EUROLINE ANA Profile 3 (IgG)

Test instruction
ORDER NO. ANTIBODIES AGAINST IG CLASS SUBSTRATE FORMAT
nRNP/Sm, Sm, SS-A, Ro-52, SS-B,
DL 1590-1601-3 G Scl-70, PM-Scl, Jo-1, CENP B, Ag-coated 16 x 01 (16)
IgG
DL 1590-6401-3 G PCNA, dsDNA, nucleosomes, immunoblot strips 64 x 01 (64)
histones, rib. P-prot., AMA M2

Indications: The EUROLINE test kit provides qualitative in vitro determination of human autoantibodies
of the immunoglobulin class IgG to the 15 different antigens nRNP/SM, Sm, SS-A, Ro-52, SS-B, Scl-70,
PM-Scl, Jo-1, CENP B, PCNA, dsDNA, nucleosomes, histones, ribosomal P-protein and AMA M2
in serum or plasma to support the diagnosis of Sharp syndrome (MCTD), systemic lupus erythematosus
(SLE), Sjögren’s syndrome, progressive systemic sclerosis, poly-/dermatomyositis, overlap syndromes,
limited form of progressive systemic sclerosis (CREST syndrome), primary biliary cholangitis.

Application: The EUROLINE ANA Profile 3 (IgG) for the detection of antibodies against 15 different
nuclear, cytoplasmic and mitochondrial antigens offers a multiplex approach for the determination of
these antibodies in a single reaction, with optimal, fully automated processing and objective evaluation of
the test results using the EUROLineScan software. These antibodies are linked to rheumatic diseases
and autoimmune liver diseases.

Principles of the test: The test kit contains test strips coated with parallel lines of highly purified
antigens. In the first reaction step, the immunoblot strips are incubated with diluted patient samples. In
the case of positive samples, the specific IgG antibodies (also IgA and IgM) will bind to the
corresponding antigenic site. To detect the bound antibodies, a second incubation is carried out using an
enzyme-labelled anti-human IgG (enzyme conjugate) catalysing a colour reaction.

Contents of the test kit:

Component Format Format Symbol
1. Test strips coated with the antigens:
nRNP/Sm, Sm, SS-A, Ro-52, SS-B, Scl-70, PM-Scl,
16 strips 4 x 16 strips .STRIPS.
Jo-1, CENP B, PCNA, dsDNA, nucleosomes,
histones, rib. P-protein, AMA M2
2. Positive control
1 x 0.02 ml 4 x 0.02 ml .POS CONTROL 100x.
(IgG, human), 100x concentrate
3. Enzyme conjugate
Alkaline phosphatase-labelled anti-human IgG (goat), 1 x 3 ml 4 x 3 ml .CONJUGATE 10x.
10x concentrate
5. Sample buffer
1 x 100 ml 3 x 100 ml .SAMPLE BUFFER.
ready for use
6. Wash buffer
1 x 50 ml 1 x 100 ml .WASH BUFFER 10x.
10x concentrate
7. Substrate solution
Nitrobluetetrazoliumchloride/5-Bromo-4-chloro-3- 1 x 30 ml 4 x 30 ml .SUBSTRATE.
indolylphosphate (NBT/BCIP), ready for use
8. Incubation tray 2 x 8 channels ---
9. Test instruction 1 booklet 1 booklet
.LOT. Lot description Storage temperature
.IVD. In vitro diagnostic medical device Unopened usable until
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The following components are not provided in the test kits but can be ordered at EUROIMMUN under the
respective order numbers.
Performance of the test requires an incubation tray:
ZD 9895-0130 Incubation tray with 30 channels
ZD 9898-0144 Incubation tray with 44 channels (black, for the EUROBlotOne and EUROBlotCamera
system)
For the creation of work protocols and the evaluation of incubated test strips using EUROLineScan
green paper and adhesive foil are required:
ZD 9880-0101 Green paper (1 sheet)
ZD 9885-0116 Adhesive foil for approx. 16 test strips
ZD 9885-0130 Adhesive foil for approx. 30 test strips
If a visual evaluation is to be performed in individual cases, the required evaluation protocol can be
ordered under: ZD 1590-0101-3 G Visual evaluation protocol ANA Profile 3 EUROLINE.

Preparation and stability of the reagents

Note: All reagents must be brought to room temperature (+18°C to +25°C) approx. 30 minutes before
use. Unopened reagents are stable until the indicated expiry date when stored at +2°C to +8°C. After
initial opening, reagents are stable for 12 months or until the expiry date, unless stated otherwise in the
instructions. Opened reagents must also be stored at +2°C to +8°C and protected from contamination.

- Coated test strips: Ready for use. Open the package with the test strips only when the strips have
reached room temperature (+18°C to +25°C) to prevent condensation on the strips. After removal of
the strips the package should be sealed tightly and stored at +2°C to +8°C.

- Positive control: The control is a 100x concentrate. For the preparation of the ready for use control
the amount required should be removed from the bottle using a clean pipette tip and diluted 1:101
with sample buffer. Example: add 15 µl of control to 1.5 ml of sample buffer and mix thoroughly. The
ready for use diluted control should be used at the same working day.

- Enzyme conjugate: The enzyme conjugate is supplied as a 10x concentrate. For the preparation of
the ready for use enzyme conjugate the amount required should be removed from the bottle using a
clean pipette tip and diluted 1:10 with sample buffer. For one test strip, dilute 0.15 ml enzyme
conjugate with 1.35 ml sample buffer. The ready for use diluted enzyme conjugate should be used at
the same working day.

- Sample buffer: Ready for use.

- Wash buffer: The wash buffer is supplied as a 10x concentrate. For the preparation of the ready for
use wash buffer the amount required should be removed from the bottle using a clean pipette tip and
diluted 1:10 with distilled water. For one test strip, dilute 1 ml in 9 ml of distilled water. The ready for
use diluted wash buffer should be used at the same working day.

- Substrate solution: Ready for use. Close bottle immediately after use, as the contents are sensitive
to light .

Storage and stability: The test kit must be stored at a temperature between +2°C to +8°C. Do not
freeze. Unopened, all test kit components are stable until the indicated expiry date.

Waste disposal: Patient samples, controls and incubated test strips should be handled as infectious
waste. Other reagents do not need to be collected separately, unless stated otherwise in official
regulations.

Warning: The control of human origin has tested negative for HBsAg, anti-HCV, anti-HIV-1 and anti-
HIV-2. Nonetheless all materials should be treated as being a potential infection hazard and should be
handled with care. Some of the reagents contain the agent sodium azide in a non-declarable
concentration. Avoid skin contact.

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Preparation and stability of the patient samples

Samples: Human serum or EDTA, heparin or citrate plasma.

Stability: Patient samples to be investigated can generally be stored at +2°C to +8°C for up to 14 days.
Diluted samples should be incubated within one working day.

Sample dilution: The patient samples for analysis are diluted 1:101 with sample buffer using a clean
pipette tip. For example, add 15 µl of sample to 1.5 ml sample buffer and mix well by vortexing. Sample
pipettes are not suitable for mixing.

Incubation

Pretreat: Remove the required amount of test strips from the package and place them
each in an empty channel (Make sure that the surface of the test strips is not
damaged!). The number on the test strip should be visible. Fill the channels of
the incubation tray according to the number of serum samples that should be
tested with 1.5 ml sample buffer each.
Incubate for 5 minutes at room temperature (+18°C to +25°C) on a rocking
shaker. Afterwards aspirate off all the liquid.

Incubate: Fill each channel with 1.5 ml of the diluted serum samples using a clean
(1st step) pipette tip.
Incubate at room temperature (+18°C to +25°C) for 30 minutes on a rocking
shaker.

Wash: Aspirate off the liquid from each channel and wash 3 x 5 minutes each with
1.5 ml working-strength wash buffer on a rocking shaker.

Incubate: Pipette 1.5 ml diluted enzyme conjugate (alkaline phosphatase-labelled anti-

(2nd step) Human IgG) into each channel.
Incubate for 30 minutes at room temperature (+18°C to +25°C) on a rocking
shaker.

Wash: Aspirate off the liquid from each channel. Wash as described above.

Incubate: Pipette 1.5 ml substrate solution into the channels of the incubation tray.
(3rd step) Incubate for 10 minutes at room temperature (+18°C to +25°C) on a rocking
shaker.

Stop: Aspirate off the liquid from each channel and wash each strip 3 x 1 minute
with distilled water.

Evaluate: Place test strip on the evaluation protocol, air dry and evaluate.

For automated incubation with the EUROBlotMaster select the program Euro01 AAb EL30.

For automated incubation with the EUROBlotOne select the program Euro 01/02.

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EUROLINE ANA Profile 3 (IgG)

Incubation protocol

Pretreat
Put the test strip into the incubation channel and fill each
channel with 1.5 ml sample buffer

5 min Shake

1. Step: Incubate
Aspirate off, pipette 1.5 ml of diluted serum sample (1:101) into
the incubation channel

30 min Shake

Wash
Aspirate off, wash 3 x 5 min with 1.5 ml working-strength
wash buffer each

2. Step: Incubate
Aspirate off, pipette 1.5 ml working-strength enzyme conjugate
into the incubation channel

30 min Shake

Wash
Aspirate off, wash 3 x 5 min with 1.5 ml working-strength
wash buffer each

3. Step: Incubate
Aspirate off, pipette 1.5 ml substrate into the incubation channel

10 min Shake

Stop
Aspirate off, rinse three times with 1.5 ml distilled water

Evaluation
EUROLineScan (digital)

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Interpretation of Results
Handling: For the evaluation of incubated test strips we generally recommend using the
EUROLineScan software. After stopping the reaction using deionised or distilled water, place the in-
cubated test strips onto the adhesive foil of the green work protocol using a pair of tweezers. The
position of the test strips can be corrected while they are wet. As soon as all test strips have been placed
onto the protocol, they should be pressed hard using filter paper and left to air-dry. After they have dried,
the test strips will be stuck to the adhesive foil. The dry test strips are then scanned using a flatbed
scanner (EUROIMMUN AG) and evaluated with EUROLineScan. Alternatively, imaging and evaluation
are possible directly from the incubation trays (EUROBlotCamera and EUROBlotOne). For general
information about the EUROLineScan program please refer to the EUROLineScan user manual
(YG_0006_A_UK_CXX, EUROIMMUN AG). The code for entering the test into EUROLineScan is
Ana3b.
If a visual evaluation must be performed, place the incubated test strips onto the respective work
protocol for visual evaluation. This protocol is available at EUROIMMUN under the order no.
ZD 1590-0101-3 G.

Note: Correct performance of the incubation is indicated by an intense staining of the control band. A
white band at the position of an antigen has to be interpreted as negative.

Antigens and their arrangement on the strips: The EUROLINE test strips have been coated with the
following antigens:
nRNP/Sm: U1-nRNP purified by affinity chromatography from calf and
rabbit thymus. nRNP/Sm
Sm: Sm antigen purified by affinity chromatography from bovine
spleen and thymus. The Sm antigen contains the core proteins of
Sm
snRNP particles. D protein is the main component of the Sm
preparation.
SS-A
SS-A: SS-A antigen (60 kDa) purified by affinity chromatography from Ro-52
bovine spleen and thymus.
Ro-52: Recombinant Ro-52 (52 kDa). The corresponding human SS-B
cDNA has been expressed with the baculovirus system in insect cells.
SS-B: SS-B antigen purified by affinity chromatography from calf and
rabbit thymus. Scl-70
Scl-70: Scl-70 (DNA-topoisomerase I) antigen purified by affinity
chromatography from bovine and rabbit thymus. PM-Scl
PM-Scl: Recombinant PM-Scl100. The corresponding human cDNA
has been expressed with the baculovirus system in insect cells.
Jo-1
Jo-1: Jo-1 (Histidyl-tRNA synthetase) antigen purified by affinity
chromatography from calf and rabbit thymus.
CENP B: Recombinant centromere protein B. The corresponding CENP B
human cDNA has been expressed with the baculovirus system in
insect cells.
PCNA: Recombinant PCNA (36 kDa). The corresponding human PCNA
cDNA has been expressed with the baculovirus system in insect cells.
dsDNA: Highly purified native, double-stranded DNA isolated from dsDNA
salmon testes. Nucleosomes
Nucleosomes: Native nucleosomes purified from calf thymus.
Histones: A mixture of individually purified histone types isolated from Histones
calf thymus.
Rib. P-protein: Ribosomal P-proteins purified by affinity Rib. P-
chromatography. protein
AMA M2: Purified M2 antigen (pyruvate-dehydrogenase complex).
AMA M2

Control

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EUROIMMUN recommends interpreting results based on the signal intensity:

Signal intensity
Signal
EUROLineScan Result
Visual evaluation
Flatbed scanner
No signal 0-5 o Negative
Very weak band 6-10 (+) Borderline
Medium to strong band 11-25 or 26-50 +, ++ Positive
Very strong band with an intensity comparable Strong
>50 +++
to the control band positive

Results in the borderline range (+) should be evaluated as increased but negative. The table above
contains values for the evaluation using a flatbed scanner. The values for other instruments supported
by EUROLineScan can be found in the EUROLineScan program. To do so mark the corresponding
assay in the test list (main menu: “Help” → “Test”) and click on details and select the corresponding
instrument in “image source”.

An indirect immunofluorescence test should always be performed in parallel with the determination of
cell nucleus antibodies by EUROLINE. On the one hand, this provides a check on plausibility as a safe-
guard against false-positive results, on the other hand, by using EUROIMMUN HEp-2 cells, and in
particular in combination with frozen sections of primate liver, immunofluorescence permits the
detection of a wider range of cell nucleus antibodies, as not all cell nucleus antigens are presently
available in the EUROLINE.

For the medical diagnosis, the clinical symptoms of the patient and, if available, further findings should
always be taken into account alongside the serological result. A negative serological result does not
exclude the presence of a disease.

Test characteristics
Calibration: The reactivity of each antigen is standardised by the human reference sera CDC-ANA #1 to
#11 of the “Centers for Disease Control and Prevention” (CDC, Atlanta, USA). The reactivity of the CDC
sera in the EUROIMMUN ANA Profile EUROLINE is summarised in the following table:

CDC-1 CDC-2 CDC-3 CDC-4 CDC-5 CDC-6 CDC-7 CDC-8 CDC-9 CDC-10 CDC-11

Antigen Homoge Speckled/ Speckled RNP Sm Nucleolar SS-A Centro- Scl-70 Jo-1 PM-Scl
neous/ SS-B mere
rim
nRNP/Sm pos. neg. pos. pos. pos. neg. neg. neg. neg. neg. neg.
Sm pos. neg. pos. neg. pos. neg. neg. neg. neg. neg. neg.
SS-A neg. pos. pos. neg. neg. neg. pos. neg. neg. neg. neg.
Ro-52 neg. pos. pos. neg. neg. neg. pos. neg. neg. pos. neg.
SS-B neg. pos. pos. neg. neg. neg. neg. neg. neg. neg. neg.
Scl-70 neg. neg. neg. neg. neg. neg. neg. neg. pos. neg. neg.
PM-Scl neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. pos.
Jo-1 neg. neg. neg. neg. neg. neg. neg. neg. neg. pos. neg.
CENP B neg. neg. neg. neg. neg. neg. neg. pos. neg. neg. neg.
PCNA neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg.
dsDNA pos. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg.
Nucleosomes pos. neg. neg. neg. neg. neg. pos. neg. neg. neg. neg.
Histones pos. neg. neg. neg. neg. neg. pos. neg. neg. neg. neg.
Rib. P-protein neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg.
M2 neg. neg. neg. neg. neg. neg. neg. pos. neg. neg. neg.
The specificity of these sera was determined at the CDC by immunofluorescence patterns (substrate:
HEp-2 cells and primate liver), by the results of double immunodiffusion or counter
immunoelectrophoresis (the sera are not in any case monospecific).

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Measurement range: The EUROLINE is a qualitative method. No measurement range is provided.

Cross reactions: The high analytical specificity of the test system is guaranteed by the quality of the
antigen substrates used (antigens and antigen sources). This EUROLINE specifically detects IgG class
antibodies to nRNP/Sm, Sm, SS-A, Ro-52, SS-B, Scl-70, PM-Scl, Jo-1, CENP B, PCNA, dsDNA,
nucleosomes, histones, ribosomal P-protein and AMA M2. No cross reactions with other autoantibodies
have been found.

Interference: Haemolytic, lipaemic and icteric sera up to a concentration of 5 mg/ml for haemoglobin, of
20 mg/ml for triglycerides and of 0.4 mg/ml bilirubin showed no effect on the analytical results of the
present EUROLINE.

Inter- and intra-assay variation: The inter-assay variation was determined by multiple analyses of
characterised samples over several days. The intra-assay variation was determined by multiple analyses
of characterised samples on one day. In every case, the intensity of the bands was within the specified
range. This EUROLINE displays excellent inter- and intra-assay reproducibility.

Sensitivity and specificity:

nRNP/Sm: For the detection of autoantibodies against RNP/Sm a sensitivity of 100% with reference to
the ELISA method was determined using 22 samples of patients with MCTD (mixed connective tissue
disease). The specificity was 100% for healthy blood donors (n = 50) and 98% in a panel of non-SLE
rheumatic diseases (Sjögren`s syndrome n = 14, systemic sclerosis n = 18, polymyositis n = 25).

Sm: For the detection of autoantibodies against Sm a sensitivity of 100% with reference to the ELISA
method was determined using 45 samples of patients with SLE. The specificity was 100% for healthy
blood donors (n = 50) and 100% in a panel of non-SLE rheumatic diseases (Sjögren`s syndrome n = 14,
systemic sclerosis n = 18, polymyositis n = 25).

SS-A: For the detection of autoantibodies against SS-A a sensitivity of 100% with reference to the ELISA
method was determined using 14 samples of patients with Sjögren`s syndrome. The specificity was
100% for healthy blood donors (n = 50) and 97.4% in a panel of non-SLE rheumatic diseases (systemic
sclerosis n = 18, MCTD n = 22).

Ro-52: The investigation of sera from 103 patients (SLE n = 23, Sjögren’s syndrome n = 77 and
neonatal lupus erythematosus n = 3) yielded a sensitivity of 100% for the detection of autoantibodies
against
Ro-52 with reference to the Westernblot method. The specificity was 100% for healthy blood donors
(n = 65). Antibodies against Ro-52 are not disease specific and can be detected in samples from
patients suffering from myositis, systemic sclerosis and other collagenoses. As an example, the
prevalence of autoantibodies against Ro-52 in sera from patients with systemic sclerosis (n = 20) was
determined to be 31.6%.

SS-B: For the detection of autoantibodies against SS-B a sensitivity of 100% with reference to the ELISA
method was determined using 14 samples of patients with Sjögren`s syndrome. The specificity was
100% for healthy blood donors (n = 50) and 100% in a panel of non-SLE rheumatic diseases (systemic
sclerosis n = 18, MCTD n = 22).

Scl-70: For the detection of autoantibodies against Scl-70 a sensitivity of 100% with reference to the
ELISA method was determined using 18 samples of patients with systemic sclerosis. The specificity was
100% for healthy blood donors (n = 50) and for a panel of non-SLE rheumatic diseases (MCTD n = 22,
Sjögren`s syndrome n = 14, myositis n = 25).

PM-Scl: In 14 of 20 sera of patients with polymyositis, having a nucleolar-positive pattern in the indirect
immunofluorescence (HEp-2 cells/primate liver), autoantibodies against PM-Scl were detected. The
specificity was 100% for healthy blood donors (n = 50) and 100% in a panel of non-SLE rheumatic
diseases (MCTD n = 22, Sjögren`s syndrome n = 14, systemic sclerosis n = 18).

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Jo-1: For the detection of autoantibodies against Jo-1 a sensitivity of 100% with reference to the ELISA
method was determined using 5 samples of patients with myositis. The specificity was 100% for healthy
blood donors (n = 50) and 100% in a panel of non-SLE rheumatic diseases (systemic sclerosis n = 18,
MCTD n = 22, Sjögren`s syndrome n = 14).

CENP B: In 19 of 20 sera of patients with systemic sclerosis, having a centromer-positive pattern in the
indirect immunofluorescence (HEp-2 cells/primate liver), autoantibodies against CENP B (sensitivity
95%) were detected. The specificity was 100% for healthy blood donors (n = 50) and 100% in a panel of
non-SLE rheumatic diseases (MCTD n = 22, Sjögren`s syndrome n = 14, myositis n = 25).

PCNA: In 13 of 20 patient sera, having a cyclin I-positive pattern in the indirect immunofluorescence
(HEp-2 cells/primate liver), autoantibodies against PCNA were detected. The specificity was 100% for
healthy blood donors (n = 50) and 100% in cyclin I-negative sera of patients with SLE (n = 83).

dsDNA: For the detection of autoantibodies against dsDNA a sensitivity of 93% with reference to the
ELISA method was determined using 36 samples of patients with SLE. The specificity was 100% for
healthy blood donors (n = 50) and for a panel of non-SLE rheumatic diseases (Sjögren`s syndrome
n = 14, systemic sclerosis n = 18).

Nucleosomes: For the detection of autoantibodies against nucleosomes a sensitivity of 97% with
reference to the EUROIMMUN Anti-Nucleosomes ELISA (IgG) method was determined using
34 samples of patients with SLE. The clinical prevalence determined by the ELISA (CE-notified test,
coated with native mononucleosomes free from histon H1 and non-histon proteins, Patent
EP1476750B1/US7566545 (B2)) amounted to 58%. The specificity was 100% for healthy blood donors
(n = 50) and in a panel of non-SLE rheumatic diseases (Sjögren`s syndrome n = 14, systemic sclerosis
n = 18).

Histones: For the detection of autoantibodies against histones a sensitivity of 75% with reference to the
ELISA method was determined using 40 samples of patients with SLE. The specificity was 100% for
healthy blood donors (n = 50) and 97% in a panel of non-SLE rheumatic diseases (Sjögren`s syndrome
n = 14, systemic sclerosis n = 18).

Ribosomal P-protein: For the detection of autoantibodies against ribosomal P-protein a sensitivity of
82% with reference to the ELISA method was determined using 46 samples of patients with SLE. The
specificity was 100% for healthy blood donors (n = 50) and in a panel of non-SLE rheumatic diseases
(Sjögren`s syndrome n = 14, systemic sclerosis n = 18).

AMA-M2: For the detection of autoantibodies against AMA-M2 a sensitivity of 100% with reference to
the ELISA method was determined using 36 samples of patients with primary biliary liver cirrhosis. The
specificity was 100% for healthy blood donors (n = 50) and 100% in a panel of other liver diseases
(autoimmune hepatitis n = 28, toxic liver damage n = 38, viral hepatitis B/C n = 69).

Limiting results were excluded from the calculation of the performance data.

Reference range: The reference range was determined using a cohort of healthy blood donors (n = 50).
All blood donors were negative.

Clinical significance
Antibodies against nuclear antigens (ANA) are directed against various cell nuclear components
(biochemical substances in the cell nucleus). These encompass nucleic acids, cell nuclear proteins and
ribonucleoproteins. The serological detection of autoantibodies against individual or several cell nuclear
autoantigens is an essential element in the diagnosis of autoimmune diseases, particularly rheumatic
diseases. The frequency (prevalence) of anti-nuclear antibodies in inflammatory rheumatic diseases is
between 20% and 100% (in rheumatoid arthritis between 20% and 40%). Therefore, differential ANA
diagnostics to detect autoantibodies against different nuclear antigens is indispensable for the
identification of individual rheumatic diseases. ANA analysis is also helpful in the diagnosis of other
autoimmune diseases, such as primary biliary cholangitis (PBC) or autoimmune hepatitis (AIH).

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The ANA profiles offer innovative test combinations based on the lineblot technology (EUROLINE).
Positive test results provide important serodiagnostic information for the diagnosis of the rheumatic
diseases below, as well as further autoimmune diseases such as PBC.
1. Systemic lupus erythematosus (SLE)
SLE is a chronic inflammatory autoimmune disease which occurs in phases and mainly affects the
connective tissue and various organic systems. Worldwide, women are ten times more frequently
affected by collagenosis than men, whereby there are regional differences, e.g. 12.5 in 100,000
women in central Europe and up to 100 in 100,000 women in the US have SLE. The predilection age
is between 15 and 30 years. The clinical symptoms vary greatly and can include butterfly erythema,
discoid hyperkeratotic skin changes, purpura, arthralgia, myalgia, kidney insufficiency,
neuropsychiatric abnormalities, polyneuropathy, pericarditis, cardiomyopathy, pleuritis, lung fibrosis,
anaemia, hepatomegaly and splenomegaly. An SLE attack is often accompanied by fever.
In drug-induced lupus around 50% to 75% of patients treated with procainamide and 25% to 30% of
those treated with hydralazine develop ANA without symptoms of SLE during long-term therapy. A
third of these patients demonstrate autoantibodies against histones and after varied duration of
therapy show polyarthalgia, pleuritis and pericarditis. These ANA persist for years after the drugs
have been discontinued and the symptoms have abated.
2. Sharp syndrome (mixed connective tissue disease = MCTD)
Sharp syndrome is a multi-symptomatic and multiform MCTD combining symptoms of rheumatoid
arthritis (RA), SLE, systemic sclerosis (SSc) and polymyositis. It has not yet been clarified if it is an
independent disease.
3. Sjögren’s syndrome (primary Sjögren’s syndrome, SS)
SS is a chronic inflammatory autoimmune disease of the exocrine glands which can be found in one
to four million people in the US alone. Nine out of ten patients are women. The main clinical feature of
primary SS is ocular and oral dryness as a result of the destruction of lachrymal and salivary glands
by lymphocytic infiltration. The pancreatic glands, the mucous secreting glands of the intestine,
bronchia or vagina and the sudoriferous glands may also be affected. Around 5% of SS patients
develop malignant lymphoma. In secondary SS the disease signs of primary SS occur as
accompanying symptoms of RA, SSc, SLE, polymyositis/dermatomyositis, PBC and AIH.
4. Systemic sclerosis (systemic scleroderma, SSc)
SSc is an autoimmune connective tissue disease, which affects the skin and the inner organs. It
affects around 2 to 50 in 100,000 persons worldwide (USA: 25 in 100,000), and is around three to four
times more common in women than in men.
Shortening of the lingual frenum and Raynaud’s syndrome are early symptoms of SSc. In the
following phase oedema of the hands and feet develop. The skin becomes stiff and in later stages
atrophic, waxy and thin. Finally, deformation of the hands occurs. The fingers become fixed in a bent
position (claw hand) and are highly tapered at the ends (Madonna fingers). Furthermore, the
characteristic masklike face with rigid mimic develops. Finally, callosity of the inner organs,
particularly of the digestive tract, lungs, heart and kidneys occurs. At present, lung involvement is the
most frequent cause of death from SSc. Manifest SSc is the collagenosis with the highest vital risk for
the patient. The 10-year survival rate is 55%.
SSc is divided into limited and diffuse forms, depending on the cutaneous distribution. In the limited
form, skin involvement is limited to the distal extremities. In the diffuse form (also proximal systemic
sclerosis) the symptoms are diffusely distributed over the trunk, the proximal and distal extremities
and the face.

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5. Myositis (poly-/dermatomyositis)
The autoimmunogenic myositides (idiopathic inflammatory myopathies) are systemic autoimmune
diseases with inflammation of the skeletal musculature, symmetric and proximal accentuated pain and
muscle weakness. They occur with an incidence of 0.1 to 1 per 100,000 per year, a prevalence of 1 to
6 per 100,000 and a ratio of men to women of 1 to 2. They can be divided into polymyositis of adults
(around 30%), dermatomyositis of adults (around 30%), paraneoplastic polymyositis of the lungs,
ovaries, mammary glands, gastrointestinal tract and in myeloproliferative diseases (around 8%),
infantile myositis/dermatomyositis with accompanying vasculitis (around 7%), as well as myositides in
association with autoimmune diseases such as RA, lupus erythematosus, MCTD and rare forms such
as granulomatosis, eosinophile, focal and inclusion body myositis (around 20%). It should be noted
that dermato-/polymyositis is often of paraneoplastic origin, particularly in elderly patients.
Dermatomyositis symptoms can occur before the tumour is even diagnostically detectable.
Polymyositis (PM) is a systemic inflammatory disease of the skeletal muscles of unknown aetiology
with perivascular lymphocytic infiltration. When the skin is involved, the disease is known as
dermatomyositis (DM). Clinical symptoms of PM are recurring bouts of fever, muscle weakness,
arthralgia, possibly Raynaud’s syndrome, trouble with swallowing and involvement of the inner
organs. In DM, skin symptoms appear as purple-coloured exanthema on the eye lids, nose bridge and
cheeks, periorbital oedema, local erythema and scaly eczema dermatitis.
6. Rheumatoid arthritis (RA)
RA is both one of the most common autoimmune disorders and also the most common chronic
inflammatory joint disease. The disease affects around 1% of the world population, whereby 75% of
patients are female. It is characterised by inflammation of the synovial membrane, which spreads
symmetrically from the small to large joints leading to the destruction of the joints in the late phase
accompanied by a systemic involvement of the soft tissue. Initial symptoms include painful swelling of
basic finger joints with morning stiffness in the joints. Reliable and earliest possible diagnosis is
indispensable to keep the disease under control with suitable therapy and to avoid irreversible joint
damage.
7. Primary biliary cholangitis (PBC)
PBC is a chronic non-suppurative destructive cholangitis with progressive inflammatory destruction of
the small biliary ducts and liver cirrhosis in the final stage. In 80% to 90% of cases the patients are
female, mainly between 20 and 60 years of age. In rare cases, the disease also affects children. In
Germany the prevalence is around 3 to 4 cases per 100,000 inhabitants. Demographic differences
(Caucasians, Africans, etc.) are minimal.
PBC can be subdivided into various stages using liver biopsy. In around 6% of cases there is an
increased risk of hepatocellular carcinoma. In the final stage of PBC (decompensated cirrhosis) only
liver transplantation will save the patient's life. In around 75% of cases the transplant patients recover
fully from PBC. Some patients, however, suffer a PBC relapse after transplantation, but only with a
very slow disease course.
In addition to the typical PBC histological characteristics, specific serodiagnostic parameters are
important for confirming suspected cases of PBC: 1. Biochemical markers of cholestasis, such as
increased levels of alkaline phosphatase (AP) and gamma-glutamyl transferase (γGT) in serum, 2.
Presence of PBC-specific autoantibodies, in particular autoantibodies against mitochondria (AMA)
which are directed against the component M2 (family of oxo-acid dehydrogenases), and 3. Additional
determination of ANA, in particular against nuclear granules (nuclear dots, sp100 and PML) and
against nuclear membrane (gp210), which are also pathognomonically relevant. Autoantibodies
against centromere proteins are found regularly in a proportion of patients with overlap syndrome with
SSc.

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Overview
Autoantibodies
Autoimmune disease Prevalence
against
nRNP/Sm MCTD 95%
Sm SLE 5% - 40%
SS-A SS or SLE 40% - 95% or 20% - 60%
Neonatal lupus erythematosus 95% - 100%
Ro-52 SS or SLE 70% - 90% or 40% - 60%
SSc or idiopathic inflammatory myopathy 20% or 20% - 40%
SS-B SS or SLE 40% - 95% or 10% - 20%
Neonatal lupus erythematosus 75%
Scl-70 SSc 25% - 75%
Diffuse or limited form of SSc 40% - 65% or 5% - 15%
PM-Scl SSc including overlap syndrome 10% - 20% or 5% - 20%
PM/SSc overlap syndrome 18%
SSc (anti-PM-Scl75 positive) 24% - 50%
SSc (anti-PM-Scl100 positive) 7%
Jo-1 Myositis (polymyositis/dermatomyositis) 25 - 35%
CENP A SSc - limited form or SSc - diffuse form 80% - 95% or 5% - 10%
CENP B SSc - limited form or SSc - diffuse form 80% - 95% or 8%
PBC 10% - 30%
PCNA SLE 3%
dsDNA SLE 40% - 90%
Nucleosomes SLE 40% - 70%
Histones Drug-induced SLE 95% - 100%
SLE or RA 50% or 15% - 50%
Ribosomal P- SLE 10%
protein
AMA M2: PBC or other chronic liver diseases up to 96% or 30%
SSc 7 - 25%
DFS70 Atopic dermatitis 4% - 10%
Rheumatic diseases 5% - 10%
Mi-2α DM approx. 20%
Mi-2β DM, associated with neoplasia approx. 10%
(e.g. colon or breast carcinoma)
Ku SLE/myositis/SSc up to 10% / 40% / 5%
RP11 SSc 5%
RP155 SSc 7%
Sp100 PBC 21%
PML PBC 13%
gp210 PBC 26%

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Version: 18/01/2018