ANA Testing

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ANA Testing

• Carrie Marshall
• 1/18/08

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

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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?

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• 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)

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• 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

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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)

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Results

• Results typically include positive/negative,
  titer and pattern of staining
• Titers less than 140 should be considered
  negative (20-30 of healthy people)
• Titers of 140 to 1160 should be considered
  positive at low titer (further workup is not
  recommended in the absence of specific symptoms)

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Results

• Titers equal to or greater than 1160 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

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What Follow-up Testing?

• Ideally this would depend on clinical symptoms,
  but often
• dsDNA
• Sm
• nRNP
• Ro/SSA
• La/SSB
• Scl-70
• Jo-1

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Patterns

• The IF pattern is still reported, but does not
  correlate well with what the antibodys
  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

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Patterns

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

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Patterns
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Patterns
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Patterns
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To summarize

• You screen for ANAs using IF on slides with HEp-2
  cells
• If its 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 dont screen for ANAs using ELISA because its
  hard to get all the various antigens (40) onto
  the well walls

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

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• 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)

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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

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Question

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

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Answer

• Sjogrens syndrome
• Dermatomyositis
• Polymyositis
• (ANA can be negative in more than 50)

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Question

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

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Answer

• Falling C3 and C4 levels

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Question

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

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Answer

• Female gender
• Anti-dsDNA and anti-Smith

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Question

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

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Answer

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

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Question

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

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Answer

• Anti-histone (H2A-H2B dimer)

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Question

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

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Answer

• Diffuse scleroderma Scl-70
• CREST anti-centromere

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Question

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

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Answer

• Transfer-RNA synthetases

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Question

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

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Answer

• Antiribonucleoproteins (either U1-RNP or nRNP)