Consultative Coagulation How to Effectively Answer Common Questions About Hemostasis Testing Session #5020

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1 Consultative Coagulation How to Effectively Answer Common Questions About Hemostasis Testing Session #5020 Dorothy M. (Adcock) Funk, M.D. Colorado Coagulation Karen A. Moser, M.D. Saint Louis University School of Medicine October 9, 2014

2 Disclosures Dr. (Adcock) Funk and Dr. Moser: In the past 12 months, I have not had a significant financial interest or other relationship with the manufacturer(s) of the product(s) or provider(s) of the service(s) that will be discussed in my presentation.

3 Topic Overview Direct Oral Anticoagulants Effects on coagulation assays Mixing Tests (Mixing Studies) Use and pitfalls Case examples will be provided during both course sections

4 Direct Oral Anticoagulant Agents Brief Overview of Effects on Coagulation Assays

5 New Oral Anticoagulant Agents Direct Thrombin Inhibitors Dabigatran Trade Name: Pradaxa Direct Xa Inhibitors Rivaroxaban Trade Name: Xarelto Apixaban Trade Name: Eliquis Edoxaban Not FDA approved as of yet Pradaxa is a registered trademark of Boehringer Ingelheim Pharma GmbH & Co. Xarelto is a registered trademark of Bayer Aktiengesellschaft. Eliquis is a registered trademark of Bristol-Myers Squibb Company.

6 Coagulation Cascade: In vitro model APTT APTT FXII FXI FIX FVIII FVII PT PT Extrinsic pathway DXa Intrinsic pathway FII FV FXa Thrombin FIIa DTI Fibrinogen TCT Fibrin

7 Dabigatran Effect on APTT and PT On therapy range Upper limit of normal range *Hawes E, Deal A, Jeanneret, et al. J Thromb Haemost. 2013;11:

8 Dabigatran TT Response Thrombin Time (s) Dabigatran measured by LC-MS/MS ng/ml *Hawes E, Deal A, Jeanneret, et al. J Thromb Haemost 2013;11:

9 Rivaroxaban Effect on APTT and PT On therapy range Upper limit normal range Francart S, Hawes E, Deal A, et al. Thromb Haemost 2014 doi /TH

10 Dabigatran and Rivaroxaban (Apixaban) Effect on Coagulation Assays Drugs act as inhibitors in the laboratory: Incomplete correction with 1:1 plasma mix Non specific inhibitor effect in factor assays Can cause a false positive Bethesda assay False positive Lupus Anticoagulant Assays Falsely elevated (normal) APCR, protein C clot-based activity and protein S activity and possibly antithrombin activity (depending on drug and method) Dabigatran: Falsely low FXIII activity No effect on: D-dimer, VWF assays, free protein S antigen, chromogenic protein C activity, reptilase time

11 Effect of Dabigatran on Factor Assays Factor Activity in IU/dL FVIII FIX FXI Intrinsic Factors APTT-Based Extrinsic Factors PT-Based * Adcock DM, et al. AJCP. 2013;139:

12 Dabigatran and Rivaroxaban Effect on Coagulation Assays % change compared to Reference Plasma Dabigatran Rivaroxaban AT assays either Laboratory IIa Xa based Assays PC and PS assays: clot-based * Adcock DM, et al. AJCP. 2013;139: ; ECAT Proficiency Data 2012.

13 Case # 1 33 y/o woman suffers a post-partum DVT and is treated with rivaroxaban. Her physician orders a thrombophilia work-up; patient has the lab work drawn 2 days after starting tx. The following assays were performed: Protein S activity (clot based): 65% (63 140%) Protein C activity (chromogenic): 72% (55 140%) APCR ratio: 2.2 ( ) drvvt confirm is positive (ratio 1.3) with negative Staclot LA and negative acl and B2GP1 IgG and IgM Staclot is a registered trademark of Diagnostica Stago.

14 Case # 1 The patient s clinician calls you to discuss the results and you respond: - PS activity may be falsely (elevated or decreased) on rivaroxaban? - PC chromogenic activity may be falsely (elevated or decreased) on rivaroxaban? - APCR ratio may be falsely (elevated or decreased) on rivaroxaban? - LA assay results may be falsely (positive or negative) on rivaroxaban?

15 Case # 2 82 year old man with atrial fibrillation is on dabigatran and presents to the ED with bleeding and an elevated APTT and PT. He is in acute renal failure. A FVIII activity with Bethesda titer is ordered: - Factor VIII Activity: 8% (50-150%) - Bethesda Titer: 1.2 BU (<0.8 BU) What do you tell the clinician?

16 Mixing Tests (Mixing Studies) Use and Pitfalls

17 Mixing Tests- Overview What are plasma mixing tests (mixing studies)? How are they performed? In what situations are they useful? How are they interpreted? Practical examples

18 Plasma Mixing Test Used in the evaluation of a prolonged APTT (most commonly) and/or PT Screen for determining whether prolongation is due to a factor deficiency or inhibitor Can be performed with other assays e.g. DRVVT, VWF activity, FXIII activity Performance and interpretation varies Very few published guidelines or standards The ART of coagulation testing!

19 Coagulation Cascade: In vitro model FXII FXI FIX APTT Intrinsic pathway FVIII FII FV FX FVII PT Extrinsic pathway Thrombin Fibrinogen TCT Fibrin

20 Mixing Tests (APTT or PT) Normal Plasma Mixing Test Results 1. Corrects 50% + 50% 1:1 2. Fails to Correct Patient APTT or PT Normal Pooled Plasma Perform APTT or PT on Mixture **Principle- at least ~50% factor is present in the mixture, which is adequate to correct APTT or PT in cases of factor deficiency

21 Mixing Tests (APTT or PT) Correction = Factor Deficiency If immediate correction observed, rule out time/temperature dependent inhibitor No/partial correction or prolongation with incubation = Factor Inhibitor

22 Mixing Tests- Technical Variables Source of normal plasma Ratio of patient plasma to normal plasma 1:1 or 4:1 (for a weak inhibitor) Method to determine time/temperature dependence Criteria for correction Based on specific value versus formula

23 Source of Normal Plasma (NP) Pooled (n > 20) normal plasmas, fresh or frozen Do not use plasma from previously normal APTT or PT sample Lyophilized plasmas generally not recommended Platelet poor (< 5-10X 10 9 /L) Well characterized Known factor activities (close to 100% of all factors) FVIII can cause false negative study Should be screened and LA negative

24 Incubated Mixing Test 50% 50% Incubate each at 37º C for 60 to 120 minutes, then mix patient with NP Patient NP Patient + NP Compare Incubated Mix to Control Separately incubated patient + NP Mix (Control) Patient + NP incubated together (Incubated Mix)

25 Mixing Test Results What is Correction?

26 Definitions of Correction No consensus or standard definition Correction in relation to reference interval Upper limit of 2 SD or 3 SD Upper limit + 5 seconds Correction in relation to normal pooled plasma (NP) NP + 5 seconds NP plus 10% Correction in relationship to mean normal clotting time Mean normal clotting time + 2SD or +3SD Rosner index of <15* Percent correction* Other laboratory-defined criteria * Index and % correction cutoff must be established by each laboratory

27 Definitions of Correction, cont. Correction in relation to reference range or normal pool Weak inhibitor with minimal prolongation of APTT or PT may correct into normal range with mixing Factor deficiency with prolonged PT may not correct to normal with mix May result from PIVKA interference Calculations (% correction or Rosner index) Cut-offs are reagent and instrument specific Each laboratory must determine own cut-offs

28 Published Calculations for Mixing Study Correction Determination Percent Correction Chang SH, Tillema V, Scherr D. Am J Clin Pathol. 2002; 117: Rosner Index Rosner E, Pauzner R, Lusky A, et al. Thromb Haemost. 1987; 57(2):

29 Percent Correction Am J Clin Pathol. 2002;117: Percent Correction = PP APTT 1:1 Mix APTT PP APTT CNP APTT X 100 PP patient plasma; CNP citrated normal plasma APTT* >75% correction - Factor deficiency < 58% correction Inhibitor PT* > 75% correction Factor deficiency < 70% correction - Inhibitor *Percent Correction cutoff should be verified by each laboratory

30 Rosner Index Thromb Haemost. 1987; 57(2): Index = B C A X 100 A = Clotting Time of Patient B = Clotting Time 1:1 Mixing Test C = Clotting Time of normal pooled plasma (NP) High index (15 or greater) suggests inhibitor* Low index suggests factor deficiency* * Index cut-off must be established by each laboratory

31 What about minimal prolongation? 3-5 s prolonged clotting time Mixing tests are difficult to interpret Are mixing tests indicated? 4:1 APTT plasma mix may be useful Weak inhibitor may correct into the normal range

32 Follow Up Prolonged clotting time (PT and/or APTT) Mixing test (PT and/or APTT, depending on initially prolonged clotting time) Corrects Does Not Correct Factor deficiency more likely Appropriate factor activity assays Correlation with all available clinical and laboratory information is essential Inhibitor more likely Lupus anticoagulant and/or specific factor inhibitor assays Adapted from Semin Thromb Haemost. 2013; 39:

33 Time to Practice!

34 Case 3 Test Result (s) Reference Interval PT APTT APTT 1:1 mix APTT 1:1 incubated mix APTT 1:1 incubated control 35.2 NA 35.5 NA TT 11.0 <20 What are the abnormalities? What is your differential diagnosis? What additional testing is indicated?

35 Case 4 Test Specific factor inhibitor Result (s) Reference Interval PT APTT APTT 1:1 mix APTT 1:1 incubated mix 70.2 NA APTT 1:1 incubated control 68.4 NA TT 13.6 <20 What are the abnormalities? What is your differential diagnosis? What additional testing is indicated?

36 Case 5 Test Result (s) Reference Interval APTT APTT 1:1 mix APTT 1:1 incubated mix 44.3 APTT 1:1 incubated control NA 36.4 NA TT 9.0 <20 What are the abnormalities? What is your differential diagnosis? What additional testing is indicated?

37 Case 6 Test Result (s) Reference Interval PT APTT APTT 1:1 mix APTT 1:1 incubated mix 54.6 APTT 1:1 incubated control NA 50.4 NA TT 13.5 <20 What are the abnormalities? What is your differential diagnosis? What additional testing is indicated?

38 Case 7 Test Result Reference Interval APTT APTT 1:1 mix TT >150 <20 APTT (heparin neutralized) TT (heparin neutralized) >150 <20 What are the abnormalities? What is your differential diagnosis? What additional testing is indicated?

39 Conclusion- Mixing Tests Mixing tests can be a useful screening tool to distinguish the presence of a factor inhibitor from factor deficiency Must be performed and interpreted with caution Guidelines and standards are needed

40 References Direct Oral Anticoagulants Adcock DM. Coagulation assays and anticoagulant monitoring. Hematology American Society of Hematology Education Program Book: Adcock DM, Gosselin B, Kitchen S, Dwyre D. The Effect of Dabigatran on Select Specialty Coagulation Assays. Am J Clin Pathol. 2013;139: Hawes EM, Deal AM, Adcock D, Gosselin R, Jeanneret C, Cook AM, Taylor JM, Whinna HC, Winkler AM, Moll S. Performance of coagulation tests in patients on therapeutic doses of dabigatran: a cross-sectional pharmacodynamics study based on peak and trough levels. J Thromb Haemost 2013;11: Francart S, Hawes E, Deal A, Adcock D, Gosselin R, Jeanneret C, Friedman K, Moll S. Performance of coagulation tests in patients on therapeutic doses of rivaroxaban. A cross-sectional pharmacodynamic study based on peak and trough plasma levels. Thromb Haem In press. Gosselin R, Hawes E, Moll S, Adcock D. Performance of various laboratory assays in the measurement of dabigatran in patients receiving therapeutic doses. A prospective study based on peak and trough levels. Am J Clin Pathol 2014;141:

41 References- Mixing Tests Chang S, Tillema V, Scherr D. A percent correction formula for evaluation of mixing studies. Am J Clin Pathol. 2002; 117: Clinical and Laboratory Standards Institute (CLSI). One stage prothrombin time (PT) test and activated partial thromboplastin time (APTT) test; approved guideline- second edition. CLSI document H47-A2. Wayne, PA: CLSI, Kershaw G, Orellena D. Mixing tests: diagnostic aides in the investigation of prolonged PT and APTT. Semin Thromb Haemost. 2013; 39: Krishnan J. Coagulation testing. In: Kottke-Marchant K, ed. An Algorithmic Approach to Hemostasis Testing. Northfield, IL: CAP Press; 2008: Ledford-Kraemer M. All mixed up about mixing studies. In: The Clotting Times: The Official Newsletter of CLOT-Ed, Inc. 2004; 3(4):1-11. Pengo V, Tripodi A, Reber G, et al. Update of the guidelines for lupus anticoagulant detection. J Thromb Haemost. Sahud MA. Laboratory diagnosis of inhibitors. Semin Thromb Haemst. 2000; 26(2): Wagenman BL, Townsend KT, Mathew P, Crookston KP. The laboratory approach to inherited and acquired coagulation factor deficiences. Clin Lab Med. 2009; 29:

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