more likely to prolong these initial reaction times beyond the reference range. Significant prolonga-tion of the R or CT by direct FXa inhibitors often re-quires supratherapeutic doses of this class of DOACs(52). Overall, the results of these prior studies demonstrate various and inconsistent ef-fects of DOACs on viscoelastic testing.

Activated clot time

The activated clotting time is a POC whole blood assay used to monitor heparin anticoagulation in cardiopulmonary bypass, percutaneous coronary intervention, and extracorporeal membrane oxy-genation(53, 54). The blood specimen is mixed with kaolin or silica, which activates the intrinsic pathway of coagulation. Clotting is detected by the movement of a magnet or photo-optically in a cart-ridge by the change in velocity of blood as the

viscosity increases. Only dibagtran significantly prolongs the activated clotting time. The direct FXa inhibitors do not have a significant effect on the activated clotting; therefore, heparin monitor-ing by activated clotting time can be performed in the presence of FXa inhibitors(55).

Summary

●POC testing does not currently have a role in monitoring the therapeutic effects of DOACs.

●DOACs can demonstrate inconsistent effects on viscoelastic testing.

●Direct thrombin inhibitors can prolong activating clotting times, but the direct Fxa inhibitors do not have a significant effect.

WHAT STRATEGIES CAN BE USED TO MITIGATE TESTING INTERFERENCES IN PATIENTS TAKING DOACS?

There are 3 general strategies that can be imple-mented to mitigate these known interferences,which include(a) discontinuing DOAC treatment,(b) using a different analytical method not subject to the interference, and(c) reversal or removal agents to neutralize the inhibition. In situations where testing must be performed while patients are prescribed DOACs,temporary discontinuation of the DOAC ideally should be considered using

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appropriate clinical judgment. Additionally, not all DOACs behave in the same manner in specific as-says, and a change in methodology may be a help-ful alternative. For example, the chromogenic antithrombin activity assay is available in 2 differ-ent forms; one form uses the inhibition of FXa,and the other uses the inhibition of thrombin.Direct FXa inhibitors interfere with assays using the inhibition of exogenous FXa, causing falsely in-creased results for antithrombin (56). In contrast,antithrombin assays that use exogenous thrombin inhibition do not show any interference from direct FXa inhibitors. The following discussion focuses on other strategies that may mitigate DOAC interfer-ence in specific assays, including a section on spe-cific products for DOAC neutralization.

Strategies for factor assays

The underestimation of factor activity and non-specific inhibitor effect seen in clot-based factor assays(see section 3a) may be mitigated by multi-dilution analysis and utilizing DOAC-insensitive re-agents(32). However, these strategies have been shown to be potentially more effective for rivarox-aban and apixaban than for dibagtran.

Strategies for LA testing

Similarly to other coagulation testing, it is prefer-able for LA testing to be performed in the absence of anticoagulants(i.e., before the initiation of DOAC therapy)(26,29).If therapy has been started and testing is required, an earnest consideration of temporarily stopping therapy should be evalu-ated. If the risk of temporary discontinuation is not excessively high, consider stopping the DOAC therapy for a minimum of 2 to 3 days before collecting the sample for LA testing(3, 28). If the risk of temporary discontinuation of anticoagula-tion is too high, consider a temporary transition to low molecular weight heparin, which may have less interference on the LA testing, and draw trough levels just before the next dose(3).

Strategies for DOAC neutralization

If anticoagulant discontinuation is not safe or feasible, consider the use of DOAC removal agents in sample preparation prior to performing coagula-tion testing. DOAC-STOPTM and DOAC-RemoveTM are products that claim to absorb or remove any DOAC in plasma samples while having a minimal ef-fect on plasma proteins involved in the clotting mechanism and are supported by some studies such as PT, aPTT, anti-Xa, TT, and LA panels (57-61). More recently, a DOAC filter has been intro-duced to remove DOACs prior to LA testing (62).These assays are classified as laboratory-developed tests, which require additional resources and a thorough evaluation by the laboratory director and staff.

Similarly, the reversal agents idarucizumab and andexanet alfa may be used as a sample pretreatment step to neutralize the impact of dibagtran and factor Xa inhibitors, respective-ly.Idarcuzumab is a monoclonal antibody frag-ment that binds to dibagtran with an affinity around 350 times stronger than its affinity for thrombin(63). Andexanet alfa is a recombinant modified human factor Xa decoy protein that has no catalytic activity but can bind factor Xa in-hibitors with high affinity(64). Both have been shown to neutralize DOACs in samples prior to coagulation testing(65, 66).

Importantly, the approach with DOAC neutral-izing agents can be cost-prohibitive and not feasible for all laboratories. Utilizing these strat-egies requires resources and direct discussions with laboratory leadership for an evaluation and implementation.

Communication strategies for laboratories

In most cases, the strategies discussed here re-quire communication between the laboratory and clinicians. The laboratories have information re-garding the potential interferences by DOACs on the laboratory assays, while clinicians are aware of

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their patient's most current clinical context, includ-ing anticoagulation status, future anticoagulation management, and laboratory testing needs.Effective communication, exchange of informa-tion, and crafting strategies between both par-ties have been shown to optimize patient care(67). The level of communication between par-ties will likely vary and be dependent on the type of medical institution and laboratory. A sim-ple route achievable for many laboratories could include attaching a comment to results where cer-tain DOACs could interfere. An example is attach-ing a comment that reads,"Factor Xa inhibitors may lead to false increases in this test" to an an-tithrombin assay that uses factor Xa. A higher level of communication could include continuing med-ical education lectures given by the laboratory dir-ector or staff to ordering providers and/or on an individual case-by-case basis if the laboratory dir-ector is involved in coagulation test interpreta-tions and direct communication with the clinical team. Another method could incorporate infor-mation technology to guide ordering based on a defined algorithmic approach to ultimately tailor the best available testing for the patient's specific scenario. The aforementioned strategies may be used in combinations or in hybrid forms to satisfy individual institutional needs.

Summary

●Alternative laboratory testing methods may be considered to assist with interpreting coagula-tion test results when DOAC interference is involved.

●Clot-based factor and inhibitor testing should be avoided in patients taking DOACs, but if testing must be performed, multidilution analysis and use of DOAC-insensitive PT and aPTT reagents are recommended if available.

●LA testing should be performed in the absence of DOAC treatment, but if testing is necessary,multiple strategies should be considered by both the clinical and the laboratory team in collaboration.

●DOAC neutralizing reagents are available and may have some potential for their utilization.However, they are cost-prohibitive and may not be possible to implement by most laboratories.

●Multiple strategies exist for laboratories to com-municate with clinicians about the impact of DOACs on coagulation testing.

SHOULD DOAC CONCENTRATIONS BE MONITORED?

DOACs have predictable pharmacokinetics and pharmacodynamics relative to warfarin with fewer interferences from food and other medications;therefore, routine monitoring is generally not ne-cessary for dose adjustments, especially when there are no clinical concerns for toxicity or lack of efficacy(5, 7, 68-70).

There are urgent and nonurgent situations pro-posed where screening for the presence of DOACs may be clinically useful. Urgent situations include severe hemorrhage(toxicity) or thrombosis(ther-apy failure), an emergent surgical procedure while on therapy, and trauma. Nonurgent situations in-clude advanced age, severe renal failure with dialy-sis dependence, prior intervention with high bleeding risk, prior small bowel resection, gastro-intestinal malabsorption, patients with extremes of body weight, failed therapy, and drug interac-tions(9, 71-74).

It is highly discouraged to test patients beyond these very limited clinical scenarios based on the available data(75). Accordingly, due to the limited clinical utility, infrequent need, and rela-tively predictable pharmacokinetics and pharma-codynamics, many clinical laboratories do not perform assays that measure DOACs.

Summary

●Patients on DOACs do not require routine labora-tory monitoring,though there may be very limited and specific urgent or nonurgent situations where DOAC plasma concentration measure-ments may help guide clinical decision-making.

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WHAT TESTS ARE AVAILABLE FOR MEASURING DOACS?

If DOAC measurement is being considered,there are indirect and direct measurements that can be strategically implemented. While widely available, the PT and aPTT are not appropriate for measuring DOACs.

Indirect measurement

Indirect DOAC assays are used to determine the presence or absence of the drug in the plasma of the patient (5,7,76,77).TT is highly sensitive to da-bigatran and can be used to detect the presence of dibagtran A. normal TT rules out the presence of dibagtran in a specimen. Note the TT is not af-fected by the FX a inhibitors. In contrast, normal PT and/or aPTT results cannot be used to definitively exclude the presence of a DOAC in a sample (3-6).

Chromogenic anti-Xa assay calibrated for un-fractionated heparin and low molecular weight heparin can detect the presence of direct FXa inhi-bitors (7). Since anti-Xa assays are widely available,they may be useful in emergency situations to de-tect the presence of a factor Xa inhibitor in the pa-tient's plasma, though they demonstrate variable sensitivity to FXa DOACs and will not provide a DOAC concentration (78). This assay is widely avail-able and may assist with DOAC detection (7).

Direct measurement

Direct DOAC assays measure the drug concen-tration usually in ng/mL (5, 7, 76, 77). For direct measurements, LC-MS/MS is considered the gold standard. Each assay is calibrated with each drug to be measured, and this method demonstrates good accuracy and precision over a broad concen-tration range, although it is not widely available(79). At this time, there are no international refer-ence materials available. Peak and trough mea-surements are preferred as they give more information than random measurements (80).

This method cannot be used in emergency settings.

Chromogenic anti-Xa assays that use the appro-priate direct FXa inhibitor calibrators and controls can be used to measure drug levels with a relative-ly short turnaround time compared to LC-MS/MS.They can be performed in as little as 30 min in some laboratories and may eliminate the need to use reversal agents or other hemostatic agents in patients with little or no anti-Xa activity(81).

Ecarin-based assays(clot based and chromo-genic) provide a direct measure of dibagtran ac-tivity. This assay is not readily available or useful in the absence of specific kits and standards (i.e.,standardization of the concentration of ecarin in the test). It has been demonstrated to have a good correlation with LC-MS/MS-based methods(5,7,76).

The chromogenic anti-Flla method is a quantita-tive measurement of dibagtran and other direct thrombin inhibitors and is based on the inhibition of a constant and defined quantity of thrombin.The method has good correlation with LC-MS/MS-based methods(7,82,83).The dilute TT using equal volumes of diluted patient plasma and thrombin with dibagtran calibrators is suitable for quantitative assessment of the anticoagulant effect of dibagtran(7, 76). A strong correlation between dilute TT and LC-MS/MS has been re-ported (84).

Summary

●There are indirect and direct methods that are available for measuring DOACs.

●The TT can be used to rule out dibagtran, and di-lute TT and ecarin-based assays can measure da bigatran concentrations.

●Heparin-calibrated anti-Xa assays can detect the presence of direct FXa inhibitors, but calibration with specific drugs is required to provide direct FXa inhibitor concentrations.

●LC-MS/MS is the gold standard for measurement of DOACs.

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HOW SHOULD DOAC CONCENTRATIONS BE INTERPRETED?

Many factors may affect DOAC concentrations,including drug-drug interactions, renal dysfunc-tion, prior bariatric surgery, concomitant food in-take in certain scenarios (e.g., therapeutic doses of rivaroxaban), and dose intensity. At the current time, standardized therapeutic ranges for DOACs have not been established. Peak and trough"on-therapy" plasma concentrations were ob-served in various pharmacokinetic and pharmaco-dynamic studies, but therapeutic reference ranges have not been established (85,86).

Given the lack of robust data that DOAC concentrates significantly change management decisions, guidelines by the International Society of Thrombosis and Haemostasis Scientific and Standardization Committee suggest not routinely monitoring DOACs concentration(87). However,there may be clinical scenarios where measuring DOAC concentrations could potentially be useful.For example, these guidelines suggest measuring a trough level when using DOACs after bariatric sur-gery(no sooner than 4 weeks postoperatively) to provide insight regarding drug absorption.Another scenario where measuring DOAC concen-trations might be helpful is in the setting of a major or life-threatening bleed. Prothrombin complex concentrates, andexanet alfa, and idarucizumab are available reversal agents for FXa inhibitors and dabigatran. International Society of Thrombosis and Haemostasis Scientific and Standardization Committee guidelines from 2016 and 2024 sug-gested that FXa inhibitor concentrations greater than 50 ng/mL in the setting of severe bleeding are likely sufficient to warrant anticoagulant rever-sal. The threshold can be lowered to 30 ng/mL in life-threatening bleeding situations(73,88).

Yet, even in these limited scenarios, the chal-lenge is that there are no clear optimal "cutoff"plasma concentrations for a trough level after

bariatric surgery or for using emergent reversal agents with serious bleeding.Additionally,few clin-ical laboratories offer rapid assays for DOAC con-centrations,further limiting the realistic utilization of a cutoff concentration for emergent reversal. Ultimately, appropriate DOAC reversal in the setting of major bleeding depends on mul-tiple factors, including timing of the most recent DOAC dose and dose intensity.

Summary

●There are currently no universal or standar-dized therapeutic ranges of DOAC plasma concentrations.

●DOAC plasma concentrations may be used in certain scenarios, including emergent anticoagu-lant reversal, but the optimal cutoff concentra-tion has yet to be determined.

CONCLUSION

The introduction of DOACs has brought major im-provements in anticoagulation management. The oral route of administration with fixed doses along with no requirement for routine therapeutic moni-toring has dramatically improved clinical care since the early 2010s. The now ubiquitous use of DOACs, however, poses challenges in understand-ing impacts on coagulation testing. This document explores these impacts to provide guidance to both the laboratory and clinical sides of patient care.

Clinicians ordering coagulation testing for pa-tients taking DOACs should approach these scen-arios thoughtfully, as this requires discretion to determine appropriate and optimal timing for testing. Communication and collaboration with the laboratory leadership and staff is strongly sug-gested prior to testing. The laboratory medical dir-ector can provide guidance to the clinical team on current methodologies and how to interpret re-sults for patients on DOACs.

Coagulation Testing and Direct Oral Anticoagulants

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Nonstandard Abbreviations:DOAC,direct oral anticoagulant;FXa, factor Xa; POC,point of care;PT,prothrombin time;apTT,activated partial thromboplastin time;PT,platelet function testing;LA,lopus anticoagulants;RVVT,Russell viper venom time;APCR, activated protein C resistance;TT, thrombin time.

Author Contributions: The corresponding author takes full responsibility that all authors on this publication have met the following required criteria of eligibility for authorship:(a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data;(b) drafting or revising the article for intellectual content;(c) final approval of the published article; and(d)agreement to be accountable for all aspects of the article thus ensuring that questions related to the accuracy or integrity of any part of the article are appropriately investigated and resolved. Nobody who qualifies for authorship has been omitted from the list.

Lindsay Bazydlo (Conceptualization-Equal, Writing-original draft-Equal, Writing-review & editing-Equal), Maximo Marin (Conceptualization-Equal, Writing-original draft-Equal, Writing-review & editing-Equal), Anna Merrill (Conceptualization-Equal,Writing-original draft-Equal, Writing-review & editing-Equal), Louise M. Man (Conceptualization-Equal, Writing-original draft-Equal, Writing-review & editing-Equal), Olajumoke Oladipo (Conceptualization-Equal, Writing-original draft-Equal, Writing-review & editing-Equal), and Neil Harris (Conceptualization-Equal, Writing-original draft-Equal, Writing-review & editing-Equal)

Authors' Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form.

Research Funding: None declared.

Disclosures: L.A.L. Bazydlote received an honorarium, travel support, and conference registration for presentation of this work at ADLM 2025. O.O. Oladipo received honoraria from Diagnostica Stago, Inc. A.E. Merrill has received consulting fees from Diagnostica Stago, Inc., an honorarium from ADLM, an honorarium from the Association for Molecular Pathology; is on an ad-visory board for Diagnostica Stago, Inc.; is on the editorial board for Annals of Laboratory Medicine; and is an associate editor for The Journal of Applied Laboratory Medicine, ADLM.

Role of Sponsor: No sponsor was declared.

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