Transcript ANA Testing
ANA Testing Carrie Marshall 1/18/08 History This is often-mentioned, never-seen LE cell. These dead nuclei are being engulfed by PMNs. 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 (nonrheumatic) and healthy people 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? 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) 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 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) 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) 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 4050 in 100,000 (but 5,000 will have + ANA) Each hospital can change these cutoffs based on their patient population What Follow-up Testing? Ideally this would depend on clinical symptoms, but often: dsDNA Sm nRNP Ro/SSA La/SSB Scl-70 Jo-1 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 Patterns Peripheral or rim = dsDNA Homogenous = dsDNA, histones Speckled = many antigens Nucleoli = associated with scleroderma Centromeric = CREST syndrome Cytoplasmic = myositis, mitochondrial Patterns Patterns Patterns 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 dsDNA Crithidia luciliae has a large mitochondrion with dsDNA (and no histones) 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 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) 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 Question In what 2-3 diseases should you continue a work-up even if the ANA is negative? Answer Sjogren’s syndrome Dermatomyositis Polymyositis (ANA can be negative in more than 50%) Question Besides a rising anti-dsDNA titer, what other lab test can help predict an upcoming SLE flare? Answer Falling C3 and C4 levels Question What is the single greatest risk factor for SLE? What 2 antibodies are the most specific for SLE (not ANA)? Answer Female gender Anti-dsDNA and anti-Smith Question Why do SLE patients test falsely positive on VDRL tests? Answer This tests uses particles coated with phospholipids, SLE patients who make anti-phospholipid antibodies will make the test look like it’s positive. Question What specific autoantibody is characteristic of drug-induced lupus? Answer Anti-histone (H2A-H2B dimer) Question What is the major autoantibody in diffuse scleroderma? In CREST syndrome? Answer Diffuse scleroderma = Scl-70 CREST = anti-centromere Question What enzyme class is the target of autoantibodies in polymyositis? Answer Transfer-RNA synthetases Question Patients with MCTD typically have a high titer of what autoantibody? Answer Antiribonucleoproteins (either U1-RNP or nRNP)