Review Article
Perspective: Widening Spectrum and Gaps in Autoantibody Testing for Systemic Autoimmune Diseases
Marvin J Fritzler*
Corresponding Author: Dr. Marvin Fritzler, Cumming School of Medicine, University of Calgary, 3330 Hospital Dr. NW, Calgary, AB, Canada T3H 1H7
Received: September 24, 2018; Revised: January 13, 2019; Accepted: October 25, 2018
Citation: Fritzler M. (2019) Perspective: Widening Spectrum and Gaps in Autoantibody Testing for Systemic Autoimmune Diseases. J Rheumatol Res, 1(1): 10-18.
Copyrights: ©2019 Fritzler M. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Share :
  • 448

    Views & Citations

Over half a century has elapsed since the introduction of ANA and autoantibody testing to confirm the diagnosis of and screen for systemic autoimmune diseases (SAID). Despite this long history, there are several gaps in the understanding, utilization and interpretation of the test results. One reason for persisting gaps is the proliferation of novel autoantibody targets described along with a widening spectrum of SAID and clinicians that use these tests. The gaps include standardization of ANA test protocols and the availability of appropriate controls. There are also persisting nomenclature gaps. A significant proportion of SAIDs are seronegative, a gap that is being closed by ongoing research and discovery.

 

Keywords: Autoantibodies, Anti-nuclear antibodies, Diagnostic assays, Systemic autoimmune diseases

 

Abbreviations: ALBIA: Addressable Laser Bead Immunoassay; ALD: Autoimmune Liver Diseases; AE: Autoimmune Encephalitis; ANA: Anti-Nuclear Antibody; ANCA: Anti-Neutrophil Cytoplasmic Antigen; APS: Anti-Phospholipid Syndrome; AQP4: Aquaporin 4; IIF: Indirect Immunofluorescence; CENP: Centromere Protein; DID: Double Immunodiffusion; DFS: Dense Fine Speckled; DNA: Deoxyribonucleic Acid; ELISA: Enzyme Linked Immunoassay; GN: Glomerulonephritis; GP1: Glycoprotein 1; IIM: Idiopathic Inflammatory Myopathies; ILD: Interstitial Lung Disease; Jo-1: Histidyl tRNA Synthetase; LIA: Line Immunoassay: LE: Lupus Erythematosus; MAA: Multi-Analyte Array; MAAAA: Multi-Analyte Array with Algorithmic Analysis; MCTD: Mixed Connective Tissue Disease; NLE: Neonatal Lupus Erythematosus; NMDAR1: N-Methyl-D-Aspartate Receptor 1; NPLE: Neuropsychiatric Lupus Erythematosus; OS: Overlap Syndrome; PCNA: Proliferating Cell Nuclear Antigen; PLA2R: Phospholipase A2 Receptor; PM/Scl: Polymyositis/Scleroderma Antigen; Rib-P: Ribosomal P Protein; RNAP: RNA Polymerase; RNP: Ribonucleoprotein; SACLE: Subacute Cutaneous Lupus; SAID: Systemic Autoimmune Diseases; Sm: Smith Autoantigen; SjS: Sjögren’s Syndrome; SLE: Systemic Lupus Erythematosus; SNP: Soluble Nucleoprotein; SPA: Solid Phase Assays; SSA: Sjögren’s Syndrome Antigen A; SSc: Systemic Sclerosis; Th/To: Mitochondrial RNA Processing Complex, Topoisomerase I (Scl-70); TRIM: Tri-Partite Motif; UCTD: Undifferentiated Connective Tissue Disease

INTRODUCTION

I recently attended a medical meeting focused on a systemic autoimmune disease (SAID) and as the meeting considered metanalyses, classification criteria, odds ratios, confidence intervals, dimethyl ‘chicken-wire’ and ‘kappaphredon’ levels, it became clear that despite more than a half century of study and experience, gaps in autoantibody testing, especially the anti-nuclear antibody (ANA) test and its clinical value, are not largely resolved (Table 1). Some of these gaps persist despite knowledge available to fill them, while other gaps require additional effort and international collaboration. The perspectives in this article focus on the drivers of the gaps, the gaps themselves and where possible, resolutions to the gaps.

Widening spectrum: ‘Clientele’, autoantibody: Discoveries and technologies

Arguably, the major change in autoantibody testing over the past half century has not been the continuous discovery of novel autoantibodies as biomarkers for various SAID or steady advances in diagnostic technologies, but the broadening spectrum of clinicians and health care providers that use and rely on autoantibody testing in their practices (Figure 1) [1]. Dating to the discovery of the LE cell and the development of the LE cell test [2] and then the indirect immunofluorescence (IIF) assay using cryopreserved rodent tissue sections [3], the initial autoantibody testing ‘clientele’ was largely restricted to rheumatologists and immunologists who were attending to patients with various stages of systemic lupus erythematous (SLE). The onset of the era of cell and molecular biology in the mid-1960s became an inflection point for a widening spectrum of autoantibodies associated with SAIDs, such as anti-Sm for SLE [4] and anti-U1-ribonucleoprotein (RNP) for mixed connective tissue disease (MCTD) [5]. By the mid-1970s, the next major inflection came when the advantages of tissue culture cells, such as HEp-2, became obvious and they were very rapidly adopted by the diagnostic industry as an alternative to rodent cryopreserved sections for the ANA test [6]. Prior to that, IIF on rodent tissue sections was considered an “insensitive” screening test for systemic sclerosis (SSc), Sjögren’s syndrome (SjS), autoimmune inflammatory myopathies (AIM) and other SARDs because, other than nuclear speckled and nucleolar patterns observed in IIF tests of SSc sera, more common autoantibodies, such as anti-centromere, anti-RNA polymerase, anti-Ro60/SSA and -La/SSB that came to light with the introduction of HEp-2 substrates, were typically not seen (Table 2). These inflection points marked the beginning of the ‘golden age of autoantibody discovery’ and along with that, the spectrum of clinicians that utilize the ANA and related autoantibody tests has expanded to include primary care providers and virtually every subspecialty in medicine including, most recently, pulmonary medicine and psychiatry (Figure 1). Hence, SAIDs and their associated biomarkers have gained prominence in virtually all branches of medicine, a spectrum that will likely continue to widen. In addition, with this widening spectrum of autoantibodies and clinician ‘clientele’, the ANA and other autoantibody tests became known as screening tests rather than ‘confirmatory diagnostic tests’ with the result being decreasing pre-test probability (Figure 1) and concerns about inappropriate use of ANA testing [7].

The broadening spectrum of SAID is intertwined with newer high-throughput solid phase assays (SPA) such as enzyme linked immunoassays (ELISA), addressable laser bead immunoassays (ALBIA) and line immunoassays (LIA) developed as substitutes for the ANA IIF [8]. Because of perceived unsatisfactory performance characteristics (i.e., lack of sensitivity) of some SPAs, the ANA IIF test was declared the ‘gold standard’ screening test in 2010 [9,10]. In that declaration it was not clear that the wide spectrum of SAIDs was taken into consideration. For example, it is well-known that the IIF HEp2 test has limited (i.e., <60%) sensitivity for SjS, AIM, anti-phospholipid syndrome (APS) and the broader spectrum of SAIDs (Table 2). Notable exceptions where the HEp-2 assay has higher (>80%) sensitivity or other clinical utility include SLE, SSc, autoimmune liver diseases and uveitis associated with juvenile idiopathic arthritis [11,12].

One of the claims that HEp-2 should be the ‘gold standard’ was based on the notion that the HEp-2 cell represented a “mini-array” of >100 target autoantigens [10]. However, this claim failed to recognize that while this is theoretically correct, in practice many well know targets (e.g. Ro52/TRIM21, Ro60/SSA, ribosomal P proteins, Jo-1, to name a few) do not give consistent IIF patterns on HEp-2 substrates [7,8]. This gap between theory and practice is reminiscent of an adage attributed to Yogi Berra, “In theory there is no difference between practice and theory; in practice there is” [13]. Hence, the notion that HEp-2 cells are a multi-analyte array (MAA) and that IIF is the preferred test platform became engrained in clinician’s thinking and even prompted some autoantibody test kit manufacturers to roll back newer diagnostic platforms and ramp up production and marketing of HEp-2 IIF kits. This had an overall beneficial effect because it challenged the diagnostic industry to close the gap between high-throughput SPA ANA screening and HEp-2 IIF. Some studies now report that SPA ANA screening assays are equivalent or superior to the HEp-2 IIF test and are also a cost-effective alternative [14-16]. Last, the ‘gold standard’ proclamation marked the advent of automated ANA IIF technologies, which further closed technical and subjective interpretation gaps in ANA IIF testing [1,17].

Another factor widening the spectrum of ANA and autoantibody testing is a concerted move to preventive medicine and precision health (PH). Until recently, it has been assumed that the primary use of ANA and autoantibody testing is to support the diagnosis of a SAID with ‘intent to treat’ [7] and as criteria for entry into clinical trials [18,19]. However, an emerging evidence-based approach to very early SAID identification and ‘case finding’ is focusing on ‘intent to prevent’ morbidity and mortality associated with SAIDs [7,20,21]. In very early SAIDs, signs and symptoms do not always point to a single ‘high pretest probability’ disease, necessitating a paradigm shift in diagnostics where the focus is on testing individuals based on evidence-based risk factors and the earliest signs and symptoms (lower pre-test probability) of SAIDs. Real-time MAA data on patients starting at the earliest onset of disease has the potential to guide further investigations (biopsy, imaging, etc.), referrals to appropriate specialists, and serve as a guide to treatment, predicting disease flares and confirming remissions. One of the anticipated benefits of an earlier and accurate diagnosis is decreasing health care expenditures [7,21]. 

Standardization gaps

A significant limitation of ANA IIF testing is the tremendous gap in standardizing the test; an issue that persists almost half a century after the adoption of this assay. Despite numerous studies and analyses, there is no universally accepted screening serum dilution (for adults or children) [22], different manufacturers use different secondary antibodies, the cells are grown and fixed with differing protocols, the assay is semi-quantitative at best and the interpretation of various IIF patterns is highly observer dependent [19,23,24]. Part of the challenge is that despite attempts at protocol standardization and regulatory protocols (i.e., 510K approval by the Food and Drug Administration USA), diagnostic laboratories often do “what is right in their own eyes” [25,26]. Thankfully, as mentioned above, advances are being made in the performance of the IIF test through automated robotics and digital image analytics accompanied by progress in standardization of the nomenclature of ANA IIF patterns [12,27]. The lingering, and seemingly unresolvable, limitations of IIF testing on HEp-2 cells portend a continuing replacement of this test with SPA and MAA that can outperform it. Indeed, akin to the proposed inverted ‘pyramid’ of reflex testing in the ANCA testing [28], there is a sense that ANA screening should follow suit and broad-spectrum screening tests be replaced by Multi-Analyte Arrays with Algorithmic Analyses (MAAAAs) [29]. The MAA component of technology platforms are well developed and increasingly available but a gap to be closed is the last AA (algorithmic analyses) using artificial intelligence to link big data to clinical care pathways [30,31]. 

Assay performance and ‘seronegative’ gaps

There should be no assumptions that a move to newer, high-throughput technologies is nirvana [32]. Indeed, some old challenges will persist and new challenges will arise. Inter-manufacturer and inter-laboratory variability will continue to be a challenge, although standardization appears to be more easily attainable because purified components providing quantitative results are typically used in newer MAA platforms. Hence, a goal of standardized performance could be based on international reference standard sera and the assignment of results in ‘international units” [33,34]. This means that for every antigen in a MAA, an internal reference standard should be required, an important technical gap that needs to be and can be addressed.

Despite the discovery and description of numerous autoantibody targets, a seronegative gap persists for many SAIDs (Table 2). It seems ironic that after more than 50 years of research that a serological gap persists despite, for example, more than 180 targets of autoantibodies being described in SLE [3,5], only a handful are used in diagnostic assays (Table 2). Akin to other discoveries, many targets perish in the innovation “valley of death” [36,37]. The reasons for this are not well studied for autoantibodies but the selection of certain autoantibody targets and the rejection of others is dependent on their ability to be independently validated and meet SMAARTT criteria: Specificity for disease balanced by acceptable Sensitivity, are they Measurable in conventional diagnostic platforms, are they Actionable or associated with a clear clinical Advantage or outcome (i.e., predictive, prognostic), are they Realistic, Timely and Titratable [38]. Therefore, as SAID-specific MAAAAs are developed, it will also be important to continue to fill the “seronegative” gap in these conditions. Fortunately, the search for novel SARD autoantibody targets that close the seronegative gap is a productive academic and industry enterprise with novel candidates continuously reported.  In addition, rescuing targets that have perished in ‘death valley’ and their incorporation into new MAAAAs may be a rewarding data mining exercise.

Nomenclature gaps

It is well known that many of the autoantibody targets in SAID are not restricted to the nucleus [12]. Hence, the term ANA is technically inaccurate and misleading because many of the SAID autoantibody targets are in the cytoplasm and/or directed to mitotic cells [39,40]. However, proposals to change the terminology from ANA to anti-cellular antibodies (ACA) have been met with resistance [12].

Another concern is persisting misunderstanding and lack of attention to well-defined autoantibody systems in SAID. For example, current literature is replete with misnomers such as confusing the SSA/Ro60 antigen system with the Ro52/TRIM21 system. This in part has been fostered by diagnostic companies who persist in combining these two antigens into a single assay. Although once thought to be part of the same subcellular macromolecule and linked to the diagnosis of SjS [41], the molecular evidence and clinical correlations no longer support either claim [42]. Indeed, anti-SSA/Ro60 and SSB/La are key autoantibody biomarkers for SjS, but anti-SSA/Ro60 is aslo seen in a broad spectrum of SAID and related conditions [42], such as subacute cutaneous and neonatal lupus [43-46]. And, while anti-SSA/Ro60 can co-exist with anti-Ro52/TRIM21, the latter is seen in an even wider spectrum of SAID [47], is the second most common antibody detected in SSc sera [48] and in IIM is particularly common in the anti-Jo-1 subset of anti-synthetase syndrome [49]. These and a number of other compelling reasons should bring to a close the notion that somehow SSA/Ro60 and Ro52/TRIM21 can be tested together or that they fit together into a clear-cut clinical paradigm.

Classification criteria closing gaps

Curiously, despite the limitations of the ANA IIF test, it continues to be a key criterion in the classification of some of SAIDs, especially SLE. The most recently revised SLE classification criteria supported by the American College of Rheumatology (ACR) and the European League Against Rheumatism (EULAR) uses the ANA IIF as a required entry criterion [50,51], apparently taking into consideration that the lack of specificity of the ANA test for SLE is counter-balanced by the other weighted clinical and serological findings. Two issues regarding the new ACR/EULAR criteria should be noted. First, analysis of the comparative performance of the three prevalent SLE classification criteria, the Revised ACR Criteria [52] and the SLICC Criteria [53], suggests that the newer EULAR/ACR criteria are a step in the right direction (Table 3) [51]. Second, with respect to the ANA HEp-2 IIF requirement, “an equivalent” assay is a permissible option. This criterion may create misunderstanding because by not defining the characteristics of an “equivalent” test, it has no technical comparator definition except the assumption that this equivalence will relate to sensitivity and specificity of the ANA at a titer of ≥ 1/80. This may be a moot point because, as discussed above, there is an apparent progressive move to high-throughput ANA testing by MAA that is not only equivalent to but exceeds the performance of the ANA IIF test. 

CONCLUSION

Gaps in autoantibody testing, especially the anti-nuclear antibody (ANA) IIF test are largely driven by the clinical approaches that focus on screening for SAIDs in patients with low pre-test probability, a wide spectrum of clinicians who order the tests and continuous evolution of diagnostic test platforms. Some gaps, such as nomenclature persist despite knowledge available to fill them, while other gaps require additional effort and international collaboration.

ACKNOWLEDGEMENT

No funding was received for this manuscript. The author acknowledges the advice and input of Dr. Michael Mahler and Ms. Patricia Swartwood (Inova Diagnostics, San Diego, CA USA).

Some images in Figure 1 were adapted from those purchased from Shutterstock.com: Account 192281638. 

1.       Mahler M, Meroni PL, Bossuyt X, Fritzler MJ (2014) Current concepts and future directions for the assessment of autoantibodies to cellular antigens referred to as anti-nuclear antibodies. J Immunol Res 2014: 315179.

2.       Pisetsky DS (2012) The LE cell: Crime scene or crime stopper? Arthritis Res Ther 14: 120.

3.       Nakamura RM, Peebles CL, Molden DP, Tan EM (1984) Advances in laboratory tests for autoantibodies to nuclear antigens in systemic rheumatic diseases. Lab Med 15: 190-198.

4.       Tan EM, Kunkel HG (1966) Characteristics of a soluble nuclear antigen precipitating with sera of patients with systemic lupus erythematosus. J Immunol 96: 464-471.

5.       Sharp GC, Irvin W, Tan EM, Gould G, Holman HR, et al. (1972) Mixed connective tissue disease - An apparently distinct rheumatic disease syndrome associated with a specific antibody to an extractable nuclear antigen (ENA). Am J Med 52: 148-159.

6.       Nakamura RM, Tan EM (1977) Recent progress in the study of autoantibodies to nuclear antigens. Hum Pathol 9: 85-91.

7.       Fritzler MJ (2016) Choosing wisely: Review and commentary on anti-nuclear antibody (ANA) testing. Autoimmun Rev 15: 272-280.

8.       Olsen NJ, Choi MY, Fritzler MJ (2017) Emerging technologies in autoantibody testing for rheumatic diseases. Arthritis Res Ther 19: 172.

9.       Meroni PL, Schur PH (2010) ANA screening: An old test with new recommendations. Ann Rheum Dis 69: 1420-1422.

10.    American College of Rheumatology (2011) Position Statement, methodology of testing for antinuclear antibodies. Internet Communication: https://www.rheumatology.org/Portals/0/Files/Methodology%20of%20Testing%20Antinuclear%20Antibodies%20Position%20Statement.pdf

11.    Damoiseaux J, Andrade LE, Fritzler MJ, Shoenfeld Y (2015) Autoantibodies 2015: From diagnostic biomarkers toward prediction, prognosis and prevention. Autoimmun Rev 14: 555-563.

12.    Damoiseaux J, von Muhlen CA, Garcia-de la Torre I, Carballo OG, de Melo CW, et al. (2016) International consensus on ANA patterns (ICAP): The bumpy road towards a consensus on reporting ANA results. Auto Immun Highlights 7: 1.

13.    https://www.snopes.com/fact-check/practice-and-theory/

14.    Perez D, Gilburd B, Azoulay D, Shovman O, Bizzaro N, et al. (2018) Anti-nuclear antibodies: Is the indirect immunofluorescence still the gold standard or should be replaced by solid phase assays? Autoimmun Rev 17: 548-552.

15.    Bizzaro N, Brusca I, Previtali G, Alessio MG, Daves M, et al. (2018) The association of solid-phase assays to immunofluorescence increases the diagnostic accuracy for ANA screening in patients with autoimmune rheumatic diseases. Autoimmun Rev 17: 541-547.

16.    Meroni PL, Borghi MO (2018) Diagnostic laboratory tests for systemic autoimmune rheumatic diseases: Unmet needs towards harmonization. Clin Chem Lab Med.

17.    Claessens J, Belmondo T, De LE, Westhovens R, Poesen K, et al. (2018) Solid phase assays versus automated indirect immunofluorescence for detection of antinuclear antibodies. Autoimmun Rev 17: 533-540.

18.    Pisetsky DS, Lipsky PE (2018) The role of ANA determinations in classification criteria for SLE. Arthritis Care Res (Hoboken).

19.    Pisetsky DS (2017) Antinuclear antibody testing - Misunderstood or misbegotten? Nat Rev Rheumatol 13: 495-502.

20.    Choi MY, Fritzler MJ (2016) Progress in understanding the diagnostic and pathogenic role of autoantibodies associated with systemic sclerosis. Curr Opin Rheumatol 28: 589-594.

21.    Choi MY, Barber MR, Barber CE, Clarke AE, Fritzler MJ, et al. (2016) Preventing the development of SLE: Identifying risk factors and proposing pathways for clinical care. Lupus 25: 838-849.

22.    Mahler M, Fritzler MJ (2014) Antinuclear antibodies in children. J Rheumatol 41: 1260-1262.

23.    Pisetsky DS, Spencer DM, Lipsky PE, Rovin BH (2018) Assay variation in the detection of antinuclear antibodies in the sera of patients with established SLE. Ann Rheum Dis 77: 911-913.

24.    Meroni PL, Chan EK, Damoiseaux J, Andrade LEC, Bossuyt X, et al. (2018) Unending story of the indirect immunofluorescence assay on HEp-2 cells: Old problems and new solutions? Ann Rheum Dis.

25.    Fritzler MJ, Wiik A, Tan EM, Smolen JS, McDougal JS, et al. (2003) A critical evaluation of enzyme immunoassay kits for detection of antinuclear antibodies of defined specificities. III. Comparative performance characteristics of academic and manufacturers' laboratories. J Rheumatol 30: 2374-2381.

26.    Fritzler MJ, Wiik A, Fritzler ML, Barr SG (2003) The use and abuse of commercial kits used to detect autoantibodies. Arthritis Res Ther 5: 192-201.

27.    Chan EK, Damoiseaux J, de Melo CW, Carballo OG, Conrad K, et al. (2016) Report on the Second International Consensus on ANA Pattern (ICAP) workshop in Dresden 2015. Lupus 25: 797-804.

28.    Syed RH, Gilliam BE, Moore TL (2008) Rheumatoid factors and anticyclic citrullinated peptide antibodies in pediatric rheumatology. Curr Rheumatol Rep 10: 156-163.

29.    Colon-Franco JM, Bossuyt PMM, Algeciras-Schimnich A, Bird C, Engstrom-Melnyk J, et al. (2018) Current and emerging multianalyte assays with algorithmic analyses-are laboratories ready for clinical adoption? Clin Chem 64: 885-891.

30.    Frohlich H, Balling R, Beerenwinkel N, Kohlbacher O, Kumar S, et al. (2018) From hype to reality: Data science enabling personalized medicine. BMC Med 16: 150-1122.

31.    Prodan Z, I, Cerne D, Mancini I, Simi L, Pazzagli M, et al. (2018) Personalized laboratory medicine: A patient-centered future approach. Clin Chem Lab Med.

32.    Spellerberg M (2017) Tests for autoimmunity: A luddite analysis. Pathology 49: 565-567.

33.    Meroni PL, Biggioggero M, Pierangeli SS, Sheldon J, Zegers I, et al. (2014) Standardization of autoantibody testing: A paradigm for serology in rheumatic diseases. Nat Rev Rheumatol 10: 35-43.

34.    Sheldon J, Dellavance A (2015) Strategies for building reference standards for autoantibodies. Front Immunol 6: 194.

35.    Yaniv G, Twig G, Shor DB, Furer A, Sherer Y, et al. (2014) A volcanic explosion of autoantibodies in systemic lupus erythematosus: A diversity of 180 different antibodies found in SLE patients. Autoimmun Rev 14: 75-79.

36.    Gamo NJ, Birknow MR, Sullivan D, Kondo MA, Horiuchi Y, et al. (2017) Valley of death: A proposal to build a “translational bridge” for the next generation. Neurosci Res 115: 1-4.

37.    Hudson J, Khazragui HF (2013) Into the valley of death: Research to innovation. Drug Discov Today 18: 610-613.

38.    Fritzler MJ, Mahler M (2018) Redefining systemic lupus erythematosus — SMAARTT proteomics. Nat Rev Rheumatol 14: 451-452.

39.    Stinton LM, Eystathioy T, Selak S, Chan EKL, Fritzler MJ, et al. (2004) Autoantibodies to protein transport and messenger RNA processing pathways: Endosomes, lysosomes, Golgi complex, proteasomes, assemblyosomes, exosomes and GW Bodies. Clin Immunol 110: 30-44.

40.    Choi MY, Clarke AE, St PY, Hanly JG, Urowitz MB, et al. (2018) Antinuclear antibody-negative systemic lupus erythematosus in an international inception cohort. Arthritis Care Res (Hoboken).

41.    Ben-Chetrit E, Chan EKL, Sullivan KF, Tan EM (1988) A 52 kD protein is a novel component of the SS-A/Ro antigenic particle. J Exp Med 167: 1560-1571.

42.    Schulte-Pelkum J, Fritzler M, Mahler M (2009) Latest update on the Ro/SS-A autoantibody system. Autoimmun Rev 8: 632-637.

43.    Siegel DM, Deng JS, Sontheimer RD (1985) Ro/SS-A antibody-associated cutaneous lupus erythematosus: Neonatal and subacute cutaneous lupus. Semin Dermatol 4: 69-78.

44.    Allaire SH, Prashker MJ, Meenan RF (1994) The costs of rheumatoid arthritis. Pharmacoeconomics 6: 513-522.

45.    Mondini M, Vidali M, Andrea MD, Azzimonti B, Airo P, et al. (2006) A novel autoantigen to differentiate limited cutaneous systemic sclerosis from diffuse cutaneous systemic sclerosis: The interferon-inducible gene IFI16. Arthritis Rheum 54: 3939-3944.

46.    Gordon P, Rosenthal E, Simpson JM, Sharland G, Brucato A, et al. (2004) Anti-52 kDa Ro, anti-60 kDa Ro and anti-La antibody profiles in neonatal lupus. J Rheumatol 31: 2480-2487.

47.    Dugar M, Cox S, Limaye V, Gordon TP, Roberts-Thomson PJ, et al. (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity. Postgrad Med J 86: 79-82.

48.    Hudson M, Pope J, Mahler M, Tatibouet S, Steele R, et al. (2012) Clinical significance of antibodies to Ro52/TRIM21 in systemic sclerosis. Arthritis Res Ther 14: R50.

49.    Mahler M, Miller FW, Fritzler MJ (2014) Idiopathic inflammatory myopathies and the anti-synthetase syndrome: A comprehensive review. Autoimmun Rev 13: 367-371.

50.    Tedeschi SK, Johnson SR, Boumpas D, Daikh D, Dorner T, et al. (2017) Developing and refining new candidate criteria for SLE classification: An international collaboration. Arthritis Care Res (Hoboken) 70: 571-581.

51.    Aringer M, Costenbader KH, Brinks R, Boumpas D, Daikh, et al. (2018) Vlaidation of new systemic lupus erythematosus criteria. Ann Rheum Dis 77: A260.

52.    Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum 40: 1725.

53.    Petri M, Orbai AM, Alarcon GS, Gordon C, Merrill JT, et al. (2012) Derivation and validation of systemic lupus international collaborating clinics classification criteria for systemic lupus erythematosus. Arthritis Rheum 64: 2677-2686.

54.    Conrad K, Schlosser U, Hiepe F, Fritzler MJ (2015) Autoantibodies in systemic autoimmune diseases: A diagnostic reference. Berlin, Pabst Scientific Publishers.

55.    Mehra S, Walker J, Patterson K, Fritzler MJ (2013) Autoantibodies in systemic sclerosis. Autoimmun Rev 12: 350-354.

56.    Mecoli CA, Casciola-Rosen L (2018) An update on autoantibodies in scleroderma. Curr Opin Rheumatol 30: 548-553.

57.    Tzioufas AG, Tatouli IP, Moutsopoulos HM (2012) Autoantibodies in Sjogren's syndrome: Clinical presentation and regulatory mechanisms. Presse Med 41: e451-e460.

58.    Pablos JL, Everett ET, Norris JS (2004) The tight skin mouse: An animal model of systemic sclerosis. Clin Exp Rheumatol 22: S81-S85.

59.    Casciola-Rosen L, Mammen AL (2012) Myositis autoantibodies. Curr Opin Rheumatol 24: 602-608.

60.    Mammen AL (2016) Autoimmune myopathies. Continuum (Minneap Minn) 22: 1852-1870.

61.    Paik JJ, Mammen AL, Wigley FM, Gelber AC (2014) Myopathy in scleroderma, its identification, prevalence and treatment: Lessons learned from cohort studies. Curr Opin Rheumatol 26: 124-130.

62.    Pericleous C, Ferreira I, Borghi O, Pregnolato F, McDonnell T, et al. (2016) Measuring IgA anti-beta2-glycoprotein I and IgG/IgA anti-domain I antibodies adds value to current serological assays for the anti-phospholipid syndrome. PLoS One 11: e0156407.

63.    Zohoury N, Bertolaccini ML, Rodriguez-Garcia JL, Shums Z, Ateka-Barrutia O, et al. (2017) Closing the serological gap in the antiphospholipid syndrome: The value of “non-criteria” antiphospholipid antibodies. J Rheumatol 44: 1597-1602.

64.    Alarcon-Segovia D, Cardiel MH (1989) Comparison between 3 diagnostic criteria for mixed connective tissue disease. Study of 593 patients. J Rheumatol 16: 328-334.

65.    Amigues JM, Cantagrel A, Abbal M, Mazieres B (1996) Comparative study of 4 diagnosis criteria sets for mixed connective tissue disease in patients with anti-RNP antibodies. Autoimmunity Group of the Hospitals of Toulouse. J Rheumatol 23: 2055-2062.

66.    Trouw LA, Rispens T, Toes RE (2017) Beyond citrullination: Other post-translational protein modifications in rheumatoid arthritis. Nat Rev Rheumatol 13: 331-339.

67.    Martinez-Prat L, Lucia D, Ibarra C, Mahler M, Dervieux T, et al. (2018) Antibodies targeting protein-arginine deiminase 4 (PAD4) demonstrate diagnostic value in rheumatoid arthritis. Ann Rheum Dis.

68.    Verheul MK, Bohringer S, van Delft MAM, Jones JD, Rigby WFC, et al. (2018) The combination of three autoantibodies, ACPA, RF and anti-CarP antibodies is highly specific for rheumatoid arthritis: Implications for very early identification of individuals at risk to develop rheumatoid arthritis. Arthritis Rheumatol.

69.    Aringer M, Dorner T, Leuchten N, Johnson SR (2016) Toward new criteria for systemic lupus erythematosus - A standpoint. Lupus 25: 805-811.