Virtual Lectures Planning Committee Disclosure Summary

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1 Mayo Medical Laboratories Virtual Lectures 2014 MFMER MFMER Virtual Lectures Planning Committee Disclosure Summary As a provider accredited by ACCME, College of Medicine, Mayo Clinic (Mayo School of CPD) must ensure balance, independence, objectivity and scientific rigor in its educational activities. Course Director(s), Planning Committee Members, Faculty, and all others who are in a position to control the content of this educational activity are required to disclose all relevant financial relationships with any commercial interest related to the subject matter of the educational activity. Safeguards against commercial bias have been put in place. Faculty also will disclose any off label and/or investigational use of pharmaceuticals or instruments discussed in their presentation. Disclosure of these relevant financial relationships will be published in activity materials so those participants in the activity may formulate their own judgments regarding the presentation. Listed below are individuals with control of the content of this program who have disclosed Relevant financial relationship(s) with industry: None No relevant financial relationship(s) with industry: Rajiv Pruthi, MBBS program presenter Curtis Hanson, MD program planning committee Sharon Preuss program planning committee Bobbi Pritt, MD, MSc, DTMH program planning committee Cara Schmidt program planning committee References to off-label and/or investigational usage(s) of pharmaceuticals or instruments in their presentation: None MFMER 1

2 Novel anticoagulants: Role of Laboratory monitoring impact on coagulation based testing. Rajiv K. Pruthi, M.B.B.S Co-Director, Special Coagulation Laboratory & Director, Mayo Comprehensive Hemophilia Center Division of Hematology/Internal Medicine Dept of Laboratory Medicine & Pathology Mayo Clinic, Rochester, MN CP MFMER Disclosures Relevant Financial Relationship(s) None Off Label Usage None 2015 MFMER

3 Learning Objectives: To know the effects of novel anticoagulants on commonly ordered coagulation assays To know the circumstances in which monitoring may be useful Understand limitations of currently available assays Review availability of assays to monitor novel anticoagulants 2015 MFMER Contact activator thromboplastin dabigatran Rivaroxaban/ Apixaban/ edoxaban TT: thrombin time; RT: reptilase time 2015 MFMER

4 Newer anticoagulants: when to monitor Monitoring is not routinely required/needed Selected situations measuring drug effect may be useful: Assess compliance Hemorrhagic or thrombotic complications Pre-operative for urgent surgery Cases of overdosing 2015 MFMER Newer Anticoagulant Drugs: Effect on laboratory tests Routine assays: Prothrombin Time Activated partial thromboplastin time Fibrinogen Thrombin Time Specialized tests AT, PC, PS, Lupus anticoagulant assays Anti-Xa assays APCR-V Coagulation factor assays Others J Thromb Haemost. 2009;7(Suppl. 1): MFMER

5 Dabigatran 2015 MFMER Effect on PT (INR): single reagent PT [INR] Multiple dose R 2 = Dabigatran plasma concentration [ng/ml] Van Rynn Thromb Haemst 2010;103:1116 5

6 Dabigatran effect on PT (INR): multiple reagents Innovin Simple simon Owren s Nycotest Owren s Dabigatran ug/l Lindahl TL et al T & H 2011;105: MFMER Potential reasons for such discrepancy INR = PT (patient) PT (Geo mean) PT reagent (thromboplastin) Recombinant vs tissue derived PT geometric mean Changes between reagents and between lots of reagents ISI: varies with reagent and regent/instrument combination ISI ISI: international sensitivity index 2015 MFMER

7 Dabigatran effect on multiple APTT reagents 160 APTT (seconds) Triniclot APTT HS PTT Automate DG APTT HS APTT SP Actin FSL Dabigatran ug/l Lindahl TL et al T & H 2011;105: MFMER Potential reasons for such discrepancy Sensitivity of aptt reagent to anticoagulant Heparin sensitivity varies Effect of instrument Mechanical endpoint vs optical endpoint 2015 MFMER

8 Effect on thrombin time TT (seconds) Multple dose R2= Dabigatran ug/l Van Rynn Thromb Haemst 2010;103:1116 Dabigatran effect on thrombin time: (in house Mayo data, spiked pooled plasma sample) seconds Dabigatran level (mcg/ml) 2015 MFMER

9 Dabigatran effect on PT and APTT Prolongs respective clotting times Degree of prolongation will vary with reagent instrument combinations Not accurately reflective of drug levels Although theoretically possible, calibration of individual labs PT and APTT reagents is possible but no advisable Would need to be revalidated with each change in lot No safe PT/APTT level for invasive procedures (in patients on dabigatran) Prolongs APTT (Mixing study inhibited) May be mistaken for presence of inhibitor FVIII Inhibitor or lupus anticoagulant 2015 MFMER Rivaroxaban 2015 MFMER

10 Rivaroxaban & Prothrombin Time 60 Prothrombin time (sec) C trough Rivaroxaban concentration Neoplastin, Diagnostica Stago Mueck W et al 2008;47: Varying effect of rivaroxaban on different prothrombin time reagents Prothrombin time (sec) Recombiplastin (ISI: 1.0) Triniclot (ISI 1.22) Neoplastin Plus ISI 1.17 Neoplastin (ISI 1.75) Thromborel (ISI 1.07) Innovin (ISI 0.93) Rivaroxaban (ug/ml) Samama MM et al T&H 103;2010: MFMER

11 Plasma concentration of rivaroxaban & Coagucheck XS PT ratio R 2 = Rivaroxaban ug/ml Samama MM et al T&H 103;2010: MFMER APTT ratio Plasma concentration of rivaroxaban & APTT Actin FS CK Prest PTT LA PTT A Rivaroxaban (ug/ml) Samama MM et al T&H 103;2010: MFMER

12 Effect of direct anti-xa inhibitors Variable prolongation of PT/aPTT Reagent/instrument dependent Cannot establish a cut off to detect or exclude presence of drug No safe level of PT/aPTT 2015 MFMER Effect on specialty assays 2015 MFMER

13 Dabigatran effect on specialized assays Prolongs and inhibits screening assays False impression of either lupus anticoagulant of specific factor inhibitors Results in artifactually reduced factor levels False positive Bethesda assays No effect or falsely lowers Clauss fibrinogen False positive drvvt Lupus anticoagulant 2015 MFMER AT activity Xa based: no effect IIa based: over estimates (miss a deficiency) PC Activity Clot based: falsely over estimates Chromogenic: no effect PS activity Clot based: falsely over estimates Activated protein C resistance (APC-R) Falsely normal ratio (miss the diagnosis) 2015 MFMER

14 Conclusions: laboratory effects of new anticoagulants Routine monitoring not required But maybe useful (selected situations) Co-morbidities affecting widely available screening tests: Liver disease Vitamin K deficiency Other (DIC, inhibitors etc) Sensitivity to trough drug concentrations Likely essential 2015 MFMER Conclusions: laboratory effects of new anticoagulants Variable thromboplastins in use Affects sensitivity and reproducibility More complex assays (anti-xa) better performance characteristics Not generally available FDA approval of various kits Validation and calibrators required 2015 MFMER

15 Differences on and off Dabigatran Jan 6th Jan 13th PT ( ) APTT (21-33) 1:1 mix DRVVT (<1.2) 1:1 mix Confirm 1.4 ND TT > (16-25) Staclot APTT Staclot 8 4 ND: not done 2015 MFMER Dabigatran and selected assays False positive test result for lupus anticoagulant DRVVT and Staclot APTT assays Falsely elevated protein S activity No significant impact on antithrombin activity Factor Xa inhibition assays may falsely lower No impact on Clauss fibrinogen PT derived fibrinogen: false reduction 2015 MFMER

16 Effect on Hemoclot Thrombin Inhibitor Kit Van Rynn Thromb Haemst 2010;103:1116 Anti-Xa assays Most reliable tests for drug levels (with appropriate calibrators) 2015 MFMER

17 Anti-Xa assays R 2 = Heptest ratio Rivaroxaban (ug/ml) Samama MM et al T&H 103;2010: LMWH calibrated anti-xa assay Stago :Liquid heparin IU/mL rivaroxaban Apixaban Drug concentration ng/ml 2015 MFMER

18 Anti-Xa effect on specialized assays Prolongs and inhibits screening assays False impression of either lupus anticoagulant of specific factor inhibitors Results in artifactually reduced factor levels False positive Bethesda assays False positive drvvt Lupus anticoagulant 2015 MFMER AT activity Xa based:over estimates (miss a deficiency) IIa based: no effect PC Activity Clot based: falsely over estimates Chromogenic: no effect PS activity Clot based: falsely over estimates Activated protein C resistance (APC-R) Falsely normal ratio (miss the diagnosis) 2015 MFMER

19 Role of laboratory monitoring Not currently recommended Dabigatran Data published on bleeding risk with varying levels No current FDA guidance Anti-Xa inhibitors No current indication for monitoring Await FDA approval of calibrators/kits 2015 MFMER Thank you for your attention Questions? 2015 MFMER

20 Lupus Anticoagulant New CLSI Guidelines for Laboratory Testing Rajiv K Pruthi, M.B.B.S. Associate Professor, Mayo Clinic College of Medicine Director, Hemophilia Center, Co Director Special Coagulation & DNA Diagnostic Laboratory Division of Hematology/Internal Medicine 2015 MFMER slide-39 Disclosure Relevant Financial Relationships None Off Label/Investigational Uses None 2015 MFMER slide-40 20

21 LA & APL Antibodies / Syndrome Diagnostic Guidelines Publications LA testing guidelines (criteria, recommendations) ISTH (1983, 1991, 1995, 2009): Thromb Haemost (&JTH 7:1737) CAP (2002): Triplett DA. Arch Pathol Lab Med 126:1424 BCSH (1991, 2000, 2012): Keeling et al. Br J Haematol 157:47 CLSI/NCCLS (4 2014): H 60A ( APS classification criteria Wilson WA et al. (1999): Arthritis Rheum 42:1309 Miyakis S et al. (2006): J Thromb Haemost 4:295 All of the above are primarily based on expert opinions together with results of limited studies; evidence based recommendations are lacking (as are standards) MFMER slide-41 When should one test for antiphospholipid antibodies: Suspicion of APS* Clinical Criteria** Vascular Thrombosis (Arterial, Venous) &/or Pregnancy Morbidity 1 fetal death 10 weeks 3 consecutive spontaneous abortions <10 weeks 1 premature birth 34 weeks Placental insufficiency, Severe pre eclampsia Laboratory Criteria (persistent 12 wks) ACL / β 2 GPI Abs (IgG, IgM) mod. high titer &/or Lupus anticoagulant (by ISTH criteria) 1 clinical + 1 lab criterion fulfills definition *Antiphospholipid antibody Syndrome **Clinical events not otherwise readily explained Wilson WA 1999;42: Miyakis S 2006;4: MFMER slide-42 21

22 APS Non Criteria Clinical Manifestations Cardiac valve lesions (vegetations, thickening) Thrombocytopenia (immune mediated: AITP) Livedo reticularis & other skin manifestations Non thrombotic neurologic manifestations, including cognitive dysfunction, chorea Small artery vasculopathy/vasculitis (APL associated nephropathy, etc.) Thrombotic microangiopathic syndromes (TTP, HUS, HELLP, etc.) 2015 MFMER slide-43 Why is it important to detect these antibodies? (Diagnostic and prognostic) Thrombosis risk LA > ACL IgG > ACL IgM* LA + IgG ACL (β 2 GPI) >> LA alone** (double positive) LA + ACL + β 2 GPI >> single/double*** (triple positive) Pregnancy loss or complications LA, ACL, β 2 GPI IgG LA, ACL, β 2 GPI IgG **** Bleeding risk LA / hypoprothrombinemia (or F X def.) Thrombocytopenia (AITP) Anticoagulation related bleeding Asymptomatic *Horbach DA TH 1996; Wahl DG Lupus 1997 & 1998; Galli M Blood 2003 **de Laat B Blood 2004 & 2005; de Groot PG JTH 2005; Galli M Blood 2007 ***Pengo V Thromb Haemost 2005; Ruffatti A TH 2006 ****Ruffatti TR MFMER slide-44 22

23 What are antiphospholipid antibodies? Misnomer Antibodies are directed against protein/phopholipid complexes Not solely directed against phospholipid 2015 MFMER slide-45 Phospholipid layer 2015 MFMER slide-46 23

24 Phospholipid layer 2015 MFMER slide-47 Why do the in-vitro findings (prolonged clotting times) differ from in-vivo events (thrombosis)? 2015 MFMER slide-48 24

25 LA & APL Antibodies: in vivo Pathophysiology Antibody inhibition of natural anticoagulant pathways (PLdependent assembly): Proteins C &/or S (APC inactivation of Factors Va & VIIIa) Tissue factor pathway inhibitor (TFPI TF VIIa >Xa inhibition) Protein Z ZPI complex (Factor Xa inhibition) Down regulation of physiological B 2 GPI functions Disruption of protective Annexin V binding to anionic phospholipids (Rand JH. Thromb Res 2004; delaat B. Blood 2007) Dysregulation of Fibrinolysis (inhibition) Platelet activation; enhanced thrombin generation Endothelial cell activation or injury 2015 MFMER slide-49 Detection of anti-phospholipid antibodies Liquid phase Lupus anticoagulants Solid phase ELISA based Anti cardiolipin and anti beta 2 glycoprotein I 2015 MFMER slide-50 25

26 Guidelines discussion Pre analytical Analytical Post analytical 2015 MFMER slide-51 Pre analytical aspects: factors resulting in false positive or indeterminate results. Dos Test before or after completion of anticoagulation Donts Test while patient is on anticoagulation Runs the risk of false positive results 2015 MFMER slide-52 26

27 aptt PT/PCT Intrinsic Extrinsic XII XI DRVVT VII aptt IX VIII X anti Xa PT V Heparin II Fibrinogen DTI 2015 MFMER slide-53 Pre analytical aspects: Sample processing: Dos Collection in M trisodium citrate Double spin (reduce platelet count to <10 x 10 9 /L) Donts Filtration through 0.22 µm cellulose acetate filters 2015 MFMER slide-54 27

28 Effect of residual platelets on results of clot based LAC testing Chantarangkul V et al Throm & Haem 2002;87: MFMER slide-55 Analytical Aspects: How many different assays to screen for LAC? Maximum of 2 tests based on 2 different assay principles dilute Russell s viper venom time (DRVVT) activated partial thromboplastin time (APTT) lupus sensitive (low phospholipid content) If strong clinical suspicion of Antiphospholipid Antibody syndrome (APS) Additional assays may be considered CLSI. Lab Testing for LAC. Approved Guideline CLSI H60-A MFMER slide-56 28

29 LAC Testing trends (EQA Programs) Program ECAT RCPA UKNEQUAS Year 2005/ / /2006/2010 Total Laboratories Methods APTT (including SCT) 495 (45%) 167 (4%) 731 (39%) drvvt 488 (45%) 170 (41%) 786 (42%) KCT 59 (5%) 72 (17%) 196 (11%) dpt 47 (4%) 1 60 (3%) others (5%) CLSI. Lab Testing for LAC. Approved Guideline CLSI H60-A MFMER slide-57 aptt SCT dpt/pct Intrinsic Extrinsic XII XI DRVVT VII IX aptt VIII X PT V II Fibrinogen 2015 MFMER slide-58 29

30 Is lupus sensitivity activator dependent? Activators of intrinsic pathway Silica (aptt and silica clot time) Kaolin (Kaolin clot time) Ellagic acid (not recommended by ISTH 2009) Sensitivity shown to be dependent on phospholipid content Kumano, O et al JTH 2012; 10:2338 Equivalent to silica based activators 2015 MFMER slide-59 Sequence of assay performance 2015 MFMER slide-60 30

31 Step 1: Prolonged phospholipid dependent screening assay No: stop Yes: Step 2: mixing study with normal pooled plasma Or a weak LAC Corrects: stop (Factor deficiency) Inhibited Step 3: phospholipid confirm Does not shorten: Alternative explanation FVIII inhibitor, drug effect etc Step 4: exclude other causes Shortens: LAC confirmed 2015 MFMER slide-61 Traditionally accepted criteria for a positive LAC Step 1. Prolongation of at least 1 of 2 phospholipid (PL) dependent clotting time tests based on different principles (APTT, DRVVT etc) Step 2. Inhibition: demonstrated by mixing study of patient and normal pooled plasma Step 3. Confirmation that the inhibitory activity is phospholpid (PL) dependent shortening of clotting time with addition of phospholipid Step 4. Exclusion/evaluation for other coagulopathies that may give similar laboratory results or yield false positive or indeterminate results (eg, anticoagulation therapy effects, coagulation factor inhibitor or deficiency) 2015 MFMER slide-62 31

32 Step 1: Prolonged phospholipid dependent screening assay No: stop Yes: Step 2: phospholipid confirm Does not shorten: Not LAC Shortens: LAC confirmed Step 3: mixing study with normal pooled plasma Corrects: factor deficiency Inhibited: Alternative explanation FVIII inhibitor, drug effect etc Step 4: exclude other causes 2015 MFMER slide-63 Analytic and Post Analytic: Calculation of ratios Screening clotting times should be normalized to the mean of reference interval (RI) Normalized screen ratio= patient APTT/mean of RI APTT But manufacturer's instructions should be followed MFMER slide-64 32

33 Analytic and Post Analytic: Calculation of ratios Confirmatory clotting times: Unpaired tests (APTT/PNP or Hex phase NT) Delta of screen and confirm Paired tests (drvvt) Normalized screen to confirm ratio = Screen patient /Screen mean of RI Confirm patient/ Confirm mean of RI 2015 MFMER slide-65 Generation of reference intervals CLSI document: EP28-A3c: Defining, establishing and verifying reference intervals in the Clinical Laboratory MFMER slide-66 33

34 Post analytical aspects: calculations and interpretation REPORTING & INTERPRETATION: 1. Numerical results of all testing should be reported with reference interval or cut off values 2. Interpretive comments that address and integrate all test results (LA panel) should be provided 3. The interpretive report should indicate whether LA is detected, not detected, or indeterminant. 4. If LA is present, the test panel should be repeated at or beyond 12 weeks to determine persistence of LA as part of the evaluation for LA 2015 MFMER slide-67 LA Testing Guidelines Review Selected Comparisons: ISTH SSC 2009 vs CLSI H60 A* ISTH SSC (2009) Cutoffs (Ref. Interval): 99% (nonparametric) drvvt first, then APTT APTT activator: Silica only, not Ellagic acid Tests not recommended: dpt, KCT, PNP, etc. Algorithm order: Screen, Mix, Confirmatory CLSI H60 A (2014) Cutoffs (Ref. Interval): +2SD (parametric) for each test Both drvvt & APTT screen APTT activator: no restriction (Silica, Ellagic acid, Kaolin) Does not restrict supplemental tests, if validated by lab Algorithm order: Screen, Confirmatory, Mix Normalized ratio reporting: relative to Normalized ratio reporting: relative to mean normal pool mean ref. interval *Table 3, Section 15: Harmonization excerpts 2015 MFMER slide-68 34

35 LA & APL Antibodies / Syndrome Summary & Recommendations LA &/or ACL positivity are relatively frequent Test for both LA and ACL (+/ supplemental β 2 GPI Abs) Important to evaluate persistence of positivity (>3 mo.) Numerous pre analytical, analytical and post analytical variables (patient, sample, AC Rx, interpretation and reporting, etc.) Know your laboratory s quality > select a good one Not all LA or ACL are pro thrombotic; no predictive tests yet Rarely, bleeding can occur with LA (II or X deficiency, AITP) APL Syndrome (APS) diagnosis challenging Thrombotic events &/or fetal losses, otherwise unexplained Persistently significantly positive LA &/or ACL (β 2 GPI) Anticoagulation management can be difficult (& Rx impacts Dx) Heparin (inhibited APTT); Warfarin (inhibited PT/INR); DOACs (false positive) 2015 MFMER slide-69 35