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ANA Testing

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					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 (non-
rheumatic) 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 40-
50 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)

				
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