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ANA Diagnostics Using

Indirect Immunofluorescence

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Table of Contents
Autoantibodies against cell nuclei (ANA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Autoantibodies against cell nuclei, homogeneous . . . . . . . . . . . . . . . . . . . . . 10
Autoantibodies against dsDNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Autoantibodies against DFS70 pattern . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Autoantibodies against nuclear membrane . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Autoantibodies against nucleoplasm, coarse granular . . . . . . . . . . . . . . . . . . 14
Autoantibodies against nucleoplasm, fine granular . . . . . . . . . . . . . . . . . . . . 15
Autoantibodies against Ku . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Autoantibodies against Mi-2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Autoantibodies against PM-Scl . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Autoantibodies against RNA polymerase I . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Autoantibodies against U3-nRNP / fibrillarin . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Autoantibodies against Scl-70 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Autoantibodies against nuclear dots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Autoantibodies against centromeres . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Autoantibodies against PCNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Autoantibodies against centrioles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Autoantibodies against spindle fibres. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
Autoantibodies against midbody . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Autoantibodies against mitochondria. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
Autoantibodies against ribosomal P-proteins . . . . . . . . . . . . . . . . . . . . . . . . . 29
Autoantibodies against Jo-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
Autoantibodies against SRP and PL-12 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Autoantibodies against Golgi apparatus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
Autoantibodies against lysosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Autoantibodies against F-actin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
Autoantibodies against vimentin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Cytoplasmic rings & rods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
Dilution scheme for immunofluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

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Autoantibodies against cell nuclei (ANA)

Definition

Anti-nuclear autoantibodies are directed against antigens of the cell nucleus.

These autoantigens are named after their biochemical characteristics (DNA,
histones, ribonucleoproteins: RNP), the disease associated with the corre-
sponding autoantibody (SS-A, SS-B: Sjögren‘s syndrome, antigens A and B;
PM-Scl: polymyositis, progressive systemic sclerosis) or, occasionally, after the
patient in whom the corresponding antibody was first detected (Sm, Ro, La).

More than 100 autoantigens are presented in HEp-2 cells.

The most important among them are:
Polynucleotides Double-stranded DNA, single-stranded DNA, RNA
Histones H1, H2A, H2B, H3, H4, H2A-H2B complex
Ribonucleoproteins U1-(n)RNP, Sm, SS-A (Ro), SS-B (La)
Nucleolar antigens U3-(n)RNP/fibrillarin, RNA polymerase I, PM-Scl (PM-1),
7-2-RNP (To), 4-6-S-RNA, NOR-90 (nucleolar organiser)
Centromeres Kinetochore proteins
Other proteins Scl-70, PCNA (cyclin I), nuclear granules, Ku, Mi-2,
lamins, lamin receptors

Analytics

Due to its high sensitivity and specificity, the indirect immunofluorescence test
(IIFT) using human epithelial cells (HEp-2) and primate liver is the gold standard
for the detection of anti-nuclear autoantibodies (ANA). The signal intensities of
a positive and a negative sample differ significantly and microscopic evaluation
allows an exact determination of how the indicator dye (usually fluorescein) is
spread in the tissue or cells. Each bound autoantibody causes a typical fluo-
rescene pattern, depending on the location of the corresponding autoantigen.

If the analysis result is positive, test substrates with defined single antigens
(ELISA, western blot, line blot) are used for further differentiation. Using mo-
nospecific test methods alone is not sufficient for the determination of anti-
nuclear autoantibodies since not all relevant antigens are available in their puri-
fied form. For verification of analysis results, monospecific tests should always
be accompanied by IIFT.
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Evaluation

Anti-nuclear autoantibodies in patient serum are characteristic in many dis-

eases, in particular, but not exclusively, rheumatic diseases. In the foreground
are the following:

Autoimmune disease ANA prevalence (%)

Systemic lupus erythematosus (SLE) 80 – 100
Drug-induced lupus erythematosus 100
Mixed collective tissue disease (MCTD, Sharp syndr.) 100
Rheumatoid arthritis 20 – 40
Other rheumatic diseases 20 – 50
Progressive systemic sclerosis 85 – 95
Polymyositis / dermatomyositis 30 – 50
Sjögren‘s syndrome 70 – 80
Autoimmune hepatitis 30 – 40
Ulcerative colitis 26

The detection of autoantibodies against cell nuclei is an important diagnostic

indicator in many autoimmune diseases. Antibodies against nuclear antigens
are directed against various cell nuclear components (biochemical substances
in the cell nucleus). These encompass nucleic acids, cell nuclear proteins and
ribonucleoproteins. They are a characteristic finding in many diseases, in par-
ticular rheumatic diseases. The frequency (prevalence) of anti-nuclear antibod-
ies in inflammatory rheumatic diseases is between 20 % and 100 %, and it is
lowest in rheumatoid arthritis at between 20 % and 40 %. Therefore, differential
antibody diagnostics against nuclear antigens is indispensible for the diagnosis
of individual rheumatic diseases and their differentiation from other autoim-
mune diseases.

Systemic lupus erythematosus

The determination of antibodies against double-stranded DNA (dsDNA) is con-

sidered the most important criterion for the diagnosis of systemic lupus ery-
thematosus (SLE). Immune complexes consisting of dsDNA and corresponding
autoantibodies cause tissue damage in the subcutis, kidneys and other organs.
The antibody titer correlates with the activity of the disease. Antibodies against
nucleosomes and Sm are also considered to be pathognomonic for autoanti-

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bodies in SLE. Antibodies against other polynucleotides, ribonucleotides, his-

tones and further nuclear antigens can also be detected in this disease.

In drug-induced lupus erythematosus with manifestations such as arthralgia,

arthritis, exanthema, serositis, myalgia, heptomegalia and splenomegalia, an-
tibodies against histones are always observed. This reversible form of SLE can
be induced by antibiotics (e.g. penicillin, streptomycin, tetracyclines), chemo-
therapeutic agents (e.g. INH, sulfonamides), anticonvulsants (e.g. phenytoin,
hydantoines), antiarrythmics (e.g. procainamide, practolol), antihypertensives
(e.g. reserpine, hydralazine), psychotropics (e.g. chlorpromazine), anti-thyroid
drugs (e.g. thiouracil derivatives), anti-rheumatoid basis therapeutics (e.g.
gold, D penicillamine) and other drugs such as contraceptives and allopurinol.

Autoantibodies in systemic lupus erythematosus

Antigen Prevalence (%)
Double-stranded DNA 60 – 90
Single-stranded DNA 70 – 95
Nucleosomes 50 – 95
RNA 50
RNA helicase A 6
Histones 50 – 80
U1-nRNP 15 – 40
Sm 5 – 40
SS-A (Ro) 20 – 60
SS-B (La) 10 – 20
PCNA (cyclin) 3
Ku 10
Ribosomal P-proteins 10
(Hsp-90: heat shock protein, 90 kDa 50)
(Cardiolipin 40 – 60)

Mixed connective tissue disease

High autoantibody titers against U1-nRNP are characteristic for mixed connec-
tive tissue disease (MCTD, Sharp syndrome). The antibody titer correlates with
the activity of the disease.

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Autoantibodies in mixed connective tissue disease

Antigen Prevalence (%)
U1-nRNP 95 – 100
Single-stranded DNA 20 – 50

Rheumatoid arthritis

In rheumatoid arthritis, antibodies against histones can be observed in up to

half of all cases, whereas antibodies against U1-nRNP are found more rarely.
Antibodies against RANA (“rheumatoid arthritis nuclear antigen”) cannot be
detected using HEp-2 cells.

Anti-nuclear antibodies in rheumatoid arthritis

Antigen Prevalence (%)
Histones 15 – 50
Single-stranded DNA 8
U1-nRNP 3
(RANA 90 – 95)

Progressive systemic sclerosis

Progressive systemic sclerosis ( PSS, scleroderma) can manifest itself in two

forms, which cannot always be clearly differentiated. Until now, antibodies
against fibrillarin, RNA polymerase I and Scl-70 have only been observed in the
diffuse form of the disease. Autoantibodies against centromeres are associated
with the limited form of PSS.

Autoantibodies in progressive systemic sclerosis (limited form)

Antigen Prevalence (%)
Centromeres 80 – 95

Autoantibodies in progressive systemic sclerosis (diffuse form)

Antigen Prevalence (%)
Fibrillarin 5 – 10
PM-Scl (PM-1): (75 kDa / 100 kDa main antigen) 13 (10 / 7)

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Autoantibodies in progressive systemic sclerosis (diffuse form)

Antigen Prevalence (%)
Scl-70 25 – 75
RNA polymerase I 4
Ku, incl. overlap syndrome 25 – 50
7-2-RNP (To) rare
NOR-90 (nucleolar organiser region) rare

Polymyositis / dermatomyositis

Autoantibodies against PM-Scl occur in polymyositis and dermatomyositis.

Other anti-nuclear antibodies (Mi-1, Mi-2 and Ku) and antibodies against Jo-1
can also be found in these diseases.

Autoantibodies in polymyositis and dermatomyositis

Antigen Prevalence (%)
PM-Scl (PM-1), incl. overlap syndrome with PSS 50 – 70
Jo-1 (histidyl-tRNA synthetase) 25 – 35
Mi-1 10
Mi-2 5
Ku, incl. overlap syndrome 50
Single-stranded DNA 40 – 50
PL-7 (threonyl-tRNA synthetase) 4
PL-12 (alanyl-tRNA synthetase) 3

Sjögren‘s syndrome

In (primary) Sjögren‘s syndrome, antibodies against SS-A and SS-B are pre-
sent, mainly in combination with one another. In addition, autoantibodies
against the salivary secretory ducts are found in 40 to 60 % of cases.

Autoantibodies in primary Sjögren‘s syndrome

Antigen Prevalence (%)
SS-A (Ro) 40 – 95
SS-B (La) 40 – 95

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Autoantibodies in primary Sjögren‘s syndrome

Antigen Prevalence (%)
Single-stranded DNA 13
(RANA 70)
(Salivary excretory ducts 40 – 60)
(Rheumatoid factors 60 – 80)

Primary biliary cirrhosis

In addition to antibodies against mitochondria, various autoantibodies against

cell nuclei are associated with primary biliary cirrhosis. Some of them can be
considered pathognomonic. Furthermore, antibodies against SS-A and cen-
tromeres can also be frequently found in PBC. The presence of these two anti-
bodies or antibodies against GP210 indicate an unfavourable prognosis.

Anti-nuclear autoantibodies in primary biliary cirrhosis

Antigen Prevalence (%)
Nuclear dots 25 – 40
Nuclear membrane 20 – 40
SS-A 20
Centromeres 20 – 30

At times, antibodies against nuclear antigens are detectable in subjectively

healthy individuals, with a prevalence of 5 % and usually at a low titer (different
immunoglobulin classes, but mainly IgM).

Anti-nuclear autoantibodies: The most important associated diseases

Antigen Disease Prevalence (%)
dsDNA Systemic lupus erythematosus (SLE) 60 – 90
SLE 70 – 95
Drug-induced SLE 60
MCTD (Sharp syndrome) 20 – 50
ssDNA
Polymyositis / dermatomyositis 40 – 50
Progessive systemic sclerosis (PSS),
Sjögren‘s syndrome, rheum. arthritis 8 – 14
SLE 50
RNA
PSS, Sjögren‘s syndrome 65

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Anti-nuclear autoantibodies: The most important associated diseases

Antigen Disease Prevalence (%)
Drug-induced SLE 95
Histones SLE 50 – 80
Rheumatoid arthritis 15 – 50
MCTD (Sharp syndrome) 95 – 100
U1-nRNP SLE 15 – 40
Rheumatoid arthritis 3
Sm SLE 5 – 40
Sjögren‘s syndrome 40 – 95
SS-A (Ro) SLE 20 – 60
Neonatal lupus syndrome 100
Sjögren‘s syndrome 40 – 95
SS-B (La)
SLE 10 – 20
Fibrillarin PSS, diffuse form 5 – 10
RNA polymerase I PSS, diffuse form 4
RNA helicase A SLE 6
Poly- / dermatomyositis / overlap syndr. 50 – 70
PM-Scl (PM-1)
PSS, diffuse form 5 – 10
Centromeres PSS, limited form 80 – 95
Scl-70 PSS, diffuse form 25 – 75
Cyclin (PCNA) SLE 3
SLE 10
Ku
Poly- / dermatomyositis, PSS 30 – 55
Mi-1, Mi-2 Dermatomyositis 5 – 10

Antibodies against cytoplasmic components of HEp-2 cells cannot always be

clearly differentiated by their immunofluorescence pattern. Only a few cyto-
plasm-reactive antibodies can be assigned to a particular disease, e.g. antibod-
ies against mitochondria in primary biliary cirrhosis and antibodies against the
proteins, Jo-1, PL-7 and PL-12 in polymyositis and dermatomyositis. Further
rare antibodies found in polymyositis are those directed against OJ, EJ and
signal recognition particles (SRP). Other cytoplasmic antibodies – against ribo-
somes, Golgi apparatus, lysosomes and cytoskeletal components such as vi-
mentin and cytokeratins – are of minor clinical significance. The diagnostic val-
ue of mitosis-associated antigens has also not yet been finally clarified. When
all these arguments are considered, the high immunological relevance and the
resulting diagnostic value of anti-nuclear autoantibodies becomes clear.

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Autoantibodies against cell nuclei, homogeneous

HEp-2 cells Primate liver

HEp-2 cells show a homogeneous fluorescence of the cell nucleus. The con-
densed chromosomes of mitotic cells are accentuated. The area surrounding
the chromosomes is dark.

On the substrate primate liver a homogeneous, partly coarse to fine clumpy

fluorescence of the cell nuclei can be observed.

Known target antigens: dsDNA, ssDNA, nucleosomes and histones.

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Autoantibodies against dsDNA

Anti-dsDNA positive Anti-dsDNA negative Anti-dsDNA negative

(kinetoplast) (cell nucleus) (basal body)
The standard substrate for the immunofluorescence test is the haemoflagel-
late Crithidia luciliae. This possesses a dsDNA-containing giant mitochondrion
(kinetoplast) which, apart from dsDNA, essentially displays no antigens which
occur also in the cell nucleus. Antibodies which react with the kinetoplast are
therefore directed exclusively at dsDNA. With C. luciliae they produce a homo-
genous, partly edge-accentuated fluorescence of the kinetoplast. Any reaction
in the cell nucleus is not evaluated; fluorescence in the basal body of the flagel-
lum is without significance. Antibodies to ssDNA cannot stain the kinetoplast.

Clinical association: Autoantibodies against dsDNA are found exclusively in

SLE and in 40 - 90 % of cases, depending on the method of investigation and the
disease activity.

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Autoantibodies in DFS70 pattern

HEp-2 cells

On the substrate HEp-2 cells autoantibodies against the DFS70 antigen depict
a homogeneous nuclear pattern of dense fine speckles with a granular fluores-
cence of the chromatin in the metaphase.

Known target antigen: DFS70 or LEDGF (lens epithelium-derived growth factor)

Clinical association: Autoantibodies against DFS70 have been found in patients

with different diseases, such as atopic dermatitis, asthma and interstitial cys-
titis, and in healthy blood donors. Due to their low prevalence in systemic au-
toimmune rheumatic diseases it had been discussed whether the detection of
these autoantibodies can be used as an exclusion criterion. It has recently been
shown, however, that anti-DFS70 antibodies also occur in rheumatic diseases
with a prevalence of up to 11 %. The clinical association is therefore unclear.

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Autoantibodies against nuclear membrane

HEp-2 cells Primate liver

On HEp-2 cells the interphase cells show a homogeneous fluorescence of the

cell nucleus, with the rim of the nucleus accentuated. The chromosomes of
mitotic cells are dark.

On tissue sections of primate liver a characteristic linear fluorescence of the

nuclear membrane can be seen.

Known target antigen: GP210, lamin A, lamin B, lamin C, lamin B receptor

Clinical association: Antibodies against nuclear membrane occur in primary

biliary cirrhosis (PBC).

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Autoantibodies against nucleoplasm, coarse granular

HEp-2 cells Primate liver

HEp-2 cells generally show a coarse granular, sometimes medium to fine gran-
ular fluorescence, which is spread over the entire cell nucleus, leaving the nu-
cleoli free. In mitotic cells the condensed chromosomes are dark, while the
periphery shows an almost homogeneous, smooth fluorescence.

Tissue sections of primate liver also show a granular fluorescence. The nucleoli
do not react. The antibodies react with primate liver to the same extent as with
HEp-2 cells.

Known target antigens: U1-nRNP and Sm.

Clinical association: SLE and mixed connective tissue disease.

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Autoantibodies against nucleoplasm, fine granular

HEp-2 cells Primate liver

HEp-2 cells show a fine granular fluorescence of the cell nucleus in the inter-
phase. The nucleoli are also reactive, but they are slightly silhouetted against
the karyoplasm. In some samples they do not react at all. Mitotic cells show a
granular fluorescence, with the chromosomes excluded.

On tissue sections of primate liver there is no granular reaction in the hepato-

cyte nuclei, but the nucleoli show a smooth fluorescence in samples with a high
antibody titer.

Known target antigens: SS-A and SS-B.

Clinical association: Sjögren‘s syndrome, SLE and neonatal LE.

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Autoantibodies against Ku

HEp-2 cells Primate liver

In the indirect immunofluorescence test with HEp-2 cells, antibodies against

Ku exhibit a fine-speckled fluorescence of the cell nuclei and the nucleoli are
positive in parts. It is difficult to recognise the difference to antibodies to SS-A,
SS-B, Sm and RNP.

However, if primate liver sections are incubated in parallel, possibly in the

same field, a typical clumpy-speckled staining of the cell nuclei is found, which
is almost certain proof of antibodies to Ku.

Clinical association: The prevalence of autoantibodies against Ku is 25 – 50 % in

overlap syndrome of poly-/dermatomyositis and progressive systemic sclero-
sis (often accompanied by primary pulmonary hypertension), 5 – 10 % in vari-
ous forms of myositis, 10 % in systemic lupus erythematosus and up to 5 % in
progressive systemic sclerosis.

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Autoantibodies against Mi-2

HEp-2 cells Primate liver

Autoantibodies to Mi-2 show a fine-speckled fluorescence of the cell nuclei in

the indirect immunofluorescence test with HEp-2 cells. The nucleoli are partly
unaffected.

With primate liver autoantibodies against Mi-2 depict a fine-granular fluores-

cence of the hepatocyte nuclei.

Clinical association: Antibodies against Mi-2 are highly specific markers for
dermatomyositis with nail fold hypertrophy. They are found in 15 – 30 % of pa-
tients with dermatomyositis and in 8 – 12 % of patients with idiopathic myositis.

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Autoantibodies against PM-Scl

HEp-2 cells Primate liver

In the immunofluorescence test with HEp-2 cells, autoantibodies against PM-Scl

exhibit a homogeneous fluorescence of the nucleoli with a simultaneous weak-
er, fine-speckled reaction of the nucleoplasm. The condensed chromosomes of
the mitotic cells are unaffected; a fine, speckled fluorescence is shown outside
of the chromosomes.

A homogeneous fluorescence of the nucleoli also appears with frozen sections

of primate liver, as well as a very weak, fine-speckled to reticular staining of the
cell nuclei.

Clinical association: PM-Scl antibodies can be detected in 18 % of patients with

polymyositis/systemic sclerosis overlap syndrome. Here, the autoantibodies
are usually directed against both main antigens: PM-Scl75 and PM-Scl100. If
progressive systemic sclerosis is exclusively present, antibodies to PM-Scl75
show a prevalence of 10 %, and antibodies to PM-Scl100 a prevalence of 7 %.
With test systems which detect only anti-PM-Scl100, some patients with pro-
gressive systemic sclerosis remain unidentified.

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Autoantibodies against RNA polymerase I

HEp-2 cells Primate liver

HEp-2 cells show a granular fluorescence of the nucleoli. The nucleoplasm

is almost dark. In mitotic cells the region of condensed chromosomes is not
stained. Outside of the chromosomes a fine granular to smooth fluorescence
can be seen. In mitotic cells one to several dots may fluoresce, which corre-
spond to the nucleolus organisator (NOR).

Clinical association: Antibodies against RNA polymerase I have so far only been
detected in progressive systemic sclerosis (diffuse form). The prevalence is 4 %.

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Autoantibodies against U3-nRNP / fibrillarin

HEp-2 cells Primate liver

On HEp-2 cells interphase cells show a granular fluorescence of the nucleoli.

Mitotic cells have a coronary perichromosomal fluorescence.

The substrate primate liver depicts a homogeneous fluorescence of the cell

nuclei.

Clinical association: Antibodies against fibrillarin have so far been observed

only in progressive systemic sclerosis (diffuse form). The prevalence is 5 – 10 %.

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Autoantibodies against Scl-70

HEp-2 cells Primate liver

HEp-2 cells show an almost homogeneous nuclear fluorescence in interphase

cells. The nucleoli are accentuated, also showing a homogeneous fluorescence.
The cytoplasm is dark. In mitotic cells only the border area of condensed chro-
mosomes fluoresces.

Clinical association: Scl-70 antibodies are detected in 25 – 75% of patients with

progessive systemic sclerosis (diffuse form), depending on the analysis meth-
od and the activity of the disease.

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Autoantibodies against nuclear dots

HEp-2 cells Primate liver

In immunofluorescence using HEp-2 cells, five to more than twenty differently

sized granules which are spread over the cell nucleus (nuclear dots) can be
seen in the nuclei during interphase. The cytoplasm is dark if antibodies against
mitochondria, which are associated with primary biliary cirrhosis, are not pre-
sent at the same time. In mitotic cells the nuclear dots are dissolved. Outside
the (unstained) chromosomes only isolated granules fluoresce.

Antibodies against nuclear dots react with primate liver to the same extent as
with HEp-2 cells. If both substrates are used in parallel, these antibodies can
even be identified if antibodies against centromeres are present at the same
time. This can occasionally be observed in cases of primary biliary cirrhosis.

Known target antigens: Sp100, Sp140, PML, SUMO

Clinical association: Autoantibodies against nuclear dots occur in 10 – 30 % of

patients with primary biliary cirrhosis.

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Autoantibodies against centromeres

HEp-2 cells Primate liver

HEp-2 cells show a very specific fluorescence pattern, which is characterised by

fine, evenly sized granules (generally 46 or 92 centromeres per cell nucleus).
The granules in interphase cells are spread evenly over the nucleus, while in
mitotic cells they are arranged either ribbon-like in the median plane (meta-
phase) or in two parallel ribbons approaching the centrioles (anaphase).

On tissue sections of primate liver 10 to 20 granules, which are spread over

the cell nuclei, can be seen. The fluorescence of these granules is significantly
weaker than the HEp-2 cell staining and is therefore easy to miss. Mitotic cells
are only rarely detected on liver substrate.

Clinical association: With a high specificity and a prevalence of 80 – 95 %, an-

tibodies against centromeres are pathognomonic for the limited form of pro-
gressive systemic sclerosis. In the limited form the extremities are favoured
and the inner organs less affected.

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Autoantibodies against PCNA

HEp-2 cells Primate liver

Autoantibodies against PCNA show a cell cycle-dependent fluorescence pattern

with HEp-2 cells. Half of the cell nuclei of all interphase cells exhibit a bright,
fine-speckled basic fluorescence with the nucleoli being unaffected. The same
fluorescence pattern is found with the other half, but the intensity is lower by
a factor of 10. The area of the condensed chromosomes is not stained in the
mitosis; the surrounding area of the chromosomes shows only a weak, fine-
speckled fluorescence, corresponding to the darker nuclei of the interphase
cells in pattern and intensity.

The reaction with primate liver is largely negative.

Clinical association: PCNA antibodies are specific for SLE. The prevalence,
however, is only 3 %.

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Autoantibodies against centrioles

HEp-2 cells Primate liver

A typical positive result is characterised by fluorescing centrioles in the cyto-

plasm of HEp-2 cells, namely one or two centrioles per cell. In mitotic cells the
centrioles are located at two opposing poles.

With primate liver, high-titer samples produce small fluorescing dots in the
cytoplasm of hepatocytes.

Clinical association: A high titer (> 1:1,000) indicates progressive systemic scle-
rosis or Raynaud’s syndrome. But the prevalence is only a few percent.

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Autoantibodies against spindle fibres

HEp-2 cells Primate liver

Using HEp-2 cells antibodies against MSA1 and MSA2 (HSAG5) antigens can
be detected. MSA1 antibodies cause a fine granular to reticular pattern of the
nuclear matrix in interphase cells. The nucleoli do not fluoresce. In the pres-
ence of MSA-2 antibodies the interphase cell nuclei are not stained.

On tissue sections of primate liver a granular fluorescence of the cell nuclei can
be observed.

Clinical association: Autoantibodies against MSA-1 are associated with

Sjögren‘s syndrome, but they are also found in other rheumatic diseases. Auto-
antibodies against MSA-2 occur predominantly in SLE.

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Autoantibodies against midbody

HEp-2 cells Primate liver

In the indirect immunofluorescence test, HEp-2 cells in the metaphase of mi-

tosis show a fine granular fluorescence of the median plane in the presence of
midbody antibodies. In contrast to the pattern found with antibodies to cen-
tromeres, this fluorescing line remains in the middle until the end of mitosis.
Their length corresponds to the whole cell width in the separation zone, and the
line increasingly shortens until only a fluorescing dot is seen in the telophase,
binding the daughter cells together (“goodbye kiss”). Half of the interphase
cells contain numerous coarser fluorescing drops; the remaining cells are dark.

On tissue sections of primate liver there is an unspecific fluorescence.

Clinical association: The diagnostic significance of these antibodies is still un-

known.

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Autoantibodies against mitochondria

HEp-2 cells Primate liver

HEp-2-cells contain the antigens M2, M3, M5 and M9; here the antibodies pro-
duce a coarse granular fluorescence of the cytoplasm which does not include
the nucleus (previously, the likewise PBC-relevant nuclear dots also reacting
were wrongly suspected of being stray mitochondria).

The primate liver shows a granular fluorescence of the cytoplasm. The cell nu-
clei are dark. The reaction of the tissue is generally weaker than that of HEp-2
cells.

Clinical association: Autoantibodies against mitochondria can be detected in

various diseases. They often occur together with other autoantibodies, e.g. with
autoantibodies against cell nuclei. Antibodies to mitochondria are of particular
significance for the diagnosis of primary biliary cirrhosis (PBC). The prevalence
is up to 98 %.

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Autoantibodies against ribosomal P-proteins

HEp-2 cells Primate liver

Autoantibodies against ribosomal P-proteins cause a smooth to fine granular

staining of the cytoplasm when using HEp-2 cells as the substrate.

Hepatocytes of the primate liver show a fluorescence of the entire surface with
patchy accentuation. There is no reaction with low-titer samples.

Clinical association: Autoantibodies against ribosomal P-proteins are a charac-

teristic marker for SLE. The prevalence is around 10 %.

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Autoantibodies against Jo-1

HEp-2 cells Primate liver

Antibodies against Jo-1 show a fine granular to homogenous cytoplasmic fluo-

rescence in the indirect immunofluorescence test (IIFT) with HEp-2 cells. The
cell nuclei also show distinct sharp dots in many cases. According to recent
findings, these enzymes are not solely localised in the cytoplasm, but are also
found in the cell nucleus in some species.

On tissue sections of primate liver the cytoplasm is only slightly stained. The
fluorescence cannot be used for diagnostics.

Clinical association: Antibodies against Jo-1 can be detected in polymyositis

with a prevalence of 25 – 35 %. They are often associated with other concurrent
autoimmune diseases such as SLE, systemic sclerosis, interstitial lung fibrosis,
Raynaud‘s syndrome and polysynovitis.

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Autoantibodies against SRP and PL-12

HEp-2 cells HEp-2 cells

Autoantibodies against SRP (left) produce a mainly cytoplasmic, smooth to fine

granular fluorescence on HEp-2 cells. In mitotic cells the fluorescence is peri-
chromosomally intensified, the chromosomes are unaffected.

Autoantibodies against PL-12 (right) show a fine granular to homogenous cy-

toplasmic fluorescence with HEp-2 cells. The cell nuclei also show distinct
clear dots in many cases. According to recent findings, these enzymes are
not solely localised in the cytoplasm, but are also found in the cell nucleus in
some species.

Clinical association: Antibodies against SRP can be found in polymyositis and

dermatomyositis in approx. 5 % of cases. They are also markers for necrotis-
ing myopathy, an autoimmune disease that differs from polymyositis, but can
manifest with skin changes typical for dermatomyositis. Antibodies against
PL-12 occur in myositis with a prevalence of 3 %.

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Autoantibodies against Golgi apparatus

HEp-2 cells Primate liver

Autoantibodies against Golgi apparatus present in the indirect immunofluores-

cence on HEp-2 cells as reticular-granular structures which are in contact with
the cell nucleus on one side. The cytoplasm of the hepatocytes is also stained.
In HEp-2 cells which are in the mitosis, the Golgi apparatus is to a large extent
dispersed. Here the antibodies show no reaction.

On primate liver the cytoplasm of hepatocytes is also stained.

Clinical association: Autoantibodies against Golgi apparatus occur in different

autoimmune diseases, particularly in SLE and Sjögren‘s syndrome. Detection
of these antibodies has little relevance due to their low disease specificity.

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Autoantibodies against lysosomes

HEp-2 cells Primate liver

On HEp-2 cells antibodies against lysosomes show a fine to medium or coarse

droplet-shaped fluorescence of the cytoplasm.

On tissue sections of primate liver there is an unspecific fluorescence.

Clinical association: The diagnostic significance of these antibodies is un-

known. They can occasionally be found in healthy individuals.

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Autoantibodies against F-actin

VSM47 HEp-2 cells Primate liver

Autoantibodies against F-actin cause a microfilamentous fluorescence pattern

using the cell line VSM47 (vascular smooth muscle).

On HEp-2 cells individual or several bunched fibre structures fluoresce. They

are located primarily in the cytoplasm, but can also stretch over the cell nuclei.

On tissue sections of primate liver there is a strong reaction of the bile canali-
culi.

Clinical association: The determination of autoantibodies against F-actin is

of particular significance for the diagnosis of AIH (prevalence around 50 %),
the exclusion of a combined liver disease (overlap syndrome) and for delimi-
tation of AIH against alcohol- or drug-induced cirrhosis and other forms of
chronic liver inflammation, such as virus-induced hepatitis, primary biliary
liver cirrhosis (PBC) and primary sclerosing cholangitis (PSC).

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Autoantibodies against vimentin

HEp-2 cells Primate liver

Antibodies against vimentin cause staining of a fine net of fibres in the cy-
toplasm of HEp-2 cells. The net is particularly dense near the cell nucleus. In
mitotic cells numerous round fluorescing droplets can be seen outside the dark
chromosomes. These are probably condensed vimentin.

On tissue sections of primate liver there is an unspecific fluorescence.

Clinical association: It is not known whether autoantibodies against vimentin

have any diagnostic relevance. The same goes for other rare autoantibodies
such as cytokeratin, tropomyosin, vinculin, etc.

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Cytoplasmic rings & rods

HEp-2 cells

Rings and rods are a cytoplasmic pattern on HEp-2 cells that has been described
only recently. These filamentous structures, which are expressed in all stages
of the cell cycle, present themselved as rings, rods or loops. It is assumed that
the reaction is directed against the autoantigen inosine monophosphate dehy-
drogenase 2 (IMPDH2) (Seelig et al.).

Clinical association: The depicted pattern was observed mainly in patients with
hepatitis C infections, particularly after treatment with interferon-alpha or riba-
virin (prevalence 35 %).

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Dilution scheme for immunofluorescence

Titration of serum samples in steps from 1:10 or 1:3.2 (square root of 10). is
recommended for all EUROIMMUN immunofluorescence test systems. The in-
dividual dilutions can be easily set up without the need for numerical acrobat-
ics (1:10, 1:32, 1:100, 1:320, 1:1,000, etc.).

Previously the precision was exaggerated by using quadratic dilution steps. On

the other hand, titrating by a factor of 4 results in too rough a framework.

For every test parameter there is a suitable starting dilution. In order to sim-
plify the test procedure and evaluation of results, two antibody categories are
differentiated at EUROIMMUN: Antibodies of group I are already diagnostically
relevant at at titer of 1:10, while those of group II first at 1:100.

The titers determined for each sample are classified using the symbols (+) to
++++. The differing clinical significance of antibody titers for the two groups is
already incorporated into this scheme.

Serum
1:10 1:32 1:100 1:320 1:1,000 1:3,200 1:10,000
dilution

Evaluation,
+ ++ ++ +++ +++ ++++ ++++
group I

Evaluation,
+ ++ +++ ++++ ++++
group II

= suitable starting dilutions

+ = weak positive, ++ = positive, +++ = strong positive, ++++ = very strong positive
Group I: most organ-specific autoantibodies (AAb), ANCA, AAb against dsDNA
Group II: ANA, AMA, ASMA, AAb against skeletal muscle

In order to make optimal use of the great potential of indirect immunofluores-

cence, experts in this area always test for the majority of autoantibodies using
two dilutions, for the following reasons:

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Blocking effect: In two out of every 100 high-titer sera an untypical result is
seen with the starting dilution. Some strongly positive sera even react as false
negative if they are not sufficiently diluted.

1:100 1:1,000 1:10 1:100

AMA (kidney) Anti-Yo (cerebellum)

1:10 1:100 1:1 1:10

Anti-epid. basal membrane (tongue) pANCA (granulocytes)

Autoantibody masking: If unspecific antibodies or additional, visually domi-

nant autoantibodies are present in too high a concentration, they can mask a
relevant antibody.

1:100 1:1,000 1:100 1:1,000

Anti-ribosomes and anti-nucleoli ANA, homog. and anti-centromeres

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Titer estimation: When two dilutions with an interval of a factor of 10 are used,
a titer can be determined for most positive results without further incubations
(see table). Results are obtained one day earlier than with stepwise titrations.

Fluorescence at
1:100 1:1,000 AAb titer
weak negative 1:100
strong negative 1:320
strong weak 1:1,000
strong moderate 1:3,200
strong strong 1:10,000

In contrast, it is not possible to quantify a positive result from a single dilution:

AAb show very different abating behaviour depending on the avidity. This is
detected by the parallel analysis. Photometric systems based on cytochemical
ELISA or fluorescence are obsolete.

1:100 1:1,000 1:100 1:1,000

ANA, homogeneous (titer 1:1,000) ANA, homogeneous (titer 1:10,000)

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Drug.-induced lupus erythematosus

SYSTEMIC

Systemic lupus erythematosus

Neonatal lupus erythematosus
AUTOANTIBODIES AND

Progressive systemic sclerosis

Paraneoplastic autoimmunity
Anti-phospholipid syndrome
ASSOCIATED

Sharp syndrome (MCTD)

Primary biliary cirrhosis

AUTOIMMUNE DISEASES

Autoimmune hepatitis
Rheumatoid arthritis

Sjögren‘s syndrome
Habitual abortions
Domains of immunofluorescence

Dermatomyositis
Diagnostically

Polymyositis
essential
relevant

Dated 19.06.2012

Cell nuclei (ANA)

CENP-F (cyclin II – mitosin)
dsDNA
Nucleosomes
Sm
U1-RNP
SS-A (Ro, 60 kDa)
SS-B (La)
Histones
PCNA (cyclin I)
RNA helicase A
MSA1 (NuMA)
MSA2 (HsEg5)
Scl-70
Fibrillarin (U3-RNP)
RNA polymerases I, II, III
NOR-90, PDGF-R
Centromeres (CENP-A, CENP-B)
Autoantibodies against

PM-Scl (1, 75, 100)

Ku
Mi-2
SRP
Jo-1
PL-7, PL-12, OJ, EJ, SC, KS
Nuclear dots
Sp100, Sp140, SUMO, PML
Nuclear membrane, lamin B recept.
GP210, NUP62
Cardiolipin
Beta-2 glycoprotein
Phosphatidylserine
Coagulation factor (lupus anticoag.)
Prothrombin, annexin A5
C1q
Mitochondria (AMA)
Mitochondria (AMA M2-3E / BPO)
Ribosomal P-proteins
ASMA
F-actin
IgG (rheumatoid factors)
Citrullinated peptides (CCP)
Sa
Filaggrin, RA keratin
Citrullinated enolase peptide (CEP-1)

EUROIMMUN AG · D-23560 Luebeck (Germany) · Seekamp 31 · Tel +49 45158550 · Fax 5855591 · E-mail euroimmun@euroimmun.de
FA_1510_I_UK_B03, 05/2013