Automated tests of ANA immunofluorescence as throughput autoantibody detection technology: strengths and limitations

  • Pier Luigi Meroni1, 2, 5Email author,

    Affiliated with

    • Nicola Bizzaro3,

      Affiliated with

      • Ilaria Cavazzana4,

        Affiliated with

        • Maria Orietta Borghi1, 2 and

          Affiliated with

          • Angela Tincani4

            Affiliated with

            BMC Medicine201412:38

            DOI: 10.1186/1741-7015-12-38

            Received: 10 February 2014

            Accepted: 10 February 2014

            Published: 3 March 2014

            Abstract

            Anti-nuclear antibody (ANA) assay is a screening test used for almost all autoimmune rheumatic diseases, and in a number of these cases, it is a diagnostic/classification parameter. In addition, ANA is also a useful test for additional autoimmune disorders. The indirect immunofluorescence technique on monolayers of cultured epithelial cells is the current recommended method because it has higher sensitivity than solid phase assays. However, the technique is time-consuming and requires skilled operators. Automated ANA reading systems have recently been developed, which offer the advantage of faster and much easier performance as well as better harmonization in the interpretation of the results. Preliminary validation studies of these systems have given promising results in terms of analytical specificity and reproducibility. However, these techniques require further validation in clinical studies and need improvement in their recognition of mixed or less common staining patterns.

            Keywords

            Anti-nuclear antibodies Indirect immunofluorescence Autoimmunity

            Background

            Anti-nuclear antibody assay (ANA) is the screening test of choice for diagnosis of almost all systemic autoimmune rheumatic diseases (SARDs) because of its greater sensitivity compared with other assays, even though its specificity is much lower (Box 1) [1]. The gold standard method for ANA detection is still indirect immunofluorescence (IIF) on human epithelial (HEp-2) cells, as the alternative tests cannot display comparable sensitivity [2]. However, the technique is time-consuming and requires skilled operators. This fact together with the widespread increase in ANA requests and the reduction of laboratory facilities because of the budget constriction generated a strong need for advanced automated platforms as in other branches of the laboratory medicine.

            ANA automated reading systems

            Currently, at least six commercial systems for the automated reading of ANA IIF are available: Aklides (Medipan, Dahlewitz, Germany), EUROPattern (Euroimmun AG, Luebeck, Germany), Helios (Aesku Diagnostics, Wendelsheim, Germany), Image Navigator (ImmunoConcepts, Sacramento, CA), NOVA View (Inova Diagnostics, San Diego, CA), and Zenit G-Sight (A. Menarini Diagnostics, Florence, Italy).

            These systems are based on a composition of different hardware modules combined with mathematical pattern-recognition software algorithms, enabling fully automated image acquisition, analysis, and evaluation of IIF ANA tests.

            Samples can be classified as positive or negative and the main IIF pattern recognized (Table 1). In addition, quantitative fluorescence intensity value (equivalent to the end-point titer) can be obtained. To date, 13 studies have been published assessing the reliability of automated IIF analysis as a standardized alternative for the conventional manual visual approach (Table 2) [314].
            Table 1

            Types of indirect immunofluorescence pattern identified by the currently available automated systems for anti-nuclear antibody assay

            System

            Pattern

            Aklides

            Homogeneous, speckled, nucleolar, centromeric, nuclear dots, cytoplasmic

            EuroPattern

            Homogeneous, speckled, nucleolar, centromeric, nuclear dots, cytoplasmic

            Helios

            Visual recognition by the operator

            Image Navigator

            Visual recognition by the operator

            Nova View

            Homogeneous, speckled, nucleolar, centromeric, nuclear dots, cytoplasmic

            Zenit G-Sight

            Homogeneous, speckled, nucleolar, centromeric, nuclear dots, mitochondrial

            Table 2

            Automated/manual positive–negative agreement (PNA) for each anti-nuclear antibody indirect immunofluorescence reading system, based on 13 published studies

            System

            Studies, n

            Patients, n

            PNA, mean

            Aklides

            3

            1801

            0.95

            EuroPattern

            2

            467

            0.97

            Helios

            1

            1005

            0.98

            Image Navigator

            1

            3185

            0.99

            Nova View

            2

            842

            0.95

            Zenit G-Sight

            3

            830

            0.92

            All systems

            1

            149

            0.96

            Total

            13

            8279

            0.97

            The reported advantages of these systems include reduction in intra-laboratory and inter-laboratory variability, improvement in correlation between staining patterns with corresponding autoantibody reactivities, higher throughput in laboratory workflows, no requirement for a darkroom, integrated file storage, and easy retrieval of scanned wells.

            Comparison of the available ANA automated reading systems

            Although comparable performance between automated and conventional ANA IIF analysis for the interpretation of negative and positive samples has been reported, discrepancies between patterns have been found, especially when systems are able to detect basic patterns only, or when mixed fluorescent patterns are present in the samples [314].

            Some automated IIF systems present misinterpretation difficulties when antibodies react with a limited and specific cell component, such as Golgi apparatus, nuclear dots, or nuclear membrane [314]. Such misinterpretation may have implications in clinical settings, emphasizing the need and importance of visual validation (Table 3).
            Table 3

            Indirect immunofluorescence patterns detected on HEp-2 cells, with, related antigens and diagnosis a

             

            Related antigens

            Related diagnosis

            Nuclear patterns

              

              Homogeneous

            DNA, histones, chromatin/nucleosomes

            SLE, drug-induced SLE, JIA

              Peripheral/rim or nuclear envelope

            Lamins, LAP1/2 gp210, nucleoporin p62; Tpr nuclear envelope and nuclear pore complex antigens

            SLE, RA, PBC, myositis, autoimmune liver disease, PAPS

              Coarse speckled

            U1-snRNP, U2-6 snRNP (Sm), nuclear matrix

            MCTD, SLE, Raynaud, SSc, SS, UCTD

              Fine speckled

            SSA/Ro, SSB/La, common to many antigens

            SLE, SS, SSc, myositis, MCTD

              Dense fine speckled

            DFS70/LEDGF-P75

            Healthy subjects and other inflammatory conditions

              PCNA

            Auxiliary protein proliferating cell nuclear antigen: elongation factor of DNA polymerase δ

            SLE, lymphoproliferative diseases, SS

              Diffuse speckled with “cloudy” mitoses

            Topoisomerase-I

            SSc

              Centromere

            Kinetochore: CENP-A, CENP-B, CENP-C, CENP-F

            SSc (limited)

              Nucleolar homogeneous

            PM/Scl, RNA polymerase, To/Th , B23 phosphoprotein/numatrin

            SSc, myositis, overlap myositis/SSc

              Nucleolar speckled

            RNA polymerase (I to III)

            SSc

              Nucleolar clumpy

            U3-RNP (fibrillarin)

            SSc

              Multiple/few nuclear dots

            Sp100/140, PML bodies, NDP53, p80-coilin, PML bodies

            PBC, CAH, SS

              Centrosome/centriole (formerly spindle apparatus)

            Enolase, ninein, pericentrin

            SSc, Raynaud’s phenomenon, inflammatory disease

              MSA

            NuMA/centrophilin Hseg5

            RA, inflammatory conditions; pneumonia (mycoplasma)

            Cytoplasmic patterns

              

              Diffuse homogeneous (nucleoli positive)

            Ribosomal proteins

            SLE

              Fine speckled

            Jo-1, SRP, PDH (mitochondria)

            Myositides, DM, PBC, interstitial lung disease

              Discrete speckled

            Endosome (early endosome antigen 1), GW/P bodies, multivesicular bodies/lysosomes

            Neurological conditions, SS, SLE, RA, PBC

              Golgi complex

            Golgi proteins

            SLE, SS, RA, overlap syndromes, cerebellar ataxia

              Cytoplasmic fibers

            Actin, cytokeratin, tropomyosin, vimentin

            CAH, DM, infections and other inflammatory diseases

            CAH, chronic autoimmune hepatitis; CENP, centromere protein; DM, dermatomyositis; DFS70/LEDGF, dense fine speckled/lens epithelium-derived growth factor; JIA, juvenile idiopathic arthritis; MCTD, mixed connective tissue disease; MSA, mitotic spindle apparatus; PAPS, primary antiphospholipid syndrome; PBC, primary biliary cirrhosis; PCNA, proliferating cell nuclear antigen; PDH, phosphate dehydrogenase; PM, polymyositis; RA, rheumatoid arthritis; Scl, scleroderma; SLE, systemic lupus erythematosus; snRNP, small nuclear ribonuclear protein; SRP, signal recognition particle; SSc, systemic sclerosis; SS, Sjögren’s syndrome; UCTD, undifferentiated connective tissue disease.

            aModified from Agmon-Levin et al. [15].

            Such IIF assays have identifed more than 50 autoantibodies against 30 different nuclear and cytoplasmic antigens [16]. The use of large cultured cells with high rates of mitosis enables adequate pattern recognition by evaluation of the fluorescence distribution during different phases of the cell cycle. In fact, identification of cell cycle dynamics (for example, interphase, mitosis) is crucial both for defining different patterns (such as the fine or large speckled patterns within a speckled staining pattern, the centromere patterns and the PCNA patterns) and for distinguishing between different patterns (for example anti-nuclear membrane from the homogeneous pattern).

            Correct identification of different IIF patterns is sometimes diagnostic (for example, the centromere pattern and the PCNA pattern) or may suggest the occurrence of autoantibodies to specific antigens (Table 3). Many sera contain more than one antibody; in such cases, accurate analysis of the different patterns often requires direct evaluation of the slides to enable exact definition of the autoantibody profile in a given patient.

            Systemic sclerosis (SSc) represents a paradigmatic example of an autoimmune disease that is characterized by the occurrence of ANA in virtually all patients, but for which interpretation of the patterns is complesx [17]. In fact, SSc ANA are mainly represented by four mutually exclusive specificities: anti-centromere (ACA), anti-topoisomerase I, anti-nucleolar, and anti-RNA polymerase III antibodies. Anti-PM-Scl, U1-RNP and anti-Ku are usually detected in overlap syndromes. About 60% of patients with SSc have ACA or anti-topoisomerase I antibodies as disease markers. Many other ANA that are present in SSc (for example, anti-RNA polymerase III, anti-Th/To, anti-PM/Scl, anti-Ku, anti-fibrillarin) are directed against different proteins localized in the nucleus and nucleolus. These antigen-antibody systems identify SSc subgroups with different evolution, organ involvement, and survival prognosis. The use of IIF for detection of ANA is mandatory for SSc diagnosis, displaying a sensitivity of 85% [1]. ACA and anti-topoisomerase I negative sera show strong anti-nuclear staining, featuring speckled or nucleolar (homogeneous, clumpy or speckled) patterns (Box 1). Therefore, the definition of the single nucleolar staining could address the suspect of specific autoantibodies, relevant for the diagnosis of SSc. A nucleolar ANA associated with new onset of Raynaud’s phenomenon could be helpful in identifying a patient with early disease, sometimes associated with severe organ involvement. It is essential that ANA results are confirmed by more specific methods such as western blotting or immunoprecipitation assays.

            All these points underline the importance of correct interpretation of a given fluorescence pattern, and the need for standardization of analysis in automated systems.

            There is one other important point about using automated systems for ANA reading. The ANA test was originally ordered predominantly by rheumatologists and clinical immunologists, but nowadays a broader range of clinical disciplines (including primary care, dermatology, nephrology, gastroenterology, neurology, oncology, hematology, obstetrics, gynecology, cardiology) are currently ordering the test. This change in test referral patterns affects the post-test probability for a given disease, as screening tests with limited specificity (such as IIF ANA) are strongly affected when the pre-test probability in a given population decreases [17]. A positive ANA test obtained outside of the rheumatologic setting displays poor predictive value for future development of a rheumatic disease, but it represents a significant risk factor for SLE. Taking into account that the prevalence of SLE is 1 in 2000 (0.05%), the observed frequency of 2.5% in individuals with a 1/80 positive ANA test represents a 50-fold relative risk for development of the disease [18, 19]. Thus, ANA testing is a useful tool for SLE diagnosis.

            Conclusions

            Current evidence from preliminary study results indicates that there is good correlation between manual and automated interpretation of ANA IIF assays, at least in the ability to discriminate between positive and negative results and in recognizing the main IIF patterns. Such systems will therefore speed up routine performance of these tests and help to harmonize interpretation of the results across laboratories. However, there is a need to have their clinical diagnostic power validated by clinical studies, in addition to the analytical studies that have already been published. In addition, these new systems could be further improved if they were better able to recognize mixed fluorescent or less common fluorescent patterns.

            Box 1 Anti-nuclear antibody assay

            Best screening test for SLE
            • ▪ Sensitivity ≥95%

            • ▪ Specificity is only 57% for SLE compared with related rheumatic and autoimmune disorders

            Key diagnostic assay for:
            • ▪ SSc (sensitivity 85%)

            • ▪ SS (sensitivity 48%)

            • ▪ Drug-induced lupus (sensitivity 100%)

            • ▪ PM/DM (sensitivity 61%)

            • ▪ JIA (sensitivity 57%)

            • ▪ MCTD (sensitivity 100%)

            • ▪ Autoimmune hepatitis (sensitivity up to 60%)

            Important role in assessing prognosis in Raynaud’s phenomenon [2].

            DM, dermatomyositis; JIA, juvenile idiopathic arthritis; MCTD, mixed connective tissue disease; PM, polymyositis; SLE, systemic lupus erythematosus; SSc, systemic sclerosis; SS, Sjögren’s syndrome.

            Author information

            PLM is Professor of Rheumatology at the University of Milan. MOB is deputy Director of the Experimental Immuno-Rheumatology Laboratory at the Istituto Auxologico Italiano. NB is Director of the Diagnostic Department at San Antonio Hospital, Tolmezzo. AT is Professor of Rheumatology at the University of Brescia and head of Rheumatology and Clinical Immunology Unit at Spedali Civili of Brescia. IC has a tenured position in Rheumatology and Clìnical lmmunology Unit at Spedali Civili of Brescia.

            Abbreviations

            ACA: 

            Anti-centromere antibodies

            ANA: 

            Anti-nuclear antibody

            CAH: 

            Chronic autoimmune hepatitis

            CENP: 

            Centromere protein

            DFS70/LEDGF: 

            Dense fine speckled/lens epithelium-derived growth factor

            DM: 

            Dermatomyositis

            IIF: 

            Indirect immunofluorescence

            JIA: 

            Juvenile idiopathic arthritis

            MCTD: 

            Mixed connective tissue disease

            MSA: 

            mitotic spindle apparatus

            PAPS: 

            Primary antiphospholipid syndrome

            PBC: 

            Primary biliary cirrhosis

            PCNA: 

            Proliferating cell nuclear antigen

            PDH: 

            Phosphate dehydrogenase

            PM-Scl: 

            Polymyositis-scleroderma

            PM: 

            Polymyositis

            PNA: 

            Positive/negative agreement

            RA: 

            Rheumatoid arthritis

            SARDS: 

            Systemic autoimmune rheumatic disease

            SLE: 

            Systemic lupus erythematosus

            snRNP: 

            Small nuclear ribonuclear protein

            SSc: 

            Systemic sclerosis

            SS: 

            Sjögren’s syndrome

            UCTD: 

            Undifferentiated connective tissue disease.

            Declarations

            Authors’ Affiliations

            (1)
            Department of Clinical Sciences & Community Health, University of Milan
            (2)
            Istituto Auxologico Italiano
            (3)
            Laboratory of Clinical Pathology, Ospedale San Antonio
            (4)
            Rheumatology Unit and Chair, Spedali Civili di Brescia, Università degli Studi di Brescia
            (5)
            Division of Rheumatology, Istituto G. Pini

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            © Meroni et al.; licensee BioMed Central Ltd. 2014

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