Report of the Subcommittee on Control of Anticoagulation on the determination of the anticoagulant effects of apixaban: communication from the SSC of the ISTH

Abstract

The oral direct factor Xa (FXa) inhibitor apixaban is approved for the prevention of ischemic stroke in patients with atrial fibrillation 1.Granger C.B. Alexander J.H. McMurray J.J. Lopes R.D. Hylek E.M. Hanna M. Al‐Khalidi H.R. Ansell J. Atar D. Avezum A. Bahit M.C. Diaz R. Easton J.D. Ezekowitz J.A. Flaker G. Garcia D. Geraldes M. Gersh B.J. Golitsyn S. Goto S. et al.Apixaban versus warfarin in patients with atrial fibrillation.N Engl J Med. 2011; 365: 981-92Crossref PubMed Scopus (6639) Google Scholar and for prophylaxis of postoperative venous thromboembolism after elective knee or hip replacement surgery 2.Raskob G.E. Gallus A.S. Pineo G.F. Chen D. Ramirez L.M. Wright R.T. Lassen M.R. Apixaban versus enoxaparin for thromboprophylaxis after hip or knee replacement: pooled analysis of major venous thromboembolism and bleeding in 8464 patients from the ADVANCE‐2 and ADVANCE‐3 trials.J Bone Joint Surg Br. 2012; 94: 257-64Crossref PubMed Scopus (83) Google Scholar. Studies have shown efficacy and safety for patients with acute deep vein thrombosis compared with initial subcutaneous low molecular weight heparin followed by laboratory adjusted warfarin 3.Agnelli G. Buller H.R. Cohen A. Curto M. Gallus A.S. Johnson M. Oral apixaban for the treatment of acute venous thromboembolism.N Engl J Med. 2013; 369: 799-808Crossref PubMed Scopus (1648) Google Scholar and for prolonged prophylaxis of recurrent events comparing warfarin, adjusted to an INR of 2 to 3 with placebo 4.Agnelli G. Buller H.R. Cohen A. Curto M. Gallus A.S. Johnson M. Porcari A. Raskob G.E. Weitz J.I. Apixaban for extended treatment of venous thromboembolism.N Engl J Med. 2013; 368: 699-708Crossref PubMed Scopus (1018) Google Scholar. Therapy with apixaban is given at fixed daily doses without laboratory‐guided adjustment. No study has proved the benefit of dose adjustment on clinical outcome 5.Ten Cate H. New oral anticoagulants: discussion on monitoring and adherence should start now!.Thromb J. 2013; 11: 8https://doi.org/10.1186/1477‐9560‐11‐8Crossref PubMed Scopus (0) Google Scholar. However, the anticoagulant effect may need to be measured in some patient populations such as before surgery, in patients with deterioration of renal function, during bleeding or thrombotic episodes, and to assess adherence to therapy 6.Harenberg J. Marx S. Erdle S. Krämer R. Determination of the anticoagulant effects of new oral anticoagulants: an unmet need.Expert Rev Hematol. 2012; 5: 107-13Crossref PubMed Scopus (32) Google Scholar, 7.Samama M.M. Amiral J. Guinet C. Le Flem L. Seghatchian J. Monitoring plasma levels of factor Xa inhibitors: how, why and when?.Expert Rev Hematol. 2013; 6: 155-64Crossref PubMed Scopus (36) Google Scholar, 8.Baglin T. The role of the laboratory in treatment with new oral anticoagulants.J Thromb Haemost. 2013; 11: 122-8Crossref PubMed Scopus (67) Google Scholar. Apixaban variably prolongs clotting times of several clotting assays 9.Douxfils J. Chatelain C. Chatelain B. Dogne J.M. Mullier F. Impact of apixaban on routine and specific coagulation assays: a practical laboratory guide.Thromb Haemost. 2013; 110: 283-94Crossref PubMed Scopus (169) Google Scholar, 10.Gouin‐Thibault I. Flaujac C. Delavenne X. Quenet S. Horellou M.H. Laporte S. Siguret V. Lecompte T. Assessment of apixaban plasma levels by laboratory tests: suitability of three anti‐Xa assays. A multicentre French GEHT study.Thromb Haemost. 2014; 111: 240-8Crossref PubMed Scopus (106) Google Scholar and inhibits FXa in specific chromogenic substrate assays 11.Barrett Y.C. Wang Z. Frost C. Shenker A. Clinical laboratory measurement of direct factor Xa inhibitors: anti‐Xa assay is preferable to prothrombin time assay.Thromb Haemost. 2010; 104: 1263-71Crossref PubMed Scopus (318) Google Scholar, 12.Samama M.M. Guinet C. Laboratory assessment of new anticoagulants.Clin Chem Lab Med. 2011; 49: 761-72Crossref PubMed Scopus (155) Google Scholar, 13.Frost C. Wang J. Nepal S. Schuster A. Barrett Y.C. Mosqueda‐Garcia R. Reeves R.A. LaCreta F. Apixaban, an oral, direct factor Xa inhibitor: single dose safety, pharmacokinetics, pharmacodynamics and food effect in healthy subjects.Br J Clin Pharmacol. 2013; 75: 476-87Crossref PubMed Scopus (268) Google Scholar. The variances of clotting and chromogenic methods have been determined in a previous international collaborative study using plasma samples spiked with rivaroxaban 14.Harenberg J. Marx S. Weiss C. Krämer R. Samama M. Schulman S. Subcommittee on control of anticoagulation of the ISTH. Report of the subcommittee of control of anticoagulation on the determination of the anticoagulant effects of rivaroxaban.J Thromb Haemost. 2012; 10: 1433-6Crossref PubMed Scopus (55) Google Scholar. However, anticoagulant effects of apixaban differ from those of rivaroxaban 12.Samama M.M. Guinet C. Laboratory assessment of new anticoagulants.Clin Chem Lab Med. 2011; 49: 761-72Crossref PubMed Scopus (155) Google Scholar. Direct oral FXa inhibitors are defined chemical agents with individual pharmacology, anticoagulant effects, and clinical development programs 15.Harder S. Graff J. Novel oral anticoagulants: clinical pharmacology, indications and practical considerations.Eur J Clin Pharmacol. 2013; 69: 1617-33Crossref PubMed Scopus (82) Google Scholar.Therefore, standardization of assays is required to determine precisely the effect of every individual oral direct FXa inhibitor. The Subcommittee on Control of Anticoagulation of the International Society on Thrombosis and Haemostasis (ISTH) elected to perform a collaborative study to analyze the anticoagulant effects specifically for apixaban on several coagulation tests using human pooled plasma samples. Special attention was given to reduce the error variances between assays using adequate control samples and validated application procedures from the manufacturers and analyzing the data for individual coagulation platforms. Platelet‐poor plasma (PPP) was prepared from blood collected in 3.2% citrate (blood/citrate, v/v, 9/1) at Diagnostica Stago (Asnières sur Seine, France) 10.Gouin‐Thibault I. Flaujac C. Delavenne X. Quenet S. Horellou M.H. Laporte S. Siguret V. Lecompte T. Assessment of apixaban plasma levels by laboratory tests: suitability of three anti‐Xa assays. A multicentre French GEHT study.Thromb Haemost. 2014; 111: 240-8Crossref PubMed Scopus (106) Google Scholar, 14.Harenberg J. Marx S. Weiss C. Krämer R. Samama M. Schulman S. Subcommittee on control of anticoagulation of the ISTH. Report of the subcommittee of control of anticoagulation on the determination of the anticoagulant effects of rivaroxaban.J Thromb Haemost. 2012; 10: 1433-6Crossref PubMed Scopus (55) Google Scholar. Apixaban was kindly provided by Bristol‐Myers Squibb (Plainsboro, NJ, USA). Study material consisted of four PPP samples with known concentrations of 0, 45, 235, and 440 ng mL−1 apixaban as reference and five samples (A, B, C, D, E) with blinded concentrations of 0, 30, 60, 100, and 330 ng mL−1. All plasma samples were freeze dried in sealed glass vials. All materials were kindly prepared by Diagnostica Stago. Twelve laboratories participated in the study (Supplementary Material). Four laboratories used Stago instrumentation, 4 ACL instrumentation, 3 Sysmex CS7000 coagulation platform, and one microtiterplate method for the chromogenic assays and the KC10 instrument for the coagulation assays. The thromboplastin reagent RecombiPlasTin 2G (Instrumentation Laboratory, Kirchheim, Germany) was selected because of its relatively good sensitivity to apixaban 9.Douxfils J. Chatelain C. Chatelain B. Dogne J.M. Mullier F. Impact of apixaban on routine and specific coagulation assays: a practical laboratory guide.Thromb Haemost. 2013; 110: 283-94Crossref PubMed Scopus (169) Google Scholar, and Technoclot (Technoclone, Vienna, Austria) was chosen as a new reagent. The prothrombin‐induced clotting time (PiCT) assay was performed using the reversed two‐stage clotting procedure (Pentapharm, Basel, Switzerland) 12.Samama M.M. Guinet C. Laboratory assessment of new anticoagulants.Clin Chem Lab Med. 2011; 49: 761-72Crossref PubMed Scopus (155) Google Scholar. Assays were performed as described by the manufacturer. Five chromogenic substrate assays were included in the study: STA Liquid Anti‐Xa (Diagnostica Stago), STA Rotachrom (Diagnostica Stago), Coamatic Heparin (Haemochrom, Essen, Germany), HemosIL Anti‐Xa liquid (Instrumentation Laboratories, Kirchheim, Germany), and Technochrome Anti‐Xa (Technoclone, Vienna, Austria). Assays were performed as described by the manufacturer for each instrument platform by the study participants. All assays were performed on all samples on four different days as duplicates 14.Harenberg J. Marx S. Weiss C. Krämer R. Samama M. Schulman S. Subcommittee on control of anticoagulation of the ISTH. Report of the subcommittee of control of anticoagulation on the determination of the anticoagulant effects of rivaroxaban.J Thromb Haemost. 2012; 10: 1433-6Crossref PubMed Scopus (55) Google Scholar. Every day, freshly reconstituted samples and reagents were prepared. The results of the analyses on the samples with the known concentrations of apixaban were plotted versus time (coagulation assays) or optical density (chromogenic assays) to calculate the concentration of apixaban in the five blinded samples. The ratios of the coagulation data were calculated centrally (CG) for the samples with known concentrations of apixaban. The mean, median, standard deviation (SD), coefficient of variance (CV), minimum (min), and maximum (max) values were calculated from the data (SAS release 9.3 program; SAS Institute, Cary, NJ, USA). Differences in the CV for each assay compared across the sites were analyzed (Maloney‐Rastogi test) 14.Harenberg J. Marx S. Weiss C. Krämer R. Samama M. Schulman S. Subcommittee on control of anticoagulation of the ISTH. Report of the subcommittee of control of anticoagulation on the determination of the anticoagulant effects of rivaroxaban.J Thromb Haemost. 2012; 10: 1433-6Crossref PubMed Scopus (55) Google Scholar, 16.Maloney C.J. Significance tests for Grubb's estimators.Biometrics. 1970; 26: 671-5Crossref Google Scholar. The level of significance was set at P<0.01. A subanalysis was determined for assays performed on nonadapted systems versus a coagulation platform on which the assays were specifically adapted (i.e., chromogenic assays adapted on coagulation instrumentation [Stago and ACL]). The ratios of the coagulation assays for the prothrombin time (PT) assays using RecombiPlasTin 2G and Technoclot and of the PiCT were plotted against known plasma concentrations of apixaban (Fig. S1, Supplementary Material). The mean, median, SD, CV, min, and max values of samples with unknown concentrations (blinded samples) are summarized in Table S1 using the sum of all values and all methods provided by participants. The relation between the error variance of the methods and the concentration of apixaban in the blinded samples are shown in Fig. 1. Statistical analysis did not indicate differences in the variances across the assays (P=0.3403) and across the slopes of each method versus the concentrations of apixaban (P=0.3615). The results of the determination of the concentration of apixaban in the five blinded samples by individual assays are given in Tables S2–S6 (Supplementary Material). The P‐values are included in these tables for the analysis for differences in the error variance between two assays. In a subanalysis, the CVs of the chromogenic tests were lower when comparing data for assays with validated versus without validated applications on the Stago (Table S7) and ACL coagulation platforms (Table S8). The results of the interlaboratory comparison show decreasing CVs across the methods for increasing concentrations of apixaban from 30 to 330 ng mL−1. Low concentrations of 30–60 ng mL−1 are expected as trough levels during lower dose treatment of apixaban and levels of 100 ng mL−1 and higher as peak levels during therapy 12.Samama M.M. Guinet C. Laboratory assessment of new anticoagulants.Clin Chem Lab Med. 2011; 49: 761-72Crossref PubMed Scopus (155) Google Scholar, 13.Frost C. Wang J. Nepal S. Schuster A. Barrett Y.C. Mosqueda‐Garcia R. Reeves R.A. LaCreta F. Apixaban, an oral, direct factor Xa inhibitor: single dose safety, pharmacokinetics, pharmacodynamics and food effect in healthy subjects.Br J Clin Pharmacol. 2013; 75: 476-87Crossref PubMed Scopus (268) Google Scholar, 17.Spyropoulos A.C. Douketis J.D. Gerotziafas G. Kaatz S. Ortel T.L. Schulman S. Periprocedural antithrombotic and bridging therapy: recommendations for standardized reporting in patients with arterial indications for chronic oral anticoagulant therapy.J Thromb Haemost. 2012; 10: 692-4Crossref PubMed Scopus (66) Google Scholar, 18.Pernod G. Albaladejo P. Godier A. Samama C.M. Susen S. Gruel Y. Blais N. Fontana P. Cohen A. Llau J.V. Rosencher N. Schved J.F. de Maistre E. Samama M.M. Mismetti P. Sié P. Management of major bleeding complications and emergency surgery in patients on long‐term treatment with direct oral anticoagulants, thrombin or factor‐Xa inhibitors: proposals of the working Group on Perioperative Haemostasis (GIHP) – March 2013.Arch Cardiovasc Dis. 2013; 106: 382-93Crossref PubMed Scopus (229) Google Scholar. No variance in the amount of apixaban detected by the different methods was observed across the concentrations of apixaban studied. This indicates that sensitive PT assays, PiCT, and chromogenic methods used in the study will determine with a similar variation the concentration of apixaban (up to 440 ng mL−1) added to human plasma samples in vitro.