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Direct Oral Anticoagulants: Laboratory Methods for Assessing Dabigatran

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Pearls of Laboratory Medicine are peer-reviewed presentations focused on a specific test or disease area relevant to contemporary laboratory medicine and pathology.

Welcome to this pearl of laboratory medicine brought to you by aacc and the clinical chemistry trainee council view this and many more pearls as well as other free educational material at trainee hi my name is dr dodd adcock i am currently the chief medical officer and a senior vice president of lab corp diagnostics welcome to this pearl of laboratory

Medicine on doax laboratory methods for assessing debigatron this program was created with bob goslin from the thrombosis and hemostasis center at the university of california davis health system this session is a joint effort between the american association for clinical chemistry and the north american specialized coagulation laboratory association i would like

To review the definition of a number of terms that will be used in this and following presentations venous thromboembolism or vte represent clots within the veins most commonly deep vein thrombosis dvt and pulmonary embolism pe pharmacokinetics is drug concentration after administration pharmacodynamics the drug effect after administration peak levels maximum drug

Concentration after administration trough levels the drug level just before the next dose and then on therapy range is a commonly used term to address doax as this class of drugs do not have therapeutic ranges an on therapy range reflects the expected drug concentration from the lowest trough to the highest peak value for a given dose an indication dabigatran

Which is administered as de bigatranitexylate brand name pradaxa is an oral direct thrombin inhibitor which is immediate acting with peak concentration one and a half to three hours after administration it inhibits both free and bound thrombin also known as activated factor 2. dabigatran is primarily excreted by the kidneys and has a half-life of about 13 hours

This table provides the expected mean peak and trough drug concentrations depending on the dose of debigatran administered note that the 25th to 75th percentile ranges are quite broad with overlap between the peak and trough ranges this cartoon of the coagulation cascade demonstrates the various targets for anticoagulant agents and depicts the laboratory

Testing pathways and assay targets the currency or dollar signs in the common pathway is a simple trick to remember those factors in this pathway and their order of reactions 10 5 2 and 1. factor 1 is also known as fibrinogen both direct 10a inhibitors and direct thrombin inhibitors can potentially cause prolongation of the ptt pt and rvvt as these drugs

Inhibit factors within these pathways as dabigatrin is a direct thrombin inhibitor the tests that may be affected by its presence include the pt ptt thrombin time ekren methods and other tests that utilize a thrombin substrate these data are from a study we performed soon after de bigatran was fda approved healthy volunteers were administered therapeutic doses

And peak levels were obtained plasma samples were evaluated with seven different commonly used ptt reagents and six pt reagents the red horizontal line depicts the upper limit of normal for most reagents and the vertical lines demonstrate the drug concentrations as measured by mass spectrometry as you can see response is reagent dependent with significantly more

Variability in pt reagents than ptt reagents i also want to point out that time in seconds for the ptt reagents plateaus as drug concentration increases finally it is important to note that both pt and ptt results can be normal when individuals have therapeutic concentrations of dabigatran in their blood normal pt and or ptt results therefore are not a reliable

Indicator of dabigatran presence the thrombin time is a simple test by combining an undiluted or slightly diluted plasma sample with the low concentration of thrombin reagent and measuring the time to clot formation as dabigatran is a direct inhibitor of thrombin a standard thrombin time is exquisitely sensitive to the presence of dabigatran such that very low

Drug concentrations far below on therapy range cause prolongation of the thrombin time with on therapy concentrations of dubigatran the thrombin clotting time is undetectable that is no clot detected and therefore this assay cannot be used to quantify the bigatran concentration the thrombin time is able to determine drug presence although prolongation of the

Thrombin time is not specific for dubigatran thromboendine prolongation can also be secondary to heparin parenteral direct thrombin inhibitors such as our gatraban and low or dysfunctional fibrinogen levels to recap dabigatran in a patient’s sample can be determined using the thrombin time although remember that prolongation of the thrombin time is not specific

For debigatron a normal pt and or aptt cannot exclude the presence of dabigatran we will now review the laboratory methods to quantify debigatron the gold method to quantify dabigatran is liquid chromatography tandem mass spectrometry lcm sms this is the only method to quantitate direct oral anticoagulant agents that is specific to the drug being measured for

Example if a patient is onto bigatran and heparin this assay will measure only the dabigatran that is present if the patient were on a different direct thrombin inhibitor such as argatrobin the argatroban would not be measured in a dubigatran mass spectrometry assay when using mass spectrometry active metabolites or conjugates of the drug must be considered as

These may contribute to overall anticoagulation but would not be measured in a debigatron mass spectrometry assay a conjugate of dabigatran dabigatran glucuronide adds about 20 anticoagulant activity but in alkaline hydrolysis sample pre-treatment splits the conjugate allowing measurement of total debigatron this assay has an excellent lower limit of detection

And measures drug over a broad range with good discrimination while lcm sms is the gold standard method to measure dubigatran the lack of an international reference for assay calibration can lead to variability between testing locations accuracy and precision however within a single testing location is quite good the dilute thrombin time can be used to quantitate

Debigatron if the assay is calibrated using a dabigatran calibrator results compare well to a mass spectrometry method currently there are no dabigatran calibrators that are fda cleared for ivd use all are for research use only an adequate lower limit of detection can be achieved if the sample is properly diluted ecrine is a thrombin like metalloprotease

From the venom of echus caronatus a saw scaled viper ekren converts prothrombin to mesothrombin mesothrombin is a potent thrombin intermediate that can be inhibited by dabigatran or other direct thrombin inhibitors such as by valorudin but not by heparin the classic acron method is a clot-based assay called the akron clotting time a chromogenic akron assay is

Now available and it has advantages over the clot-based method in that the chromogenic assay is not affected by low prothrombin or low fibrinogen levels both of which will falsely prolong the akron clotting time the chromogenic method demonstrates good accuracy and reproducibility this slide depicts the akron clotting time assay without on the left and with

Calibration on the right using a debigatron calibrator as compared to a mass spectrometry method the assay demonstrates a linear response to drug over a broad therapeutic range both the akron reagent and the dabigatran calibrator are labeled r-u-o one disadvantage of this assay is the lattilat variability that can occur with the akron reagent these are data

From the akron chromogenic assay calibrated with a dabigatran calibrator performed in three different laboratories as compared to a mass spectrometry method there is good agreement between testing locations with a linear response to drug over a broad drug concentration this assay is labeled r-u-o as well the chromogenic anti-2a method is exactly like the heparin

Anti-10 assay except the reagent is thrombin and not activated factor 10. the kid is labeled ru research use only the assay is easily automatable a dual curve is needed to measure drug concentration adequately over the on therapy range the assay performance regarding precision inaccuracy appears quite good although there is limit limited published data on the

Method in summary screening tests such as the pt and aptt are insufficient for assessing debigatran anticoagulation and cannot be used to exclude the presence of drug a normal thrombin time virtually excludes dabigatran presence mass spectrometry methods are considered the gold standard for measuring debigatron although there is no international reference for

Calibration alternative quantitative methods have been demonstrated to be equivalent to mass spectrometry including the drug calibrated dilute thrombin time akron clotting time and akron chromogenic assays more data is required for chromogenic anti-2a methods but they appear adequate alternative methods for quantifying debigatron can be adapted to open systems

That are programmable automated coagulation analyzers however there are no fda approved methods thank you for your attention for more like this as well as articles podcasts and more please visit the trainee council at

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Direct Oral Anticoagulants: Laboratory Methods for Assessing Dabigatran By AACC