Autoantibodies as Diagnostic Markers

Connective Tissue Disease-Specific Autoantibodies

The loss of immune tolerance characteristic for connective tissue diseases (CTDs) such as systemic lupus erythematosus (SLE), systemic sclerosis (SSc), poly/dermatomyositis (PM/DM), Sjögren’s syndrome (SjS), and mixed connective tissue disease (MCTD) brings about the generation of various nonorgan-specific autoantibodies (autoAbs) [1–3]. Although the triggering factors for the occurrence of autoAbs and their role in the pathogenesis of CTD are still not entirely understood, autoAbs are widely used as diagnostic markers in clinical routine nowadays [4, 5]. The L.E. cell phenomenon described by Hargraves in the late 1940 in patients suffering from SLE proved to be a result of autoAb binding to nuclear material of polymorphs and marked the beginning of a rapidly evolving autoAb era in clinical diagnostics [6]. Indirect immunofluorescence (IIF) was the first assay technique employed to reveal autoAbs in patients with CTD [7]. The groundbreaking works of Holborow and Friou et al. led to the discovery of so-called antinuclear antibodies (ANAs) as marker autoAbs of CTD like SLE [8, 9]. In the following years, clinicians made tremendous efforts to understand the clinical significance of autoAbs and their potential use for the serological diagnosis of CTD and beyond [10]. This process was greatly driven by novel emerging assay techniques used for autoAb testing and their respective assay performance characteristics (Fig. 1; Table 1). The ensuing discourse has led to the definition of various diagnostic strategies for the serological diagnosis of autoimmune disorders and continues to date. Of note, ANA detected by IIF was included into the diagnostic criteria of SLE and autoimmune hepatitis (AIH) later [11–13]. In this context, the discovery of autoAbs to extractable nuclear antigens (ENAs) apart from autoAbs to dsDNA or histones in the search for disease-specific autoAbs provides an intriguing example for the change in the understanding of the clinical meaning of autoAbs as diagnostic markers [14–16]. Thus, the seminal paper of E.M. Tan and H.G. Kunkel on the identification of Sm as an autoantigenic target of SLE and the use of double radial immunodiffusion (DRID; Ouchterlony technique) for its detection ushered in a new era in autoAb diagnostics and its clinical application [17]. Although ANA turned out to be a sensitive marker for SARD as a whole disease group, its specificity for distinct SARD entities was not satisfactory despite being defined as a diagnostic marker for SLE [11]. Thus, the clinical need for more specific “ANA” was met by the pioneering work of H.G. Kunkel, E.M. Tan, and others discovering more and more novel autoAbs to ENA with clinical significance [14, 18]. However, not all ENAs identified as targets for CTD-specific autoAbs could be isolated by the saline extraction technique reported previously and should not be termed ENA [19]. Furthermore, apart from autoAbs recognizing nuclear autoantigens, anticytoplasmic autoAbs (ACyA) have been introduced into the autoAb panel for SARD serology [20]. Thus, the anti-SjS antigen A (SS-A) autoAbs also termed Ro have been shown to interact with its respective target in the cytoplasm [21]. As a fact, the progress in proteomics enabled the identification of cytoplasmic autoantigenic targets interacting with for instance myositis-specific autoAbs like anti-histidyl tRNAse autoAbs (Jo-1) or SLE specific autoAb against ribosomal proteins [22–24]. Obviously, this created confusion among clinical and laboratory experts and called for clarification. In terms of ANA testing, the introduction of human epidermoid laryngeal carcinoma (HEp-2) cells as improved autoantigenic substrate in IIF has encouraged the reporting of CTD-specific cytoplasmic patterns over the years [2]. This contradiction in terminology was addressed by a recent consensus recommending the use of anticellular antibodies instead of ANA [4]. Notwithstanding, the use of ANA and ENA is well established particularly among clinicians and it remains to be seen how this issue will be solved adequately in the years to come [25]. In summary, autoAb testing is an integral part in the serological diagnosis of CTD and may also assist in the prognosis, subclassification, as well as monitoring of disease activity [4, 10, 26–29].

Fig. 1
figure 1

Evolving autoantibody (autoAb) testing and strategies for the serological diagnosis of systemic autoimmune rheumatic diseases. ANA antinuclear antibody, autoAb autoantibody, CIE counterimmunoelectrophoresis, D/LIA dot/line immunoassay, ELISA enzyme-linked immunosorbent assay, ENA extractable nuclear antigen, IB immunoblot/westernblot, ID/DRID immunodiffusion/double radial immunodiffusion, IIF indirect immunofluorescence, IP immunoprecipitation, MIA microbead immunoassay, RIP radioimmunoprecipitation

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Table 1 Autoantibody (AAB) detection methods in routine diagnostics of systemic rheumatic diseases
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As mentioned earlier, not only the discovery of novel SARD-specific autoAbs has challenged the diagnostic skills of clinicians but the introduction of novel assay techniques with differing assay performance, too [30]. Thus, the change from immunodiffusion-based detection techniques like DRID or counterimmunoelectrophoresis (CIE) detecting precipitating autoAbs to enzyme-linked immunosorbent assay (ELISA) regarding the analysis of autoAbs to Sm or SS-A called the specificity of these distinct markers suddenly into question [31–33]. The solid-phase ELISA brought about a significantly elevated sensitivity which in turn is related to a diminished diagnostic specificity [34]. Furthermore, with the better understanding of the chemical structure of for instance the small nuclear ribonucleoprotein (snRNP) complex representing the Sm autoantigen, six different protein structures (B, B’, D, E, F, G) were identified as autoantigenic targets with SmD being apparently the most specific one for SLE [35–37]. Alone, these critical aspects require a comprehensive knowledge on the interpretation of assay characteristics by clinicians which were not always conveyed by laboratorians adequately [1, 3]. The badly needed comprehension of pretest and posttest probabilities of presence of disease and its relation to the diagnostic performance of autoAb analysis such as ANA testing appears not satisfactorily developed in clinicians [19, 38, 39]. Thus, novel diagnostic strategies translating the progress in autoAb testing proved difficult to get in line with established diagnostic pathways [27, 40, 41]. The recent attempt to substitute ANA IIF testing as screening assay within the two-tier strategy by novel multiplex techniques failed or met with great resistance among rheumatologists [4, 42, 43]. Consequently, the two-stage strategy recommending ANA testing by IIF as screening and appropriate confirmation of ANA positives by a different analysis was confirmed by expert consensus for CTD serology recently [4].

Autoimmune Vasculitis-Specific Autoantibodies

Of note, like revealed for the L.E. phenomenon in patients with SLE, patients suffering from autoimmune vasculitides demonstrate loss of tolerance to polymorphs, too [44]. In contrast, the occurring autoAbs recognize specific neutrophil cytoplasmic and not nonspecific nuclear components and were described first in association with glomerulonephritis in 1982 by Davies et al. [45]. Van de Woude’s group reported so-called antineutrophil cytoplasmic antibodies (ANCAs) to be associated with granulomatosis with polyangiitis (GPA, formerly Wegener’s granulomatosis) shortly later and, consequently, the term ANCA-associated vasculitides (AAV) was coined [44, 46, 47]. Thus, this group of autoimmune vascular disorders comprises GPA, microscopic polyangiitis (MPA), and eosinophilic granulomatosis with polyangiitis (EGPA, formerly Churg-Strauss syndrome) [48, 49]. Their leading clinical characteristics are microvascular inflammation, tissue necrosis, and the appearance of ANCAs [20, 194–196]. However, few systems distinguish between positive and negative screening results only (Helios, Aesku.Diagnostics, Wendelsheim, Germany; Image Navigator, Immuno Concepts, Sacramento, USA; Cytospot, Autoimmun Diagnostika, Straßberg, Germany) [185, 197]. In summary, all systems were reported to meet the demand for automated interpretation and satisfactory system performances were obtained by comparative studies at least for qualitative ANA evaluation [197, 198].

The fully automated interpretation system AKLIDES® was the first platform which performance was evaluated in clinical studies successfully [199–201]. Egerer et al. published the first clinical evaluation in 2010 by comparing the use of the new technology for ANA assessment of 1222 sera in the routine laboratory environment of both a university and a private referral laboratory [199]. An agreement of 93.0 % (859/924) and of 90.6 % (270/298) between automated AKLIDES® interpretation and classical ANA reading in the university and the private laboratory were reported, respectively. Remarkably, end-titer analysis based on quantitative fluorescence reading was shown for the first time, which overcomes a crucial shortcoming of IIF and levels it with other quantitative assay techniques established in routine clinical laboratories. Thus, the application range of the novel interpretation systems (AKLIDES®, Europattern®, NovaView®) was enlarged by adding ANCA and anti-dsDNA autoAb testing on human neutrophils and Crithidia luciliae, respectively [191, 202–204].

In summary, the intriguing development of these novel automated IIF interpretation systems strengthened the position of IIF as screening technique within the two-tier strategy for ANA and ANCA analyses. Thus, the demand of even large laboratories in terms of automated autoAb testing by IIF with modern data management could be addressed adequately. Tozzoli et al. concluded that a new technological era in the routine autoimmune laboratory was reached by the introduction of fully automated IIF in 2009 [180]. Furthermore, this technology may also stimulate clinical research regarding larger population studies, e.g., the prevalence of the dense-fine speckled (DFS) pattern, and hence, of the DFS70 autoAbs, in different apparently healthy and diseased populations [205].

Combination of Screening and Confirmatory Testing

Irrespectively of the tremendous progress in automated autoAb testing by IIF at the beginning of this millennium, the constraint to use two different assay techniques for the recommended two-stage strategy of ANA and ANCA analyses has not been abolished yet [4]. This strategy enables a plausibility control of the obtained results because specific autoAb assays may give false-positive findings. For instance, a positive anti-dsDNA finding in ELISA in combination with ANA negativity cannot be regarded as relevant regarding diagnosis of SLE. However, the possibility of false-negative findings using the two-tier strategy especially for ANA reading in terms of sera positive for autoAbs to SS antigen A (SS-A/Ro) is still eminent at hand and represents an essential drawback of such approach [206]. Only the combination of both stages in one multiplex test would overcome these shortcomings and provide an ideal solution for autoAb testing addressing key clinical and laboratory needs. As a fact, this intriguing idea is quite simple, and thus, it appears astonishing that no such attempt was undertaken earlier. Hence, combination of the advantages of cell-based assays and the potential for multiplexing by microbead immunoassay (MIA) employing IIF within one reaction environment could revolutionize autoimmune diagnostics (Fig. 2).

Fig. 2
figure 2

Multiplexing strategy of CytoBead® technology exemplified for CytoBead® ANA assay. Combination of ANA screening with HEp-2 cells (middle part) and anti-ENA testing with antigen-coated microbeads (peripheral parts I–IV) in one reaction environment. Example of an ANA positive serum with positive homogeneous fluorescence pattern on HEp-2 cells and positive signal on dsDNA-coated microbeads presented as green fluorescence halo (small red microbeads in part III). ANA antinuclear antibody, CENP centromere protein, Da Dalton, dsDNA double-stranded DNA, ENA extractable nuclear antigen, hom homogeneous, RNP ribonuclear protein, Scl-70 DNA-Topoisomerase I, Sm Smith, SS Sjögren-Syndrome, (+) positive, (−) negative

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Second-Generation ANA Testing

To realize the idea of combining autoAb screening and confirmation, we started develo** a unique IIF reaction environment encompassing classical ANA analysis on HEp-2 cells and simultaneous multiplex detection of autoAbs by MIA. Indeed, merging screening and confirmatory testing for disease-specific autoAbs could generate many benefits including shorter hands-on times, better reproducibility of autoAb findings, and higher cost-effectiveness especially for larger sample series.

First, a MIA which utilizes multiple carboxylated polymethylmethacrylate bead populations differing in size and/or concentrations of fluorescent dye for multiplexing was developed [207]. The classification of bead populations and measurement of corresponding ligand fluorescence intensity was readily performed by AKLIDES® enabling the detection of six different antinuclear autoAbs to Scl-70, Sm, SS-A (Ro60), SS-B (La), CENP-B, and, dsDNA. This assay development created the basis for the design of a unique IIF reaction environment which could integrate the classical ANA testing on HEp-2 cells in one test [102]. The new assay technique combining classical ANA testing with confirmatory analysis by MIA was termed CytoBead® technology (Fig. 3a). Intriguingly, the novel options of digital fluorescence enabling quantitative analysis not only of specific autoAb testing by MIA but also of classical ANA reading on HEp-2 cells can be readily employed by CytoBead® assays. Thus, they can be standardized by calibrated interpretation systems for automated autoAb testing. Consequently, this is a new age of standardization of ANA testing as a whole which was not feasible with classical ANA testing by IIF in the past.

Fig. 3
figure 3

CytoBead® assays for the detection of a antinuclear antibodies (ANA) with CytoBead® ANA assay, b antineutrophil cytoplasmic autoantibodies (ANCA) with CytoBead® ANCA assay, and c celiac disease (CD)-specific (auto)antibodies (auto/Abs) with CytoBead® CeliAK assay. Matching principle of specific fluorescence patterns on HEp-2 cells (a), neutrophil granulocytes (b), and esophagus tissue (c) with positive reactions of antigen-coated microbeads immobilized in peripheral compartments. CENP centromere protein, Da Dalton, dsDNA double-stranded DNA, EmA endomysial antibody, GBM glomerular basement membrane, MPO myeloperoxidase, PR3 proteinase 3, RNP ribonuclear protein, Scl-70 DNA-Topoisomerase I, Sm Smith, SS Sjögren-Syndrome, (+) positive, (−) negative

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Altogether, a new generation of autoAb testing could be established that can meet the demand of modern routine service laboratories for the serology of SARD/CTD by addressing the key disadvantages of the currently recommended two-stage autoAb testing.

Recently, this new assay referred to as second generation ANA testing was evaluated in a comprehensive serological study comprising inter alia 174 patients with SLE, 103 with SSc, 46 with SjS, 36 with RA, 13 with MCTD, 21 with DM/PM, 21 with infectious disease, 93 with autoimmune liver diseases, 78 with inflammatory bowel disease, and 101 blood donors [102]. The CytoBead® ANA simultaneously determines ANA on HEp-2 cells and autoAbs to dsDNA, CENP-B, SS-A/Ro52, SS-A/Ro60, SS-B/La, RNP-Sm, Sm, and Scl-70. The obtained good agreement of the CytoBead® ANA with classical ANA reading by IIF and ELISA supports the notion that the novel combined reaction IIF environment for one-step ANA analysis employing HEp-2 cells and autoantigen-coated fluorescent beads as respective targets can provide at least the same assay performance like classical two-tier ANA testing.

Furthermore, simultaneous detection of ANA and specific autoAbs such as to SS-A/Ro by CytoBead® ANA can almost eliminate the risk of false-negative findings and increase the already high negative predictive value of ANA testing. Of note, this is especially in the interest of rheumatologists who would like to exclude the presence of autoimmunity in their differential diagnosis of SARD by ordering ANA testing. In this study, 4/267 (1.5 %) ANA-negative patients with positive anti-SS-A or anti-CENP-B autoAbs were determined by second-generation ANA analysis. As a fact, these distinct patients with RA and SjS would have been missed by the currently recommended two-tier strategy since ANA negativity and positivity for anti-SS-A and anti-CENP-B autoAbs were confirmed by classical testing.

New-Generation ANCA Testing

The CytoBead® technology was also applied for the comprehensive analysis of ANCA and the resulting CytoBead® ANCA was evaluated in terms of its assay performance [208]. Indeed, the combination of both IIF and antigen-specific assays was found in several studies to be the optimal strategy for ANCA detection and led to the recommendation of a two-stage ANCA testing.

Alike CytoBead® ANA development, after having designed a multiplex addressable MIA detecting MPO-ANCA, PR3-ANCA, and autoAbs against the noncollagen region of the alpha-3 subunit of collagen IV representing the glomerular basement membrane (GBM) antigen, a unique reaction environment for the additional detection of ANCA on fixed neutrophils was generated (Fig. 3b). The novel CytoBead® ANCA is a unique combination of a classical cell-based assay with multiplexing microbead technology [204, 208].

Sowa et al. recruited 592 patients including 118 patients with AAV, 133 with RA, 49 with infectious diseases, 77 with inflammatory bowel disease, 20 with autoimmune liver diseases, 70 with primary sclerosing cholangitis (PSC), and 125 blood donors and compared multiplex CytoBead® ANCA testing with classical methods such as IIF and ELISA [208]. Quantitative PR3- and MPO-ANCA analysis by multiplex CytoBead® technology turned out to be at least equal or better compared to classical ELISA testing for specific ANCA. Remarkably, automated endpoint ANCA titer analysis by only one serum dilution employing the automated interpretation system AKLIDES® revealed a very good agreement with the classical ANCA IIF on neutrophils. Another intriguing finding was the detection of PR3-ANCA in patients suffering from ulcerative colitis (UC) and PSC apart from those with GPA. These data appear to confirm a recent report of PR3-ANCA positive patients suffering from UC and PSC detected by another sensitive MIA technique [138]. Thus, the new reaction environment of the CytoBead® ANCA enables highly sensitive PR3-ANCA testing and might compete with third-generation ELISA in terms of assay performance.

Consequently, automated multiplex IIF combining screening and confirmatory ANCA testing in one test may replace the time-consuming current two-stage ANCA testing strategy by a one-step multiplexing CytoBead® analysis [206]. In context of the emergency diagnostics required for rapidly progressive glomerulonephritis, the novel multiplex ANCA analysis by CytoBead® appears to be an attractive approach to meet the clinical need for comprehensive ANCA testing in the fastest way possible.

Comprehensive CD Serology

The serological diagnosis of CD comprises the detection of EMA and auto/Abs against deamidated gliadin and TG2 of the IgA isotype. As a fact, EmA detected by IIF is still considered the gold standard for (auto)Ab testing in CD [65]. To address the need for comprehensive CD-specific (auto)Ab testing in terms of workload and cost reduction in routine autoimmune laboratories, we developed a multiplex CytoBead® CeliAK assay (Fig. 3c) [209]. Multiplex CD-specific (auto)Ab testing might even be an attractive diagnostic tool in the context of the novel diagnostic criteria published by the European Society for Paediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) recently [65]. These criteria obviously strengthen the role of CD serology within the workup of patients with the suspicion of CD. Thus, CD can be diagnosed without histology by waiving duodenal biopsy in case of anti-TG2 autoAb IgA levels 10 times higher than the upper limit of normal (ULN) in patients positive for HLA-DQ2 or HLA-DQ8 and a positive response to gluten-free diet or confirmation by EmA testing.

Hence, the novel CytoBead® CeliAK was evaluated by investigating in total 380 patients and controls comprising 155 CD patients, 5 with IgA-deficiency, 68 with cystic fibrosis, 59 with eye diseases, and 93 blood donors [209]. Findings were compared with classical IgA-(auto)Ab analyses by ELISA and IIF. As a fact, the difference between CytoBead® and classical testing was only significant for anti-TG2 autoAb testing whereas the eight discrepant sera with anti-TG2 autoAb positivity by ELISA and negative levels by CytoBead® CeliAK belonged to four CD patients and four controls. Altogether, the CytoBead® CeliAK represents the first multiplex quantitative IgA anti-TG2 autoAb and anti-DG Ab multiplex assay which provides simultaneous EmA analysis as reference method and IgA deficiency testing. This comprehensive approach has the potential to improve CD serology and demonstrated excellent results regarding the great number of CD patients with anti-TG2 autoAb levels >10× ULN due to its high sensitivity. Additionally, due to the flexibility of the technique, further autoAbs such as those to GP2 stratifying CD patients further might be included [210, 211].

Conclusion

Hitherto, the history of autoAb testing has been characterized by an intriguing development of several assay techniques to keep up with the tremendous progress in the understanding of autoimmune diseases and their appropriate diagnostics [180, 197]. Today, autoAb analysis is an integral part in the serological diagnosis of SARD like CTD and AAV and organ-specific autoimmune disorders [4, 26, 60]. Hence, there is no doubt that the introduction and further evolvement of IIF as one of the first autoAb-detecting assay techniques had and have an essential impact on this process [162, 197]. In the history of autoAb testing, various techniques emerged and were replaced by newer ones providing better assay performance and benefits regarding higher sample throughput and standardization [34, 80]. In this context, it is astonishing to note that IIF is still one of the key techniques to analyze autoAbs and even recommended as screening assay within the two-stage strategy for ANA and ANCA testing. In addition, IIF remains a reference method for the detection of distinct autoAbs like EmA in the serology of organ-specific autoimmune disorders indeed [65].

Despite the obvious benefits of IIF, this assay technique has been characterized by time consuming and subjective evaluation, insufficient automation, as well as poor standardization since its introduction [162]. In particular, pattern reading for ANA and ANCA testing was prone to inconsistencies in description and classification of respective staining patterns.

As a consequence, novel assay techniques based on solid-phase immunoassays like ELISA or multiplexing technologies creating the basis for different commercial platforms evolved and were introduced into routine autoimmune laboratories [149]. Nonetheless, IIF is still recommended to be used as the gold standard method for instance for ANA testing due to the unsatisfactory assay performance of even the latest multiplex technologies in this important area of autoAb analysis [4].

This situation changed dramatically by the development of digital fluorescence and its implementation in IIF testing. The breathtaking new options of pattern recognition combined with progress in automated fluorescence microscopy paved the way for the evolvement of an entirely new generation of automated interpretation systems [206]. Different commercially available IIF platforms for autoAb testing were designed and applied for ANA and ANCA reading in particular. First evaluation studies support the good performance of these systems and high agreement between visual and automated autoAb interpretation [212].

Of note, this enormous technology development comprising digital fluorescence image acquisition and automatic pattern recognition could be extended to other cell-based IIF assays in the search for new biomarkers. Thus, the quantification of γH2AX foci for DNA damage analysis, which used to be time consuming, subjective, and not suitable for high-throughput screening, could be standardized and automated [213, 214]. Successful evaluation studies support the introduction of this new DNA damage marker into clinical routine for cytostatic resistance development diagnostics [215].

Nevertheless, since the majority of clinical immunology laboratories follow the two-stage strategy for ANA and ANCA testing, substantial constraints regarding high-throughput and cost-effectiveness remain. The expansion of automated IIF interpretation systems like AKLIDES® to assess addressable MIAs created a unique novel assay platform allowing fully automated evaluation of cell-based screening tests and antigen-specific multiplex assays in one reaction environment for the first time. The evolvement of the CytoBead® technology combining quantitative autoAb screening and confirmatory testing in one IIF analysis enables second-generation autoAb detection in one test. This intriguing multiplex reaction environment addresses key needs for an effective standardized autoAb testing in laboratory routine. Major disadvantages of classical autoAb analysis by IIF were overcome by this new technique. First diagnostic applications for second-generation ANA and ANCA testing as well as comprehensive serology of CD-specific (auto)Abs were developed and successfully evaluated.