ANA IFA: HEp-20-10

Test instruction
For In Vitro Diagnostic Use CLIA Complexity: High
ANTIBODIES FORMAT
ORDER NO. SUBSTRATE SPECIES
AGAINST SLIDES x FIELDS
FA 1522-1003 10 x 03 (030)
FA 1522-1005 10 x 05 (050)
FA 1522-1010 10 x 10 (100)
cell nuclei
FA 1522-1050 HEp-20-10 cells human 10 x 50 (500)
(ANA)
FA 1522-2003 20 x 03 (060)
FA 1522-2005 20 x 05 (100)
FA 1522-2010 20 x 10 (200)

Intended use: The EUROIMMUN ANA IFA: HEp-20-10 is an indirect immunofluorescence

antibody test for the qualitative or semi-quantitative detection of antibodies against cell nuclei
(ANA) in human serum and EDTA-plasma. This test system is used as an aid in the diagnosis of
systemic rheumatic diseases in conjunction with other laboratory and clinical findings

Summary and explanation

The diagnostic value of autoantibodies against cell nuclei (ANA) for many autoimmune diseases
has been well documented. Antibodies against nuclear antigens are directed against various
nucleic components (biochemical substances of the cell nucleus) [1, 2, 3]. These include nucleic
acids, nucleic proteins and ribonucleoproteins. The antibodies are characteristic of various
diseases, particularly of those of the rheumatic form [4, 5, 6]. The prevalence of antinuclear
antibodies in inflammatory rheumatic diseases is between 20% and 100%, it is lowest in
rheumatoid arthritis with 20% and 40% [7]. Therefore, differentiation of antibodies against nuclear
antigens is indispensable in the diagnosis of rheumatoid diseases [4, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20]. In the foreground are the following rheumatoid diseases [3, 5, 21, 22, 23, 24,
25]:

Autoimmune disease Prevalence ANA

Systemic lupus erythematosus (SLE)
active 95% - 100%
inactive 80% - 100%
Drug-induced lupus erythematosus 100%
Mixed connective tissue disease (MCTD or
100%
Sharps syndrome)
20% - 40%
Rheumatoid arthritis
20% - 50%
Other rheumatic diseases
Progressive systemic sclerosis 85% - 95%
Polymyositis and dermatomyositis 30% - 50%
Sjgrens syndrome 70% - 80%
Autoimmune hepatitis 30% - 40%
Ulcerative colitis 26%

FA_1522_A_US_D06.doc
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Systemic lupus erythematosus

Autoantibodies in systemic lupus erythematosus

Antigen Prevalence
Double-stranded DNA 60% - 90%
Single-stranded DNA 70% - 95%
RNA 50%
Histones 95%
U1-nRNP 30% - 40%
Sm 20% - 40%
SS-A (Ro) 20% - 60%
SS-B (La) 10% - 20%
Cyclin (PCNA) 3%
Ku 10%
RNP: Ribosomal P proteins 10%
(Hsp-90: Heat shock protein, 90 kDA 50%)
(Cardiolipin 40% - 60%)

For systemic lupus erythematosus (SLE), the determination of antibodies against double-stranded
DNA is considered to be one of the most important criteria for diagnosis [22, 24, 26, 27, 28].
Immune complexes of double-stranded DNA and the corresponding autoantibodies cause tissue
damage in the subcutis, the kidneys and other organs [29, 30, 31]. The antibody titer correlates
with the clinical activity of the disease [32]. Equally, antibodies against Sm are considered to be
pathognomonic of systemic lupus erythematosus [22, 24, 25, 28, 33]. Additionally, antibodies
against other polynucleotides, ribonucleotides, histones and other nucleic antigens can be found
[7, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 27, 28, 34, 35, 36, 37].
In drug-induced lupus erythematosus with manifestations such as arthralgia, arthritis, exanthema,
serositis, myalgia, heptomegalia and splenomegalia, antibodies against histones are observed.
This reversible form of SLE can be induced by antibiotics (e.g. penicillin, streptomycin,
tetracyclines), chemotherapeutic agents (e.g. INH, sulfonamides), anticonvulsants (e.g. phenytoin,
hydantoines), antiarrythmics (e.g. procainamid, practolol), antihypertensives (e.g. reserpin,
hydralazin), psychotropics (e.g. chlorpromacin), antithyroid drugs (e.g. thiouracilderivates),
antirheumatoid basic therapeutics (e.g. Gold, D-penicillamin) and other drugs such as
contraceptives and allopurinol [38, 39].
Sharps syndrome
High autoantibody titers against U1-nRNP are characteristic for Sharps syndrome (MCTD = Mixed
connective tissue disease). The antibody titer correlates with the clinical activity of the disease [3,
40, 41].

Autoantibodies in Sharps syndrome

(Mixed connective tissue disease = MCTD)
Antigen Prevalence
U1-nRNP 95% - 100%
Single-stranded DNA 20% - 50%

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Rheumatoid arthritis
In rheumatoid arthritis, antibodies against histones can be observed in more than half of all cases,
while antibodies against U1-nRNP are rarely found. Antibodies against RANA ("rheumatoid arthritis
nuclear antigen") cannot be detected with HEp-2 cells [42, 43].

Cell nuclei 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 noticed in the diffuse form [2]. Autoantibodies against centromeres are
associated with the limited form of progressive systemic sclerosis [3, 40, 41, 42, 44, 45].

Autoantibodies in progressive systemic sclerosis

(diffuse form)
Antigen Prevalence
Fibrillarin 5% - 10%
PM-Scl (PM-1), including overlap syndrome 50% - 70%
Scl-70 25% - 75%
RNA polymerase I 4%
7-2-RNP (To) rare
NOR-90 (nucleolus organiser region) rare

Autoantibodies in progressive systemic sclerosis

(limited form)
Antigen Prevalence
Centromeres 80% - 95%

Polymyositis / dermatomyositis
Autoantibodies against PM-Scl occur in polymyositis and dermatomyositis, but other nuclear
antibodies (against Mi-1, Mi-2, Ku) and antibodies against Jo-1 can also be identified in these
diseases [2, 40, 42, 46, 47, 48].

Autoantibodies in polymyositis and dermatomyositis

Antigen Prevalence
PM-Scl (PM-1), including overlap syndrome 50% - 70%
Jo-1 (histidyl-tRNA Synthetase) 25% - 35%
Mi-1 10%
Mi-2 5%
Ku 50%
Single-stranded DNA 40% - 50%
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Autoantibodies in polymyositis and dermatomyositis

Antigen Prevalence
PL-7 (Threonyl-tRNA Synthetase) 4%
PL-12 (Alanyl-tRNA Synthetase) 3%

Sjgrens syndrome
In Sjgren's syndrome (primary Sjgren's syndrome), antibodies against SS-A and SS-B are
present, mainly in combination with one another [1, 42]. In addition, autoantibodies against the
secretory ducts of the salivary gland are found in 40 to 60% of all cases.

Autoantibodies in primary Sjgrens syndrome

Antigen Prevalence
SS-A (Ro) 40% - 95%
SS-B (La) 40% - 95%
Single-stranded DNA 13%
(RANA 70%)
(Salivary gland excretory ducts 40% - 60%)
(Rheumatoid factors 60% - 80%)

Autoantibodies against nuclear antigens occur in many other diseases, such as primary biliary
liver cirrhosis ("Nuclear Dots", SS-A) and chronic active autoimmune hepatitis
(SS-A, Lamine) [42, 43]. At times, antibodies against nuclear antigens are detectable in
subjectively healthy individuals, usually at a low titer (various immunoglobulin classes, mainly IgM).

Autoantibodies against cell nuclei:

Important associated diseases
Antigen Disease Prevalence
Double-stranded Systemic lupus erythematosus 60% - 90%
DNA
Single-stranded Systemic lupus erythematosus 70% - 95%
DNA Drug-induced lupus erythematosus 60%
Mixed connective tissue disease (MCTD or Sharps 20% - 50%
syndrome)
Polymyositis / dermatomyositis 40% - 50%
Scleroderma, Sjrgrens syndrome, rheum. arthritis 8% - 14%
RNA Systemic lupus erythematosus 50%
Scleroderma, Sjrgrens syndrome 65%
Histones Drug-induced lupus erythematosus 95%
Systemic lupus erythematosus 30% - 70%
Rheumatoid arthritis 15% - 50%
U1-nRNP Mixed connective tissue disease (MCTD, Sharps 95% - 100%
syndrome)
Systemic lupus erythematosus 30% - 70%
Rheumatoid arthritis 3%
Sm Systemic lupus erythematosus 20% - 40%
SS-A (Ro) Sjgrens syndrome 40% - 95%
Systemic lupus erythematosus 20% - 60%
Neonatal lupus syndrome 100%
SS-B (La) Sjgrens syndrome 40% - 95%
Systemic lupus erythematosus 10% - 20%
Fibrillarin Progressive systemic sclerosis, diffuse form 5% - 10%

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Autoantibodies against cell nuclei:

Important associated diseases
Antigen Disease Prevalence
RNA polymerase I Progressive systemic sclerosis, diffuse form 4%
PM-Scl (PM-1) Polymyositis/dermatomyositis/overlap syndrome 50% - 70%
Progressive systemic sclerosis, diffuse form 5% - 10%
Centromeres Progressive systemic sclerosis, limited form 80% - 95%
Scl-70 Progressive systemic sclerosis, diffuse form 25% - 75%
Cyclin (PCNA) Systemic lupus erythematosus 3%
Ku Systemic lupus erythematosus 10%
Poly-/dermatomyositis, progressive systemic sclerosis 30% - 55%
Mi-1, Mi-2 Dermatomyositis 5% - 10%

Antibodies against components of the cytoplasm of HEp-2 cells cannot always be clearly
differentiated. Only a few cytoplasm-reactive antibodies can be assigned to a particular disease,
e.g. antibodies against mitochondria in primary biliary liver cirrhosis and against the proteins PL-7
and PL-12 in polymyositis and dermatomyositis [43]. Further rare antibodies found in polymyositis
are those directed against OJ, EJ and signal recognition particles (SRP). Other cytoplasmic
antibodies against ribosomes, Golgi apparatus, lysosomes and cytoskeletal components such
as actin, vimentin and cytokeratins are of minor clinical significance [2]. The diagnostic value of
mitosis-associated antigens has also not yet been finally clarified.
Antigen: For the detection of antinuclear antibodies (ANA) by means of indirect
immunofluorescence, human epithelial cells (HEp-2) are mainly preferred today. When compared
to the conventional cell line the HEp-20-10 cell line, which is of identical genetic origin as the
HEp-2, shows an increased spectrum of cells in the mitotic phase and of human nuclear antigens.
Principles of the test: HEp-20-10 cells are incubated with diluted patient sample. If the reaction is
positive, specific antibodies attach to the antigens. In a second step, the attached antibodies are
stained with fluorescein-labelled anti-human antibodies and made visible with a fluorescence
microscope.

Materials
Contents of a test kit for 50 determinations: FA 1522-1005 (IgG)
Description Format Symbol
1. Slides, each containing 5 BIOCHIPs coated with HEp-20-10
10 slides .SLIDE.
cells
2. Fluorescein-labelled anti-human IgG (goat), ready for use 1 x 1.5 ml .CONJUGATE.
3. Positive control: autoantibodies against cell nuclei (ANA
homogeneous), control serum with titer information, human, 1 x 0.25 ml .POS CONTROL.
ready for use
4. Negative control: autoantibody-negative, human, ready for use 1 x 0.1 ml .NEG CONTROL.
5. Salt for PBS pH 7.2 2 packs .PBS.
6. Tween 20 2 x 2.0 ml .TWEEN 20.
7. Embedding medium, ready for use 1 x 3.0 ml .GLYCEROL.
8. Cover glasses (62 mm x 23 mm) 12 pieces .COVERGLASS
9. Instruction booklet 1 booklet ---
.LOT. Lot description Storage temperature
.IVD. In vitro diagnostics Unopened usable until

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Additional materials and equipment (not supplied):

Performance of the test with TITERPLANE technology requires reagent trays TRAY, which
are not provided in the test kits. These reagent trays are reusable. They are available from
EUROIMMUN under the following order number:
- ZZ 9999-0110 Reagent trays for slides containing up to 10 fields
Fluorescent Microscope: Equipped with a 488 nm excitation filter; 510 nm color separator; &
520 nm blocking filter with a 100 W mercury vapour lamp light source or LED bluelight.
Distilled or de-ionized water for wash buffer production
Pipettes with a range of 10l to 200l
Cuvettes or wash/staining dishes for PBS wash step
Lint free towelling

Warnings and precautions

For in vitro diagnostic use.
Warning: Potentially biohazardous material. The BIOCHIPs coated with antigen substrates
have been treated with a disinfecting fixing agent. Neither HBsAg nor antibodies against HIV-1,
HIV-2, and HCV could be detected in the control sera using FDA-cleared or European CE-
approved test systems. Nevertheless, all test system components should be handled as
potentially infectious materials. Patient samples, controls and slides are to be handled as
potentially infectious materials. All reagents are to be disposed of in accordance with official
disposal regulations.

Some of the reagents contain sodium azide at a concentration of 0.09%. Sodium azide has been
reported to form lead or copper azides in laboratory plumbing which may cause explosions. Rinse
sink thoroughly with water after disposing of solutions containing azide. Avoid skin contact.
The individual reagents of one lot are matched with one another and should not generally be
swapped with reagents of another lot or with reagents from another manufacturer.

Preparation and stability of the reagents

Storage and stability: The slides and the reagents should be stored at a temperature of between
+2C and +8C. The test system is stable for a period of 18 months after the date of manufacture if
stored properly. Do not use beyond the expiration date noted on the kit label. After initial opening,
the reagents are stable until the expiry date when stored between +2C and 8C and protected
from contamination, unless stated otherwise below. Protect from exposure to heat and light.
Indications of instability: Do not use if reagents appear cloudy.
- Slides: Ready for use. Remove the protective cover only when the slides have reached room
temperature (condensed water can damage the substrate). Mark with a felt-tip pen. Do not
touch the BIOCHIPs. After the protective cover has been opened, the slide should be
incubated within 15 minutes. If the protective cover is damaged, the slide must not be used for
diagnostics.
- Fluorescein-labelled secondary antibody (FITC): Ready for use. Before using for the first
time, mix thoroughly. The conjugate is sensitive to light. Protect from sunlight .

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- Positive and negative controls: Ready for use. Before using for the first time, mix
thoroughly.
Positive control with titer information: The label contains the target value and the substrate
used to determine the target value. The lower tolerance limit is one titer level below the target
value, the upper tolerance level limit lies two titer levels above the target value. The control is
to be diluted with PBS-Tween. After opening, the control has a shelf-life of 6 months when
stored at +2C to +8C. Diluted controls must be incubated within one working day.
- PBS-Tween: 1 pack of Salt for PBS should be dissolved in 1 liter of distilled water and mixed
with 2 ml of Tween 20 (stir for 20 min until homogeneous). The prepared PBS-Tween can be
stored at +2C to +8C, generally for 1 week. PBS-Tween should not be used if the solution
becomes cloudy or contamination appears.
- Embedding medium: Ready for use.

Preparation and stability of the patient samples

Samples: Human sera or EDTA plasma.
Stability: CLSI (formerly NCCLS) Document H18-A2 recommends the following storage conditions
for samples: Samples should be stored at room temperature no longer than 8 hours. If the assay
will not be completed within 8 hours, the samples should be refrigerated at 2-8C. If the assay will
not be completed within 48 hours, or for shipment of the sample, samples should be frozen at
-20C or lower. Frozen samples must be mixed well after thawing and prior to testing. Diluted
samples should be incubated within 8 hours. Do not use bacterially contaminated samples.
Recommended sample dilution for qualitative evaluation: The sample to be investigated is
diluted 1:100 in PBS-Tween. For example, dilute 10.1 l sample in 1000 l PBS-Tween and mix
thoroughly, e.g., vortex for 4 seconds.
Recommended sample dilution for semi-quantitative evaluation: The dilution of samples to be
investigated is performed using PBS-Tween. For each add 100 l of PBS-Tween to the tube and
mix with 11.1 l of the next highest concentration, e.g. vortex for 2 seconds. EUROIMMUN
recommends incubating samples from a dilution of 1:100.
Dilution Dilution scheme

1:10 100 l PBS-Tween + 11.1 l undiluted sample

11.1 l
After every two
1:100 100 l PBS-Tween + 11.1 l 1:10 diluted sample dilution steps, a new
pipette tip should be
11.1 l used to prevent
carryover.
1:1000 100 l PBS-Tween + 11.1 l 1:100 diluted sample
...

...

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Procedure
The TITERPLANE Technique was developed by EUROIMMUN as an aid in standardizing
immunological analyses: Patient samples, controls and in separate steps conjugate and
embedding medium are applied to the reaction fields of a reagent tray. The BIOCHIP Slides are
then placed into the recesses of the reagent tray, where all BIOCHIPs of the slide come into
contact with the fluids, and the individual reactions commence simultaneously. The fluids are
confined to the recessed wells eliminating the need to use a conventional humidity chamber.
*Reaction fields of the reagent tray must be hydrophilic and surrounding area hydrophobic. The
reagent tray may be used repeatedly as long as the hydrophilic and hydrophobic properties are
maintained. Clean with mild laboratory glassware detergent and rinse thoroughly with DI water.
The tray can be checked for these properties by adding a defined amount of PBS (25l) to each
well to be sure it is restricted to the well.
Prepare: The preparation of the reagents and of the serum and plasma samples is
described in this test instruction.
Pipette: Apply 30 l of diluted sample to each reaction field of the reagent tray, avoiding
air bubbles. Transfer all samples to be tested before starting the test to a reagent
tray.
Incubate: Start reactions by fitting the BIOCHIP Slides into the corresponding recesses of
the reagent tray. Ensure that each sample makes contact with its BIOCHIP and
that the individual samples do not come into contact with each other. Incubate for
30 min at room temperature (+18C to +25C).
Wash: Rinse the BIOCHIP Slides with a gentle flush of PBSTween using a beaker and
immerse them immediately afterwards in a cuvette containing PBSTween for
5 min. Shake with a rotary shaker if available. Wash max. 16 slides then replace
PBS-Tween with new buffer.
Pipette: Apply 25 l of fluorescein-labelled anti-human globulin to each reaction field
of a clean reagent tray. Fill all the fields needed before continuing incubation. The
labelled anti-human serum should be mixed before use.
Incubate: Remove one BIOCHIP Slide from cuvette. Within five seconds blot only the back
and the sides with a lint free paper towel and immediately fit the BIOCHIP Slide
into the recesses of the reagent tray. Do not dry the areas between the reaction
fields on the slide. Check for correct contact between the BIOCHIPs and liquids.
Then continue with the next BIOCHIP Slide. Protect the slides from direct
sunlight. Incubate for 30 min at room temperature (+18C to +25C).
Wash: Fill cuvette with new PBS-Tween. Rinse the BIOCHIP Slides with a gentle flush
of PBSTween using a beaker and place them into the cuvette filled with the new
PBS-Tween for 5 min. Optional: shake with a rotary shaker if available. 10 drops
of Evans Blue for each 150 ml phosphate buffer can be added for
counterstaining. Wash max. 16 slides then replace PBS-Tween with new buffer.
Embed: Place embedding medium onto a cover glass drops of max. 10 l per reaction
field. Use a reagent tray. Remove one BIOCHIP Slide from PBSTween and dry
the back, all four sides, as well as the surface around, but not between the
reaction fields with a lint free paper towel. Put the BIOCHIP Slide, with the
BIOCHIPs facing downwards, onto the prepared cover glass. Check immediately
that the cover glass is properly fitted into the recesses of the slide. Gently correct
the position if necessary.
Evaluate: Read the fluorescence with the microscope.
General recommendation: Objective 20x (tissue sections, infected and
transfected cells), 40x (cell substrates).
Excitation filter: 488 nm, color separator: 510 nm, blocking filter: 520 nm.
Light source: mercury vapor lamp, 100 W, EUROIMMUN LED, EUROStar
Bluelight.

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BIOCHIP slide
TITERPLANE Technique
BIOCHIPs reagent tray

diluted samples
Pipette: 30 l per field

Incubate: 30 min

Wash: 1 s flush
PBS-Tween
5 min cuvette

labelled antibody
Pipette: 25 l per field

Incubate: 30 min

Wash: 1 s flush
5 min cuvette PBS-Tween

Embed: max. 10 l per field embedding medium

cover glass

20 x
40 x
Evaluate: fluorescence microscopy

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Interpretation of results
Fluorescence pattern (positive reaction): Antibodies against nuclear antigens (ANA) can be
found on numerous substrates. For the targeted determination and differentiation of antinuclear
antibodies, a substrate consisting of human epithelial cells (HEp-20-10) is used. The cell nuclei
show a distinct fluorescence, which is characterized by certain patterns. In the case of negative
samples, the nuclei show no specific fluorescence. In each field evaluated, both interphase nuclei
and mitotic cells be examined, and this in several areas if possible.
If the positive control shows no specific fluorescence pattern or the negative control shows a clear
specific fluorescence the results are not to be used and the test is to be repeated.
A large range of fluorescence images can be found on the EUROIMMUN website
(www.euroimmun.com)
Qualitative evaluation: A titer of 1:100 or greater that results in a positive reaction is considered
positive.
Semi-quantitative evaluation: The endpoint titer is defined as the highest sample dilution factor
for which specific fluorescence of is identifiable.
Reported results should include titers and staining pattern.
For diagnosis the clinical symptoms of the patient should always be taken into account along with
the serological results.
Some of the most important fluorescence patterns are the homogeneous and granular nuclear
fluorescence as well as staining of the nucleoli and the centromeres (clearly identifiable especially
in the mitotic cells). The relevant binding patterns frequently correspond with biochemically defined
nuclear antigens:

Autoantigens of the cell nuclei Fluorescence pattern

Polynucleotides Double-stranded DNA Homogeneous
Single-stranded DNA Homogeneous
RNA Partly homogeneous
Histones H1, H2A, H2B, H3, H4, H2A- Homogeneous
H2B complex
Ribonucleoproteins of U1-nRNP Coarse granular, nulceoli negative
the nucleoplasm (ENA) Sm Coarse granular, nulceoli negative
SS-A (Ro) Granular
SS-B (La) Granular
Antigens of the nucleolus U3-nRNP/fibrillarin Nucleoli granular
RNA polymerase I Nucleoli granular
PM-Scl (PM-1) Nucleoli homogeneous
7-2-RNP (To) Nucleoli homogeneous
4-6-S-RNA Nucleoli homogeneous
Centromeres Proteins of kinetochores Typical granular
Other proteins Scl-70 Nearly homogeneous, marked nucleoli
Cyclin (PCNA) Granular, 50% 10 times brighter
Nuclear dots Nuclear dots
NOR-90 Metaphase 1-2 granula
Ku Reticular
Mi-1 Fine granular
Mi-2 Fine granular
Lamins Nuclear membrane

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Limitations of the procedure

1. A diagnosis should not be made on a single test result. The clinical symptoms of the patient
should always be taken into account along with the serological results by the physician.
2. In the case of positive and questionable immunofluorescence reactions, an enzyme
immunoassay with defined target antigens should be performed subsequently for the
verification and differentiation of the results.
3. In addition to ANA antibodies, certain other autoantibodies (e.g. AMA, SMA etc.) may also
react with the substrates. Only specific ANA fluorescence patterns should be evaluated.
4. Adaptation of this assay for use with automated sample processors and other liquid handling
devices, in whole or in part, may yield differences in test results from those obtained using the
manual procedure. It is the responsibility of each laboratory to validate that their automated
procedure yields test results within acceptable limits.
5. Mishandling of slides during the staining procedure, especially allowing slides to dry between
steps, may result in a washed out" pattern appearance and/or a high level of background
staining.
6. Coplin jars used for slide washing should be free from all dye residues. Use of coplin jars
containing dye residue may cause staining artifacts.
7. The light source, filters and optical equipment of the fluorescence microscope can influence the
sensitivity of the assay. Using traditional mercury vapour lamp systems, the performance of the
microscope is significantly influenced by correct maintenance, especially alignment of the lamp
and replacement of the lamp after the recommended period of time. The EUROIMMUN
EUROStar fluorescence microscope with LED-Bluelight as the light source offers many
advantages. Contact EUROIMMUN for details.
8. Cross reactivity: 11 characterized CDC samples were incubated which did not produce any
unexpected fluorescence pattern. The results showed no cross reactivity with these sera.
9. Interferences: Normal and positive sera samples were spiked with fixed concentrations of
hemoglobin, triglycerides and bilirubin. Interferences testing against rheumatoid factor was
performed by spiking of three ANA positive samples and a negative sample with varying
amounts of a high positive RF serum. None of these components were found to affect the test.

Expected values
Reference range: Serum samples of 200 normal healthy adult blood donors of mixed age and
gender were examined with the EUROIMMUN ANA IFA HEp-20-10 kit at the recommended
dilution. A prevalence of 10% was found which is in concordance of data reported in the literature.
Reference range: Less than 1: 100
Note: It is recommended that each laboratory determine its own normal range based on the
population and equipment used.

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Performance characteristics
Measurement range: The dilution starting point for this measurement system is 1:100. Samples
can be further diluted by a factor of 10 so that the dilution series is 1:1000, 1:10000 etc. There is
no upper limit to the measurement range.

Reproducibility: Intra-assay reproducibility was determined by 10fold repeated measurements of

9 dilutions of a characterized positive serum sample and a negative sample. Inter-assay
reproducibility was determined by repeated measurements of a characterized serum sample in 9
dilutions and a negative sample at 5 different times. Inter-lot reproducibility was determined by
measurements of 9 dilutions of a characterized serum sample and a negative sample using
different kit lots within a period of over two years.
The results did not exceed the acceptable deviation of fluorescence intensity of +/- 1 intensity level
for all samples. The fluorescence intensity level is the intensity of the specific fluorescence
expressed as a numeric value. These values can vary from 0 (no specific fluoescence) to 5
(extremely strong specific fluorescence).
Comparison to predicate kit: A study was conducted at a US hospital clinical laboratory
comparing the performance of the EUROIMMUN ANA IFA HEp-20-10 kit and an FDA-cleared IFA
kit in current distribution. 198 prospective samples from all over the USA, sent to the laboratory for
ANA testing, were tested with both test systems. The panel consisted of 58 men and 138 women
(and 2 unknown) samples. Age ranged from 0 to 91 with an average age of 50.5 years. The results
are shown in the table below. The overall agreement was 98% with a positive agreement of 97%
and a negative agreement of 100%.

Predicate IFA
n = 198
positive negative
EUROIMMUN positive 135 0
IFA negative 4 59

The agreement of pattern evaluation is also excellent. Patterns of 111 ANA positive samples were
found identical on both devices.

Literature references

1. Al Attia HM, Al Ahmed YH, Chandani AU. Serological markers in Arabs with lupus
nephritis. Lupus 7 (1998) 198-201.

2. Genth E, Mierau R. Diagnostic significance of scleroderma and myositis-associated

autoantibodies. [Article in German] Z Rheumatol 54 (1995) 39-49.

3. Jolly M, Smaron M, Olsen Utset T, Ellman M. Are isolated antinucleolar antibodies a

marker of scleroderma? J Clin Rheumatol 9 (2003) 291-295.

4. van Venrooij WJ, Charles P, Maini RN. The consensus workshops for the detection of
autoantibodies to intracellular antigens in rheumatic diseases. J Immunol Methods 5
(1991) 181-189.

5. James K, Carpenter AB, Cook L, Marchand R, Nakamura RM. Development of the

antinuclear and anticytoplasmic antibody consensus panel by the Association of
Medical Laboratory Immunologists. Clin Diagn Lab Immunol 7 (2000) 436-443.

6. Schmidt KL, Hellmich B, Manger B, Tillmann K, Truckenbrodt H. Checkliste Rheumatologie.

Thieme Verlag 2000.

7. Fritsch C, Hoebeke J, Dali H, Ricchiuti V, Isenberg DA, Meyer O, Muller S. 52-kDa Ro/SSA
epitopes preferentially recognized by antibodies from mothers of children with neonatal
lupus and congenital heart block. Arthritis Res Ther 8 (2005) R4 [Epub ahead of print]
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AG

8. Schlumberger W, Meyer W, Proost S, Dhnrich C, Mller-Kunert E, Sonnenberg K, Olbrich S,

Stcker W. The new EUROBLOT technology: Differentiation of Autoantibodies against
cell nuclei. European Journal of Clinical Chemistry and Clinical Biochemistry 33 (1995) 116.

9. Buyon, JP, Winchester RJ, Slade SG, Arnett F, Copel J, Friedman D, Lockshin MD.
Identification of mothers at risk for congenital heart block and other neonatal lupus
syndromes in their children: comparison of ELISA and immunoblot to measure anti-
SSA/Ro and anti-SSB/La antibodies. Arthritis and rheumatism 36 (1993) 1263.

10. Metskula K, Salur L, Mandel M, Uibo R. Demonstration of high prevalence of SS-A

antibodies in a general population: association with HLA-DR and enterovirus antibodies.
Immunol Lett 106 (2006) 14-18.

11. Stcker W, Teegen B, Meyer W, Mller-Kunert E, Proost S, Schlumberger W, Sonnenberg K.

Differenzierte Autoantikrper-Diagnostik mit BIOCHIP-Mosaiken. In: Conrad, K. (Hrsg.):
Autoantikrper. Pabst-Verlag (1998) 78-99.

12. Stcker K, Stcker W, Ritter-Frank Y, Scriba PC. Chemically activated glass slides for
frozen sections and their use in autoantibody diagnosis [Article in German: Chemisch-
aktivierte Glasobjekttrger fr Gefrierschnitte und ihre Anwendung in der
Autoantikrperdiagnostik.] Acta histochem Jena 31 (1985) 283-294.

13. Stcker W. Rational histochemistry using a new microanalytic technic [Article in German:
Rationelle Histochemie mit einer neuen Mikroanalysemethode.] Acta histochem Jena 31 (1985)
269-281.

14. Stcker W, Fauer, Krause, Barth, Martinetz (Erfinder). Verfahren zur Optimierung der
automatischen Fluoreszenzmustererkennung in der Immundiagnostik. Aktenzeichen DE
102006027516.0 (2006).

15. Steller U, Stcker W (Erfinder). Verfahren zur Erzeugung perfekter Macro- und Microarrays
durch Kombinieren vorselektierter beschichteter Festphasen-Fragmente. Aktenzeichen
DE 12006027517.9 (2006).

16. Mller M, Wessel S, Morrin M (Erfinder). Konstante Lichtquelle fr die

Fluoreszenzmikroskopie. Aktenzeichen DE 10 2006 027 518.7 (2006).

17. Stcker W, Rateike M, Morrin M. (Erfinder). Verfahren zur Herstellung Festphasen-

gebundener Bioreagenzien. Aktenzeichen PCT/EP2005/000974 - EP 1718 948 (2005).

18. Stcker W. Moderne Techniken fr die Histochemie. Verh Anat Ges 81 (1987) 473-474.

19. Stcker W. TITERPLANE Vorrichtung zur Durchfhrung von Mikroanalysen. Europisches

Patent Nr. EP 0 018 435 (1979).

20. Stcker W. TITERPLANE Apparatus and method for simultaneously mixing specimens
for performing microanalyses. USA-Patent Nr. 4,339,241 (1980/1982).

21. Fritzler MJ. Autoantibodies in Scleroderma. Journal of Dermatology 20 (1993) 257-268.

22. Goulvestre C. Antinuclear antibodies. [Article in French] Presse Med 35 (2006) 287-295.

23. Suer W, Dhnrich C, Schlumberger W, Stcker W (Erfinder). In vitro-Diagnosekit zum

Nachweis von Lupus erythematodes disseminatus (LED). Aktenzeichen: 20212524.6
(2002).

24. Tomer Y, Buskila D, Shoenfield Y. Pathogenic significance and diagnostic value of lupus
autoantibodies. International Archives of Allergy and Immunology 100 (1993) 293-306.

13
 Medizinische
EUROIMMUN Labordiagnostika
AG

25. Mahler M, Stinton LM, Fritzler MJ. Improved serological differentiation between systemic
lupus erythematosus and mixed connective tissue disease by use of an SmD3 peptide-
based immunoassay. Clin Diagn Lab Immunol 12 (2005) 107-113.

26. Rabbani MA, Shah SM, Ahmed A. Cutaneous manifestations of systemic lupus
erythematosus in Pakistani patients. J Pak Med Assoc 53 (2003) 539-541.

27. Bruns A, Blass S, Hausdorf G, Burmester GR, Hiepe F. Nucleosomes are major T and B cell
autoantigens in systemic lupus erythematosus. Arthritis Rheum 43 (2000) 2307-2315.

28. Al Attia HM, iAl Ahmed YH, Chandani AU. Serological markers in Arabs with lupus
nephritis. Lupus 7 (1998) 198-201.

29. Forger F, Matthias T, Oppermann M, Becker H, Helmke K. Clinical significance of anti-

dsDNA antibody isotypes: IgG/IgM ratio of anti-dsDNA antibodies as a prognostic
marker for lupus nephritis. Lupus 13 (2004) 36-44.

30. van den Berg L, Nossent H, Rekvig O. Prior anti-dsDNA antibody status does not predict
later disease manifestations in systemic lupus erythematosus. Clin Rheumatol 25 (2006)
347-352.

31. Alba P, Bento L, Cuadrado MJ, Karim Y, Tungekar MF, Abbs I, Khamashta MA, D'Cruz D,
Hughes GR. Anti-dsDNA, anti-Sm antibodies, and the lupus anticoagulant: significant
factors associated with lupus nephritis. Ann Rheum Dis 62 (2003) 556-560.

32. Parodi A, Drosera M, Barbieri LB, Babbini G, Rebora A. Antidouble-stranded DNA isotypes
in lupus erythematosus patients with prevalent cutaneous presentation. Br J Dermatol
147 (2002) 754-756.

33. Houman MH, Smiti-Khanfir M, Ben Ghorbell I, Miled M. Systemic lupus erythematosus in
Tunisia: demographic and clinical analysis of 100 patients. Lupus 13 (2004) 204-211.

34. Suer W, Dahnrich C, Schlumberger W, Stocker W. Autoantibodies in SLE but not in

scleroderma react with protein-stripped nucleosomes. J Autoimmun 22 (2004) 325-334.

35. Soto ME, Vallejo M, Guillen F, Simon JA, Arena E, Reyes PA. Gender impact in systemic
lupus erythematosus. Clin Exp Rheumatol 22 (2004) 713-721.

36. Yao Z, Seelig HP, Renz M, Hartung K, Deicher H, Keller E, Nevinny-Stickel C, Albert ED. HLA
class II genes and antibodies against recombinant U1-nRNP proteins in patients with
systemic lupus erythematosus. SLE Study Group. Rheumatol Int 14 (1994) 63-69.

37. Manderson AP, Carlucci F, Lachmann PJ, Lazarus RA, Festenstein RJ, Cook HT, Walport MJ,
Botto M. The in vivo expression of actin/salt-resistant hyperactive DNase I inhibits the
development of anti-ssDNA and anti-histone autoantibodies in a murine model of
systemic lupus erythematosus. Arthritis Res Ther 8 (2006) R68.

38. Sarzi-Puttini P, Atzeni F, Capsoni F, Lubrano E, Doria A. Drug-induced lupus

erythematosus. Autoimmunity 38 (2005) 507-518.

39. Atzeni F, Marrazza MG, Sarzi-Puttini P, Carrabba M. Drug-induced lupus erythematosus.

[Article in Italian] Reumatismo 55 (2003) 147-154.

40. Hartung K, Seelig HP. Laboratory diagnostics of systemic autoimmune diseases. Part 1.
Collagenoses. [Article in German] Z Rheumatol 65 (2006) 709-724.

41. Chung L, Flyckt RL, Colon I, Shah AA, Druzin M, Chakravarty EF. Outcome of pregnancies
complicated by systemic sclerosis and mixed connective tissue disease. Lupus 15 (2006)
595-599.

14
 Medizinische
EUROIMMUN Labordiagnostika
AG

42. Lyons R, Narain S, Nichols C, Satoh M, Reeves WH. Effective use of autoantibody tests in
the diagnosis of systemic autoimmune disease. Ann N Y Acad Sci 1050 (2005) 217-228.

43. Granito A, Muratori P, Muratori L, Pappas G, Cassani F, Worthington J, Guidi M, Ferri S, DE

Molo C, Lenzi M, Chapman RW, Bianchi FB. Antinuclear antibodies giving the 'multiple
nuclear dots' or the 'rim-like/membranous' patterns: diagnostic accuracy for primary
biliary cirrhosis. Aliment Pharmacol Ther 24 (2006) 1575-1583.

44. Pakunpanya K, Verasertniyom O, Vanichapuntu M, Pisitkun P, Totemchokchyakarn K, Nantiruj

K, Janwityanujit S. Incidence and clinical correlation of anticentromere antibody in Thai
patients. Clin Rheumatol 25 (2006) 325-328.

45. Respaldiza N, Wichmann I, Ocana C, Garcia-Hernandez FJ, Castillo MJ, Magarino MI,
Magarino R, Torres A, Sanchez-Roman J, Nunez-Roldan A. Anti-centromere antibodies in
patients with systemic lupus erythematosus. Scand J Rheumatol 35 (2006) 290-294.

46. Frank MB, McCubbin V, Trieu E, Wu Y, Isenberg DA, Targoff IN. The association of anti-
Ro52 autoantibodies with myositis and scleroderma autoantibodies. Journal of
Autoimmunity 12 (1999) 137-142.

47. Martin-Domenech R, Turrin AI, Lpez-Longo FJ, Vzquez Galeano CM, Carreo Prez L,
Zea Mendoza AC. Anti-Ro/SSA response in myositis with anti-Jo-1 antibodies. Poster
Abstract (1995).

48. Rutjes SA, Vree Egberts WT, Jongen P, Van Den Hoogen F, Pruijn GJ, Van Venrooij WJ. Anti-
Ro52 antibodies frequently co-occur with anti-Jo-1 antibodies in sera from patients with
idiopathic inflammatory myopathy. Clinical and experimental immunology 109 (1997) 32-40.

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