ANA Test

What is being tested?

Antinuclear

antibodies (ANA) are a group

ofantibodies produced by a person's
immune system when it fails to adequately
distinguish between "self" and "nonself."
These antibodies, known as auto antibodies,
attack the body's own healthy cells and
cause autoimmune disease.
The ANA test identifies the presence of these
auto antibodies in the blood that target
antigens contained inside the nucleus.

When is it ordered?
The ANA test is ordered when
someone shows signs and
symptoms that are associated
with a systemic autoimmune
disease such as systemic lupus
erythematosus (SLE)and Sjogren
syndrome , or ruling out other
conditions with similar signs and
symptoms.

Laboratory methods used to

detect
ANA:
There are different methods to detect
ANA.
Two common methods are commonly
performed:
-Immunoassays:
enzyme linked

immunosorbent assay (ELISA).

-Indirect immunofluorescent
technique (IIF): is considered
the gold standard semiquantitative method.

Principle of the IIF

technique:
The procedure is carried out in two basic reaction steps:
Step 1 - Human serum is reacted with human

epithelial laryngeal carcinoma cells (HEp-2) that are

affixed to a slide. Antibodies, if present, will bind to
the antigens forming stable antigen-antibody
complexes. If no antibodies are present, the complex
will not be formed and serum components will be
washed away.
Step 2 - Fluorescein labeled (FITC) antihuman
antibodies are added to the reaction site which bind
with the complexes formed in step one. This results in
a positive reaction of bright apple-green fluorescence
when viewed with a fluorescence microscope.

Figure 1: Indirect immunofluorescence technique.

Figure 2: ANA test by IIF ( tissue used is HEp-2 which have

been shown to have greater sensitivity than tissue sections and
yield sharper pattern recognition).

QUALITY
CONTROL:

Both a positive and negative

control should be included with each
assayrun.
positive control: is used as a
monitor of assay sensitivity and
pattern identification.
Negative control: there should be
no distinct pattern visible or any
clear difference between the nucleus
and cytoplasm .

Figure 3: Negative control (left): Cells display low level nonspecific fluorescence, but no specific nuclear staining. Positive
control (right): Cells display apple green nuclear fluorescence.

INTERPRETATION OF
RESULTS:
Four major staining
patterns have been
described and associated
with different autoimmune
disorders.

1.Homogeneous (diffuse)
pattern:

Figure 4: Diffuse staining of the entire

nucleus.

Homogeneous
Nuclear antigens:
ds DNA and
(diffuse)

histone
or histone
alone .
pattern
:
Disease association:
i. High titers are suggestive of SLE.
ii. Antibody to histone alone has a
high association with druginduced lupus and may be
ordered to support the diagnosis.

2.

Peripheral (rim) pattern:

Figure 5 :Smooth staining primarily around the

outer region of the nucleus with weaker staining in
the center.

Peripheral (rim) pattern:

Nuclear antigens: dsDNA.
Disease association:

High titers to dsDNA are

suggestive of active SLE.

3. Speckled pattern :

Figure 6: Fluorescent aggregates

throughout the nucleus.

Speckled pattern :

Figure 7: Fluorescent aggregates

throughout the nucleus.

Speckled
pattern
:
Nuclear
antigens:
extractable nuclear
antigens [Smith antigen,
ribonucleoprotein, Ro, La and
Scleroderma-70].
Disease association:
Scleroderma.
Sjogrens syndrome.
Mixed connective tissue disease.
Rheumatoid arthritis (RA).
SLE.

4. Nucleolar pattern:

Figure 8: Fluorescent staining of the

nucleoli within the nucleus.

Nucleolar pattern:

Figure 9: Fluorescent staining of the nucleol

within the nucleus.

Nucleolar
pattern:
Nuclear
antigens: nucleolar
antigens.
Disease association:
Scleroderma.
Dermatomyositis and poly myositis.
Sjogrens syndrome.
Less common in SLE and RA.