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INOVA NEWS
Antinuclear antibodies:
From past to present
In 1957, George J Friou first described an indirect immunofluorescence (IIF) test system
for the detection of anti-nuclear antibodies (ANA) – thus beginning a new generation of
ANA testing. The test uses HEp-2 cells, a cell line which was established in 1952 by Alice E
No. 7
IN THIS ISSUE
p2 ANA immunofluorescence: Resurrection of
an old test | Pier Luigi Meroni, MD, PhD
p4 Detection of antinuclear antibodies
Moore et al. from tumors that had been produced in irradiated-cortisonized weanling rats
Xavier Bossuyt, MD, PhD
after injection with epidermoid carcinoma tissue from the larynx of a 56-year-old male. The
p7 Digital image analysis results show high
HEp-2 cell – a native protein array, with hundreds if not thousands of antigens, provides
the ideal substrate for the detection of ANA. Since the inception of utilizing HEp-2 cells for
ANA screening, the diagnosis of systemic autoimmune rheumatic diseases (SARDs), has
evolved.
The IIF on HEp-2 cells has been replaced in some laboratories with multiplex or
ELISA screening methods. Due to concerns over ‘false negative’ results, the lack of
transparency to clinicians, and absence of the newer test algorithms, the American
College of Rheumatology (ACR) formed a Task Force to recommend the use of the
traditional IIF method for ANA screening. This initiated a renaissance of the method
which is reflected by entire sessions dedicated to HEp-2 ANA testing at international
scientific meetings.
During the last years, the first digital imaging systems have been developed which eliminate major drawbacks of the method – the subjectivity and the lack of automated reading.
In this issue of the INOVA Newsletter we are delighted to present novel insights and
updates on ANA detection using IIF on HEp-2 cells, authored by experts in the field.
Enjoy reading!
Michael Mahler, PhD
Director of Research-Immunopathology
INOVA Diagnostics
reproducibility and agreement with human
interpretation on HEp-2 cells | Carol Buchner, MT (ASCP)
p10 High impact of the dense fine speckled
pattern on HEp-2 cells on the diagnosis of systemic
autoimmune diseases | Michael Mahler, PhD
p14 Autoantibodies that cannot be identified on
HEp-2 cell need tissue substrate | Thorsten Krieger, MD, PhD
p19 NOVA Lite® IFA slide kits |
Carol Buchner, MT
ANA immunofluorescence: Resurrection of an old test
Pier Luigi Meroni, MD, PhD
Professor of Rheumatology
University of Milan-Italy
Detection of autoantibodies is vital in the diagnosis and management of patients with autoimmune
diseases (AIDs). The use of new assays (i.e. automated, high-thoroughput solid phase methods, multiarray systems) and the better knowledge of the
physiopathology of AIDs have improved our
diagnostic power. As a consequence the impact of
AID in the daily practice is increased both from a
clinical and a laboratory point of view.1, 2
The spreading of autoimmunity testing from reference to general laboratories raises several practical problems. Among them, the most important
are the correct performance and the clinical interpretation of the assays. This is particularly true
owing to the increasing need of early diagnosis,
a prerequisite for a successful treatment for many
AIDs. Besides the problem of the treatment delay,
a wrong diagnosis, either through false positive
or false negative tests may also be responsible for
additional costs due to the repetition of confirmatory tests and/or to consequent unnecessary
diagnostic investigations.1-4
The use of new autoantibody assays raises also the
problem of the clinical interpretation of their results
in comparison with those detectable by “historical” methods. In fact, there are no well planned
studies that compared old and new methods in
terms of sensitivity/specificity or positive and
negative predictive value. Hence, one of the tasks
of the international committees for autoantibody
standardization in the future will be to draw up
specific guidelines regarding how to use and interpret these new assays. These issues are related to
2| INOVA NEWS No. 7
Ta b l e 1
P R O B L E M S I N A N A S TA N D A R D I Z AT I O N
Isotype of the fluoresceinated antiserum (IgG vs IgG/IgM/IgA)
Efficiency of the fluorescent microscope
Starting serum dilution (1:80 is the widely suggested starting dilution: with positive results > at 1:160 considered to be
pathological)
Recognition of standard patterns (nuclear and cytoplasmic)
Correct preparation of the slides for reading
any autoantibody assay5,6, and have been reported
even for a basic screening test for ANA detection.
The methodology for detection of ANAs has
changed over the years from the lupus erythematosus (LE) cell test, to indirect immunofluorescence
(IIF) utilizing sections of various rodent organs (e.g.
rat or mouse liver or kidney, etc.) to cell lines, in
particular HEp-2.
HEp-2 cells contain a large variety of autoantigens (approximately 100 to 150), most of them still
undefined. ANAs are detected by IIF, in which both
pattern and titer can be described. Although ANA
IIF has been used for a long time and does represent classification criteria for several AIDs, still there
are problems in its standardization7-9 (Table 1).
Over the years, numerous solid phase immunoassays have been developed in order to offer
methods for ANA detection much easier, faster,
cheaper and better standardized compared to
IIF using fixed HEp-2 cells as a substrate. Many
commercial laboratories and some hospital labora-
tories have recently switched their ANA screening tests to the new assays. Unfortunately, such a
decision was made without solid evidence that the
new tests could be able to replace the standard IIF
assay. As a result, inaccurate results for ANA tests
have been reported and the ACR created an ANA
Task Force to evaluate the extent of the problem
and to recommend solutions. A review of the literature by the committee indicates that up to 35%
of patients with SLE and a positive ANA by IIF were
negative on solid phase assays.10 Accordingly the
committee prepared a position report addressing
this problem and suggesting specific recommendations (Table 2).
Both physicians taking care of AID patients and
people working in autoimmunity diagnostic laboratories should be kept informed of such problems
and on the possible solutions to avoid misdiagnosis. Comparative studies on the new and the “old”
techniques are mandatory to better define their
use and limitations.
ANA screening assay:
Take home messages
R E C O M M E N D AT I O N S O F T H E A C R A N A TA S K F O R C E *
Laboratories using bead-based multiplex platforms or other solid
phase assays for detecting ANA must provide data to ordering
physicians that their assay has the same or improved sensitivity
and specificity compared to the IFA ANA
In-house assays for detecting ANA as well as anti-DNA, anti-Sm,
anti-RNP, anti-Ro/SS-A, anti-La/SS-B, etc. should be standardized
according to national (e.g., CDC) and/or international
(e.g.,WHO, IUIS) standards
Laboratories should specify the utilized methods for detecting
ANA when reporting their results
IIF ANA test should remain the gold standard for ANA testing
*Members of the ACR ANA Task Force: Peter Schur (chair), Donald Bloch, Joe
Craft, John A. Goldman, Pier Luigi Meroni, Eileen Moynihan, Morris Reichlin,
Westley Reeves, Eng Tan, Dan Wallace, and Mark Wener.
•
IIF ANA test is still the recommended method for ANA screening
•
IIF ANA positivity is required for the classification of several AIDs
•
Solid-phase or multiplex assays can detect only the specific autoantibodies directed against the limited number of autoantigens that are
displayed
References
1. Wiik A, Cervera R, Haass M, et al. European attempts to set guidelines for
improving diagnostics of autoimmune rheumatic disorders. Lupus 2006;
15: 391-396
2. Fritzler MJ, Fritzler ML. The emergence of multiplexed technologies as
diagnostic platforms in systemic autoimmune diseases. Curr Med Chem
2006; 13: 2503-2512
3. Shoenfeld Y, Cervera R, Haass M et al. A new initiative that can contribute to agreed diagnostic models of diagnosing autoimmune disorders
throughout Europe. Ann NY Acad Sci 2007; 1109: 138-144
4. Bossuyt X, Louche C, Wiik A. Standardisation in clinical laboratory medicine: and ethical reflection. Ann Rheum Dis 2008; 67: 1061-1063
5. Savige J, Gillis D, Benson E, et al. International Consensus Statement on
Testing and Reporting of Antineutrophil Cytoplasmic Antibodies (ANCA).
Am J Clin Pathol. 1999; 111: 507-513
Ta b l e 2
6.
Andreoli L, Rizzini S, Allegri F, et al. Are the current attempts at standardization of antiphospholipid antibodies still useful? Emerging technologies
signal a shift in direction. Semin Thromb Hemost 2008; 34: 356-360
7. Verstegen G, Duyck MC, Meeus P, Ravelingien I, De Vlam K. Detection
and identification of antinuclear antibodies (ANA) in a large community
hospital. Acta Clin Belg. 2009; 64: 317-323
8. Van Blerk M, Van Campenhout C, et al. Current practices in antinuclear
antibody testing: results from the Belgian External Quality Assessment
Scheme. Clin Chem Lab Med. 2009; 47: 102-108
9. Wiik AS, Høier-Madsen M, Forslid J, Charles P, Meyrowitsch J. Antinuclear
antibodies: A contemporary nomenclature using HEp-2 cells. J Autoimmun
2010; 35: 276: 290
10. Meroni PL, Schur PH. ANA screening: an old test with new recommendations. Ann Rheum Dis. 2010; 69: 1420-1422
TESTING FOR ANTINUCLEAR ANTIBODIES |
3
Detection of antinuclear antibodies
Xavier Bossuyt, MD, PhD
Professor of Medicine
University of Leuven – Belgium
Antinuclear antibodies (ANAs) are found in
patients with rheumatic diseases, such as SLE,
systemic sclerosis (SSc), Sjögren’s syndrome (SjS)
and polymyositis-dermatomyositis (PM/DM).
Antinuclear antibodies, though, are also found
in patients with non-rheumatic diseases such as
infectious disease, malignant disease and thyroid
disease, and even in individuals with no medical
condition, particularly women >40 years old and
elderly people.1, 2
Evidence-based guidelines for the use of ANA
testing proposed by the Amercican College
of Rheumatology (ACR) ad hoc committee on
immunologic testing state that ANA testing is
useful to varying degrees for the diagnosis and
monitoring of certain systemic autoimmune
rheumatic diseases (SARDs).
Ta b l e 1 1
ANTINUCLEAR ANTIBODY TESTING IS:
Very useful for the diagnosis of SLE and SSc
Somewhat useful for the diagnosis of SjS and PM/DM
Very useful for the monitoring or prognosis of juvenile
chronic arthritis (to stratify the risk for uveitis)
A critical part of the diagnosis of drug-associated lupus, mixed
connective tissue disease, and autoimmune hepatitis
The guidelines also state that ANA testing is not
useful for the diagnosis, monitoring, or prognosis
of other diseases including rheumatoid arthritis
and thyroid disease.1
Antinuclear antibodies are directed against
various nuclear antigens
Traditionally, IIF on HEp-2 cells is used for ANA
screening followed by more specific second line
4| INOVA NEWS No. 7
tests which are performed to identify the target
antigen of the antibodies (e.g. dsDNA or extractable nuclear antigens [ENAs]).
The term ENA generally includes, but is not limited to Sm, RNP, Ro (SS-A), La (SS-B), Jo-1 and Scl-70.
Anti-dsDNA and anti-ENA antibodies are clinically
important in patients with SARDs.
Some major patterns can be discerned by IIF on
HEp-2 cells: homogenous, speckled, centromeric, nucleolar, speckled or diffuse cytoplasmic.
Although not absolute, there exists a relationship
between the pattern observed on HEp-2 cells by IIF
and the presence of anti-dsDNA and/or anti-ENA
antibodies.2 There is an association between antidsDNA/ENA antibodies and specific autoimmune
diseases.2
HEp-2 patterns, the target antigens, and their
associated disease states:
•
A homogenous pattern is associated with antibodies
to dsDNA (SLE) or histones (drug-induced lupus)
•
A speckled pattern is associated with antibodies to
U1-RNP (mixed connective tissue disease, SLE), Sm
(SLE), and Ro (SS-A)/La (SS-B) (SjS, SLE)
•
A centromere pattern is associated with anticentromere antibodies (limited cutaneous form of
SSc)
•
A nucleolar pattern is associated with antibodies to
PM/Scl, RNA-polymerase III, and Scl-70 (SSc)
•
A speckled cytoplasmic pattern is associated
with antibodies to mitochondria (primary biliary
cirrhosis) or Jo-1 (PM), whereas a diffuse cytoplasmic
pattern is associated with anti-ribosome antibodies
(SLE)
Anti-ENA and anti-dsDNA antibodies occur with the
highest prevalence in samples with high ANA titers.3
The ENA specificities that are most related to high ANA
titers are U1-RNP and Sm.3 Patients with no autoimmune
disease and healthy individuals usually have low ANA
titers.2
Quantitative automated solid phase methods have in
some settings replaced IIF methods for the detection
of ANA. Because solid phase methods employ only
a limited number of autoantigens, they have a lower
sensitivity than IIF. For example, the sensitivity of IIF for
SLE is reported to be 90-95%, whereas the sensitivity of
antibodies to dsDNA, Sm, Ro (SS-A), and La (SS-B) for
SLE is reported to be 50-70%, 8-20%, 30-50%, and 20%,
respectively.2 In our own experience, 18 (29%) of 62 SLE
patients were negative for anti-dsDNA, anti-Sm, anti-U1RNP, or anti-Ro (SS-A) antibodies (unpublished data). Of
these 18 patients, 15 were ANA positive by IIF on HEp-2
cells. The low sensitivity of solid phase methods has also
been illustrated by a case report in which a diagnosis of
SLE was delayed because of a false negative ANA result
by solid phase method.4
The sensitivity of IIF for SSc is reported to be
85-90%, whereas the sensitivity of anti-Scl-70 and
anti-centromere antibodies is reported to be 15-20%
and 40-60%, respectively.2 In our own experience,
23 (33%) of 70 SSc patients had no antibodies to
centromere, Scl-70, PM-Scl-70 or RNA-Pol-III.5 Of
these 23 patients, 20 were positive by IIF. Anti-Scl-70,
anti-centromere, anti-RNA Pol-III, and anti-PM-Scl-100
antibodies were found in 21%, 37%, 7%, and 4% of
patients with SSc, respectively.5
of the ACR has recently concluded that solid phase
immunoassays may not be appropriate at present to
replace IIF as a screening test for detection of ANA.6
They recommend that IIF ANA testing should remain
the gold standard.6
Antinuclear antibodies are not only important for the
diagnosis of SARDs, but also for autoimmune hepatitis.
It should be mentioned that the target antigens of
the ANAs in autoimmune hepatitis are diverse and/or
unknown and that no solid phase methods are available
to screen for autoimmune hepatitis-related ANAs at
present.7
Although indirect immunofluorescence has a
high sensitivity, the specificity is low
Antinuclear antibodies are also found in patients with
non-rheumatic diseases and in healthy individuals.
Moreover, one should appreciate that anti-ENA
antibodies (especially anti-Jo-1, anti-ribosomal P and
anti-Ro (SS-A) may be overlooked by IIF on HEp-2 cells. In
a prospective study in which we evaluated antinuclear
antibodies in 2405 consecutive samples by IIF on HEp-2
cells and by solid phase assays we found the sensitivity
of antinuclear antibody testing by IIF for detection
of anti-ENA antibodies to be 82.9%.8 Recently, we
reported the prozone phenomenon for the ANA test by
IIF in a case of SjS with anti-Ro (SS-A) antibodies.9 Thus,
although IIF on HEp-2 cells has a high sensitivity, it may
miss clinically important autoantibodies. When there is
a high clinical suspicion, irrespective of the ANA result,
focused testing for specific autoantibodies should be
performed.
Given the low sensitivity of solid phase immunoassays
as a screening test for detection of ANA, a task force
TESTING FOR ANTINUCLEAR ANTIBODIES |
5
Other challenges facing IIF on HEp-2 include intra- and
inter- laboratory variance. Many sources contribute
to the variability of indirect immunofluorescence on
HEp-2 cells:
•
Different sources of HEp-2 cell lines
•
Different fixations
•
Different reading systems and optics
•
Different secondary antibodies, and heterogeneous
assays10
•
Visual evaluation is subjective and requires
considerable expertise of the technician9
References
1. Solomon DH, Kavanaugh AJ, Schur PH. American College of Rheumatology Ad Hoc Committee on Immunologic Testing Guidelines. Evidencebased guidelines for the use of immunologic tests: antinuclear antibody
testing. Arth Rheum 2002; 47: 434-444
2. Bizzaro N, Wiik A. Appropriateness in anti-nuclear antibody testing: from
clinical request to strategic laboratory practice. Clin Exp Rheum 2004; 22:
349-355
3. Bossuyt X, Hendrickx A, Frans J. Antinuclear antibody titer and antibodies
to extractable nuclear antigens. Arth Rheum 2005; 53: 987-988
4. Kroshinsky D, Stone JH, Bloch DB, Sepehr A. Case records of the Massachusetts General Hospital. Case 5-2009. A 47-year-old woman with a rash
and numbness and pain in the legs. N Engl J Med. 2009; 360: 711-720
5. Maes L, Blockmans D, Verschueren P, Westhovens R, De Beéck KO, Vermeersch P, Van den Bergh K, Burlingame RW, Mahler M, Bossuyt X. AntiPM/Scl-100 and anti-RNA-polymerase III antibodies in scleroderma. Clin
Chim Acta. 2010; 411: 965-971
6| INOVA NEWS No. 7
Taken together, IIF on HEp-2 cells remains an important
and established laboratory method in a multi-step
diagnostic approach to systemic rheumatic diseases
and autoimmune hepatitis. The advent of digital IFA
systems will undoubtedly result in a more standardized
approach to antinuclear antibody testing.10
6.
Meroni PL, Schur PH. ANA screening: an old test with new recommendations. Ann Rheum Dis. 2010; 69: 1420-1422
7. Alvarez F, P.A. Berg, F.B. Bianchi, L. Bianchi, A.K. Burroughs, E.L. Cancado,
R.W. Chapman, W.G.E. Cooksley, A.J. Czaja, V.J. Desmet, P.T. Donaldson, A.L.W.F. Eddleston, L. Fainboim, J. Heathcote, J.-C. Homberg, J.H.
Hoofnagle, S. Kakumu, E.L. Krawitt, I.R. Mackay, R.N.M. MacSween, W.C.
Maddrey, M.P. Manns, I.G. McFarlane. International Autoimmune Hepatitis
Group Report: review of criteria for diagnosis of autoimmune hepatitis. J
Hepatol 1999; 31(5): 929-938
8. Bossuyt X, Luyckx A. Antibodies to extractable nuclear antigens in antinuclear antibody-negative samples. Clin Chem. 2005; 51: 2426-2427
9. Bossuyt X, Mariën G, Vanderschueren S. A 67-year-old woman with a systemic inflammatory syndrome and sicca. Clin Chem. 2010; 56: 1508-1509
10. Hiemann R, Büttner T, Krieger T, Roggenbuck D, Sack U, Conrad K. Challenges of automated screening and differentiation of non-organ specific
autoantibodies on HEp-2 cells. Autoimmun Rev. 2009; 9: 17-22
Digital image analysis results show high reproducibility and agreement
with human interpretation on HEp-2 cells
Carol Buchner, MT (ASCP)
Manager, IFA Development
INOVA Diagnostics, San Diego, CA, USA
The term antinuclear antibody (ANA) describes a variety
of autoantibodies that react with constituents of cell
nuclei including DNA, RNA, proteins and riboproteins.1
The detection of ANA in human serum is an important
tool for diagnosing connective tissue diseases, especially
Indirect
systemic lupus erythematosus (SLE).1-3
immunofluorescence (IIF) is the reference method
for ANA testing which detects a wide range of
autoantibodies to nuclear and cytoplasmic antigens.1,2
A negative test virtually rules out SLE.3 Currently, the
American College of Rheumatology recommends IIF
on HEp-2 as the method of choice for ANA screening.
In conjunction with the patient history and physical
condition, IIF on HEp-2 offers excellent sensitivity (95%)
for SLE.3
Lack of standardization for IIF ANA testing still remains
a concern.4 Sources of variability include, but are not
limited to, the microscope and the interpretation by
the operator. The introduction of automation can
eliminate these sources of variability as it provides
an objective output.5,6 NOVA View® digital IFA system
contains a microscope with an automated stage, a CCD
(charge-coupled device) digital camera, a LED light
source and software that controls the motorized stage.
This system takes digital images, archives the images,
preliminarily categorizes the samples as positive or
negative and provides pattern recognition for positive
samples. The automated reading is followed by human
visual interpretation of the archived images that allows
review and user confirmation of the automated results.
The archived images facilitate training and allow for the
exchange of results between labs and clinicians.6 NOVA
View reduces variability and provides an approach to
standardize ANA interpretation.
Benefits of an Automated Digital Image Analysis Analyzer
Reduces hands-on time
•
Automated scanning and digital imaging of HEp-2 slides increases
productivity by reducing hands on time
Supports standardization
•
Proprietary algorithms provide objective and consistent output
Prompts appropriate analysis
•
Helps recognize samples requiring additional review
Facilitates case review
•
Creates digital image database that allows review, follow-up and
consultation
TESTING FOR ANTINUCLEAR ANTIBODIES |
7
In order to assess the performance of NOVA View,
studies were conducted to evaluate precision based
on light intensity units and endpoint titration data. In
addition, agreement was determined by comparing
results obtained by NOVA View on clinical samples to
visual human interpretation of the captured images.
two lots of HEp-2 slides, two lots of conjugates and
three operators (Table 1b).
•
Endpoint titration studies were performed by diluting
five sera 1:40 to 1:81,920 in PBS for 25 separate runs.
(Fig. 1).
•
204 clinically defined sera were used for comparison.
The output of NOVA View was compared to the visual
human interpretation of the archived images (Fig. 2).
•
The study was conducted using NOVA View digital IFA
system (INOVA Diagnostics, Inc.) with pre-production
software version 1.0.1.**
Methodology
•
Intra-assay variability was determined by running
five sera with five different patterns 36 times each
(Table 1a).
•
Total variability was determined by running five sera
45 times. The 45 individually run assays integrated
Table 1
Assay Variability
b)
a)
Intra-assay Variability
Pattern
Average LIU*
Homogeneous
486.2
Speckled
421.6
Centromere
343.1
Nucleolar
451.6
Nuclear Dots
422.1
N=36
% CV
12.3
16.9
14.5
9.0
13.1
*light intensity units
**Upgraded software version is available at time of print
8| INOVA NEWS No. 7
Total Assay Variability
Pattern
Average LIU*
Homogeneous
1054.3
Speckled
1859.4
Centromere
672.2
Nucleolar
625.6
Nuclear Dots
877.3
N=45
% CV
19.1
13.1
13.8
16.7
14.8
Fig. 1
Endpoint Titration Data on NOVA View
20
18
16
14
12
# of Assays 10
n=25
Minus One Dilution
Mid Endpoint Titer
Plus One Dilution
8
6
4
2
0
HOMO
1:2560
Mean Dilution
SPECK
1:1280
CENT
1:2560
NUC
1:5120
DOTS
1:1280
•
5 samples were diluted 1:40 to 1:82,000 on 25 independent assays.
•
All endpoint titers determined by the NOVA View were plus or minus one dilution compared to the mid endpoint
titer or to each other.
Fig. 2
Percent Agreement between Archived Images and NOVA View Interpretation
•
NOVA View correlates very well with human
interpretation of archived images.
Positive agreement
97%
Negative agreement
88%
Total agreement
92%
INInSUMMARY
summary
•
NOVA View results are highly reproducible and precise
•
Results obtained using clinical samples demonstrate the capability of
NOVA View to correctly discriminate between positive and negative
•
Archived images can be stored, reviewed, and shared at any time
References
1. Tan EM. Autoantibodies to nuclear antigens (ANA): Their immunobiology and medicine. Adv Immunol 1982: 33: 167-240
2. Tan EM, et al. The 1982 Revised criteria for the classification of systemic lupus erythematosus. Arth Rheum 1982; 25: 1271-1277
3. Rippey JH, Carter S, Hood P, Carter JB. Problems in ANA test interpretation: a comparison of two substrates. Diag Immunol 1985; 3: 43-46
4. Sack U, Conrad K et al. Autoantibody Detection Using Indirect Immunofluorescence on HEp-2 Cells. Contemporary Challenges in Autoimmunity: Ann NY Acad. Sci 2009; 1173: 166–173
5.
6.
Flessland A, Landicho H,et al. Performance Characteristics of the
PolyTiter Immunofluorescent Titration System for Determination of
Antinuclear Antibody Endpoint Dilution. Clin Diag Laby Immunol; 9:
329–332
Egerer et al. Automated evaluation of autoantibodies on human
epithelial-2 cells as an approach to standardize cell-base immunofluorescence tests. Arth Research Ther 2010; 12: R40
TESTING FOR ANTINUCLEAR ANTIBODIES |
9
High impact of the dense fine speckled pattern on HEp-2 cells
on the diagnosis of systemic autoimmune diseases
Michael Mahler, PhD
Director of Research-Immunopathology
INOVA Diagnostics, San Diego, CA, USA
History of ANA antibodies
The presence of autoantibodies against intracellular
antigens, especially antinuclear antibodies (ANAs), is a
hallmark of systemic autoimmune rheumatic diseases
(SARD).1 The indirect immunofluorescence (IIF) assay is
one of the most commonly used routine tests for the
detection of ANA and was recently recommended by
a task force of the American College of Rheumatology
(ACR).2 However, approximately 20% of serum samples
from healthy individuals (HI) have been reported to
yield a positive ANA test3,4, the majority of which are
caused by autoantibodies to dense fine speckles 70
(DFS70) antigen. Anti-DFS70 antibodies were initially
identified in a patient with interstitial cystitis5, but were
later associated with various disease conditions and
especially atopic dermatitis.6
Clinical association of anti-DFS70 antibodies
Since the first description, anti-DFS70 antibodies have
been found in the sera of patients with a variety of
chronic inflammatory conditions, cancer7, and even
in HI.3 Dellavance, et al. evaluated over 10,000 ANA
positive samples by IIF and then by immunoblot,
reporting that anti-DFS70 antibodies were common
among ANA-positive individuals with no evidence of
SARD and that among autoimmune patients with this
autoantibody, over half had evidence of autoimmune
thyroiditis.8 Although the clinical association and the
root cause of anti-DFS70 antibodies are still unclear, it
has been confirmed by different research teams that
anti-DFS70 antibodies are more prevalent in apparently
HI vs. patients with SARDs.3,6 Considering the prognostic
and long term outcome of individuals that have
anti-DFS70 antibodies, it was recently reported that,
out of 40 anti-DFS70 positive HI, none had developed
SARD over an average 4-year clinical follow-up.9 Based
on this observation, it has been suggested that the
10| INOVA NEWS No. 7
presence of isolated anti-DFS70 antibodies could
be used to exclude the diagnosis of SARD, such as
systemic lupus erythematosus (SLE).3,9,10 The decreased
prevalence of anti-DFS70 autoantibodies in SARD
patients is interesting, and the reasons underlying this
observation are unclear, but may include demographic,
genetic11, racial and/or technologies used to detect this
autoantibody.
IIF pattern and cellular function
The typical IIF staining pattern has been described as
dense fine speckles that are distributed throughout
the nucleus and on metaphase chromatin.12 Since a
70-kDa protein was recognized by immunoblotting,
the antigen was initially termed DFS70 but, the primary
target autoantigen was later identified as the lens
epithelium–derived growth factor (LEDGF)13 or DNA
binding transcription coactivator p75.6 This protein
has a number of physiological functions including
serving as a cofactor for human immunodeficiency
virus replication through an interaction with the viral
integrase14 and it is highly expressed in prostate tumor
tissue.7
Consequences for ANA testing – a new algorithm
In a previous study, 172/21,512 (0.8%) of consecutive
samples tested for ANA showed the typical DFS pattern
by IIF.15 This pattern was one of the most common in the
routine laboratory setting. Since the presence of ANA
are considered a reliable screening biomarker for SARD
and are included in the classification criteria for SLE,
ANA–HEp-2 testing outside a proper clinical framework
may yield a sizable portion of ANA-positive individuals
with no consistent evidence of SARD, potentially
causing some concern and anxiety in patients and
physicians alike.9 This becomes even more crucial with
the now compelling evidence that autoantibodies may
precede the clinical onset of SARD by many years.16 As
pointed out by Fritzler MJ, not all sera demonstrating the
DFS pattern are from HI and it remains unclear whether
this staining pattern is universally recognized in clinical
diagnostic laboratories.17 In particular, the discrimination
between DFS and the so-called `quasihomogeneous
pattern` might be a challenging task for routine diagnostic
laboratories.18 This underlines the importance of a better
understanding of anti-DFS70 antibodies and the inclusion
of testing for anti-DFS70 antibodies into the diagnostic
algorithm for ANA testing (Fig. 1).
Fig.
a.)1
ANA HEp‐2
Negative
Positive
Homogeneous
coarse speckled
Centromere
Nucleolar
Dense fine speckled (DFS)
Testing for ANA Screen (ELISA), dsDNA ENA and
dsDNA, ENA and other antibodies
Negative test for dsDNA, ENA and other disease‐
related antibodies
SARD inconclusive$
SARD inconclusive
Positive test for ANA, dsDNA, ENA or other disease‐
related antibodies
SARD very likely
SARD very likely
Testing for DFS70 and ANA Screen
and ANA Screen (ELISA)
DFS70 negative
ANA Screen positive
SARD likely#
DFS70 positive
ANA Screen positive
SARD inconclusive*
DFS70 negative
ANA Screen negative
SARD inconclusive
DFS70 positive ANA Screen negative
SARD unlikely
Likelihood depends on IIF pattern obtained. $ $ Likelihood
depends on IIF pattern obtained.
= Testing
according to Mariz
et al., SARD in patients with a mono‐specific DFS70 antibody is unlikely. Further studies are needed to * *According
to Mariz
et al., SARD in patients with a mono-specific DFS70
= SARD likely
#determine the likelihood.
antibody is unlikely. Further studies are needed to determine the likelihood.
= SARD unlikely
SARD is likely if results can be confirmed (e.g. ENA sub‐differentiation). Further studies are needed to determine the # likelihood. SARD is likely if results can be confirmed (e.g. ENA sub-differentiation).
= SARD inconclusive
Further studies are needed to determine the likelihood.
Fig. 1 Characteristic staining pattern and proposed test algorithm considering anti-DFS70 antibodies
(modified from Mahler et al.3) The characteristic dense fine speckled (DFS) staining pattern of interphase cells is indicated by the red
arrow and the strong chromatin staining of mitotic cells by the blue arrow. Samples with a DFS pattern could be tested for anti-DFS70 antibodies
by a confirmatory test and by ANA Screen ELISA (QUANTA Lite® ANA Screen) containing various SARD associated autoantigens. In this context, it
is important to mention, that the majority of monospecific DFS70 samples are negative on the QUANTA Lite® ANA Screen. Patients with negative
ANA Screen ELISA and positive DFS70 result have a lower likelihood for having SARD. Patients with a positive ANA Screen ELISA result, identified ENA specificity and negative DFS70 test result have an increased likelihood of having SARD. The likelihood for SARD in patients with a DFS
pattern and a positive ANA Screen ELISA is less understood. However, the data presented by Mariz et al.9, indicates that SARD is unlikely in those
patients since a DFS pattern with confirmed DFS70 reactivity (indicating mono-specific DFS70 reactivity) is negatively associated with SARD.
Further studies performed in routine settings are required to analyze the likelihood ratios of this proposed algorithm.
It is suggested that samples with a DFS staining pattern identified by IIF should be tested for anti-DFS70 antibodies
using a specific immunoassay. The test results need to be reported and clearly explained to clinicians.
TESTING FOR ANTINUCLEAR ANTIBODIES | 11
Immunoadsorption of anti-DFS70 antibodies
In a recent study, it has been shown that DFS pattern
is found in 33.1% of ANA positive HI compared to 0.0%
of ANA positive patients with SARD (p<0.0001), which
significantly affects the diagnostic power and efficiency
of the IIF assay.3 Thus, accurate pattern recognition,
interpretation and reporting of results to clinicians
are of high importance because it could decrease the
referral of patients with a positive ANA for unnecessary
consultation and evaluation. Since the identification
of the DFS pattern might be challenging for routine
Centromere
Scl 70
DFS70 (1)
DFS70 (2)
DFS70 (3)
adsorbed
b)
Non-adsorbed
adsorbed
a)
RNP
Non-adsorbed
Fig. 2
diagnostic laboratories and inaccurate interpretation
can have significant consequences, a method that can
prevent anti-DFS70 antibodies from binding to their
cognate target and producing the DFS pattern would
significantly improve the performance characteristics of
ANA by IIF.17 This led us to develop a method, allowing
for the immunoadsorption of anti-DFS70 antibodies
(Fig. 2), which offers considerable cost-savings in both
the laboratory and the medical care system.
Fig. 2 Immunoadsorption of anti-DFS70 antibodies.
Immunoadsorption of autoantibodies associated with systemic
autoimmune rheumatic diseases (SARD) are shown in a). No significant change can be observed in samples with anti-RNP, anti-Centromere
and anti-Scl-70 antibodies. In contrast, as shown in panel b.), anti-DFS70 antibodies are blocked by immunoadsorption with recombinant
DFS70 antigen [DFS70 (1)-DFS70 (3)]. In DFS70 (3) a different pattern becomes identifiable after immunoadsorption.
12| INOVA NEWS No. 7
KEY
POINTS
Key points
•
The dense fine speckled (DFS) pattern is rarely produced by autoantibodies in sera from patients with
SARD. Therefore, it is important to recognize this pattern and confirm the reactivity to DFS70 by a
specific assay.
•
Adsorption of anti-DFS70 antibodies prior to the ANA IIF test significantly reduces the false positive
rate for SARD and thus overcomes one of the major limitations of ANA using HEp-2 cells.
References
1. Mahler M, Fritzler MJ. Epitope specificity and significance in systemic
autoimmune diseases. Ann N Y Acad Sci 2010; 1183: 267-287
2. Meroni PL, Schur PH. ANA screening: an old test with new recommendations. Ann Rheum Dis 2010; 69: 1420-1422
3. Mahler M, Hanly JG, Fritzler MJ. Importance of the dense fine speckled
pattern on HEp-2 cells and anti-DFS70 antibodies for the diagnosis of
systemic autoimmune diseases. Autoimmunity Reviews, in press
4. Watanabe A, Kodera M, Sugiura K et al. Anti-DFS70 antibodies in 597
healthy hospital workers. Arthritis Rheum 2004; 50: 892-900
5. Ochs RL, Muro Y, Si Y, Ge H, Chan EK, Tan EM. Autoantibodies to DFS 70
kd/transcription coactivator p75 in atopic dermatitis and other conditions. J Allergy Clin Immunol 2000; 105: 1211-1220
6. Ganapathy V, Casiano CA. Autoimmunity to the nuclear autoantigen
DFS70 (LEDGF): what exactly are the autoantibodies trying to tell us?
Arthritis Rheum 2004; 50: 684-688
7. Daniels T, Zhang J, Gutierrez I et al. Antinuclear autoantibodies in prostate cancer: immunity to LEDGF/p75, a survival protein highly expressed
in prostate tumors and cleaved during apoptosis. Prostate 2005; 62:
14-26
8. Dellavance A, Viana VS, Leon EP, Bonfa ES, Andrade LE, Leser PG. The
clinical spectrum of antinuclear antibodies associated with the nuclear
dense fine speckled immunofluorescence pattern. J Rheumatol 2005; 32:
2144-2149
9. Mariz HA, Sato EI, Barbosa SH, Rodrigues SH, Dellavance A, Andrade LE.
Pattern on the antinuclear antibody-HEp-2 test is a critical parameter
for discriminating antinuclear antibody-positive healthy individuals and
patients with autoimmune rheumatic diseases. Arthritis Rheum 2011;
63(1):191-200
10. Muro Y, Sugiura K, Morita Y, Tomita Y. High concomitance of disease
marker autoantibodies in anti-DFS70/LEDGF autoantibody-positive
patients with autoimmune rheumatic disease. Lupus 2008; 17: 171-176
11. Muro Y, Ogawa Y, Sugiura K, Tomita Y. HLA-associated production of antiDFS70/LEDGF autoantibodies and systemic autoimmune disease.
J Autoimmun 2006; 26: 252-257
12. Ochs RL, Stein TW, Jr., Peebles CL, Gittes RF, Tan EM. Autoantibodies in
interstitial cystitis. J Urol 1994; 151: 587-592
13. Shinohara T, Singh DP, Chylack LT, Jr. Review: Age-related cataract:
immunity and lens epithelium-derived growth factor (LEDGF). J Ocul
Pharmacol Ther 2000; 16: 181-191
14. Maertens G, Cherepanov P, Pluymers W et al. LEDGF/p75 is essential for
nuclear and chromosomal targeting of HIV-1 integrase in human cells.
J Biol Chem 2003; 278: 33528-33539
15. Bizzaro N, Tonutti E, Visentini D et al. Antibodies to the lens and cornea in
anti-DFS70-positive subjects. Ann N Y Acad Sci 2007; 1107: 174-183
16. Arbuckle MR, McClain MT, Rubertone MV et al. Development of autoantibodies before the clinical onset of systemic lupus erythematosus. N Engl
J Med 2003; 349: 1526-1533
17. Bizzaro N, Tonutti E, Villalta D. Recognizing the dense fine speckled/lens
epithelium-derived growth factor/p75 pattern on HEp-2 cells: not an
easy task! Arth Rheum 2011:63(12):4036-4037
18. Fritzler MJ. The antinuclear antibody test: last or lasting gasp? Arthritis
Rheum 2011; 63: 19-22
TESTING FOR ANTINUCLEAR ANTIBODIES | 13
Autoantibodies that cannot be identified on
HEp-2 cell need tissue substrate
Thorsten Krieger, MD, PhD
Associate Professor Immunology
AescuLabor-Hamburg, Germany
Analysis of autoantibodies (AABs) by indirect
immunfluorescence (IIF) is an important first step in the
diagnosis of autoimmune diseases. Non-organ specific
AABs, important in the diagnosis of connective tissue
disease, are routinely screened by IIF on HEp-2 cells.
Organ specific AABs are directed against highly conserved
antigens. Diagnostically relevant target structures are
located in organ specific cells such as parietal cells in
stomach epithelium, or actin in stomach epithelium and
kidney tissue. In contrast to the HEp-2 testing where the
Different AABs can stain the same cells in tissue sections
if both antigens are expressed in the cells. This leads
to a similar fluorescence pattern in the tissue. An
expert combination of different organ tissue helps to
distinguish different AABs from each other. For instance,
antimitichondrial antibodies (AMA) and PCA both
react with parietal cells in the stomach epithelium.1 To
differentiate these AABs an additional liver tissue testing
is very helpful because anti mitochondrial antibodies
(AMA) lead to a clear fluorescence in the liver. This is in
Table 1
Tissue
Stomach
Kidney
Liver
Pancreas
Cerebellum
Detectable antibody
Further differentiation/
confirmation
Clinical Association
PCA
H+/K+ATPase
Pernicious Anaemia
AMA
AMA-M2 (MIT3)
Primary Biliary Cirrosis
SMA
Actin
LKM
LKM-1
LC
LC-1
ICA
GAD
IA-2
ANNA 1
Hu
ANNA 2
Ri
Ma
Ma2
Yo
Yo (Blot)
Autoimmune Hepatitis
Type 1 Diabetes Mellitus
Paraneoplastic neurological
syndrome
PCA=parietal cell antibodies; AMA=anti mitochondrial antibodies; SMA=smooth muscle antibodies; LKM=liver-kidney-microsomal
antibodies; GAD=glutamic acid decarboxylase; LC=liver cytosol; ICA=islet cell antibody; ANNA=anti-neuronal nuclear antibodies
focus lies on the cell organelles that can be stained in the
cell and allow a correlation to specific antigens, the focus
in tissue testing lies more on the fluorescence pattern of
stained cells in the tissue. AABs that can be detected on
tissues are often named after the structure in the tissue
that is stained by the AABs (Table 1). This is because
the antigen was unknown when the AAB was detected.
For example, parietal cell antibodies (PCA) are directed
against, H+/K+ATPase, and endomysium antibodies are
directed against tissue transglutaminase.
14| INOVA NEWS No. 7
contrast to PCA antigen, which do not, due to the fact
that the H+/K+ATPase is not expressed in the liver.
A common combination of organ tissue sections is
stomach, liver and kidney. This slide allows a screening
for gastrointestinal antibodies such as PCA, AMA,
smooth muscle antibodies (SMA)/Actin, liver kidney
microsomal (LKM) and liver cytosol 1 (LC1).
“
AMA stain the cytoplasm of hepatocytes, kidney cells and stomach tissue with an
enhancement of parietal cells. A titer of 1:40 or higher is considered as positive.2
AMA can be detected also as a granular fluorescence in the cytoplasma of HEp-2
cells. AMA positive samples should be tested additionally with AMA-M2 ELISA.
To obtain optimal sensitivity, the M2-ELISA should contain PDC-E2, OGDC-E2,
BCOADC-E2 as antigen.2 Recombinant mitochondrial antigens have been made
available, in the case of pMIT3, the three main autoepitopes are conjugated in
one molecule. My personal observation is that AMA testing on HEp-2 cells is
more sensitive than triple tissue testing and correlates better to the AMA-M2MIT3 ELISA results.
For detection
of organ specific
antigens that
are not
expressed in
HEp-2 cells such
as PCA, ASMA or
LKM, additional
tests on tissue
sections are
required.
AMA on HEp-2 Cell
“
PCA antibodies can be detected in stomach tissue. The H+/K+ATPase in the
parietal cells are stained by PCA, all other cells in the stomach epithelium, in
the liver and kidney are negative. The test is capable to exclude PCA, due to the
fact that other unspecific AABs can stain parietal cells on rodent tissue that is
commonly used in the test as well. Positive results should be confirmed with
H+/K+ATPase ELISA. PCA can be found frequently in patients without pernicious
anaemia because of the long latency of 20-30 or more years before clinical
manifestation.1
AMA on mouse kidney/stomach tissue
AMA on mouse liver/stomach tissue
TESTING FOR ANTINUCLEAR ANTIBODIES | 15
SMA are directed against structures of the cytoskeleton such as actin, troponin or tropomyosin.3 F-actin is the blank
antigen associated with autoimmune hepatitis. SMA reacts with the wall of small arteries present in all three tissues, the
stomach muscular layer and interglandular fibres of the stomach. If additional fluorescence is detected peritubularly in
the kidney, the pattern is specific for F-actin.3 Due to the fact that F-actin positive SMA antibodies are more specific for
autoimmune hepatitis than SMA alone, the result is reported as F-actin positive SMA if the peritubular fluorescence can
be detected.
Positive interglandular fibers on mouse stomach tissue
SMA with a specificity for actin on monkey kidney tissue
(red arrow denotes actin)
LKM antibodies are directed against cytochromes in the endoplasmatic reticulum. In IIF, this leads to a fluorescent
staining of the cytoplasm in the liver and the proximal tubuli in the kidney.4 The distal tubuli and stomach tissue are
negative.4
Fine granular fluorescence in the cytoplasm of proximal renal tubules but
not in distal tubules
LKM antibodies can be subclassified in LKM-1, LKM-2 and LKM-3 by ELISA or line immunoassay. LKM-1 autoantibodies
recognize a major linear epitope between amino acid 263 and 270 of the CYP 2D6 protein.4 These autoantibodies
inhibit CYP 2D6 activity in-vitro and are capable of activating liver infiltrating T-lymphocytes indicating autoimmune
hepatitis.4 LKM-2 autoantibodies are found in ticrynafen induced hepatitis; however, it is less frequent since the diuretic
is not longer in use. LKM-3 antibodies can be found in patients with viral hepatitis infection, especially in hepatitis D.4
16| INOVA NEWS No. 7
Liver cytosolic autoantibodies (LC-1) recognize
formiminotransferase cyclodeaminase as antigen, a
metabolic enzyme that is highly expressed in the liver. The
antibody is found in patients with autoimmune hepatitis
and hepatitis C infection. LC-1 antibodies alone lead to
a clear fluorescence pattern of the liver in IIF. Since LC-1
antibodies are frequently present together with LKM-1
antibodies, which also appear in the liver, it is very easy
to overlook the LC1 antibody. However, IIF allows the
exclusion of the presence of the antibody if no liver staining
is observed.
Islet cell antibodies (ICA) can be found in patients
with type1 diabetes mellitus (T1DM) on pancreas tissue
sections. The antibodies react against antigens in the
pancreatic ß cells. In 80% of cases, the antibodies are
directed against glutamic acid decarboxylase (GAD).6 ICA
are a powerful predictor of islet cell autoimmunity. Another
common antigen is islet cell antigen 512 (IA-2). GAD and
IA-2 antibodies can be found alone or in combination in
patients with T1DM. In pre-diabetic patients the risk of
clinical manifestation increases if both antibodies can be
LC1 antibodies on rat liver (left) and monkey liver (right) tissue
Islet cell antibodies on primate pancreas tissue
detected. In patients with late onset autoimmunity diabetes in the adult (LADA) with ICA, the antibody cannot be
confirmed by GAD or IA-2 ELISA in every case. This suggests that a further antigen plays a role in this cohort. Insulin
autoantibodies (IAA) can be found in children, the concentration of which, at the time of diagnosis, is inversely related
to the age of the patient, being highest in those less than 5 years of age.6
Screening for T1DM antibodies with rodent pancreatic tissue sections is an inexpensive and simple procedure to
exclude T1DM antibodies, because the majority of the tested samples are negative for these antibodies. Especially
in patients with LADA, ICA is an important marker because GAD and IA-2 testing does not identify all antibodies. In
children younger than 5 years of age, additional IAA testing should be performed.
TESTING FOR ANTINUCLEAR ANTIBODIES | 17
Paraneoplastic neuronal antibodies (PNA) are markers for paraneoplastic neurological syndromes. Only IgG
antibodies are considered to be clinically relevant.7 Indirect immunofluorescence on cerebellar tissue is the initial
screen for antineuronal antibodies.7 Western blots with cerebellar extracts can be used to confirm the specificities.
Antibodies that target neuronal nuclei of the central nervous system are Hu-Ab (also called anti-neuronal nuclear
antibody 1 (ANNA-1)), Ri-Ab (ANNA-2), ANNA-3 and Ma-Ab.
The most common and widely investigated PNA is Hu-Ab.
Hu antibodies on monkey cerebellum tissue
Hu antibodies on kidney stomach liver tissue
An antibody that targets purkinje cell cytoplasm is
Yo-Ab. This antibody is the second most common
onconeuronal antibody. Three proteins are recognized
by the antibodies and are known as cerebellar
degeneration-related proteins. The Yo-antibody is
associated with neoplasms of the breast and ovaries.
If Yo-Ab cannot be confirmed on Western blot, the
PCA2-Ab associated with small cell lung cancer (SCLC)
and Tr-Ab associated with Hodgkin´s disease should be
excluded because these rare antibodies lead to a similar
IIF pattern on cerebellar tissue sections.
anti-Yo antibodies on monkey cerebellum showing a granular staining of
Purkinje cell
References
1. Conrad K. Anti-intestinal goblet cell antibodies. Autoantibodies 2007;
417-422
2. Fussey SP et al. Identification and analysis of the major M2 autoantigens in primary biliary cirrhosis. Proc Natl Acad Sci 1988; 85: 86548658
3. Selmi C. Smooth muscle antibodies. Autoantibodies 2007; 487-491
4. Strassburg CP et al. Autoantibodies against glucuronosyltransferase
differ between viral hepatitis and autoimmune hepatitis. Gastroenterology 1996; 111 (6): 1576-1586
18| INOVA NEWS No. 7
5.
6.
7.
Lapierre P et al. Formiminotransferase cyclodeaminase is an organ
specific autoantigen recognized by sera of patients with autoimmune
hepatitis. Gastroenterology 1999; 116: 643-649
Pietropaolo M et al. Humoral immunity in type 1 diabetes mellitus.
Autoantibodies 2007; 379-387
Honnorat, J et al. Advances in paraneoplastic neurological syndromes
2004; 16: 614-620
NOVA Lite® IFA Slide Kits
Carol Buchner, MT (ASCP)
Manager, IFA Development
INOVA Diagnostics, San Diego, CA, USA
NOVA Lite has long been synonymous with quality immunofluorescence assays. Proprietary methods used
to craft our slides make NOVA Lite assays easy to read and interpret, helping your lab provide the reliable and
consistent results demanded by the current healthcare environment.
NOVA Lite IFA Slide Kits
NAME
PACKAGE
PART #
10 X 6 wells
(classic formulation)
20 X 12 wells
(classic formulation)
708299
708290
708298
708296
NOVA Lite IgG F-Actin*
NOVA Lite ANCA
(Ethanol Fixed)
NOVA Lite; ANCA
(Formalin Fixed)
10 X 6 wells
20 X 12 wells
708295
708297
NOVA Lite HEp-2
5 X 12 wells
20 X 12 wells
708101
708100
NOVA Lite; HEp-2 ANA IgG
(H+L)
Immunoglobulin
10 X 6 wells
20 X 12 wells
704230
704235
NOVA Lite HEp-2 ANA with
DAPI
20 x 12 wells
708102
NOVA Lite ANA KSL Mouse
Kidney/Stomach/Liver
10 X 4 wells
25 X 8 wells
708390
708380
NOVA Lite ANA Plus
Mouse Kidney & Stomach
10 X 4 wells
20 X 8 wells
708155
708150
NAME
PACKAGE
PART #
5 x 6 wells
708255
NOVA Lite dsDNA Crithidia
luciliae
10 X 6 wells
20 X 12 wells
708200
708205
NOVA Lite Monkey
Esophagus
(IgG and IgA conjugates)
10 X 5 wells
25 X 10 wells
704145
704150
NOVA Lite Monkey
Esophagus
(IgA conjugate only)
25 X 10 wells
704155
NOVA Lite Monkey
Esophagus (skin antibodies
only)
10 X 5 wells
25 X 10 wells
704160
704165
NOVA Lite Rat Liver, Kidney,
Stomach
10 X 5 wells
25 X 10 wells
704170
704180
NOVA Lite Skin Antibody
Primate Esophagus
10 X 8 wells
708330
NOVA Lite Thyroid
10 X 8 wells
708350
NOVA Lite Monkey Kidney*
10 X 5 wells
704240
* Available Outside The USA Only
TESTING FOR ANTINUCLEAR ANTIBODIES | 19
INOVA NEWS
No. 7
For more information on INOVA Diagnostic’s
complete product offerings visit www.inovadx.com
INOVA NEWSLETTERS ON OTHER AUTOIMMUNE TESTING TOPICS ARE AVAILABLE UPON REQUEST
•
•
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Celiac Disease Serology with Deamidated Gliadin Peptide (DGP) Assays (No. 3)
Third Generation CCP ELISA in Rheumatoid Arthritis Serology (No. 4)
Diagnosis of Primary Biliary Cirrhosis - Utilizing MIT3 Antigen Assays (No. 5)
Testing for Antiphospholipid Syndrome (No. 6)
Published by
Authors
Editors
INOVA Diagnostics, Inc.
Pier Luigi Meroni, MD, PhD
LeoPoldine Steindl
9900 Old Grove Road
Xavier Bossuyt, MD, PhD
Anna Eslami
San Diego, CA 92131
Carol Buchner, MT (ASCP)
toll free: (800) 545-9495 (US only)
Michael Mahler, PhD
phone: (858) 586-9900 (outside the US)
Thorsten Krieger, MD, PhD
Fax (858) 586-9911
[email protected]
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©2012 INOVA Diagnostics, Inc.
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