1. Testing in the Rheumatic Diseases Salahuddin Kazi, M.D.

2. Questions to Answer When Applying a Valid Diagnostic Test to a Specific Patient* Is the test available, affordable, accurate and precise in our setting? Is the test available, affordable, accurate and precise in our setting? Can we generate a clinically sensible estimate of our patient’s pre-test probability? Can we generate a clinically sensible estimate of our patient’s pre-test probability? Will the resulting post-test probability affect our management and help our patient? Will the resulting post-test probability affect our management and help our patient? *Evidence Based Medicine: 2 nd Edition, Sacket et al, 2000

3. Test Statistics - A Review Sensitivity - The proportion of affected individuals with a positive test Sensitivity - The proportion of affected individuals with a positive test Specificity - The proportion of unaffected individuals with a negative test Specificity - The proportion of unaffected individuals with a negative test Utility lies at the extremes - SpPin “High specificity; positive test rules in” and SnNout “High sensitivity; negative test rules out” Utility lies at the extremes - SpPin “High specificity; positive test rules in” and SnNout “High sensitivity; negative test rules out”

4. Gold Standard Test Result Positive Positive Negative Negative ab cd True Pos False Pos True Neg False Neg Sensitivity = a/ a+c Specificity = d/ b+d Positive Predictive Value = a/ a+b Negative Predictive Value = d/ c+d Prevalence = a+c/ a+b+c+d

5. Likelihood Ratios The likelihood that a given test result would be expected in a patient with the target disorder compared with the likelihood that the same result would be expected in a patient without the target disorder The likelihood that a given test result would be expected in a patient with the target disorder compared with the likelihood that the same result would be expected in a patient without the target disorder +LR = sensitivity/(1-specificity) +LR = sensitivity/(1-specificity) -LR = (1-sensitivity)/specificity -LR = (1-sensitivity)/specificity

6. 1000 500 200 100 50 20 10 5 2 1 0.5 0.2 0.1 0.05 0.02 0.01 0.005 0.002 0.001 99 95 90 80 70 60 50 40 30 20 10 5 2 1 0.5 0.2 0.1 0.2 0.5 1 2 5 10 20 30 40 50 60 70 80 90 95 99 Post-test probability Pre-test probability Likelihood ratio Likelihood Ratio Normogram

7. Case #1 A 32 y/o woman describes a 6 week history of pain and stiffness in her hands A 32 y/o woman describes a 6 week history of pain and stiffness in her hands No history of fever, rash, dysuria, conjunctivitis, travel or exposure. No prior renal disease, seizures, or serositis. Her mother has deforming arthritis. No history of fever, rash, dysuria, conjunctivitis, travel or exposure. No prior renal disease, seizures, or serositis. Her mother has deforming arthritis. On exam there is warmth and soft-tissue swelling of the 2 nd and 3 rd MCPs bilaterally, the 3 rd right PIP, and the left wrist. There seems to be a small effusion in the left knee. On exam there is warmth and soft-tissue swelling of the 2 nd and 3 rd MCPs bilaterally, the 3 rd right PIP, and the left wrist. There seems to be a small effusion in the left knee. Labs: SMA6 and U/A - normal; mild anemia; CRP 2.7; ANA - 1:40, diffuse; Rheumatoid Factor - 320 Labs: SMA6 and U/A - normal; mild anemia; CRP 2.7; ANA - 1:40, diffuse; Rheumatoid Factor - 320

8. Rheumatoid Factor Anti-IgG - can be all Ig classes Anti-IgG - can be all Ig classes Specific for Fc portion of IgG Specific for Fc portion of IgG Can be polyclonal (typical of autoimmunity) or monoclonal (typical of lymphoid malignancy) Can be polyclonal (typical of autoimmunity) or monoclonal (typical of lymphoid malignancy) Causes immune complex damage Causes immune complex damage Reported as “units” or titer Reported as “units” or titer

9. RF Test Characteristics Sensitivity for RA is ~80% Sensitivity for RA is ~80% Specificity is 85-95% Specificity is 85-95% +LR of 5-16 depending on population studied +LR of 5-16 depending on population studied High titer is associated with more severe RA with extra-articular manifestations High titer is associated with more severe RA with extra-articular manifestations Monitoring titer as an indicator of disease activity is not appropriate Monitoring titer as an indicator of disease activity is not appropriate

10. Conditions Associated with RF Normal individuals (5%), especially with age (15%) Normal individuals (5%), especially with age (15%) Rheumatoid arthritis (85%), Sjögren’s Syndrome, SLE (25-50%) Rheumatoid arthritis (85%), Sjögren’s Syndrome, SLE (25-50%) Viral Infections: Hepatitis C (25-50%), mononucleosis, HIV, influenza Viral Infections: Hepatitis C (25-50%), mononucleosis, HIV, influenza Bacterial Infections: IE (25-50%), TB (10-25%), leprosy, syphilis, brucellosis Bacterial Infections: IE (25-50%), TB (10-25%), leprosy, syphilis, brucellosis Parasites: Typanosomiasis, malaria, schistosomiasis, etc. Parasites: Typanosomiasis, malaria, schistosomiasis, etc. Other: Sarcoidosis, pulmonary fibrosis (10-25%), chronic liver disease Other: Sarcoidosis, pulmonary fibrosis (10-25%), chronic liver disease

11. Case #1 – Using the Test Results Chronic, inflammatory, symmetrical polyarthritis of the hands in a young woman Chronic, inflammatory, symmetrical polyarthritis of the hands in a young woman What’s your pre-test probability that this patient has RA? What’s your pre-test probability that this patient has RA?

12. 1000 500 200 100 50 20 10 5 2 1 0.5 0.2 0.1 0.05 0.02 0.01 0.005 0.002 0.001 99 95 90 80 70 60 50 40 30 20 10 5 2 1 0.5 0.2 0.1 0.2 0.5 1 2 5 10 20 30 40 50 60 70 80 90 95 99 Post-test probability Pre-test probability Likelihood ratio In this case, a test with a moderate +LR makes the diagnosis almost certain in a patient with a high pre-test probability Highly positive RF takes a 50% pre-test probability to a >95% post-test probability

13. Case #2 A 64-year-old female was evaluated for generalized joint pain and muscle pain, fatigue, fever and chills for the past 6-8 weeks A 64-year-old female was evaluated for generalized joint pain and muscle pain, fatigue, fever and chills for the past 6-8 weeks No rash, Raynaud’s, weight loss No rash, Raynaud’s, weight loss Graves’ disease 18 years ago - radioactive iodine Graves’ disease 18 years ago - radioactive iodine Family history: SLE, thyroid disease Family history: SLE, thyroid disease PE: Tender joints but no joint swelling PE: Tender joints but no joint swelling Labs: Labs: –CBC, Chem 7, LFT’s, UA, TSH - all normal –ESR 18 mm/h, CRP <0.8, RF negative, ANA positive 1:80, homogeneous pattern

14. Anti-Nuclear Antibodies Began with the demonstration of the “LE cell” by Hargraves in 1948 Began with the demonstration of the “LE cell” by Hargraves in 1948 Includes antibodies to a number of antigens, including native DNA Includes antibodies to a number of antigens, including native DNA Performed by indirect immunofluorescence Performed by indirect immunofluorescence Reported as “negative” - usually less than a certain titer, or as a titer and pattern Reported as “negative” - usually less than a certain titer, or as a titer and pattern

15. ANA - Characteristics Sensitivity - 95-100% Sensitivity - 95-100% Specificity - Depends on titer used as cut-off Specificity - Depends on titer used as cut-off –15-30% of normals have ANA of 1:40 –5% have ANA of 1:160 +LR is ~20; utility for SLE is based on prevalence: +LR is ~20; utility for SLE is based on prevalence: –General population 50/100,000 –Young, African-American women 400/100,000 –Children/elderly men 1/100,000

16. Immunofluorescence Patterns of ANAs

17. Homogeneous Rim or Peripheral Nucleolar Speckled

18. Causes of a Positive ANA

19. Interpreting a Positive ANA

20. Other Causes of Positive ANA Non Rheumatic Disease Infections Inflammatory bowel disease Autoimmune hepatitis Pulmonary fibrosis Endocrine diseases Hematologic diseases Neoplastic diseases End-stage renal disease Post-transplant Healthy People Pregnancy Older people Family history of rheumatic disease Drug induced

21. Case #2:Why is the ANA Positive? History and PE: Does not suggest a CTD History and PE: Does not suggest a CTD Labs: normal except for positive ANA Labs: normal except for positive ANA Pretest probability of SLE is low Pretest probability of SLE is low Posttest probability for SLE remains low Posttest probability for SLE remains low Look for an alternative explanation Look for an alternative explanation –Elderly female –Positive family history of rheumatic disease Reassure: ANA result is a normal finding Reassure: ANA result is a normal finding

22. 1000 500 200 100 50 20 10 5 2 1 0.5 0.2 0.1 0.05 0.02 0.01 0.005 0.002 0.001 99 95 90 80 70 60 50 40 30 20 10 5 2 1 0.5 0.2 0.1 0.2 0.5 1 2 5 10 20 30 40 50 60 70 80 90 95 99 Post-test probability Pre-test probability Likelihood ratio Although an ANA >1:160 has a high +LR, it should not be used to screen patients without clinical evidence of autoimmune disease

23. Ordering an ANA To confirm the diagnosis of SLE when the clinical suspicion is high To confirm the diagnosis of SLE when the clinical suspicion is high To exclude SLE when the clinical suspicion is moderate (2 or 3 lupus criteria) To exclude SLE when the clinical suspicion is moderate (2 or 3 lupus criteria) Avoid ordering it when the clinic suspicion for SLE is low - a positive result can cause diagnostic confusion and unnecessary anxiety Avoid ordering it when the clinic suspicion for SLE is low - a positive result can cause diagnostic confusion and unnecessary anxiety

24. Anti-DNA Antibodies Detect antibodies to native (double stranded) DNA Detect antibodies to native (double stranded) DNA Typical methods are ELISA and immunofluorescence on Crithidia Typical methods are ELISA and immunofluorescence on Crithidia Can have both diagnostic and prognostic significance Can have both diagnostic and prognostic significance

25. Anti-DNA - Characteristics Sensitivity - 60% for SLE Specificity - 97% Low titers seen in 2-5% of RA, Sjögren’s, scleroderma, relatives of SLE pts., etc. Average +LR of 16 and -LR of 0.49 means that a positive anti-DNA has a large impact, but lack of one doesn’t exclude SLE

26. Anti-DNA - Prognosis SLE Disease activity: Useful, but with small +LR (~4) SLE Disease activity: Useful, but with small +LR (~4) Nephritis: Associated, but with very small +LR (~1.7) Nephritis: Associated, but with very small +LR (~1.7) Rising titers may predict a flare of disease activity in some, but not all, patients Rising titers may predict a flare of disease activity in some, but not all, patients Clinical correlation is advised Clinical correlation is advised

27. Anti-ENA Small nuclear RNP Small nuclear RNP –Sm: Seen in 15-30% of SLE; specific –U1-RNP: 30-40% of SLE; also RA, Sjögren's, scleroderma, and overlap syndromes Anti-Ro and anti-La Anti-Ro and anti-La –Subacute cutaneous LE –Sjögren's syndrome –Neonatal lupus with congenital heart block

28. 5’ 3’ Sm Antigens RNP Antigens 33kDa (A) EF G 28kDa (B) 16kDa (D) 70kDa C U1RNA 28kDa (B’) “ENA”-Extractable Nuclear Antigens

29. Anti-Scl-70/Anti-Centromere Scl-70 = Topoisomerase I; seen in 40-70% of patients with diffuse scleroderma; worse prognosis with more organ involvement Scl-70 = Topoisomerase I; seen in 40-70% of patients with diffuse scleroderma; worse prognosis with more organ involvement Centromere - 70-85% of patients with limited scleroderma; associated with Raynaud’s syndrome Centromere - 70-85% of patients with limited scleroderma; associated with Raynaud’s syndrome Neither is diagnostic by themselves Neither is diagnostic by themselves

30. Case #3 A 48-year-old male has chronic sinusitis with occasional bloody drainage A 48-year-old male has chronic sinusitis with occasional bloody drainage You order a c-ANCA You order a c-ANCA –Positive at 1:80 The chest radiograph, creatinine and urinalysis are normal The chest radiograph, creatinine and urinalysis are normal What is the likelihood that he has Wegener’s granulomatosis? What is the likelihood that he has Wegener’s granulomatosis?

31. 6 5 4 3 2 1 Positive Predictive Value 100 0 50 100 Disease Prevalence 1. Documented WG 2. Pulmonary-Renal Syndrome 3. Systemic Necrotizing Vasculitis 4. Rapidly Progressive GN 5. GN 6. Hospitalized Patient Jeanette: Amer J Kidney Dis 18:164, 1991 Positive Predictive Value of ANCA This Patient

32. 1000 500 200 100 50 20 10 5 2 1 0.5 0.2 0.1 0.05 0.02 0.01 0.005 0.002 0.001 99 95 90 80 70 60 50 40 30 20 10 5 2 1 0.5 0.2 0.1 0.2 0.5 1 2 5 10 20 30 40 50 60 70 80 90 95 99 Post-test probability Pre-test probability Likelihood ratio Wegener's is rare (~0.4/100,000). Without signs of progressive, necrotizing vasculitis, even a test with a high likelihood ratio is not helpful

33. ANCA Characteristics C-ANCA (Proteinase-3) C-ANCA (Proteinase-3) –90% specificity and 50-90% sensitivity for active Wegener's granulomatosis P-ANCA P-ANCA –MPO - 60% of microscopic polyangiitis, Churg- Strauss –Cathepsins, lactoferrin, elastase Should not take the place of tissue biopsy Should not take the place of tissue biopsy

34. ANCAs and Rheumatic Autoimmune Diseases P-ANCA (not directed against MPO) reported in: P-ANCA (not directed against MPO) reported in: –RA, SLE, PM/DM, Sjögren's syndrome, Juvenile chronic arthritis, Reactive arthritis, Relapsing polychondritis* C-ANCA C-ANCA –very rare in these diseases ANCA is not associated with increased frequency of vasculitis in the autoimmune rheumatic diseases ANCA is not associated with increased frequency of vasculitis in the autoimmune rheumatic diseases *Ann Intern Med 126:866-873, 1997

35. ANCA and Inflammatory Bowel Disease P-ANCA and some atypical patterns (not directed at MPO) P-ANCA and some atypical patterns (not directed at MPO) –Ulcerative colitis - 40% to 80% –Crohn’s Disease - 10% to 40% Does not facilitate the differential diagnosis of patients with inflammatory bowel disease Does not facilitate the differential diagnosis of patients with inflammatory bowel disease Correlation of titers with disease activity is not sufficiently reliable Correlation of titers with disease activity is not sufficiently reliable

36. How are ANCAs detected? Indirect immunofluorescence (IIF) Indirect immunofluorescence (IIF) –c-ANCA or p-ANCA pattern Enzyme linked immunosorbent assay (ELISA) Enzyme linked immunosorbent assay (ELISA) –specific antigens detected –PR3: (c-ANCA on IIF) –MPO: (p-ANCA on IIF) ANCA testing is problematic because of lack of standardization between laboratories ANCA testing is problematic because of lack of standardization between laboratories

37. Summary Connective tissue diseases have a low prevalence Connective tissue diseases have a low prevalence Unselected “screening” of patients with “arthritis panels” will result in large numbers of false positives Unselected “screening” of patients with “arthritis panels” will result in large numbers of false positives Estimation of clinical pre-test probability and the knowledge of test characteristics are useful tools for rationally ordering and interpreting the results of diagnostic tests Estimation of clinical pre-test probability and the knowledge of test characteristics are useful tools for rationally ordering and interpreting the results of diagnostic tests