David L. McGlasson, MS MLS(ASCP)

Posted: January 28, 2020

The Latest Research on Direct Oral Anticoagulant (DOAC) Interferences in Testing

Can DOACs be removed from plasma samples to prevent interference with plasma coagulation and chromogenic testing?

DOACs may affect almost all of the clotting coagulation methods results and some chromogenic assays.

In the last few years, a research product has been developed that can remove the interference of DOACs from plasma to delete the effect that these anticoagulants have on almost all of measurable assays that include: PT, APTT, one-stage factor assay and inhibitors that depend on either the PT and APTT methods. The results may vary depending on the reagent/instrument combinations and the concentration of the DOACs present.

Chromogenic factor assays that are based on FXa generation such as the FVIII, FIX and FX assays may be underestimated without any effect on the IIa inhibitors. However, the chromogenic FXIII levels in the presence of dabigatran may generate falsely low levels.1,2  Low levels of DOACs can also interfere with thrombophilia research, depending on the concentration, reagent/instrumentation methodology which may be used to detect lupus anticoagulants (LA) such as the DRVVT assay both low and high phospholipid concentrations.1,2  Chromogenic antithrombin assays based on both the FXa and FIIa generation can be overestimated depending on the anticoagulant concentrations as well as clot-based Protein C and S methods. Activated Protein C resistance methods may also be falsely elevated in the presence of any of the DOACs.4,5

The addition of DOAC-Stop (Haematex Research, Hornsby, Australia) to plasma has been shown in studies to remove the effects of DOACs to include dabigatran, apixaban, rivaroxaban and edoxaban from spiked and test samples. It was found to remove false positive results in lupus anticoagulants, APTT, DRVVT Screen and DRVVConfirm, PT/INR, Thrombin clotting time, Anti-FXa, Antithrombin, Protein C, Protein S, APC-R and Plasminogen.6-8

The testing was performed by adding one tablet of DOAC-Stop (Haematex) to 1.0 ml of citrated plasma. The specimen is gently mixed for 5 minutes and centrifuged for 2 minutes at 2,000 g to precipitate DOACs with the adsorbent tablets. The supernatant is then separated for analysis.

It was found to remove false positive results in lupus anticoagulants, APTT, DRVVT Screen and DRVV Confirm, PT/INR, Thrombin clotting time, Anti-FXa, Antithrombin, Protein C, Protein S, APC-R, Factor VIII (clottable and chromogenic), and Plasminogen.

The DOAC-Stop Procedure was also compared by determining the residual activity of dabigatran, rivaroxaban and apixaban against a LC-MS/MS assay. This study looked at LA testing by the DRVVT methods which are based on FXa activation.

The study used 60 test specimens (20 apixaban, 20 dabigatran, and 20 rivaroxaban). All of the plasmas were tested for the presence of the individual DOACs, and treated with the DOAC-Stop reagent. The LC-MS/MS method was then performed on all post treatment specimens. The DOAC-Stop procedure eliminated dabigatran from 99.5% rivaroxaban, 97.9% rivaroxaban and 97.1% apixaban. The residual amounts did not exceed 2.7 ng/mL dabigatran, 10.9 ng/mL rivaroxaban, 13.03 ng/mL apixaban. These levels were shown not to affect screening or special coagulation methods.9

Another protocol also examined the effect of DOAC-Stop on LA testing in subjects receiving DOACs. This setting used plasma specimens from 75 subjects receiving DOACs, which included 50 subjects on rivaroxaban, 20 on dabigatran and 5 on apixaban. LA testing was performed at baseline and post DOAC-Stop treatment. A positive LA was defined as the normalized (test/standard plasma clotting time) LA screening and screening (LA1)/confirmation (LA2) ratios exceeding 1.2.

LA diluted Russell’s viper venom time (DRVVT) normalized screening test revealed abnormal results in 73 (97.3%) and activated partial thromboplastin time (APTT)-LA in 49 (65.3%) subjects. In six (8%) subjects, antiphospholipid syndrome (APS) was found. DRVVT LA1/LA2 was abnormal in 35 (50.7%) subjects taking DOACs. The APTT ratio was normal in all studied subjects. DOAC-Stop completely removed dabigatran and reduced by 98% rivaroxaban and by 92.3% apixaban concentrations (all p<0.05). After DOAC-Stop screening DRVVT remained prolonged in 34 (49.3%) subjects (p<0.001), while DRVVT LA1/LA2 was abnormal in six (8.7%) subjects, with no association with DOAC concentrations at baseline and after DOAC-Stop. The APTT-LA screening test remained prolonged in five (7.2%) subjects, while the APTT LA1/LA2 ratio was normal in those subjects. DOAC-Stop did not influence LA testing in APS plasma. The authors concluded that application of DOAC-Stop effectively reduced plasma DOAC concentrations leading to appropriate DRVVT results in up to 97% of tested subjects.10

Another interesting study was conducted performing a comparison of 82 laboratories in determining if rivaroxaban (200 ng/mL) and andexanet alfa (200 μg/mL) interference in LA testing could be neutralized to prevent false positive/negative results. Four specimens were prepared in the following manner: (A) Pooled normal plasma (PNP) which served as the negative LA control; (B) the PNP specimen spiked with rivaroxaban in the concentration stated above; (C) specimen B treated with DOAC Stop; (D) sample B treated with andexanet alfa in the concentration stated above. These 4 specimens were sent to all of the laboratories participating in this study.

Specimen (B) caused prolongation of most LA assays and gave a falsely prolonged DRVVT screen/confirm ratio (median 1.37, compared to 0.97 for specimen (A) the negative control plasma. The sample (C) treated with DOAC Stop gave a correction in LA-test clotting times. It also neutralized the false positive LA (median DRVVT screen/confirm ratio of 0.99). The andexanet alfa treated sample (D) gave a low median DRVVT screen/confirm ratio of 0.88 but it failed to completely correct LA-test clotting times. All of the participating institutions interpreted samples A and C as being LA-negative. For sample B (rivaroxaban), 45.3% identified it as an LA-positive and 38.7% stated they had LA interference. Most of the labs (61.3%) also identified sample D as LA negative with the rest (38%) as displaying LA interference.

Their conclusions showed that DOAC-Stop could neutralize the false LA results induced by rivaroxaban in terms of the clotting times and the LA ratios. Andexanet alfa neutralized the prolonged LA ratio. However, it did not fully correct clotting times which still left some LA interference. This caused further testing to identify the cause of the lengthy clotting times.11

DOACs can also cause problems with a variety of global hemostasis evaluation methods. In this protocol investigators used the DOAC-Stop to see if the reagent can be used to eliminate anti-FXa and anti-IIa drugs such as rivaroxaban, apixaban, edoxaban and dabigatran in spiked plasma samples.

Thrombin generation tests have been used to research anticoagulant effects of DOACs. In this protocol, the authors examined if there is added value of DOAC-Stop in thrombin generation tests. All specimens were evaluated using the thrombin generation test with 5 pM tissue factor. The authors found that DOAC-Stop effectively removed DOACs from plasma, leaving the DOAC-Stop treated plasma slightly more procoagulant compared to sham treated, non-anticoagulated plasma.

Examining levels of natural coagulation inhibitors revealed a slight reduction in tissue factor pathway inhibitor upon DOAC-Stop treatment. When DOAC-Stop treated plasma was used in the calibrator wells, normal unaffected calibration curves were observed, even when dabigatran was present. These research studies indicate the DOAC-Stop reagent gives the laboratory a way to test subjects on DOACs without the interferences they can cause on the above methods. This could be beneficial in anticoagulant research and development.12

Is there a rapid test (POCT) available for detection of DOACs? DOAC Dipstick (DOASENSE GMbH)

Recently in a series of publications, authors have described a rapid test on urine for detecting anti-FIIa and anti-FXa DOACs using a dipstick test that only takes 10 minutes.13-14

DOACs are excreted in variable amounts in the urine, but without the presence of coagulation factors and inhibitors (specific and non-specific) that may interfere with the plasma coagulation testing. Most of the DOACs are excreted into the urine (25-80%), but without the presence of coagulation factors and inhibitors (specific and non-specific) that may interfere with the plasma coagulation testing. Betrixiban is the exception, with <7% being cleared by renal function. Therefore, creatinine levels can be run in parallel with the DOAC measurement. Note: because of the low renal clearance of Betrixiban this assay may not be able to pick up the presence of this DOAC. Information is needed to verify this observation.

The test is simple. The DOAC Dipstick is dipped in the urine sample for 2-3 seconds and then allowed to dry for 10 minutes and observe for a color change.

The color changes can be visually observed to determine the positive or negative results. These color changes are available in the testing package insert. An abnormal color of the urine specimen could interfere with the color determination to detect the presence of DOACs.

direct oral anticoagulant doac

In a recent publication, the accuracy of the DOASENSE was investigated in a Urine -A Multicenter Trial. The researchers aimed to show that the method demonstrated at least 95% positive and negative correct results for II-a and anti-Xa inhibitors that may or not be present in the urine of tested specimens.

The study was performed using the DOASENSE pads. There were 914 subjects included in the study, and the results were compared against a liquid chromatography-tandem mass spectrometry (LC-MS/MS) (NCT03182829) method. Of those, 880 were assessed in the protocol. They included the anti-FXa inhibitors apixaban, edoxaban and rivaroxaban (n=451), and anti-IIa inhibitor dabigatran (n=429) at 18 centers.

The results for sensitivity, accuracy, predictive values and agreement between the two methods for anti-FXa inhibitors were noninferior to 95% with a 0.5% margin and of the anti-IIa inhibitor superior to 97.5%. The comparison to the LC-MS/MS results in the analyzed groups were all showing a p value of <0.05. The receiver operating curve showed c-values of 0.989 with the anti-Xa inhibitors and 0.995 with the anti FII-a inhibitor. There was no difference in the visual evaluations of the dipstick pads between the study centers.

The qualitative determinations of both types of DOACS were accurate using the DOASENSE dipsticks in comparison with the LC-MS/MS.15

*Depending on the vendor and the country the product may be named under the DOAC Dipstick or the DOASENSE.

These products are for research use only in the U.S. and Canada. Not for diagnostic purposes.

  1. Gosselin RC, Adcock DM, Douxfils J. an update on laboratory assessment for direct oral anticoagulants (DOACs). Int J Lab Hematol. 2019;41(Suppl1)33-39.
  2. Platton S, Hunt C. Influence of DOAC Stop on coagulation assays in samples from patients on rivaroxaban or apixaban. Int J Lab Hematol 2018. DOI 10.111/ijlh 12950.
  3. Gessoni G, Valverde S, Valle L et al: effect of dabigatran on prothrombinase-based assay for detecting activated protein C resistance: an ex vivo and in vitro study in normal subjects and factor V Leiden carriers. Blood Transfus 2017;15:563-568
  4. Gosselin RC, Grant RP, Adcock DM. Comparison of the effect of the anti-Xa direct oral anticoagulants apixaban, edoxaban and rivaroxaban on coagulation assays. Int J Lab Hematol. 2016;38(5):505-513.
  5. Favresse J et al: Evaluation of the DOAC-Stop Procedure to Overcome the Effect of DOACs on Several Thrombophilia Screening Tests TH Open 2018:2e202-e209.
  6. Exner T et al: Simple method for removing DOACs from plasma samples. Thromb Res. 2018 Mar;163:117-122.
  7. Platton S, Hunt C. Influence of DOAC Stop on coagulation assays in samples from patients on rivaroxaban or apixaban. Int J lab Hematol 2018. 41 (2), 227-233.
  8. Favresse J et al: Evaluation of the DOAC-Stop Procedure to Overcome the Effect of DOACs on Several Thrombophilia Screening Tests. TH Open 2018. PMID 31249943 Free PMC article.
  9. Slavik L et al: Evaluation of the DOAC-Stop Procedure by LC-MS/MS Assays for Determining the Residual Activity of Dabigatran, Rivaroxaban, and Apixaban. Clin Appl Thromb Hemost 2019. PMID 31523979 Free PMC article.
  10. Zabczyk M, Kopytek M, Natorska J, Umndas A. the effect of DOAC-Stop on lupus anticoagulant testing in plasma samples of venous thromboembolism patients receiving direct oral anticoagulants. Clin Chem Lab Med 2019.;57(9):1374-1381.
  11. Favaloro EJ et al: Neutralising rivaroxaban induced interference in laboratory testing for lupus anticoagulant (LA): A comparative study using DOAC Stop and andexanet alfa. Thromb Res. 2019;180:10-19.
  12. Kopatz WF, brinkman HJM, Meijers JCM. Use of DOAC Stop for elimination of anticoagulants in the thrombin generation assay. Thromb Res. 2018;170:97-101.
  13. Harenberg J et al: Detecting Anti-IIa and Anti-Xa Direct Oral Anticoagulant (DOAC) Agents in Urine using a DOAC Dipstick. Semin Thromb Hemost 2018 DOI 10.1055/s-0038-1668098.
  14. Harenberg J, Schreiner R, Hetjens S, Weiss C. Detecting anti-IIA and Anti-Xa Direct Oral Anticoagulants (DOAC) Agents in Urine using a DOAC Dipstick. Semin Thromb Hemost 2019. Apr;45(3):275-284.
  15. Harenberg J et al: Accuracy of a Rapid Diagnostic Test for the Presence of Direct Oral Factor Xa of Thrombin Inhibitors in Urine-A Multicenter Trial. Thromb Haemost. 2019 Nov *. Doi: 10.1005/s-0039-1700545 [Epub ahead of print].