1. Laboratory testing for antiphospholipid syndrome Vittorio Pengo M.D. Clinical Cardiology, Thrombosis Center, University of Padua, Italy

2. Disclosures: Advisory Board: Daiichi-Sankyo, Bayer HealthCare Lecture fees: : Daiichi-Sankyo, Bayer HealthCare, Boehringer Ingelheim, Instrumentation Laboratories, Roche Diagnostics

3. A preconference workshop, preceding the Eleventh International Congress on antiphospholipid antibodies (aPL), Sydney consensus conference established the update classification criteria for APS One clinical criteria (thrombosis or pregnancy loss) + One laboratory criteria: Lupus Anticoagulant, IgG/IgM anticardiolipin antibodies, AND IgG/IgM anti β2-GPI antibodies. [Positive 12 week apart] SYDNEY, 2005 Miyakis S et al. J Thromb Haemost 2006; 4: 295–306

4. Investigators are strongly advised to classify APS patients in studies into one of the following categories. SYDNEY, 2005 I: More than one Laboratory criteria present (any combination) IIa: Lupus Anticoagulant present alone IIb: Anti-cardiolipin antibody present alone IIc: Anti-  2 glycoprotein-I antibody present alone Miyakis S et al. J Thromb Haemost 2006; 4: 295–306 To this end, all the three tests must be performed

5. Topics in lab diagnosis of antiphospholipid syndrome (APS) In whom and when checking for the presence of antiphospholipid antibodies How to read the results How definite is the diagnosis of APS

6. Checking for aPL: In whom? Young subjects (less than 50 yrs of age) Idiopathic VTE and CTPH VTE in uncommon sites If present an associated autoimmune diseases Cryptogenic stroke in general and when: Other concomitant conditions are present (i.e. dementia, livedo reticularis, epilepsy, valvular heart disease) in the absence of overt atherosclerosis In case of unexplained recurrence In women with pregnancy loss (especially in case of fetal death) Unexpected a PTT prolongation in otherwise healthy subjects

7. MASSIVE POLMONARY EMBOLISM MC/ Male 18 yrs of age Unprovoked Proximal DVT/PE Clinical Cardiology, Padua

8. Multiple cerebral infarctions in 48 years old female ( N. C.) with hemiparesis and epilepsy Courtesy of Clinical Reumatology, Padua

9. LIVEDO RETICULARIS Cerebral ischemia + livedo reticularis: Sneddon’s syndrome Courtesy of Clinical Reumatology, Padua

10. Minor stroke and multiple mitral valve thickening in 20 year of age young women Clinical Cardiology, Padua

11. Checking for aPL: when? In patients with venous thromboembolism (VTE):  Before the initiation of anticoagulant treatment (difficult)  When deciding to prolong or discontinue anticoagulant treatment In patients with arterial thromboembolism (ATE):  Initially, to decide the use of antiplatelet or anticoagulant drugs or both In women with pregnancy losses:  Initially, to establish an appropriate treatment during pregnancy After 12 weeks to confirm the initial positivity

12. CONFIRM DATA AFTER 12 WEEKS TO EXCLUDE TRANSIENT ANTIBODIES Consecutive patients referred to our Center initially positive to one or more tests exploring the presence of aPL were tested after 3 months. During a four-years period 225 patients were initially positive to one or more test and 161 were available for confirmation after 3 months. Patients were classified as triple positive (n=54: LAC+, aCL+, aβ2GPI+, same isotype), double positive (n=50: LAC-, aCL+, aβ2GPI+, same isotype) and single positive (n=53: LAC or aCL or aβ2GPI antibodies as the sole positive test). Pengo V et al. JTH 2013

13. Characteristics at initial testing Triple positive n=54 Double positive n=50 Single positive* n=53 P value Age –yrs (mean±SD)42±1445±1338±120.02 Female––no.(%)42 (78)48 (96)49 (92)<0.01 Predominant IgG isotype––no.(%)42 (78)23 (46)NA0.001 Autoimmune disorders—no.(%)6 (11)12 (24)16 (30)0.0504 Venous or Arterial Thrombosis––no.(%)42 (78)15 (30)4 (7)<0.0001 Pregnancy loss––no.(%)7 (13)28 (56)7 (13)<0.0001 Pengo V et al. JTH 2013

15. How to read the results: Are they Associated with thromboembolic events?

16. Patients fulfilling classification criteria are put together in clinical studies Thus APS is diagnosed in the presence of a single positive test* Classification criteria (I,IIa,IIb,IIc) are often mistaken for diagnostic criteria * The other two tests either negative or not performed

17. β2-Glycoprotein 1 is the main antigen implicated in APS Beta-2 glycoprotein I (β2-GPI), an apolipoprotein with an approximate molecular weight of 50 kD. Discovered more than 50 years ago it consists of five short consensus repeats, also called sushi domains. The crystal structure of this protein was first reported in 1999 and has a J-shaped conformation, with a vertical ascending part called domain I and a positive area in the bottom part of the molecule called domain V. Domains I, II, III, and IV are each composed of approximately 60 amino acids whereas domain V differs from the other four domains and consists of 82 amino acids including a C-terminal, hydrophilic, positively charged patch enabling β2-GPI to bind to anionic surfaces, for example as activated blood cells.

18. No association with clinical manifestation of APS Sole LA positivity (negative aCL and aβ2GPI ) Pengo V, Thromb Haemost 2005; 93: 1147–52. de Groot PG J Thromb Haemost 2005; 3: 1993–7. A LAC without a positive anti-b2GPI or antiprothrombin test was not associated with a risk for DVT (OR 1.3, 95% CI: 0.3–6.0) Pengo V, Arterioscler Thromb Vasc Biol 2007;27: 309–10.

19. I II III IV V + + LAC + aCL - abeta2 - ?

20. Carriers of sole LA positivity Pengo V et al. Thromb Res 2014

21. No association with thrombosis Sole aCL positivity (negative LA and aβ2GPI ) Ruffatti A J Thromb Haemost 2008; 6: 1693–6. Runchey SS Br J Haematol 2002;119:1005–10. Proven A Mayo Clin Proc 2004;79:467–75.

22. IgG aCL titre and laboratory profile Ruffatti A, Pengo V et al. JTH 2008 Low aCL titre: higher probability that aCL is the only positive test

23. IgG aCL titre and clinical features Ruffatti A, Pengo V et al. JTH 2008 Low aCL titre: higher probability of association with pregnancy morbidity

24. Plate CL C4 Factor H LAC - aCL + abeta2 - I I IIIIII IVIV V + +

25. LAC - aCL - abeta2 + I II III IV V + + Plate Anti-Domain 4/5 antibodies have been detected in non-thrombotic conditions, like atherosclerosis, leprosy and in children with atopic dermatitis or those born to mothers with systemic autoimmune diseases.

26. IgG anti β-GPI/Domain 4/5 Pengo V et al. Thromb Res 2015

27. Lupus anticoagulant/ Anti-cardiolipin antibodies/ Anti-  2-glycoprotein I antibodies Thrombosis (N=340) no.(%) No thrombosis (N=278) no.(%) Odds Ratio univariat e 95% CIMultivariate ° 95% CI LA+/aCL+/ab2+ LA+/aCL-/ab2- LA-/aCL+/ab2+ LA-/aCL+*/ab2- LA-/aCL-/ab2+ 34 (10)2 (1)14.93.5-62.733.37.0-157.6 0 (0)5 (2)NA- - 18 (5)13 (5)1.20.6-2.52.21.0-5.2 7 (2)13 (5)0.50.2-1.20.80.3-2.1 4 (1) 0.90.2-3.51.30.3-5.7 Antiphospholipid antibody profiles as risk factors of thrombosis Antiphospholipid antibody profiles as risk factors of thrombosis * > 40 GPL/MPL V Pengo et al, 2005

28. Cumulative incidence of thromboembolic events in high risk triple positive APS patients (n=160) Pengo V, JTH 2010

29. Antiphospholipid profile and subsequent TE in obstetric APS LA positive, aCL and a  2GPI positive aCL and a  2GPI positive Ruffatti A, Pengo V et al. 2006

30. TRAPS Study Title: A prospective, randomized international trial comparing Rivaroxaban vs warfarin in high-risk patients with antiphospholipid syndrome (TRAPS). EudraCT: 2013- 004575-13 Author Prof. Vittorio Pengo Associate Professor of Cardiology vittorio.pengo@unipd.it tel./fax. +39.049.8215658 mobile +39.329.8324844 Primary Study Objective(s) The primary objective is to demonstrate the non-inferiority of Rivaroxaban 20 mg (or 15mgqd in case of moderate renal insufficiency) versus warfarin (INR 2.0-3.0) with respect to the occurrence of the cumulative end point of incident acute thrombosis (arterial or venous) confirmed by appropriate imaging studies, major bleedings, and death in triple aPL-positive APS patients.

31. Cumulative incidence of thromboembolic events in the follow up period of 104 carriers of triple positivity for antiphospholipid antibody tests. Pengo V, Blood 2011

32. Risk FactorThrombosis (N=25) Controls (N=79)Hazard Ratio Univariate95%CIMultivariate95% CI Age--yr42.4 ± 14.245.3 ± 14.71.00.97-1.02-- Male sex—no.(%)8 (32)14 (18)3.31.4-8.04.41.5-13.1 aCL titre (GPL) Units95 ± 8584 ± 911.00.99-1.01-- G isotype—no. (%)22 (88)64 (81)1.70.5-5.6-- Associated autoimmune diseases—no.(%) 10 (40)39 (49)0.60.2-1.1-- Arterial risk factors-- no. (%) 8 (32)26 (33)0.90.4-2.2-- Venous risk factors—no.(%)10 (40)12 (15)2.71.2-6.33.31.3-8.5 Aspirin prophylaxis— no.(%) 7 (28)30 (38)0.40.1-1.1-- Risk factors for thrombosis in the follow up period Pengo V, Blood 2011

33. Why triple positivity is unique? Answer: In contrast to single test positivity, triple positivity arises from the presence of a single (possible pathogenic) antibody

34. aβ 2 GPI + aCL + LAC + β 2 GPI Triple positive plasma passed through a beta2GPI affinity column Affinity purified abeta2GPI Spiked into normal plasma V.Pengo et al. Thromb Haemost 1999

35. aCL ELISA and LAC activity of affinity-purified anti human ß2-GPI autoantibodies from triple positive patients Affinity purified anti β2-glycoprotein I antibody preparations Control IgG 12345 Protein [µg/ml] 756139496834 β2GPI ELISA 0.0152.3782.6582.2972.2422.255 aCL ELISA0.0052.0812.0371.6451.8061.648 dRVVT ratio 0.951.61.31.61.41.5 V.Pengo et al. Thromb Haemost 1999

36. IgG antibodies that recognize epitope Gly40-Arg43 in domain I of 2- glycoprotein I cause LAC and their presence correlates strongly with thrombosis Bas de Laat et al., Blood 2004 I II II I IV V + + Phospholipids IgG aβ2GP1-Dm1

37. IgG aβ2GP1-Dm1 and risk categories Pengo V et al. (JTH 2015)

38. Association of positive IgG aβ2GP1-Dm1 with thrombosis Pengo V et al. (JTH 2015)

39. Animal models of APS Developing thrombus after laser injury in wild type mouse Anti-b2GPI antibodies used for these experiments express anti- cardiolipin, anti-b2GPI activity, and lupus anticoagulant activity measured by the dilute Russell’s viper venom time. + aβ2GPI - aβ2GPI

40. APS with triple positivity: An Autoimmune disease the corresponding Ag is known and the specific epitope is partially known an autoimmune reaction is identified in the form of autoantibody an analogous response causes a similar disease in experimental animals Witebsky, E., 1957 Rose, N. R. 1993,

41. IgG Anti Phosphatidylserine/Prothrombin

42. Which test (possibly in the same plasma sample) Lupus anticoagulant (dRVVT and an aPTT-based test) IgG and IgM anti-cardiolipin (aCL) antibodies IgG and IgM anti-β2GPI (aβ2GPI) antibodies IgG and IgM anti PS/PT: promising but undetermined significance Anti-β2GPI-Domain I and anti-β2GPI-Domain 4/5 could be useful IgA aCL and IgA anti-β2GPI: insufficient data

43. Summary: Definite thrombotic APS Triple positive patients (LAC positive, IgG or IgM aCL> 99 th percentile, IgG or IgM ab2GPI> 99th percentile) and proven venous/arterial thrombosis.

44. Definite thrombotic APS Remarks All the following reinforce the diagnosis of definite APS Young age (less than 50 years), unprovoked VTE or VTE in unusual site or in microcirculation, cryptogenic stroke IgG isotype, High titre of antibodies and strong LAC. Positive anti-β2GP1-Domain 1