Understanding trends in autoantibody testing through the lens of systemic lupus, celiac disease, and the antiphospholipid syndrome • Michael L. Astion, MD, PhD, HTBE • Medical Director, Dept of Pathology and Laboratories, Seattle Children’s Hospital Acknowledgements • Bio-Rad • Immunology Laboratory at the Univ. of Washington Dept. of Laboratory Medicine • Colleagues at Seattle Children’s Hospital • Mark Wener, M.D. • Kathy Hutchinson, M.S. Overview • Introduction to autoimmune diseases • Illustration of autoantibody testing principles using: • Systemic Lupus Erythematosus • Celiac Disease • Anti-phospholipid syndrome • Conclusions Autoimmune Diseases • Definition: Immunologic destruction of tissues by the body’s own immune system • Complex, interrelated, multi-factorial diseases with genetic and environmental components • About 80 autoimmune diseases have been described • • • • Common ones include autoimmune thyroid disease and celiac disease Most of the 80 are uncommon Some of the 80 are primary and some are secondary to other causes Patients with 1 autoimmune Dx are at higher risk for others. Autoimmune Diseases • Hard to Dx, sometimes taking years • Usually diagnosed by specialists • Tend to have patterns of flares and remissions • Often treated by suppressing the immune system Autoimmune diseases are associated ---in complex and varying ways--- with circulating autoantibodies. • Diagnosis 3 kinds of autoantibodies causing) • Those that are an effect of disease • Naturally occurring • Thus, interpretation of autoantibody testing is difficult! • Autoantibodies present before disease onset # Autoantibodies • Pathogenic (disease Arbuckle et al. Development of Autoantibodies before the clinical onset of Systemic Lupus Erythematosus. NEJM: 2003; 349:1526-1533 Autoimmune Disease: More than 80 diseases/syndromes that can be classified into 3 overlapping categories • Systemic Rheumatic Diseases • Organ Specific Diseases • an oversimplification as usually effects more than one organ • E.g. celiac disease, Hashimoto’s thyroiditis • Intermediate group associated with pulmonary – renal syndromes Systemic Rheumatic Diseases • Chronic, multi-organ inflammatory diseases; causes largely unknown. • Diagnosis based on: • Many clinical features, and • ANA testing • RF / Anti-CCP testing • Rheumatologic Dx is difficult Systemic Rheumatic Diseases • Systemic Lupus Erythematosus (SLE) • Sjögren’s Syndrome • Progressive Systemic Sclerosis • Dermatomyositis / Polymyositis • Mixed Connective Tissue Disease • Undifferentiated CTD • Rheumatoid Arthritis • (Tests = anti-CCP and Rheumatoid factor rather than the ANA test) SLE: the prototypical systemic rheumatic disease • Multisystem disease with variable presentation/ course. Often includes arthritis, rashes, and renal, neuropsychiatric disease. • Lab tests: • + ANA in 99% of cases • Brisk, broad autoantibody response; average of 3 of 7 commonly tested antibodies present at Dx • dsDNA, Sm, RNP, SSA, SSB, Ribosomal P, APL • Specific ANA markers include Sm and dsDNA • ↓complement in active disease American College of Rheumatology (ACR): 11 criteria for classification* of SLE 1. 2. 3. 4. 5. 6. 7. malar rash, discoid rash photosensitive rash oral ulcers arthritis (non-erosive) serositis renal disorder (proteinuria, casts) 8. Neurologic: seizures, psychosis 9. Hematologic disorder ( ↓WBCs, hemolytic anemia) 10. Lab test: +ANA 11. Lab test: + dsDNA or + Sm Alopecia in SLE: common but not diagnostic *www.rheumatology.org/publications/classification/index.asp?aud=mem • Google search for “joint pain” on January 19, 2013. • This is the # 1 listing (after the ads) • What tests might an insured “worried well” patient want? • “I’m waiting for my lab results.” Methods currently used in the UW Immunology Lab ANA screen (IFA) Anti-CCP: multiplex ANA follow-up testing: • ENA screen: Sm/RNP, Sm, SSA, SSB, (Multiplex) • dsDNA (EIA, multiplex) • RiboP, histones/chromatin, Scl-70, Jo-1 : multiplex) • Anti-centromere (IFA, multiplex. • PCNA (IFA, CIE) • Anti-phospholipids • Cardiolipins: IgG, IgA, IgM all (EIA) • ß2GP1(IgG, IgM): (EIA) Rheumatoid Factor: Nephelometry Anti-cytoplasmic antibody screen (IFA) Thyroid: TPO, thyroglobulin (EIA) ANCA: • IFA • MPO, PR-3 (IFA,multiplex) GBM: (multiplex) Celiac (Tissue Transglutaminase by EIA) Sensitivity of ANA-IFA for various conditions: • SLE: 88-99% • Sjogren’s Syndrome: 60-70% • PSS (Scleroderma): 60-70% • MCTD (>95%) • Normal adults < 60 years old: 5% (No way, more like 7% - 35%) Methods of ANA testing • ANA-IFA: ~ 65% of labs, the “? gold standard” • EIA: ~ 25% of labs, but > 40% of assays (volume labs often choose EIA) • Multiplexing (beads): ~ 10% of labs, but > 20% of assays as higher volume labs are switching to this. • Microarrays: currently for research only, not yet available for clinical autoantibody testing Pyrite (fool’s gold) ANA-IFA: a problematic test that is still very useful • • Subjectivity affects precision and accuracy High false + rate is common • Methods not standardized: • Substrates • Microscopes • Photobleaching • Time consuming • Fatiguing if done in high volume • Requires a commitment to training and competency assessment Photobleaching (right part of microscope field) ANA-IFA Results from “Experienced” Labs: Results from a Classic Study • Tan et al., Arthritis Rheum, 1997, 40:1601- 1611 • Methods: • 15 different labs testing 125 normal sera • each lab used their own HEp-2 IFA method: Normal sera were positive in 32% at • Authors’ recommendations: • Report all results at 1:40 and 1:80 • Report lab’s false + rate with the test result ANA-EIA: Types of Tests •Hep-2 nuclear extract (1st generation) •Hep-2 nuclear extract spiked with extra antigens (2nd and 3rd generation kits) •Recombinant antigens only Multiplexing: Flow cytometric immunoassay based on multiplexed fluorescent microspheres (beads). Multiple antibodies can be detected in 1 reaction using multiple antigen beads. Patient’s SSA antibody=Y; Detection antibody = Y SSB Sm RiboP Scl70 Sm Y Y RNP SSA SSA Control SSB RNP dsDNA CenB Scl70 dsDNA Anatomy of a bead used in the multiplex assay Illustrations courtesy of Bio-Rad laboratories, used by permission. Multiplex assay: The detection system counts many beads of each autoantibody specificity, and determines which autoantibodies are present in the patient’s serum. A particular ANA screen may have up to 13 autoantibody specificities depending on the manufacturer. Illustrations courtesy of Bio-Rad laboratories, used by permission. Multiplex testing is becoming part of the mix of methods for autoantibody testing • It is significantly impacting auto-antibody testing. • Multiplex instruments will become part of lab instrumentation used by many labs. • Automated • Multiple results from one sample • Allows consolidation of many assays onto 1 platform • Many assays now available, or soon will be available • vasculitis panel with anti-MPO, anti-PR3, anti-GBM • celiac IgA panel, celiac IgG panel • various infectious disease panels IFA vs EIA vs Multiplex Key points about ANA testing. There is more than 1 path to the top of the mountain. • No gold standard method • There are differences between methods (EIA, IFA, multiplex). and within a method (e.g., “multiplex” assays not the same.) • Assay components differ • Assay cutoffs differ • Assay quality is variable • Literature is confusing: • Methods are difficult to compare/contrast • Patient populations are different and have biases • It is easy to find patients negative by one method and positive by another. • You can’t make lab policy and procedure based on 1 or 2 patients. • Labs need more than 1 method available to them. • Clinicians should retest by the same and a different method when results do not match clinical findings. • When switching methods, communicate to care providers the differences between the new and old methods. Individualize the differences when possible!! • The ACR position on IFA is important but flawed. Should a lab implement a solid-phase assay (ANAEIA or multiplex)? • • A complex question; the answer depends on the lab. Clear Advantages of solid-phase: • objective measurement, fast, easy to automate • Clear Disadvantages of solid-phase assays: • can require MD re-education due to loss of pattern/titer • Am Coll of Rheumatology loves IFA. This matters. • Complex issues: • volume fatigue, cost, historical momentum, training, cutoffs for positive results, change management (especially with rheumatologists) Comparing / Contrasting: EIA vs Multiplexing • Multiplexing is the most automated method • Multiplexing requires the least amount of sample • EIAs based on HEp-2 cell extracts have a broader representation of antigens. Is this an advantage or disadvantage? • Complex issues: • cost, volume fatigue, historical momentum, training, cutoffs for positive results, change management with rheumatologists When the ANA-IFA, ANA-EIA, or ANA-multiplex screen is positive, what are some approaches to confirmatory testing? • Screen with IFA, EIA or multiplex assay • If (+), test (or release) panel of individual autoantibodies • • • • • • • • • • • • dsDNA Sm RNP SSA SSB Ribosomal P Scl-70 Centromere B Chromatin /Histones Jo-1 Anti-phospholipids More… Conservative vs aggressive ANA testing sequence using solid phase (EIA or multiplex) screen • Start with solid phase assay • If negative, report result and you are finished. • If positive • Conservative: Do ANA-IFA to get pattern and titer, then do follow-up testing for individual autoantibodies • Aggressive: NO pattern/titer. Just do follow-up testing. Send out ANA-IFA when needed. Multiplex testing: ANA testing algorithm • For confirmatory testing: • “Release” the results of the individual beads • Perform additional autoantibody tests if not covered by the multiplex ANA screen Celiac Disease: Mechanism • Gluten= wheat storage protein. It is eaten. • Gliadin = alcohol soluble fraction of wheat gluten • Gliadin + TTG in gut Deamidated gliadin Deamidated gliadin Immune rxn in those with disease Tissue injury in small bowel Symptoms Celiac Disease: Clinical features Diarrhea, Steatorrhea (fat malabsorption) Abdominal cramping, pain, and distention Vitamin, mineral deficiencies if symptoms are not treated Occasionally see anemia, arthritis, rash…  risk: other autoimmune diseases, intestinal lymphoma Celiac Disease: Diagnosis Gold standard: • Symptoms, abnormal small bowel biopsy (villous atrophy, lymphocytes) in response to gluten challenge • Research method, not done in practice • Celiac disease afflicts 0.5% - 1% of the population, but alternative practitioners think it is much higher. Actual method of Dx: • Symptoms + antibody tests on gluten-containing diet • If Ab tests are +, do small bowel biopsies (not just endoscopy). • Biopsy + = celiac disease if Ab tests and biopsy improve on gluten-free diet. Rubio-Tapia A et al. ACG Clinical Guideline…celiac disease. Am J. Gastroenterol. 2013; 108:656-676 Celiac Disease: Lab Testing Autoantibodies in Celiac Disease • IgA, Anti-tissue transglutaminase (ATTG). • IgG Anti-tissue transglutaminase • IgA, Anti-endomysial antibodies (AEMY) are directed against tissue transglutaminase, so equivalent to ATTG • IgA, Anti-deamidated gliadin peptide (DGP) • IgG, Anti-deamidated gliadin peptide (DGP) *Recommended first test is IgA ATTG • Sensitivity, Specificity of IgA, ATTG is ~95% *NIH Consensus Statement on Celiac Disease. NIH Consens State Sci Statements. 2004 Jun 28-30;21(1) 122. Rubio-Tapia A et al. ACG Clinical Guideline…celiac disease. Am J. Gastroenterol. 2013; 108:656-676 Evidence-based testing for celiac disease Rubio-Tapia A et al. ACG Clinical Guideline…celiac disease. Am J. Gastroenterol. 2013; 108:656-676 Current Laboratory Testing Methods for Celiac Disease • EIA or Multiplex methods • IgA-based detection • ATTG • DGP • IgA level • IgG based detection • ATTG • DGP • Advantage of multiplexing is that it requires less specimen. • IFA (monkey esophagus) for AEMY EIA Multiplex approaches to celiac disease • Start with a panel consisting • IgA (to detect IgA deficiency) • IgA, Anti-tissue transglutaminase • IgA, Anti-DGP • Only If IgA deficient, or child < 2 years, run kit for: • IgG, Anti-tissue transglutaminase • IgG, Anti-DGP Celiac disease: Treatment • Gluten-free diet for life (no wheat, barley, or rye) • Difficult Rx! (no birthday cake) • Thus, unwise to Dx this disease unless symptomatic • Also, treat Vitamin D deficiency and other nutritional problems caused by small bowel injury 2 kids enjoying birthday cake Celiac Disease: Monitoring Treatment Follow up titers: IgA, Anti-tissue transglutaminase 1 follow-up small bowel biopsy Individual values of IgA, Anti-tissue transglutaminase CS= controls CD = patients with celiac disease (CD), GFD = celiac disease patients on gluten-free diet. Basso D et al. J Pediatr Gastroenterol Nutr. 2006;43:613-618 Overbundling in autoantibody testing: Example, Celiac disease (CD) Guidelines: in vast majority of cases only 1 - 3 tests needed. • IgA anti-tissue transglutaminase or IgA anti-endomysial antibody • IgA level • IgA deamidated gliadin peptide Occasionally needed but not frontline tests • HLA DQ2, HLA DQ8 are rarely needed to rule out CD • IgG versions of autoantibody tests useful in IgA deficiency states *Rubio-Tapia A et al. ACG Clinical Guideline…celiac disease. Am J. Gastroenterol. 2013; 108:656-676 National Institute for Health and Clinical Excellence (NICE). Coeliac disease. Recognition and assessment of coeliac disease. London (UK): National Institute for Health and Clinical Excellence (NICE); 2009 May. 86 p. (Clinical guideline; no. 86). [87 references] NIH Consensus Statement on Celiac Disease. NIH Consens State Sci Statements. 2004 Jun 28-30;21(1) 1-22. Celiac overbundling from 1 specialty lab. In >95% of cases, 1 – 3 tests are needed, but 5-test bundles were used 98% of the time. • • • 3 insurance databases (2008, 2009) > 4 million members N= 760 had celiac testing from this specialty lab Average age 32 years (range 0 – 82) Most common diagnosis codes: Diarrhea (n=135); Abdominal pain (n=104), Celiac disease (n=82); flatulence (n =29), constipation (n=23). Total Amount spent on: • celiac autoantibody tests • Total IgA level related to celiac testing $195,215 Portion of the total spend accounted for by 5-test celiac panels consisting of •4 celiac autoantibody tests •Total IgA level related to celiac testing $192,025 % of celiac tests in 5 test bundles 98.4% Causes of unusual or unexplained venous thrombosis (venous thrombophilia) 1. Activated protein C resistance (30 - 40% of patients) 2. Increased Factor VIII activity (20 - 25%) 3. Prothrombin mutation (5 - 10%) 4. Protein C deficiency (3 - 5%) 5. Protein S deficiency (3 - 5%) 6. Antiphospholipid antibody syndrome (3 - 5%) 7. Antithrombin deficiency (1 - 3%) Antiphospholipid Syndrome (APS): Diagnosis requires at least 1 clinical and 1 laboratory criteria. Clinical criteria • Recurrent arterial or venous thrombosis such as stroke, DVT, pulmonary embolism… • Pregnancy loss or premature birth Lab criteria (Persistent, medium to high level results are key!) • Persistently positive lupus anticoagulant • 2 positive EIA tests (12 weeks apart) for anticardiolipin, IgG or IgM • 2 positive EIA tests (12 weeks apart) for anti-beta2GP1, IgG or IgM Miyakis S et al. International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS). J Thromb Haemost. 2006; 4(2):295-306. APS continued… Secondary APS is associated with systemic lupus. Primary APS is not. The Rx of APS is anticoagulation to prevent further thrombotic events. A patient with alopecia in SLE. This person is also at high risk for APS. The following lab tests are NOT currently included in APS classification*. Although associated with APS. they do not add (and might hinder) diagnostic power… • IgA, anti-cardiolipin • IgA, anti-beta2GP1 • Antibodies to: • Prothrombin • Phosphatidylserine • Phosphatidylserine-prothrombin complex • Phosphatidylethanolamine • Phosphatidylinositol *Miyakis S et al. International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS). J Thromb Haemost. 2006; 4(2):295-306. Keeling D. et al. British committee for standards in hematology. Guidelines…antiphospholipid syndrome. Br J Haematol 2012;157:47-58. Methods for APS testing EIA or Multiplexing for… •IgG, IgA, IgM anticardiolipin •IgG, IgA, IgM antibeta2GP1 •Advantage of multiplexing is that it requires less specimen and reagents. Univ. Washington test requisition Conclusions 1. No gold standard for ANA testing. Labs should be able to explain their ANA testing strategy –including potential problems-- to ordering physicians. Labs should have > 1 ANA methods available, and should encourage care providers to test by >1 method when test results do not match clinical findings. 2. The foundation of celiac disease testing is the IgA, anti-Tissue Glutaminase test. Celiac testing is the most interesting testing from a social perspective because it is underused and overused at the same time! 3. The foundation of testing for anti-phospholipid syndrome are IgG, and IgM assays for anti-cardiolipins and anti-beta2GP1, as well as testing for the lupus anticoagulant. Overall trends in autoantibody testing • More automation: • movement from IFA to EIA to multiplex flow cytometry and other automated methods • More assays: •  of EIA, ,multiplex tests will be available • Gradual progression: • Steady, slow  in ANA-IFA, but still a very useful test! • EIA holds steady • Steady, slow in multiplexing Thanks! •