Choosing the Correct ANA Technology for your Laboratory

1. Choosing the Correct ANA Technology for your Laboratory • Michael L. Astion, MD, PhD, HTBE • Professor and Director of Reference Laboratory Services • University of Washington • Dept of Laboratory Medicine

2. University of Washington Acknowledgements • Bio-Rad including Ginger Weedin, Steve Binder • Immunology Laboratory at the Univ. of Washington Dept. of Laboratory Medicine • Mark Wener, M.D. • Kathy Hutchinson, M.S.

3. Astion: Biases, Disclosures Univ Washington: • Director, Reference Laboratories • Associate Director: Immunology, Chemistry, Medical Informatics • Intellectual property holder: • The “Tutor” series including ANA-Tutor, ANCA-Tutor… (Medical Training Solutions) • Grants: Optimizing use of lab tests (CareCore National Insurance Co) Consultant: • Bio-Rad, since 1992 when ANA-Tutor was 1st launched. • Various non-profit hospitals 1992: Two happy faculty celebrate launch of ANA-Tutor 2006: Bald guy discussing lab errors on UWTV

4. Overview • ANA: Am Coll Rheum vs clinical lab perspective • Review of selected systemic rheumatic diseases • ANA methods for screening and follow-up testing: IFA, EIA and multiplexing • ANA testing algorithms: dsDNA, ENA, and the others • Testing in Rheumatoid Arthritis (IgM-RF, Anti-CC) • Specialist vs. primary care perspectives on systemic rheumatic disease testing • Conclusions

5. Perspective of the Amer Coll RheumatologyACR position statement on ANA testing, February 2009www.rheumatology.org/publications/position/ana_position_stmt.asp?aud=mem • IFA-ANA test should remain the gold standard for ANA testing. • Labs using multiplex or EIA assays for ANAs must provide data to ordering physicians on request that their assay has the same or improved sensitivity and specificity compared to the IFA ANA. • In-house assays for ANA, anti-DNA, anti-Sm…should be standardized according to CDC or WHO standards. • Report your ANA method when reporting results.

6. ANA testing: Clinical Lab Perspective • No gold standard for ANA testing. • Labs should be able to explain their ANA testing strategy –including problems-- to ordering physicians. • ANA testing is one of the least standardized areas of lab testing, for example different IVD kits for the “same” antigen(s) vary significantly. Modest standardization remains a goal. • At the care provider’s request, labs should describe their ANA methods. • 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.

7. Binder SR. Autoantibody detection using multiplex technologies. Lupus. 2006;15: 412–421.

8. Systemic Rheumatic Diseases • Chronic, multi-organ inflammatory diseases; causes largely unknown. • Autoantibodies (antibodies against “self” antigens) present. Some damage is immune - mediated. • Diagnosis is based on: • Many clinical features, and • Antinuclear antibody (ANA) testing • RF, Anti-CCP testing

9. 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

10. SLE: As regards ANA testing, it is the prototypical systemic rheumatic disease • Multisystem disease with variable presentation and course. Manifestations include arthritis, skin rashes, renal disease, neuropsychiatric disease. • Lab tests: • + ANA in >90% of cases on initial presentation • Brisk, broad autoantibody response; average of 3 of 7 commonly tested antibodies present at Dx • dsDNA, Sm, RNP, SSA, SSB, Ribo P, antiphospholipid… • Specific ANA markers include Sm and dsDNA/chromatin • Decreased complement in active disease

11. American College of Rheumatology (ACR):11 criteria for classification* of SLE • malar rash, 2. discoid rash, 3. photosensitive rash • oral ulcers • arthritis (non-erosive) • serositis • renal disorder (proteinuria, casts) • Neurologic: seizures, psychosis • Hematologic disorder ( ↓WBCs, hemolytic anemia) • Lab test: +ANA or +ACL • Lab test: + dsDNA or + Sm Alopecia in SLE: common but not diagnostic *www.rheumatology.org/publications/classification/index.asp?aud=mem

12. Sjögren’s syndrome (SS) • Characteristic triad: • dry eyes, • dry mouth • arthritis / arthralgia • Pathology: lymphocytic infiltrates in exocrine glands • Lab tests: • + ANA • + SSA • + SSB • + Rheumatoid Factor in 50 - 90% of cases Salivary gland enlargement

13. Progressive Systemic Sclerosis (Scleroderma) • Signs/ symptoms due to fibrosis • Variants: • Diffuse = bad prognosis • CREST = more limited form) • Lab tests: + ANA (nucleolar or centromere) + anti-Scl70 in the diffuse variant + anti-centromere in CREST + anti-nucleolar antibodies (e.g. RNA polymerase, fibrillarin), but not commonly used clinically. Upper: Digital pitting scars Lower: Raynaud’s

14. Main teaching points about systemic rheumatic diseases • Most Dx are based on an ACR “laundry list” and lab testing is only one component of the Dx. • ANA test: • Screens for most, but not all, of the diseases. • Confirmatory testing beyond the ANA screen is often (but not always) a part of Dx, Rx, and prognosis. • Autoantibodies are + before clinical onset and Dx • The disease symptoms (e.g. fatigue, hair loss) are common but the diseases are not. This leads to over testing.

15. 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%)

16. Autoantibodies occur before clinical onset of disease Diagnosis This is true in: • SLE • Rheumatoid Arthritis (e.g. anti-CCP) • SjÖgren’s syndrome • Antiphospholipid Syndrome • Type 1 diabetes • Probably many others # Autoantibodies Time before Dx (years) Arbuckle et al. Development of Autoantibodies before the clinical onset of Systemic Lupus Erythematosus. NEJM: 2003; 349:1526-1533

17. 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

18. ANA screen (IFA) ANA follow-up testing: ENA screen: Sm/RNP, Sm, SSA, SSB, (Multiplex) RiboP, Chromatin (Multiplex) dsDNA (multiplex and EIA) Scl-70, Jo-1 : (EIA, switching to multiplex) Anti-centromere (IFA and CEN B by multiplex) PCNA (IFA, CIE) Anti-phospholipids Cardiolipins: IgG, IgA, IgM all (EIA) ß2GP1(IgG, IgM): (EIA) Anti-cytoplasmic antibody screen (IFA) Thyroid: TPO, thyroglobulin (EIA) ANCA: IFA MPO, PR-3 (multiplex) GBM: multiplex Sendouts Histones Myositis autoantibodies (Mi-2, PL-7, PL-12 and 9 others) RNA Polymerase I, II, III Methods currently used in the UW Immunology Lab

19. Microscope-based ANA-IFA(Cultured HEp-2 cells are the substrate for antibody binding)

20. ANA-IFA: two criteria for a positive test:1) intensity > endpoint control, 2)identifiable staining pattern +, Homogeneous pattern Negative End-point Control

21. ANA-IFA patterns loosely correlate with specific antibodies and diagnoses Homogeneous Speckled Centromere Nucleolar

22. ANA-IFA titers 1:80 1:40 • The higher the titer the more likely the + ANA test is clinically significant. • In patients with disease, higher titers suggest more active disease. 1:160 1:320 1:640 control

23. ANA-IFA: a problematic test • Subjective: poor precision, poor accuracy • High false + rate • Methods not standardized: • Substrates • Microscopes • Photobleaching • Time consuming • Fatiguing • Intense training and competency assessment Photobleaching (right part of microscope field)

24. ANA-IFA results from “experienced” labs: • 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. • Results: Normal sera were positive in 32% at 1:40; 13% at 1:80, and 5% at 1:160 • Authors’ recommendations: • Report all results at 1:40 and 1:80 • Report lab’s false + rate with the test result

25. 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

26. Scl70 Sm Y Y dsDNA SSB SSA CenB RiboP Control RNP SSB Scl70 Y Y RNP Sm dsDNA SSA 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

27. Anatomy of a bead used in the multiplex assay Illustrations courtesy of Bio-Rad laboratories, used by permission.

28. 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.

29. Multiplex testing: future • 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 panel with IgA tissue transglutaminase, IgA • various infectious disease panels

30. IFA vs. EIA vs. Multiplex

31. 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 flawed and unrealistic. Key points about ANA testing

32. ANA-IFA vs. ANA-EIA vs. ANA-Multiplex: A sampling of the large and expanding literature EIA vs. IFA • Carey JL. Clin Lab Med, 1997, 17: 355-365 • Emlen W et al. Arthritis Rheum, 1997, 40: 1612-1618 • Homburger HA et al. Arch Pathol Lab Med, 1998, 122: 993-999 • Russell AS et al. Clinical Exp Rheum, 2003, 21: 477-480 • Tonuttia E et al. Autoimmunity,2004, 37: 171-176 • … Multiplexing • Shovman O. et al. Ann N Y Acad Sci. 2005;1050:380-8. • Zandman-Goddard et al. Clin Dev Immunol. 2005;12:107-111. • Nifli A. et al. J Immunol Methods. 2006;311:189-197. • Desplat-Jego S. et al. Ann N Y Acad Sci. 2007;245-255. • Bardin N. et al. Autoimmunity. 2009;42:63-68.

33. Fact: A lab can successfully switch to a solid-phase assay for ANA screening and confirmatory testing. The keys to success are: • Understanding the performance characteristics of the new vs. old method as performed by your lab. Communicating this understanding to care providers. • Encourage rheumatologists to test by more than 1 method in difficult cases. • dsDNA…

34. dsDNA assays: a difficult area • A pathogenic autoantibody • dsDNA is a specific test for SLE, part of ACR classification criteria • disease severity marker in SLE patients with nephritis • dsDNA Assays •     Farr assay •     EIA - regular (primary method used at UW) •     EIA – high salt (primary method used at UW) •     Crithidia • Multiplex (secondary method at UW) • Different dsDNA assays do not measure exactly the same thing. • It is useful to have >1 assay available for problem cases. • Best to follow a dsDNA+ patient by comparing results within the same method. • If switching methods, establish patient-specific baselines using the new method.

35. Should a lab implement a solid-phase assay (ANA-EIA 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 • Complex issues: • volume fatigue, cost, historical momentum, training, cutoffs for positive results, change management (especially with rheumatologists)

36. 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

37. When the ANA-IFA, ANA-EIA, or ANA-multiplex screen is positive, what are some approaches to confirmatory testing? • Doc chooses the specific autoantibodies to test • Lab offers “disease” panels, e.g. SLE panel • E.g, Scleroderma panel would include Scl-70 and Centromere B • SLE panel would include multiple autoantibodies (ENA, dsDNA, antiphospholipid, RiboP, etc) • Create specific panels for individual physicians.

38. Screen with IFA, EIA or multiplex assay If (+), test (or release) panel of individual autoantibodies Panels customized for particular clients UW standard panel: dsDNA, SSA, SSB, Sm, RNP UW custom panels also include Scl70, Jo-1, complement, others dsDNA Sm RNP SSA SSB Ribosomal P Scl-70 Centromere B Chromatin /Histones Jo-1 Anti-phospholipids More… ANA test sequence

39. Conservative 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 • Do ANA-IFA to get pattern and titer • Do follow-up testing for individual autoantibodies

40. Aggressive ANA testing sequence using solid phase assay (EIA or multiplex) • Start with solid phase ANA assay • If negative, report result and you are finished. • If positive, do individual autoantibody identification. • Do not do a HEp-2 pattern and titer. • This means physicians do not get “their pattern”.

41. 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

42. Am Coll Rheum Criteria for Rheumatoid ArthritisArnett F, et al. ArthRheum. 1988;31:315-24www.rheumatology.org/publications/classification/ra/ra.asp?aud=mem 1. Morning stiffness > 1 hr 2. Joint swelling ≥ 3 joint areas at once 3. Swelling in hand (wrist, metacarpal phalangeal (knuckle), or proximal interphalangeal joint) 4. Symmetric arthritis 5. Rheumatoid nodules 6. Positive test for rheumatoid factor (RF; most assays measure IgM-RF, which is autoantibody IgM directed against IgG) * 7. Erosions or bony decalcification on x-ray of the hand and wrist At least 4 of 7 criteria, with criteria 1 through 4 present for > 6 weeks. *Anti-CCP, though commonly used and newer, is not currently in the criteria

43. Disease progression in RA can be severe with erosive (deforming) arthritis and organ involvement. Therefore, the goal in RA is early detection and prognostication to choose Rx that will prevent disease progression.

44. Lab Tests in Rheumatoid Arthritis • Diagnostic / Prognostic • IgM- RF (Rheumatoid factor) • Anti-CCP (cyclic citrillunated peptide) • Also useful • CRP • ESR Nishimura K et al. Meta-analysis:… Accuracy of anti-CCP and RF for Rheumatoid Arthritis. Ann Intern Med 2007; 146: 797-808.

45. Antibody directed against the Fc portion of IgG Most assays measure IgM-RF May be involved in disease pathogenesis Higher levels tend to be associated with poorer prognosis Found in other rheumatologic (e.g. Sjogren’s syndrome) and non-rheumatologic conditions, especially Hepatitis C. Rheumatoid Factor: Introduction

46. Anti-CCP Diagnostic Test • Similar role as RF, similar sensitivity but more specific • !! Negative in patients with Hepatitis C • Predicts severe and erosive disease • Present in early synovitis • Not perfectly correlated with RF, i.e. somewhat different diagnostic information so rheumatologists usually order both tests.

47. Clinical lab perspective on ANA testing • ANA-IFA is subjective, requiring intensive training • Some labs have little ANA-IFA expertise. • Biggest ANA-IFA problem is hi false positive rates • Survey specimens show large variations -and no official “correct” answer- in reported pattern and titer. • Methods: No standardization of substrate, reagents, microscope, training, competency assessment • Looking for better, automated methods

48. Perspective on ANA testing: Internists and Family Practitioners (1) • Not knowledgeable about: • ANA testing • Systemic rheumatic diseases • Usually order the ANA test to rule out SLE • They want the result to be negative.

49. Perspective on ANA testing: Internists and Family Practitioners (2) • They would love for your lab to change your cutoff from 1:40 to 1:160. • If ANA test is +, they will refer to a Rheumatologist

50. Utilization issues regarding ANA testing • Why are too many ANA screening tests ordered in ambulatory settings? • Patient pressure via googlification. • Systemic rheumatic diseases are rare diseases with common symptoms. • The main impact on patients is the medical adventure caused by false positive results.