M1722 Hot Topics in Hemostasis

Transcription

1 M1722 Hot Topics in Hemostasis Russell A. Higgins, MD, FCAP Karen A. Moser, MD, FCAP

2 Objectives Describe the laboratory assays currently available to measure DOAC effect in individual patients. Evaluate DOAC interferences in common and esoteric hemostasis assays. Assess the performance of your factor assays in the presence of new extended half-life FVIII and FIX replacement products. Review FVIII and FIX replacement product package inserts for information pertaining to laboratory performance of factor assays. 2

3 Agenda Hot Topics in Hemostasis Opening/Introductions Measuring direct oral anticoagulants (DOAC) in the clinical laboratory Measuring extended half-life FVIII and FIX replacement products in the clinical laboratory Summary and Closing 3

4 Measuring Direct Oral Anticoagulants (DOAC) Karen A. Moser, M.D. Assistant Professor, Pathology University of Utah Medical Director, Hemostasis and Thrombosis ARUP Laboratories

5 Direct Oral Anticoagulant Agents Approval timeline Direct thrombin inhibitor Direct Xa inhibitors 5

6 Terminology Matters What to call these drugs? New oral anticoagulants (NOAC) Novel oral anticoagulants (NOAC) Non-VKA oral anticoagulant (NOAC) At least one literature report where NOAC interpreted as no anticoagulation Oral direct inhibitor (ODI) Specific oral direct anticoagulant (SODA) Target-specific oral anticoagulants (TSOAC) Direct oral anticoagulants (DOAC) Recommended by ISTH Barnes GD, Ageno W, Ansell J, Kaatz S for the Subcommittee on the Control of Anticoagulation. J Thromb Haemost. 2015; 13:

7 Coagulation Cascade (simplified) Negatively charged surface Tissue Factor Contact Factors (XII, PK, HMWK) XI XIIa XIa VIIa VII Intrinsic Pathway IX IXa Extrinsic Pathway VIIIa X Xa Direct Xa inhibitors Va II IIa (thrombin) Direct thrombin inhibitors Fibrinogen Fibrin monomer Common Pathway 7

8 Direct Oral Anticoagulants Benefits No laboratory monitoring required per FDA labeling Rapid onset and cessation of anticoagulant effect Less risk of dietary interactions than warfarin May have less risk of bleeding complications than warfarin Shantsila E, Lip GYH. Non-Vitamin K Antagonist Oral Anticoagulants: A Concise Guide. New York: Springer: Baglin T. Br J Haematol. 2014; 163(2): Makam RCP, Hoaglin DC, McManus DD, et al. PLoS One. 2018; 13(5):e doi: /journal.pone

9 Direct Oral Anticoagulants Challenges No laboratory monitoring required per FDA labeling Laboratory testing for these agents developing after they are already on market Rapid onset and cessation of anticoagulant effect Less cushion for missed doses as with warfarin Twice daily dosing for dabigatran Shantsila E, Lip GYH. Non-Vitamin K Antagonist Oral Anticoagulants: A Concise Guide. New York: Springer: Baglin T. Br J Haematol. 2014; 163(2): Tripodi A. J Thromb Haemost. 2016; 14(7):

10 Case 1 A 57 year old man presents to the emergency department of your hospital with symptoms concerning for an acute stroke. He is unresponsive, but his wife tells the ED team that he takes dabigatran for atrial fibrillation. His last dose was this morning. 10

11 Case 1 The ED physician calls the laboratory to ask how to tell whether the patient has dabigatran on board prior to initiating thrombolysis. You do not have a specific test for measuring dabigatran validated in your laboratory. What is your advice to your colleague? 11

12 When might we want to measure DOACs? Emergent Results Needed Patients with acute bleeding or thrombosis on DOAC Overdose Trauma or emergent operation needed in patient on DOAC Routine TAT Acceptable Impaired renal function (adjusted dosing recommended for dabigatran, rivaroxaban, edoxaban) Extremes of body weight Elective operative procedure in patient on DOAC Suspected interactions with concomitant drug Patients also receiving dual antiplatelet therapy Assessment of compliance Tripodi A. Blood. 2013; 121(20): Tripodi A, Ageno W, Ciaccio M, et al. Blood Transfus. 2017; 13:1-9. Douxfils J, Gosselin RC. Semin Thromb Hemost. 2017; 43: Baglin T, Hillarp A, Tripodi A, et al. J Thromb Haemost. 2013; 11:

13 Time Matters Choice of assay in emergent versus routine clinical situations will differ Emergent Favor qualitative assays* Routine clotting times Available in most hospital laboratories Less accurate Variability between reagents Routine Favor quantitative assays More accurate Gives measurement of concentration Usually requires sendout to reference laboratory * Unless quantitative assays validated locally. Use clotting times with caution, as shown in subsequent slides. Douxfils J, Ageno W, Samama C-M, et al. J Thromb Haemost. 2018; 16:

14 Qualitative Assays- Options APTT Relatively sensitive to dabigatran Normal APTT does not exclude presence of on-therapy levels of dabigatran Not very sensitive to direct Xa inhibitors Insensitive to apixaban Significant variability between reagents Baglin T, Hillarp A, Tripodi A, et al. J Thromb Haemost. 2013; 11: Douxfils J, Ageno W, Samama C-M, et al. J Thromb Haemost. 2018; 16: Gosselin RC, Adcock DM, Bates SM, et al. Thromb Haemost. 2018; 118(3): Kitchen S, Gray E, Mackie I, et al. Br J Haemotol. 2014; 166:

15 Qualitative Assays- Options PT Not very sensitive to dabigatran Somewhat more sensitive to direct Xa inhibitors Rivaroxaban > edoxaban > apixaban Insufficiently sensitive, even if paired with normal APTT, to completely exclude presence of DOAC Significant variability between reagents Baglin T, Hillarp A, Tripodi A, et al. J Thromb Haemost. 2013; 11: Douxfils J, Ageno W, Samama C-M, et al. J Thromb Haemost. 2018; 16: Gosselin RC, Adcock DM, Bates SM, et al. Thromb Haemost. 2018; 118(3): Kitchen S, Gray E, Mackie I, et al. Br J Haemotol. 2014; 166:

16 Qualitative Assays- Options TT (thrombin time) Incredibly sensitive to dabigatran Normal TT excludes the presence of any level of dabigatran Not sensitive to direct Xa inhibitor Thrombin Time (s) Dabigatran measured by LC-MS/MS ng/ml Hawes E, Deal A, Jeanneret, et al. J Thromb Haemost 2013;11: Note: There are currently limited data regarding performance of betrixaban in hemostasis assays. Baglin T, Hillarp A, Tripodi A, et al. J Thromb Haemost. 2013; 11: Douxfils J, Ageno W, Samama C-M, et al. J Thromb Haemost. 2018; 16: Gosselin RC, Adcock DM, Bates SM, et al. Thromb Haemost. 2018; 118(3): Kitchen S, Gray E, Mackie I, et al. Br J Haemotol. 2014; 166:

17 Qualitative Assays- Options Anti-Xa activity (calibrated for UFH or LMWH) Use with caution! However, this may be a reasonable option in an emergent situation with limited tests available Preferred to calibrate assay for direct Xa inhibitor you are trying to measure At least one study suggests that anti-xa assays calibrated for UFH and LMWH could detect significant levels of DOAC 1 Others suggest that response may vary by kit 2 1. Gosselin R, Grant RP, Adcock DM. Int J Lab Hematol. 2016; 38: Sabor L, Raphael M, Dogne JM, et al. Thromb Res. 2017; 156:

18 Potential qualitative use of anti-xa activity assays Key- Apixaban (diamond) Edoxaban (triangle) Rivaroxaban (circle) Gosselin R, Grant RP, Adcock DM. Int J Lab Hematol. 2016; 38:

19 Quantitative Assays- Options for Dabigatran Dilute thrombin time Dilute refers to patient plasma diluted in buffer and mixed with NPP Linear increase directly proportional to increase in dabigatran concentration Commercially available, not FDA approved Good accuracy reported between ng/ml Adaptations to method may decrease LLOD Baglin T, Hillarp A, Tripodi A, et al. J Thromb Haemost. 2013; 11: Douxfils J, Ageno W, Samama C-M, et al. J Thromb Haemost. 2018; 16: Gosselin RC, Adcock DM, Bates SM, et al. Thromb Haemost. 2018; 118(3): Kitchen S, Gray E, Mackie I, et al. Br J Haemotol. 2014; 166:

20 Quantitative Assays- Options for Dabigatran Ecarin-based methods Clotting time (ECT) or chromogenic assay (ECA) One commercial ECA kit available, not FDA approved LLOD ~14-46 ng/ml Not sensitive to heparins Chromogenic anti-factor II assay One commercial kit available, not FDA approved LLOD ~15 ng/ml Prothrombin Ecarin Meizothrombin Inhibited by DTI Not inhibited by heparin Baglin T, Hillarp A, Tripodi A, et al. J Thromb Haemost. 2013; 11: Douxfils J, Ageno W, Samama C-M, et al. J Thromb Haemost. 2018; 16: Gosselin RC, Adcock DM, Bates SM, et al. Thromb Haemost. 2018; 118(3): Kitchen S, Gray E, Mackie I, et al. Br J Haemotol. 2014; 166:

21 Quantitative Assays- Options for Direct Xa inhibitors Anti-Xa chromogenic assays with DXa inhibitor-specific calibrators Measures wide range of concentration, results ng/ml LLOQ ~30 ng/ml (varies by kit and drug) Some kits may require adaptation to measure ng/ml No FDA approved calibrator materials Can also be elevated by heparin Complicates monitoring in patients transitioning to DXa inhibitor Baglin T, Hillarp A, Tripodi A, et al. J Thromb Haemost. 2013; 11: Douxfils J, Ageno W, Samama C-M, et al. J Thromb Haemost. 2018; 16: Gosselin RC, Adcock DM, Bates SM, et al. Thromb Haemost. 2018; 118(3): Kitchen S, Gray E, Mackie I, et al. Br J Haemotol. 2014; 166:

22 Quantitative Assays- Option for all DOAC LC/MS-MS Considered gold standard for quantification Reportable range ng/ml Only available in small number of reference laboratories No international reference standard available Timing of measurement matters Prefer peak or trough instead of random Baglin T, Hillarp A, Tripodi A, et al. J Thromb Haemost. 2013; 11: Douxfils J, Ageno W, Samama C-M, et al. J Thromb Haemost. 2018; 16: Gosselin RC, Adcock DM, Bates SM, et al. Thromb Haemost. 2018; 118(3): Kitchen S, Gray E, Mackie I, et al. Br J Haemotol. 2014; 166:

23 Case 1, revisited What is your advice to your colleague? Thrombin time (TT) can be used qualitatively to assess presence/absence of dabigatran. If TT is not available, APTT may be useful qualitatively, but responsiveness depends on your local reagents. TT must be tested within 4 hours of sample collection (not a problem in this emergent situation!) 23

24 Case 2 A 30 year old woman experiences a DVT and is treated with rivaroxaban. Her physician orders a thrombophilia work-up; the patient has the lab work drawn 2 days after starting therapy. The following assays were performed: Protein S activity (clot based): 65% (63 140%) Protein C activity (chromogenic): 72% (55 140%) APCR ratio: 2.2 ( ) Positive drvvt confirm (ratio 1.3) Negative APTT-based LA confirmatory assay Negative anticardiolipin and β2gp1 IgG and IgM How do you help her physician interpret these results? 24

25 DOAC Interferences- Selected Assays Assay Dabigatran DXa inhibitors* APCR ratio False Increase False Increase AT activity - (Xa-based) False Increase (IIa-based) False Increase (Xa-based) - (IIa-based) PC activity- clot based False Increase False Increase PC activity- chromogenic - - PS activity False Increase False Increase Free PS antigen - - LA assays False Positive False Positive APTT-based factor assays (VIII, IX, XI, XII) PT-based factor assays (II, V, VII, X) False Decrease False Decrease * Limited data on betrixaban False Decrease False Decrease Mani H. Int J Lab Hematol. 2014; 36: Gosselin RC, Adcock DM, Bates SM, et al. Thromb Haemost. 2018; 118(3):

26 Case 2- revisited Results Interpretation in Presence of Rivaroxaban PS activity may be falsely increased. Risk of misclassifying PS deficiency as normal PC chromogenic activity is not affected. APCR ratio may be falsely increased. Risk of misclassifying APCR as normal LA assay results may be falsely positive. Anticardiolipin and β2gp1 IgG and IgM are not affected. 26

27 Reversal Options First reversal agent for dabigatran FDA approved October, 2015 Praxbind (idarucizumab) Humanized Fab Binds dabigatran to neutralize effect First reversal agent for rivaroxaban and apixaban FDA approved May, 2018 AndexXa (andexanet alfa) Recombinant FXa decoy molecule (inactive enzyme) Binds DXa inhibitor in place of Fxa Additional agent in development Ciraparantag (phase 2 study- reversal of apixaban and rivaroxaban) Almegren M. Vasc Health Risk Manag. 2017; 13: clinicaltrials.gov, ANNEXA-4 (identifier NCT ) Greinacher A, Thiele T, Selleng K. Thromb Haemost. 2015; 113(5): cinicaltrials.gov, ciraprantag (identifier NCT , NCT ) 27

28 Summary While routine monitoring of DOAC is not required, several clinical situations call for measuring DOAC in the laboratory Choice of assay for measuring DOAC depends on clinical question and how emergently the result is needed DOAC may interfere in a variety of routine and esoteric coagulation assays, causing false negative or positive results. 28

29 Monitoring New Factor VIII and IX Replacement Products Russell Higgins, MD Clinical Professor Department of Pathology and Laboratory Services University of Texas Health Science Center San Antonio Medical Director, University Hospital System Pathology Services

30 Factor VIII Variability Comparison of an old versus new factor VIII method Main Variables Calibrator Reagent Instrument (mechanical versus optical) Factor VIII deficient plasma Difference Factor VIII:C Bias Plot (AMAX + TOP1)/2

31 2017 CAP Survey (CGS3-02): Factor VIII Activity (ISTH Assigned Value = 88 IU/dL) BIAS -- ISTH Lot 4 Versus CAP % Diffence (CAP -ISTH)/ISTH CAP 2012 CAP 2013 CAP 2014 CAP 2015 CAP 2016 CAP 2017 Factor VIII Activity Reagent Factor VIII Activity Reagent Factor VIII Activity Reagent ISTH Assignment (IU/dL)

32 The Problem: variability of one-stage factor assays for measuring modified factor products Native Factor VIII Some Variability Interlaboratory CV% ~12 (CAP CGE) Recombinant Factor VIII Concentrates Some Variability Interlaboratory CV% 10, 10.9, 11.4 (Advate; 3 studies) Modified Recombinant Factor VIII Concentrates High Variability Interlaboratory CV% 16 (Eloctate) 14.7 (Adynovate) 122 (Bay ) High reagent and instrument dependent recovery

33 Modified factor VIII replacement products Goal = Decrease immunogenicity or extend half-life Fusion proteins (e.g. albumin or Fc portion of IgG1) Pegylated factor VIII Single chain products (heavy and light chains covalently linked)

34 Factor VIII Assays One-stage factor assay Diluted patient plasma (FVIII source) mixed with factor deficient plasma aptt-based(clot based) Most common factor assay Chromogenic (2 stages) Diluted patient plasma (FVIII source) incubated with reagent to activate Xa (Stage 1) Xa cleaves chromogenic substrate (Stage 2) Not common in US laboratories

35 Modified Coagulation Factor Products Some extended half-life (EHL) recombinant products Factor VIII Product Modification Half-Life (hours) *Eloctate ; rfviiifc Fc fusion BDD rfviii 19 *Adynovate ; BAX855 Pegylated rfviii *Afstyla ; rfviii-singlechain Single chain BD truncated rfviii 14.5 **N8 GP (late stage) glycopegylated rfviii 19 **Bay (late stage) PEGylated BDD rfviii 18.2 Factor IX Product Modification Half-Life (hours) *Alprolix ; rfixfc Fc fusion protein 82 *Idelvion, rfix-fp Albumin fusion 102 *Rebinyn ; N9 GP Glycopegylated rfix 93 Normal FVIII Half-Life 12 hours Normal FVIX Half-Life hours *Graf L. Transfus Med Hemother 2018;45: **Kitchen S, Tiefenbacher S, Gosselin R. Semin Thromb Hemost 2017;43:

36 What difference from labeled potency is acceptable? No definition of acceptability Typically ~25% to 30% is acceptable Eloctate One-stage and chromogenic assays found to be acceptable Reflected in package insert Eloctate Sommer JM, Moore N, Mcguffie-Valentine B, et al. Haemophilia (2014), 20,

37 You receive a call from a Hematologist Can your the laboratory monitor Afstyla? A) Yes B) No C) Let me investigate and I will call you back 37

38 Afstyla Field Study *one-stage factor VIII methods underestimate Afstyla by ~50% FDA approved with a conversion factor of x2 across all one-stage reagents Blue bars are Afstyla Red bars are Advate *Ledger KST, Feussner A, Kalina U, et al. Journal of Thrombosis and Haemostasis, 16:

39 N8-GP: recovery at two sites Most one-stage methods adequate APTT SP underestimates N8 GP Some overestimation by chromogenic methods Hillarp A, Bowyer A, Ezban M. Haemophilia (2017), 23,

40 BAY PTT-A and APTT-SP underestimate spiked samples (right) Chromogenic methods had adequate performance FVIII Chromogenic (Siemens) Electrochrome (IL) Coamatic (IL) Church N, Leong L, Katterle Y. Haemophilia. 2018;1 10.

41 Modified Factor FVIII Products: Monitoring Factor VIII Product Modification Half-Life (hours) Monitoring Method *Eloctate ; rfviiifc Fc fusion BDD rfviii 19 Most one-stage or chromogenic *Adynovate ; BAX855 Pegylated rfviii Most one-stage or chromogenic *Afstyla ; rfviii- SingleChain Single chain BD truncated rfviii 14.5 Most one-stage x 2 or chromogenic **N8 GP (late stage) glycopegylated rfviii 19 Most one-stage (avoid APTT-SP; SynthasIL on Siemens Instrument) or chromogenic **Bay (late stage) PEGylated BDD rfviii 18.2 Most one-stage (avoid APTT-SP and APTT- A) or chromogenic *Graf L. Transfus Med Hemother 2018;45: **Kitchen S, Tiefenbacher S, Gosselin R. Semin Thromb Hemost 2017;43:

42 Modified FIX: A Bigger Problem

43 Rebinyn (rfix-gp): one-stage FIX recovery at two sites Overestimated by Pathromtin SL and APTT-SP!!!!!! Underestimated by Actin, Actin FS, Actin FSL, and SynthaSil Bowyer AE, Hillarp A, Ezban M, et al. Journal of Thrombosis and Haemostasis, 14:

44 Alprolix -rfixfc Kaolin method underestimates Alprolix Silica-based reagent moderately underestimates Alprolix Manufacturer uses silica-based reagent Authors concluded silica-based reagent was okay Sommer JM, Buyue Y, Bardan S, et al. Thrombosis and Haemostasis 112.5/2014

45 Modified Factor FVIX Products: Monitoring Rebinyn Alprolix Idelvion Kitchen S, Tiefenbacher S, Gosselin R. Semin Thromb Hemost 2017;43:

46 Potency Labeling: methodology European Pharmacopoeia FVIII labeled with chromogenic FIX labeled with one-stage Other Countries For US, either chromogenic or one-stage *SSC/ISTH Recommendations *Dodt J, Hubbard AR, Wicks J, et al. Haemophilia (2015), 21,

47 Which WHO? In the laboratory, reference plasmas are traceable to WHO IS for Plasma rather than Concentrate Differences exist btw standards by design* WHO International Standard for Concentrate Manufacturer s internal reference material Product labeled in IU by appropriate test Post-infusion samples measured by clinical laboratories against WHO IS for factor IX in Plasma **~+20% overestimation of Refacto Laboratory Standard discovered, 2003 ***10%-20% Difference with BeneFIX * Lollar P. Journal of Thrombosis and Haemostasis 2003;1: **Thromb Haemost Dec;90(6): ***Sommer JM, Buyue Y, Bardan S, et al. Thrombosis and Haemostasis 112.5/2014

48 Solutions? Product specific calibrators Additional method (chromogenic assay or additional one-stage) Correction factors when appropriate Send to reference laboratory when product cannot be measured

49 Solutions: product specific calibration European Experience Refacto AF Laboratory Standard *BAX 855 Product specific calibrator improved recovery and precision across 11 laboratories (right) Lab must know what product is infused Difficult to manage multiple standards *Bulla O, Poncet A, Alberio L, et al. Haemophilia (2017), 23, e335 e339

50 Solutions: Use chromogenic methodology Chromogenic assays have?less variability? Diminished sensitivity at low factor levels No FDA approved chromogenic kits in US Chromogenic assays may not be expensive Aliquoting reagent reduces cost * ** *Sommer JM, Moore N, Mcguffie-Valentine B, et al. Haemophilia (2014), 20, **Kitchen S, Blakemore J, Friedman Kd, et al. Journal of Thrombosis and Haemostasis, 14:

51 Solutions: correction factors Blanket correction of one-stage methods (x2) approved for Afstyla Some concern [e.g. Synthafax or Actin FS (right)] *Ledger KST, Feussner A, Kalina U, et al. Journal of Thrombosis and Haemostasis, 16: **Bowyer A, Key N, Dalton D, et al. DOI: /hae.13290

52 Solutions: send to designated reference laboratory Long turn around time not appropriate for emergencies May be adequate for PK studies

53 Real World Solutions: Summary Modified coagulation factors are not recovered by all one-stage assays Use product inserts and literature to identify gaps in laboratory testing Develop relationship with treating physicians and pharmacists Communicate gaps in laboratory testing for products that cannot be measured accurately Develop a local strategy to cover the gaps

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