AUTO IMMUNE DISEASE , IMMUNOFLUORESCENCE , DIFFERENT PATTERNS IN IF

1. ANA IN
AUTOIMMUNITY
- Dr. Anamika.
D

2. Autoantibodies
O Directed against
O Components of the cell surface,
O Cytoplasm components
O Nuclear components

3.  Antibodies to cell nucleus component
ANA, anti-dsDNA, antibodies to extractable
nuclear antigen (ENA), (anti-Sm, anti-RNP)
 Antibodies to cytoplasmic antigens
anti-SSA, ANCA
 Cell-specific autoantibodies
lymphocytotoxic antibodies, anti-neurone
antibodies, anti-erythrocyte antibodies, anti-
platelet antibodies
 Antibodies to serum components
antiphospholipid antibody,antiglobulin,
rheumatoid factor

4. Anti-nuclear Antibodies
O Also known as antinuclear factor or ANF
O 4 types:
O Anti- nuclear antibodies to DNA
O ANA to histones
O ANA to non-histone proteins bound to
RNA
O ANA to nucleolar antigens

5. Many sub-types
 anti-Ro/SS-A antibodies Sjogrens syndrome
 anti-La/SS-B antibodies
 anti-dsDNA antibodies
 anti-Sm antibodies SLE
 anti-nRNP antibodies – Mixed connective
tissue disease
 anti-Scl-70 antibodies - scleroderma
 Anti-Jo-1 antibodies – Polymyositis and
dermatomyositis
 anti-histone antibodies – drug induced lupus
 anti-centromere antibodies – CREST syndrome
 anti-sp100 antibodies – primary biliary
cirrhosis

6. ANA TESTING
Three main methods:
1. Indirect Immunofluorescence Assay
(IFA)
2. Enzyme-linked immunosorbent assay
(ELISA)
3. Multiplex Bead Immunoassays
O Microarrays

7. Sample preparation
O Collect blood specimens, usually from arm
of pt , at any time.
O No special instructions given
O Separate the serum.
O Specimens may be refrigerated at 2–8°C
for up to seven days or frozen for up to six
months. Avoid repetitive freezing and
thawing.

8. Indirect immunofluorescence
O Immunofluorescence is commonly used
O 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
O What problems does this cause?

9. O Human and rodent cells differ (slightly),
and so some people with obvious
rheumatic disease would be negative on
this test. “ANA-negative lupus”
O Now there are human tumor cell lines that
are used (HEp-2 are preferred)

10. How is the test done?
O Whole HEp- 2 cells containing these antigens are attached to
microscope slide (cells fixed into separate dots on the slide)
O Patient serum is diluted and dropped onto HEp-2 slides
O If antibody is resent , it binds to antigen on Hep -2 cells
O Incubate for 20 mins at room temerature.
O Wash to remove unreacted antibody.
O Add secondary antibody -anti-human globulin labeled with
fluorescent tag or enzyme bind to pts antibody
characteristic fluorescent pattern
O Read using an IF scope

14. O Another source of false negatives includes
how the tissues are fixed onto the slides
O Ethanol and methanol fixation may
remove Ro/SSA antigens from cells, so
the cells are fixed with acetone

15. Results
 Depend on
 Titer of antibody (highest dilution of serum at which
auto- antibodies are still deectable)
 IFpattern
 Titers less than 1:40 should be considered
negative (20-30% of healthy people)
 Titers of 1:40 to 1:160 positive at low titer
(further workup is not recommended in the
absence of specific symptoms)

16. Results
O Titers equal to or greater than 1:160
positive

17. IF Patterns
O Peripheral or rim staining pattern = dsDNA
O Homogenous/ diffuse pattern = chromatin,dsDNA,
histones
O Speckled = many antigens like Sm antigen,
Ribonucleo-protein, SS-A& SS-B reactive antigens
O Nucleolar = RNA
O Centromeric = centromere

18. Ro
La
Smith
RNP
Jo-1
Scl-70
Ro
Nucleolar
dsDNA
Rim
Speckled
Homogenous
Nucleosomes
Ro
La
Smith
RNP
Jo-1
Scl-70
Ro
dsDNA
Nucleolar proteins

19. Homogenous pattern
 Smooth, even staining
of the nucleus with or
without
apparent masking of
the nucleoli
 Seen in Systemic lupus
– anti – DNA, anti-
histone

20. Rim/ Peripheral pattern
 Fluorescence is most
intense at the periphery of
the nucleus with a large
ring starting from the
internal nuclear membrane
and the rest of the nucleus
showing weaker yet
smooth staining.
 Not seen on Hep-2
Seen in SLE – anti
-DNA

21. Nucleolar pattern
O 23 or 46 (or some
multiple of 46) bright
speckles or ovoid
granules spread over
the nucleus of
interphase cells
O Seen in Diffuse
Scleroderma
O Scl-70

23. Speckled pattern
O Large speckles
covering the whole
nucleoplasm,
interconnected by a
fine fluorescent
network.
O m/c observed
O Least specific
O Seen in sjogrens
syndrome, SLE,
polymyositis,
dermtomyositis

25. Patterns

26. IFA- False Negatives:
o Rodent / animal cells used
o Methanol /ethanol fixation
o In Sjogren’s Syndrome,polymyositis, and
dermatomyositis (ANA -ve in >50%)
o If single antibody, at very low level, is
present: - SSA, subacute cutaneous lupus
- dsDNA, ANA negative lupus

27. False Positives:
O 33% of normals can be positive
O > 20% healthy relatives
O 75% elderly population
O In other diseases: viral infections, cirhosis,
Chronic Pulmonary Fibrosis, Chronic Infection,
Chronic Hepatitis ,Cancer

28. ELISA
O Also k/a ANA BLOT test or ANA ELISA TEST
O Amount of antibodies in units per given amount of
blood
O Whole Hep-2 cells are lysed, centrifuged to
concentrate the nuclei
O Other purified antigens are added, boost signal
for all autoantigens (e.g., SSA, Scl-70, Jo-1)
O Coated onto high-affinity binding wells to retain
all signals during processing

29. O Diluted human serum is added to wells coated
with purified nuclear antigens.
O ANA IgG specific antibody, if present, binds to
the antigen.
O All unbound materials are washed away and the
enzyme conjugate is added to bind to the
antibody-antigen complex, if present.
O Excess enzyme conjugate is washed off and
substrate is added.
O The plate is incubated to allow the hydrolysis of
the substrate by the enzyme.
O The intensity of the color generated is
proportional to the amount of IgG specific
antibody in the sample.

30. O ELISA detects the same ANA antibodies
as IFA, with improved sensitivity/specificity
O Results in one determination (no
repeating to titrate)
O Objective, automatable, requires no
specialized training
O ELISA reports out Index Values

31. Multiplex Bead Immunoassay
O Individual antigens, cell components are
coated onto multiple beads
O All activities are detected and identified in
parallel
O High cost of test and automation

32. To summarize…
O You screen for ANAs using IF on slides
with HEp-2 cells
O If it’s positive look for the specific
antigen using ELISA
O We don’t screen for ANAs using ELISA
because it’s hard to get all the various
antigens (40+) onto the well walls