1. ANA Testing
Carrie Marshall
1/18/08

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 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)