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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

Moore et al. from tumors that had been produced in irradiated-cortisonized weanling rats

after injection with epidermoid carcinoma tissue from the larynx of a 56-year-old male. The

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 elimi-

nate 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!

Antinuclear antibodies: From past to present

IN THIS ISSUE

INOVA NEWS

No. 7p2 ANA immunofluorescence: Resurrection of an old test | Pier Luigi Meroni, MD, PhD

p4 Detection of antinuclear antibodies Xavier Bossuyt, MD, PhD

p7 Digital image analysis results show high 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

Michael Mahler, PhDDirector of Research-Immunopathology

INOVA Diagnostics

2 | INOVA NEWS No. 7

Detection of autoantibodies is vital in the diagno-sis and management of patients with autoimmune diseases (AIDs). The use of new assays (i.e. automat-ed, high-thoroughput solid phase methods, multi-array 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 refer-ence to general laboratories raises several practi-cal problems. Among them, the most important are the correct performance and the clinical inter-pretation 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 confir-matory 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 “histori-cal” 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 inter-pret these new assays. These issues are related to

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 erythema-tosus (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 autoanti-gens (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 repre-sent classification criteria for several AIDs, still there are problems in its standardization7-9 (Table 1).

Over the years, numerous solid phase immuno-assays 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-

Pier Luigi Meroni, MD, PhD Professor of RheumatologyUniversity of Milan-Italy

ANA immunofluorescence: Resurrection of an old test

T a b l e 1

P R O B L E M S I N A N A S T A 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 start-ing 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

TESTING FOR ANTINUCLEAR ANTIBODIES | 3

References1. 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 contrib-ute 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 medi-cine: 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

6. Andreoli L, Rizzini S, Allegri F, et al. Are the current attempts at standardiza-tion 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 recommenda-tions. Ann Rheum Dis. 2010; 69: 1420-1422

tories have recently switched their ANA screen-ing 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 liter-ature 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 recommen-dations (Table 2).

Both physicians taking care of AID patients and people working in autoimmunity diagnostic labora-tories should be kept informed of such problems and on the possible solutions to avoid misdiagno-sis. Comparative studies on the new and the “old” techniques are mandatory to better define their use and limitations.

T a b l e 2

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 T A 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

• 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 autoanti-bodies directed against the limited number of autoantigens that are displayed

*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.

ANA screening assay:Take home messages

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).

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

tests which are performed to identify the target antigen of the antibodies (e.g. dsDNA or extract-able nuclear antigens [ENAs]).

The term ENA generally includes, but is not limit-ed 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, centromer-ic, 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 anti-dsDNA/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 anti- centromere 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)

Xavier Bossuyt, MD, PhDProfessor of MedicineUniversity of Leuven – Belgium

Detection of antinuclear antibodies


T a b l e 1 1

A N T I N U C L E A R A N T I B O D Y T E S T I N G I S :

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

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-U1-RNP, 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

Given the low sensitivity of solid phase immunoassays as a screening test for detection of ANA, a task force

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.


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

References1. Solomon DH, Kavanaugh AJ, Schur PH. American College of Rheumatol-

ogy Ad Hoc Committee on Immunologic Testing Guidelines. Evidence-based 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 Massa-chusetts 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, Ver-meersch P, Van den Bergh K, Burlingame RW, Mahler M, Bossuyt X. Anti-PM/Scl-100 and anti-RNA-polymerase III antibodies in scleroderma. Clin Chim Acta. 2010; 411: 965-971

6. Meroni PL, Schur PH. ANA screening: an old test with new recommenda-tions. 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. Donald-son, 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 antinu-clear antibody-negative samples. Clin Chem. 2005; 51: 2426-2427

9. Bossuyt X, Mariën G, Vanderschueren S. A 67-year-old woman with a sys-temic inflammatory syndrome and sicca. Clin Chem. 2010; 56: 1508-1509

10. Hiemann R, Büttner T, Krieger T, Roggenbuck D, Sack U, Conrad K. Chal-lenges of automated screening and differentiation of non-organ specific autoantibodies on HEp-2 cells. Autoimmun Rev. 2009; 9: 17-22

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


Digital image analysis results show high reproducibility and agreement with human interpretation on HEp-2 cells


Carol Buchner, MT (ASCP)Manager, IFA DevelopmentINOVA 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 systemic lupus erythematosus (SLE).1-3 Indirect 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

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

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.


Table 1Assay Variability a) b)

Intra-assay VariabilityPattern Average LIU* % CVHomogeneous 486.2 12.3Speckled 421.6 16.9Centromere 343.1 14.5Nucleolar 451.6 9.0Nuclear Dots 422.1 13.1N=36

Total Assay VariabilityPattern Average LIU* % CVHomogeneous 1054.3 19.1Speckled 1859.4 13.1Centromere 672.2 13.8Nucleolar 625.6 16.7Nuclear Dots 877.3 14.8N=45

*light intensity units

**Upgraded software version is available at time of print

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.

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

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.**


IN SUMMARY

Fig. 1

Endpoint Titration Data on NOVA View

Fig. 2

Percent Agreement between Archived Images and NOVA View Interpretation

• NOVA View correlates very well with human interpretation of archived images.

• 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.

# of Assaysn=25

Minus One DilutionMid Endpoint TiterPlus One Dilution

Mean DilutionHOMO1:2560

SPECK1:1280

CENT1:2560

NUC1:5120

DOTS1:1280

20

18

16

14

12

10

8

6

4

2

0

References1. Tan EM. Autoantibodies to nuclear antigens (ANA): Their immunobiol-

ogy and medicine. Adv Immunol 1982: 33: 167-2402. Tan EM, et al. The 1982 Revised criteria for the classification of sys-

temic lupus erythematosus. Arth Rheum 1982; 25: 1271-12773. Rippey JH, Carter S, Hood P, Carter JB. Problems in ANA test interpreta-

tion: a comparison of two substrates. Diag Immunol 1985; 3: 43-46 4. Sack U, Conrad K et al. Autoantibody Detection Using Indirect Immu-

nofluorescence on HEp-2 Cells. Contemporary Challenges in Autoim-munity: Ann NY Acad. Sci 2009; 1173: 166–173

5. 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

6. Egerer et al. Automated evaluation of autoantibodies on human epithelial-2 cells as an approach to standardize cell-base immunofluo-rescence tests. Arth Research Ther 2010; 12: R40

In 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

Positive agreement 97%

Negative agreement 88%

Total agreement 92%


High impact of the dense fine speckled pattern on HEp-2 cells on the diagnosis of systemic autoimmune diseases

Michael Mahler, PhDDirector 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

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).

ANA HEp‐2

a.)

Positive Negative

Dense fine speckled (DFS)

Testing for DFS70 and ANA Screen

Homogeneouscoarse speckledCentromereNucleolar

Testing for ANA Screen (ELISA), dsDNA ENA and and ANA Screen

(ELISA)

DFS70 positive ANA Screen negative

SARD unlikely

DFS70 negativeANA Screen negative

SARD inconclusive

DFS70 positiveANA Screen positive

SARD inconclusive*

DFS70 negativeANA Screen positive

SARD likely#

dsDNA, ENA and other antibodies

Negative test for dsDNA, ENA and other disease‐

related antibodiesSARD inconclusive$

Positive test for ANA, dsDNA, ENA or other disease‐related antibodiesSARD very likelySARD inconclusive SARD very likely

$ Likelihood depends on IIF pattern obtained. * according to Mariz et al., SARD in patients with a mono‐specific DFS70 antibody is unlikely. Further studies are needed to determine the likelihood.# SARD is likely if results can be confirmed (e.g. ENA sub‐differentiation). 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, identi-fied 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.

Fig. 1

$ Likelihood depends on IIF pattern obtained. * According to Mariz et al., SARD in patients with a mono-specific DFS70 antibody is unlikely. Further studies are needed to determine the likelihood.# SARD is likely if results can be confirmed (e.g. ENA sub-differentiation). Further studies are needed to determine the likelihood.

= Testing= SARD likely= SARD unlikely= SARD inconclusive


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

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.

Fig. 2RNP Centromere Scl 70

DFS70 (1) DFS70 (2) DFS70 (3)

a)

b)

Non

-ads

orbe

dN

on-a

dsor

bed

adso

rbed

adso

rbed


References1. Mahler M, Fritzler MJ. Epitope specificity and significance in systemic

autoimmune diseases. Ann N Y Acad Sci 2010; 1183: 267-2872. Meroni PL, Schur PH. ANA screening: an old test with new recommenda-

tions. Ann Rheum Dis 2010; 69: 1420-14223. 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 condi-tions. 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 pros-tate 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 anti-DFS70/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 autoanti-bodies 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

KEY POINTSKey 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.


Autoantibodies that cannot be identified on HEp-2 cell need tissue substrate

Thorsten Krieger, MD, PhD Associate Professor ImmunologyAescuLabor-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

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.

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

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).

Table 1

Tissue Detectable antibodyFurther differentiation/

confirmationClinical Association

Stomach Kidney

Liver

PCA H+/K+ATPase Pernicious Anaemia

AMA AMA-M2 (MIT3) Primary Biliary Cirrosis

SMA Actin

Autoimmune HepatitisLKM LKM-1

LC LC-1

Pancreas ICAGAD

Type 1 Diabetes MellitusIA-2

Cerebellum

ANNA 1 Hu

Paraneoplastic neurological syndrome

ANNA 2 Ri

Ma Ma2

Yo Yo (Blot)

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


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.

“

“

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 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-M2-MIT3 ELISA results.

AMA on HEp-2 Cell AMA on mouse kidney/stomach tissue

AMA on mouse liver/stomach tissue


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)

Fine granular fluorescence in the cytoplasm of proximal renal tubules but not in distal tubules

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

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


LC1 antibodies on rat liver (left) and monkey liver (right) tissue

Islet cell antibodies on primate pancreas tissue

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

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.


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

anti-Yo antibodies on monkey cerebellum showing a granular staining of Purkinje cell

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.

References1. Conrad K. Anti-intestinal goblet cell antibodies. Autoantibodies 2007;

417-4222. Fussey SP et al. Identification and analysis of the major M2 autoan-

tigens in primary biliary cirrhosis. Proc Natl Acad Sci 1988; 85: 8654-8658

3. Selmi C. Smooth muscle antibodies. Autoantibodies 2007; 487-4914. Strassburg CP et al. Autoantibodies against glucuronosyltransferase

differ between viral hepatitis and autoimmune hepatitis. Gastroenter-ology 1996; 111 (6): 1576-1586

5. Lapierre P et al. Formiminotransferase cyclodeaminase is an organ specific autoantigen recognized by sera of patients with autoimmune hepatitis. Gastroenterology 1999; 116: 643-649

6. Pietropaolo M et al. Humoral immunity in type 1 diabetes mellitus. Autoantibodies 2007; 379-387

7. Honnorat, J et al. Advances in paraneoplastic neurological syndromes 2004; 16: 614-620


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690243 April 12 Rev. 0

Published by

INOVA Diagnostics, Inc.

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toll free: (800) 545-9495 (US only)

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Authors

Pier Luigi Meroni, MD, PhD

Xavier Bossuyt, MD, PhD

Carol Buchner, MT (ASCP)

Michael Mahler, PhD

Thorsten Krieger, MD, PhD

Editors

LeoPoldine Steindl

Anna Eslami

INOVA NEWSLETTERS ON OTHER AUTOIMMUNE TESTING TOPICS ARE AVAILABLE UPON REQUEST

• 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)

INOVA NEWS No. 7

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