, Antiphospholipid (APL) Antibodies & APL Syndrome: Review & Update William L. Nichols, MD Mayo Clinic College of Medicine Rochester, Minnesota USA Disclosures & Objectives (Nichols) Disclosures Relevant Financial Relationships: NONE Off-Label Drug Usage: NONE Objectives Understand laboratory methods & criteria for LA & APL Ab screen and confirmation Review criteria for APL syndrome Dx Appreciate anticoagulation management issues for APL syndrome OUTLINE TERMINOLOGY Background (Definitions, History) Laboratory testing & issues (LA & APL Abs) Diagnostic criteria (Clinical, Laboratory) Illustrative case example Anticoagulation management & issues Summary and recommendations Term ACL APL APS LA Name Anticardiolipin antibodies Antiphospholipid antibodies Antiphospholipid syndrome Lupus anticoagulant Definition Antibodies (IgG, M, A) to cardiolipin (+/- β2gpi), detected by ELISA Antibodies to phospholipid and protein cofactor antigens (eg, β2gpi, prothrombin, etc.) APL (persistent) and thrombosis or recurrent fetal loss (unexplained by other causes or diagnoses) APL, detected and confirmed by phospholipid- dependent clot-based testing HISTORY Biologic false positive syphillis serology testing since 1938 (reagent contains cardiolipin) - association with SLE 1948-52: Conley & Hartman describe circulating anticoagulants in SLE 1963: Bowie et al. define thrombosis association with LA in SLE patients Feinstein & Rapaport coined name Lupus Anticoagulant (1972) HISTORY - 2 1975: Nilsson identified association of LA with spontaneous fetal loss 1983: Harris et al. described solid phase radioimmunoassay using cardiolipin as antigen -> ELISA test for ACL antibodies 1983-87ff: Concept of Antiphospholipid Antibody Syndrome proposed by Graham Hughes et al. (London) and supported by evolving studies from many investigators 1
Bowie EJW et al. J Lab Clin Med 1963;62:416-430 430 Lupus Anticoagulant (LA) DEFINITION Lupus anticoagulants (LA) are antiphospholipid antibodies (IgG, IgM, IgA?) Directed against antigens comprised of a combination of anionic (negatively charged) phospholipids and a protein cofactor (eg, prothrombin or β-2- glycoprotein I [β2gpi]) Inhibit in vitro phospholipid-dependent clotting time tests (eg, aptt, drvvt, KCT, PCT [PRP recalcification], dpt) Heterogeneous antibodies (each pt unique) Antiphospholipid Antibodies (APL Abs) CLASSIFICATION Identified by functional coagulation testing Qualitative, interpretive Anticardiolipin Antibodies (ACL) Identified by immunoassays (eg, ELISA) IgG, IgM, IgA(?) isotyping & titering LA & ACL testing must both be performed to identify or exclude APA LA +, ACL + LA-, ACL+ LA +, ACL - LA-, ACL- Antiphospholipid Antibodies (APL Abs) CLASSIFICATION Autoantibodies (persistent) Primary Anti-phospholipid Syndrome Secondary APS (eg, SLE or other AI Dz) Alloantibodies (transient) Infections Inflammation Drug-induced APL Antibodies Procainamide, Quinidine, Quinine Phenothiazines Antibiotics 2
Antiphospholipid Antibodies (APL Abs) SIGNIFICANCE Thrombosis risk LA ACL IgG > ACL IgM Pregnancy loss or complications Bleeding risk (uncommon) LA / hypoprothrombinemia (or F X def.) Thrombocytopenia (AITP) Anticoagulation -related bleeding Cardiac valvulopathy, livedo, skin ulcers Asymptomatic Antiphospholipid Antibody Syndrome DIAGNOSTIC CRITERIA (Definite APL Ab Syndrome) Clinical Criteria* Vascular Thrombosis (Arterial, Venous) &/OR Pregnancy Morbidity 1 fetal death 10 weeks 3 spontaneous abortions <10 weeks 1 premature birth 34 weeks Placental insufficiency, Severe pre-eclampsia Laboratory Criteria (persistent 12 wks) ACL / B2GP1 Abs (IgG, IgM) mod.- high titer &/OR Lupus anticoagulant (by ISTH criteria) *Clinical events not otherwise readily explained Wilson WA et al. Arth Rheum 1999;42:1309-1311 1311 Miyakis S et al. J Thromb Haemost 2005;4:295-306 Lupus Anticoagulant (LA) ISTH Diagnostic Criteria (1995) 1. Prolongation of at least 1 phospholipiddependent clotting time assay. 2. Inhibition shown by mixing patient and normal pooled plasma. 3. Phospholipid-dependent inhibition demonstrated. 4. Evaluate for other coagulopathies Lupus Anticoagulant (LA) Other Diagnostic Recommendations Two or more tests should be used to screen for LA. Inhibition and PL-dependence must be shown using the same method giving an abnormal screening assay. Both patient and normal plasma should be as platelet-free as possible. Routine clotting tests, such as PT & APTT, should be performed to evaluate for other coagulation disorders that may interfere with LA testing. Factor assays should be performed if factor deficiency or factor inhibitor is suspected. Brandt JT, Triplett DA, Alving B, Scharrer I. Thromb Haemost 1995;74(4):1185-1190. 1190. Brandt JT, Triplett DA, Alving B, Scharrer I. Thromb Haemost 1995;74(4):1185-1190. 1190. Lupus Anticoagulant Testing WHAT TESTS ARE AVAILABLE? APTT (activated partial thromboplastin time) DRVVT (dilute Russell s viper venom time) KCT (kaolin clot time) PCT (plasma clot time) STACLOT-LA (hexagonal phase phospholipid) DPT (dilute prothombin time) Textarin-Ecarin Time (venoms) Lupus Anticoagulant Testing Screening, Mixing & Confirmatory Tests SCREENING TEST APTT DRVVT Screen STACLOT-LA (Integral Mix) MIXING TEST (INHIBITION) Mix w. NPP (1:1, 4:1, etc.) DRVVT Mix (1:1) CONFIRM TEST PNP (Pltlt Neutral.) DRVVT Confirm STACLOT- HEX 3
Activated Partial Dilute Russell Thromboplastin Prothrombin Time Viper Venom Time Time ( APTT ) ( DRVVT ) Is result within Is result within Is result within normal range? normal range? normal range? APTT Mixing Test Thrombin PT Mixing Test Thrombin DRVVT Mixing ( 1 :1 mix with Time ( 1 : 1 mix with Time Test ( 1 : 1 mix with normal plasma ) normal plasma ) normal plasma ) Does result Does result correct correct Is TT sufficiently? sufficiently? rmal? Reptilase Time Platelet DRVVT Neutralization Confirmatory Procedure ( PNP ) Testing Mayo LA Testing Algorithm Is further clarification or confirmation needed? Yes Staclot LA, Factor Assays, Factor Inhibitors Screen and Titer, Clinical information, Interpretation of all results is performed. Variables Affecting Sensitivity & Specificity of LA Testing (partial listing) Pre-analytical (specimen & patient conditions) Residual platelets in frozen-thawed plasma Anticoagulants (warfarin, heparin, DTIs) Analytical (testing conditions) Reagent sensitivity to LA Phospholipid composition & concentration Activator (silica, ellagic acid, kaolin, etc.) Reference range cutoffs (validation) rmal plasma for mixing ( platelet-free ) Post-analytical (interpretation & reporting) Interpretation of all results is performed. Clinical History 19 yr-old woman presents with several day history of pleurisy and increasing dyspnea General health excellent; taking OCP x 6 mo. Family history: Mother & older sister - SLE Physical exam: right pleural rub, appears SOB CT angio exam: c/w with multifocal PEs Leg ultrasound exam negative bilaterally Assessment: Acute PEs assoc. with OCP use Thrombophilia test panel obtained Hospitalized for anticoagulation, oxygen, etc. Thrombophilia Test Panel Factor V Leiden (APC-R): Prothrombin G20210A: Antithrombin activity: Protein C activity: Protein S antigen (free): Plasminogen activity: Fibrinogen level: D-Dimer level: Cardiolipin Abs (G & M) Negative Negative rmal rmal rmal rmal Mildly increased Marked increase IgG mod. titer Lupus Anticoagulant Test Panel PT 14.0 sec (8.4-12 sec) PT mix 1:1 13.0 sec APTT 65 sec (21-33 sec) 1:1 mix 50 sec PNP 46 sec PNP buffer 56 sec drvvt 70 sec (ratio 2.0) (<1.2 ) 1:1 mix 55 sec (ratio 1.6) (<1.2 ) Confirm 47 sec (ratio 1.5) (<1.2 ) STACLOT LA 100 sec (>60-70 sec) STACLOT HEX D 20 sec (D < 8 sec) TT 18 sec (16-25 sec) LA Reflexive Test Panel PT Factor Assays: II = normal V = normal VII = normal X = normal Overall Interpretation: Moderately strong LA, confirmed by APTT/PNP, DRVVT & Staclot-LA, also inhibiting the PT, with no factor deficiency Conclusion concerning PT: Mildly prolonged PT (14s) likely reflects LA inhibition 4
Heparin A/C monitoring (prolonged APTT)? APTT?; Heparin assay (anti-xa)? Heparin A/C monitoring (prolonged APTT)? APTT; Heparin assay (anti-xa) LMW heparin - no monitoring (usually) 5
Heparin A/C monitoring (prolonged APTT)? APTT; Heparin assay (anti-xa) LMW heparin - no monitoring (usually) Warfarin A/C target INR & monitoring? INR 2-3; INR 2.5-3.5; INR 3-4 PT/INR; F II or X; Chromogenic X Heparin A/C monitoring (prolonged APTT)? APTT; Heparin assay (anti-xa) LMW heparin - no monitoring (usually) Warfarin A/C target INR & monitoring? INR 2-3; INR 2.5-3.5; INR 3-4 PT/INR; F II or X; Chromogenic X ISSUES Target INR for pts with LA & Thrombosis? Standard Intensity INR (2.0-3.0)? Higher Intensity INR (e.g., 2.5-3.5)? Some LA inhibit some PT reagents (false INR) Depends on the specific pt. LA (heterogeneous) Depends on the thromboplastin used for INR How frequently does this occur? How to recognize? Target INR for pts with LA & Thrombosis? Higher INR (2.5-3.5 or 3.0-4.5) Khamashta MA et al. NEJM 1995;332:993 (retrospective) Rosove MH et al. Ann Int Med 1992;117:303 (retrospective) Ruiz-Irastorza G et al. Arch Intern Med 2002;162:1164 Standard INR (2.0-3.0) Ginsburg JS et al. Blood 1995;86:3685 (prospective) Crowther MA et al. NEJM 2003;349:1133 (prospective) Conclusions (Pending more data / studies!) Target INR 2.0-3.0 appropriate for most LA patients with thrombosis (?) Exceptions, and how to identify? Some LA can inhibit some PT reagents (false INR) Frequently? Moll S & Ortel TL. Ann Inten Med 1997;127:177 Robert A et al. Thromb Haemost 1998;80:99 Infrequently? Lawrie AS et al. Br J Haematol 1997;98:887 Tripodi A et al. Br J Haematol 2001;115:672 Conclusions (Preliminary; more data needed) Sensitive thromboplastins (low ISI) more likely to be inhibited by LA Important to evaluate specific instrument / reagent combinations, and INR calibration International normalized ratios of 16 patients (designated by L) who had lupus anticoagulants and were receiving warfarin and 10 patients (designated by N) who did not have lupus anticoagulants and were receiving warfarin Moll, S. et. al. Ann Intern Med 1997;127:177-185 6
Tripodi A et al. Laboratory control of oral anticoagulant treatment by the INR system inpatients with antiphospho-lipid syndrome and lupus anticoagulant: Results of a collaborative study of 9 thromboplastins. BJH 2001;115:672 Factor II assay (clot-based)* Factor X assay (clot-based) Factor X assay (chromogenic substrate)** Prothrombin-Proconvertin (P & P) clot time Other: (e.g., select a different PT reagent) *F II assay currently used @ Mayo **F X chromogenic assay - evolving availability Factor II assay (clot-based) F II: longest biological half-life (48-96 hrs.) F II levels more stable (vs. F VII) w. warfarin INR influenced most by F VII, least by F II F II assay relatively available F II level correlates best with thrombosis protection or risk? F II assay can be inhibited by some LA F II therapeutic level : INR 2.0-3.0: 20-30% (15-35%) Correlate F II & PT; then follow PT Half-Lives for Vitamin K-Dependent K Factors 100 90 80 70 60 50 Plasmatic concentration 40 of factors VII, IX, X, II (% 30 activity) 25 20 10 VII Time to 50% activity Mean Range rate of (hr) (%/hr) Factor VII = 4-6 10.5 Factor IX = 10-30 2.3 Factor X = 40-60 1.1 Factor II = 48-96 0.8 IX X II Lines shown are mean values 0 20 40 60 80 100 120 140 160 180 200 220 240 Hours after warfarin administration [Modified from Frank Kazmier PhD Thesis, University of Minnesota, 1966] William R. Swaim,, MD, May 18, 1993 Therapeutic range for warfarin Day 1 2 3 4 5 6 7 8 9 10 CP1098122-9 Factor X assay (clot-based) F X has relatively long biological half life (40-60 hrs) F X levels relatively stable w. warfarin Rx (like F II) F X assay relatively available F X assay can be inhibited by some LA F X level may correlate with thrombosis protection? F X level typically lower than F II w. warfarin (why?) F X therapeutic level = 8-20%? for INR 2.0-3.5? Small differences in F X activity more difficult to assay accurately? Factor X assay (Chromogenic Substrate) RVV activation of X => Xa Chromogenic substrate cleavage by Xa => color phospholipid Assay less available t FDA approved for diagnostic testing More expensive than clot-based F X assay F X therapeutic level = 15-40%? for INR 2.0-3.5? (Few data) 7
Tripodi A et al. Laboratory control of oral anticoagulant treatment by the INR system inpatients with antiphospho-lipid syndrome and lupus anticoagulant: Results of a collaborative study of 9 thromboplastins. BJH 2001;115:672 Prothrombin-Proconvertin (P &P) clot time Thromboplastin also contains bovine fibrinogen and factor V (and phospholipid, calcium, tissue factor) Test dilutions (1:10 & serial) - pt. & normal pool Derive regression of clot time vs. % normal Therapeutic range (INR 2.0-3.5) = 10-30% (?) Assay used somewhat in Europe, but not USA Limited sources of reagents in USA Therapeutic and nontherapeutic results obtained by using the prothrombin-proconvertin time, chromogenic factor X, and factor II assays and by using international normalized ratios Moll, S. et. al. Ann Intern Med 1997;127:177-185 Summary & Recommendations LA & ACL positivity is relatively frequent Titer or degree of positivity important, as is Ig isotype Important to evaluate persistance of positivity (>3 mo.) Laboratory evaluation is complex; standardization issues, etc. Numerous pre-analytical and analytical variables (pt, sample) Know your laboratory s quality - (select a good one) t all LA or ACL are pro-thrombotic; no predictive tests yet Rarely, bleeding can occur with LA (II or X deficiency, etc.) APL Syndrome - diagnosis can be challenging Thrombotic events &/or fetal losses, otherwise uneplained Persistently significantly positive LA or ACL (B2GP1) Anticoagulation management can be difficult Selected References Brandt JT et al. Criteria for diagnosis of lupus anticoagulants: update (ISTH SSC). Thromb Haemost 1995;74:1185-1190 Greaves M et al. Guidelines for diagnosis & management of antiphospholipid syndrome. Br J Haematol 2000;109:704-715 Triplett DA. Antiphospholipid antibodies (CAP consensus conference). Arch Pathol Lab Med 2002;126:1424-1429 Galli M et al. Lupus anticoagulants stronger thrombosis risk than anticardiolipin: meta-analysis. Blood 2003;101:1827-1832 Crowther M et al. Comparison of 2 warfarin intensities in APL syndrome. N Engl J Med 2003;349:1133-1138 Proven A et al. Clinical importance of positive LA & ACL tests. Mayo Clin Proc 2004;l79:467-475 Miyakis S et al. International consensus update: criteria for antiphospholipid syndrome. J Thromb Haemost 2006;4:295-306 8