UPDATE IN THE EVALUATION OF ANTIPHOSPHOLIPID ANTIBODIES SILVIA S. PIERANGELI, PhD LOUISVILLE APL DIAGNOSTICS, INC DORAVILLE, GA USA Louisville APL Diagnostics, Inc

Louisville APL Diagnostics n Problems with false positive results n aCL positive in a wide variety of infectious diseases and in non-APS related autoimmune diseases.

Louisville APL Diagnostics

ANTIPHOSPHOLIPID SYNDROME Diagnostic Testing n 1.) Laboratory confirmation is vital for diagnosis of APS. n 2.) Anticardiolipin antibody and lupus anticoagulant tests together diagnose vast majority of APS patients-these tests are relatively standard and well understood. n 3.) More specific test for APS should be developed and broadly tested (include anti-cardiolipin positive/APS negative sera in testing). n 4.) More specific tests may enable confirmation of diagnosis in equivocal and unusual clinical presentations to APS.

Louisville APL Diagnostics ANTIPHOSPHOLIPID SYNDROME Laboratory Tests n Tests should be positive in most patients with disorder (sensitivity). n Tests should be largely confined to patients with disorder (specificity) n Tests should be performed reproducibly in most laboratories.

Louisville APL Diagnostics APhL ® ELISA Kit - Principle n Based on observation that antiphospholipid antibodies crossreact with negatively charged phospholipids but syphilis and other infectious diseases sera largely limited to cardiolipin binding (no crossreactivity) n Construction of a kit with negatively charged phospholipids might eliminate non-specific binding.

Louisville APL Diagnostics ANTI-CARDIOLIPIN ANTIBODIES n BINDS n Cardiolipin n Phosphatidylserine n Phosphatidylglycerol n Phosphatidic Acid n DOES NOT BIND n Phosphatidylcholine n ?phosphatidylethanolamine

Louisville APL Diagnostics Sensitivity of the assays Relative Sensitivity/Specificity

Louisville APL Diagnostics Prevalence of aCL, APL and anti-  2 GPI in various infectious diseases

Louisville APL Diagnostics Evaluation of different assays to diagnose APS: Multicenter study. n Samples tested: n APS=56 n Healthy controls=145 n Non-APS including infectious diseases (syphilis, HIV, other autoimmune diseases)= 206.

Louisville APL Diagnostics Evaluation of different assays to diagnose APS. n *Centers involved and assays utilized: n Univ Texas San Antonio: APhL ® ELISA test. Morehouse School of Medicine: aCL (in-house) and anti-  2 GPI (INOVA) Morehouse School of Medicine: aCL (in-house) and anti-  2 GPI (INOVA) University of Milan: in-house anti-  2 GPI and anti-prothrombin. University of Milan: in-house anti-  2 GPI and anti-prothrombin. n * participating centers were blind to the identity of the samples n Univ of Utah: Coordinating center.

Louisville APL Diagnostics Sensitivity and Specificity of the assays Assay APS samples # positives (sensitivity) Healthy controls # positives Other diseases # positives Non-APS combined # positives Specificity Positive Predictive value aCL(in-house) 42/56 (75%) 1/150 (0.6%) 27/206 (13%) 28/356 (7.8%) 91.8%59.1% a   GPI (INOVA) 44/56 (78.5%) 15/143 (10.5%) 58/197 (29.4%) 73/340 (21.4%) 78.6%37.6% aPT (in- house) 13/54 (24%) 15/132 (11.3%) 19/178 (10.7%) 34/310 (10.9%) 88.4%25.4% a   GP(in- house) 43/54(79.6%)13/145(8.9%) 9/192 (4.6%) 22/337 (6.5%) 93.2%65.1% APhL ELISA kit 45/56 (80.3%) 4/147 (2.7%) 14/204 (6.8%) 18/351 (5.1%) 94.0%68.1%

Louisville APL Diagnostics Evaluation of CAP survey samples in FDA aCL approved assays Sample Kit 1 Kit 2 Kit 3 APhLResult 99-4negnegnegnegneg >200>200pos 00-1negnegnegnegneg 00-2negnegnegnegneg pos pos 01-5negnegnegnegneg pos 02-1negnegnegnegneg 02-2negnegnegnegneg 02-3negnegnegnegNeg

Louisville APL Diagnostics CAP survey results: IgG APhL ELISA

Louisville APL Diagnostics CAP survey: IgM APhL

Louisville APL Diagnostics APhL ® ELISA kit n Six pre-diluted calibrators (ready to use) n 3 x 30 minutes incubation steps n Peroxidase and alkaline phosphatase systems available n All other reagents in “ready-to-use” form n Determination of IgG and IgM aPL antibodies n 12 month expiration date. n Good to be used in automated systems.

Louisville APL Diagnostics APhL ® ELISA kit n Antigen composed fo mixture of phospholipids - ß 2 GP1 n Sensitivity of APS (greater than 90%) n More specific than anticardiolipin test and at least as specific (or more) compared to anti-ß 2 GP1 n Incorporation of an in-house positive control n Can be utilized for first line testing, and certainly important in confirmation of APS

Louisville APL Diagnostics Conclusion n Since sensitivity of APhL ® ELISA kit is comparable to anticardiolipin test, it can be used for first line testing in place of the anticardiolipin test. The APhL ® ELISA kit will enable greater specificity - APS diagnosis

Clinical Features of APS Anticardiolipin (IgG, IgM) and Lupus anticoagulant tests (APhL ELISA ® test) Negative Anticardiolipin and Negative Lupus anticoagulant but Highly Suggestive Clinical Features Low Positive Anticardiolipin High/Medium Positive Anticardiolipin or Positive Lupus anticoagulant anti-  2 GP1 or APhL ELISA ® Test NegativePositive aCL Antiphospholipid Syndrome Confirmation with anti-  2 GP1 or APhL ELISA ® test Both NegativeEither Positive Consider IgA Anticardiolipin Antiphospholipid Syndrome Repeat test NegativePositive Antiphospholipid Syndrome

Louisville APL Diagnostics Anticardiolipin ELISA publications n Harris EN. Annotation: antiphospholipid antibodies. Br J Haematology. 1990; 74:1-9 n Harris EN, Pierangeli SS. Anticardiolipin and lupus anticoagulant testing and significance. J Clin Immunol 1994; 17:1-8. n Harris EN, Pierangeli SS, Birch D. Anticardiolipin wet workshop report: Vth International Symposium on Antiphospholipid antibdies. AM J Clin Pathol 1994: 101: n Harris EN, Pierangeli SS. “Equivocal” Antiphospholipid Syndrome. J Autoimm. 2000; 15: n Pierangeli SS, Gharavi AE, Harris EN. Testing for antiphospholipid antibodies: problems and solutions. Clin Obstet Gynecol 2001:44: n Harris EN, Pierangeli SS. Revisiting the anticardiolipin test and its standardization. Lupus 2002; 11: n Pierangeli SS, Harris EN. Anticardiolipin testing. Clin Chim Acta. 2005;

Louisville APL Diagnostics ELISAs for IgG and IgM aPL

Louisville APL Diagnostics ELISAs for IgG or IgM aPL

Louisville APL Diagnostics ELISAs for IgA aPL

Louisville APL Diagnostics Louisville APL Diagnostics, Inc web site:

Louisville APL Diagnostics Silvia S. Pierangeli, Ph.D. Silvia S. Pierangeli, Ph.D.