ANA Testing

1. ANA Testing BY ABDULLAH RADWAN

2. History This is often-mentioned, never-seen LE cell. These dead nuclei are being engulfed by PMNs.

3. ANA Testing • Guideline #1 Test for autoantibodies only when a consistent clinical suspicion of rheumatic disease is present. • Not a good screening test for patients with vague symptoms • ANA can be positive in sick people (non-rheumatic) and healthy people

4. ANA Testing • Anti-Nuclear Antibodies, but they can also be anti-cytoplasmic • Immunofluorescence is commonly used • In the past patients serum was placed on to slides with rodent (or other animal) cells and IF was performed to look for antibodies binding to cellular components • What problems does this cause?

5. Human and rodent cells differ (slightly), and so some people with obvious rheumatic disease would be negative on this test. “ANA-negative lupus” • Now there are human tumor cell lines that are used (HEp-2 are preferred)

6. Another source of false negatives includes how the tissues are fixed onto the slides • Ethanol and methanol fixation may remove Ro/SSA antigens from cells, so the cells are fixed with acetone

7. How is the test done? • Patient serum is diluted and dropped onto HEp-2 slides (cells fixed into separate dots on the slide) • Incubated, washed, secondary antibody added • Read by a tech using an IF scope (takes specialized training and there is inherent variability between individuals)

8. Results • Results typically include positive/negative, titer and pattern of staining • Titers less than 1:40 should be considered negative (20-30% of healthy people) • Titers of 1:40 to 1:160 should be considered positive at low titer (further workup is not recommended in the absence of specific symptoms)

9. Results • Titers equal to or greater than 1:160 should be considered positive and further workup should be done (only 5% of healthy people). Prevalence of SLE is 40-50 in 100,000 (but 5,000 will have + ANA) • Each hospital can change these cutoffs based on their patient population

10. What Follow-up Testing? • Ideally this would depend on clinical symptoms, but often: • dsDNA • Sm • nRNP • Ro/SSA • La/SSB • Scl-70 • Jo-1

11. Patterns • The IF pattern is still reported, but does not correlate well with what the antibody’s specificity is. • It was the most you could do “back in the day” • Now with ELISA testing for specific antigens possible, the ANA pattern has a low relevance

12. Patterns • Peripheral or rim = dsDNA • Homogenous = dsDNA, histones • Speckled = many antigens • Nucleoli = associated with scleroderma • Centromeric = CREST syndrome • Cytoplasmic = myositis, mitochondrial

13. Patterns

14. Patterns

15. Patterns

16. To summarize… • You screen for ANAs using IF on slides with HEp-2 cells • If it’s positive you look for the specific antigen that the antibody is reacting to using ELISA (the antigen is stuck to the well) or other methods • We don’t screen for ANAs using ELISA because it’s hard to get all the various antigens (40+) onto the well walls

17. dsDNA Crithidia luciliae has a large mitochondrion with dsDNA (and no histones)

18. dsDNA Guidelines suggest checking for anti-dsDNA antibodies only when the symptoms are suspicious of SLE AND the ANA is positive The “ANA-negative lupus” patients are REALLY rare now that we test with HEp-2 cells rather than animal cells

19. Guidelines suggest that the only antibodies that need to be quantified are dsDNA (to predict a flare, and nephritis risk) • Active disease (q 6-12 weeks) • Less active disease (q 6-12 months) Report quantitative results on isolated U-RNP antibodies (part of criteria for MCTD)

20. Anti-CCP • IgG against Cyclic Citrullinated Peptide (CCP) • Is a very specific marker, 98%, (very low rate of false negatives) for Rheumatoid Arthritis • Will be + in 70% of RA patients in early dz • Not found in other diseases (contrast to RF) • Should be a one time test, does not need to be repeated or followed • Indicates pts at high risk of progressive erosive disease, should be treated aggressively

21. Question • In what 2-3 diseases should you continue a work-up even if the ANA is negative?

22. Answer • Sjogren’s syndrome • Dermatomyositis • Polymyositis • (ANA can be negative in more than 50%)

23. Question • Besides a rising anti-dsDNA titer, what other lab test can help predict an upcoming SLE flare?

24. Answer • Falling C3 and C4 levels

25. Question • What is the single greatest risk factor for SLE? • What 2 antibodies are the most specific for SLE (not ANA)?

26. Answer • Female gender • Anti-dsDNA and anti-Smith

27. Question • Why do SLE patients test falsely positive on VDRL tests?

28. Answer • This tests uses particles coated with phospholipids, SLE patients who make anti-phospholipid antibodies will make the test look like it’s positive.

29. Question • What specific autoantibody is characteristic of drug-induced lupus?

30. Answer • Anti-histone (H2A-H2B dimer)

31. Question • What is the major autoantibody in diffuse scleroderma? • In CREST syndrome?

32. Answer • Diffuse scleroderma = Scl-70 • CREST = anti-centromere

33. Question • What enzyme class is the target of autoantibodies in polymyositis?

34. Answer • Transfer-RNA synthetases

35. Question • Patients with MCTD typically have a high titer of what autoantibody?

36. Answer • Antiribonucleoproteins (either U1-RNP or nRNP)