Autoantibodies, detection methods and panels for diagnosis of Sjögren's syndrome

Accepted Manuscript Autoantibodies, detection methods and panels for diagnosis of Sjögren's syndrome

Long Shen, Lakshmanan Suresh PII: DOI: Reference:

S1521-6616(17)30010-4 doi: 10.1016/j.clim.2017.03.017 YCLIM 7828

To appear in:

Clinical Immunology

Received date: Accepted date:

4 January 2017 31 March 2017

Please cite this article as: Long Shen, Lakshmanan Suresh , Autoantibodies, detection methods and panels for diagnosis of Sjögren's syndrome. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Yclim(2017), doi: 10.1016/j.clim.2017.03.017

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ACCEPTED MANUSCRIPT Autoantibodies, Detection Methods and Panels for Diagnosis of Sjögren’s Syndrome

Long Shen1,3* and Lakshmanan Suresh2,3

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1. Department of Rheumatology and Clinical Immunology, The First Affiliated Hospital of Xiamen University, Xiamen University, Xiamen, 361003, China

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2. Department of Oral Diagnostic Sciences, School of Dental Medicine, University at Buffalo,

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Buffalo, NY 14214, USA

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3. Autoimmune Division, Trinity Biotech, 60 Pineview Drive, Buffalo, NY 14228, USA

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* Corresponding author.

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Keywords: Autoantibodies, Sjögren’s syndrome, Detection Methods,

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Abbreviations: ACA, anti-centromere antibody; AMA, anti-mitochondrial antibody; ANA, Antinuclear Antibodies; CA6, carbonic anhydrase 6; CCP, citrullinated cyclic peptide; PSP,

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parotid secretory protein; RF, rheumatoid factor; SP1, salivary gland protein 1; SS, Sjögren’s syndrome; pSS, Primary Sjögren’s syndrome; *This work was supported in part by NSFC (Natural Science Foundation of China) grant 81571585 to Dr. Long Shen

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ACCEPTED MANUSCRIPT Abstract: The presence of autoantibodies is one of several hallmarks of Sjögren’s Syndrome, the detection of serum autoantibodies has a central role in the diagnosis and classification of Sjögren’s syndrome. In this review, we will discuss autoantibodies that are helpful in the

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diagnosis of Sjögren’s syndrome. This includes the traditional autoantibodies for disease classification (ANA, Anti-Ro/SSA, Anti-La/SSB, RF), autoantibodies identified from mouse

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models (Anti-SP1, Anti- PSP, Anti-CA6, and anti-alpha fodrin) and autoantibodies associated

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with other autoimmune disease (ACA, AMA, and Anti-CCP). We will also review the methods for the detection of autoantibodies and associated challenges for clinical results reporting. The

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significance of using an autoantibody panel for the diagnosis of SS will be also be reviewed.

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ACCEPTED MANUSCRIPT 1. Introduction Sjögren’s syndrome (SS) is a chronic, systemic inflammatory disorder that mainly affects the exocrine glands with a typical focal lymphocytic infiltration potentially leading to dry

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mouth(xerostomia) and dry eyes (xerophthalmia)[1]. While sicca symptoms are the hallmarks

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of the syndrome, during the disease development, patients might experience various systemic

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clinical manifestations (i.e. fatigue, arthritis, skin vasculitis, hematological disorders, lung interstitial diseases, kidney failure, peripheral and central neuropathies and gastrointestinal

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tract disorders)[1, 2]. As a result, SS was considered a heterogeneous autoimmune disease

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possessing both organ-specific and systemic features and encompassing a wide spectrum of clinical/serological abnormalities and scattered complications [1, 2].

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Sjögren’s syndrome is one of the most common autoimmune disease in adults affecting

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up to 3.2 M cases in the United States[3]. Previous studies demonstrated that about 1 in 10

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patients with clinically significant dry eye have underlying SS[4]. However, SS is greatly under recognized in clinical practice, mostly due to diverse symptomatic expressions making the initial

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diagnosis difficult. It is estimated that the disease remains undiagnosed in more than half of

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affected adults[4, 5]. While currently there is no cure for SS, results from recent clinical studies with rituximab for patients with primary SS and severe systemic complications were promising[6, 7]. The studies showed rituximab can improve salivary gland function, diminish fatigue, and reduce the number of extra-glandular manifestations, especially when the treatment was initiated early in the disease course[6, 7]. This underlines the importance of early diagnosis to identify SS patients before irreversible damage to the affected organs and tissues occur. 3

ACCEPTED MANUSCRIPT In this review, we will discuss autoantibodies that are helpful in the diagnosis of Sjögren’s syndrome. This includes the traditional autoantibodies for disease classification (ANA, AntiRo/SSA, Anti-La/SSB, RF), autoantibodies identified from mouse models (Anti-SP1, Anti- PSP, Anti-CA6, and anti-alpha fodrin) and autoantibodies associated with other autoimmune disease

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(ACA, AMA, and Anti-CCP). We will also review the methods for the detection of autoantibodies and associated challenges for clinical results reporting. The significance of using an

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2. Autoantibodies in Sjögren’s Syndrome

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autoantibodies panel for the early diagnosis of SS will be also be evaluated.

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2.1 Traditional biomarkers for disease classification: Our knowledge of SS and autoimmune diseases has expanded greatly during the past

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half century and diagnosis and classification criteria for SS have continued to evolve. The

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presence of autoantibodies has always been considered as one of the criteria for the diagnosis

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of SS. Four autoantibodies: Anti-Ro/SSA, Anti-La/SSB, ANA and RF have been extensively studied for their prevalence and association with different clinical features of SS.

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Anti-Ro/SSA, Anti-La/SSB: The most common autoantibodies found in patients with SS are

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those directed against the autoantigens Ro/La ribonucleoprotein complex. Depending on the method used, anti-Ro/SSA and anti-La/SSB antibodies are found in approximately 50-70% of pSS patients[8]. Anti-Ro/SSA antibodies can be detected either solely or concomitantly with anti-La/SSB antibodies, whereas exclusive anti-La/SSB positivity is rare[9]. A recent study showed that the presence of anti-La/SSB, without anti-Ro/SSA antibodies, had no significant association with SS phenotypic features, relative to seronegative participants[10]. In general, anti-Ro/SSA and anti-La/SSB antibodies have been correlated with younger age at diagnosis, 4

ACCEPTED MANUSCRIPT longer disease duration, more severe dysfunction of the exocrine glands, recurrent parotid gland enlargement and higher intensity of the lymphocytic infiltrates invading the minor salivary glands[11, 12]. Studies also suggested a higher prevalence of extraglandular manifestations in SS patients positive for anti-Ro/La antibodies[11, 13], including splenomegaly,

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lymphadenopathy, vasculitis and Raynaud’s phenomenon. The anti-Ro/SSA and anti-La/SSB antibodies profile that is presented at the diagnosis of pSS seems to remain constant

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throughout the course of the disease[14], even after the administration of B-cell depletion

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therapy with rituximab[15].

Anti-nuclear antibodies (ANA): detected by indirect immunofluorescence on HEp-2 cells

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have been found positive in 59-85% of pSS patients[13, 16-18]. It has been shown that SS

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patients positive for ANA are more frequently female and have a lower mean age at diagnosis

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than males. They are also characterized by a higher prevalence of recurrent parotidomegaly and an increased frequency of extraglandular features, such as Raynaud phenomenon,

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cutaneous vasculitis, articular and renal involvement, fever, adenopathies, cytopenias and

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ESR >50 mm/h[13, 16, 18]. Furthermore, ANA in pSS have been associated not only with a greater number of involved organs, but also with a higher prevalence of

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hypergammaglobulinemia, positive RF, anti-Ro/SSA, anti-La/SSB and antiphospholipid antibodies[18]. In addition, it has been found that positive ANA titers correlate with the number of positive autoantibodies against specific nuclear antigens, as well as with the percentage and levels of serum gammaglobulins[16, 18]. Rheumatoid factors (RF): are antibodies directed against the Fc portion of IgG immunoglobulin. They can belong to any isotype, although they are most commonly IgM. RF 5

ACCEPTED MANUSCRIPT levels are found elevated in 36-74% of SS patients[16-18]. RF in SS has been associated with younger age, female predominance and positive salivary gland biopsy[18, 19]. Several studies further correlated RF in pSS to an active serological profile, characterized by markers such as anti-La/SSB, anti-Ro/SSA, cryoglobulins, ANA, hypocomplementemia and

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hypergammaglobulinemia[13, 17, 18, 20]. Both the presence and the concentration of RF have been positively correlated to the number of extraglandular manifestations found in SS[17, 18,

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20]. Compared to RF negative patients, RF positive pSS patients had an increased frequency of

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articular manifestations, cutaneous vasculitis, salivary gland enlargement, cytopenias, Raynaud phenomenon, renal involvement and central nerve system involvement[13, 18]. In pSS patients

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both IgM and IgA RF have been found elevated[17]. Specifically, for IgA RF, an association has

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been reported not only with renal disease and with the levels of other autoantibodies, but also

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with focus scores in minor salivary gland biopsies[21].

2.2 Autoantibodies identified from animal models of SS and confirmed in

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SS patients.

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Anti-salivary gland protein 1 (anti-SP1), anti-carbonic anhydrase 6 (anti-CA6), and anti-

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parotid secretory protein (anti-PSP) autoantibodies were first identified from interleukin 14 alpha transgenic mouse (IL14αTG), an animal model for SS that develops many of the clinical features of SS in the same relative time frame as patients: hypergammaglobulinemia, autoantibodies, loss of salivary gland function, infiltration of salivary and lachrymal glands with lymphocytes, lymphocytic interstitial pneumonia, mild renal disease and eventually lymphoma[22, 23]. These novel antibodies have been found in patients with SS both together and without anti-Ro/SSA and anti-La/SSB, as well as in patients with idiopathic dry mouth and 6

ACCEPTED MANUSCRIPT dry eye disease[23-25]. It was proposed that antibodies to SP1, PSP, and CA6 may be useful for identifying early SS, particularly among patients who are anti-Ro/SSA and anti-La/SSB negative. In a cohort analysis of 20 patients that met the 2002 ACR diagnostic criteria for SS (including positive salivary biopsy) but who were anti-Ro/SSA and anti-La/SSB negative, 45% were positive

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for anti-SP1 and 5% were positive for anti-CA6[23]. In a separate cohort of 29 patients considered to have early idiopathic xerostomia and xerophthalmia (symptoms for less than 2

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years), who met at least three criteria for SS 76% had anti-SP1 and anti- CA6 antibodies present

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compared with only 31% with anti-Ro/SSA and Anti-La/SSB anitbodies[26]. While promising, additional larger studies are needed to understand the role of these autoantibodies in humans

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and their possible role in the pathophysiology of SS

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Anti-alpha-fodrin antibodies were initially identified from a mouse model of SS (NFS/sld

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mutant mice thymectomized 3 days after birth (3d-Tx)) and later confirmed in the serum of human SS patients with a prevalence of 38-42%[27]. The antigen recognized by the anti-alpha-

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fodrin antibodies was a 120 kDa cleavage product of alpha-fodrin, which is formed during

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apoptosis in the inflamed salivary gland tissue and represents an organ-specific autoantigen that may be responsible for the development of autoimmune lesions and the perpetuation of

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tissue destruction[28]. Differential clinical and immunological characteristics were observed in primary SS patients with and without antibodies to alpha-fodrin. Research has shown a positive correlation of alpha-fodrin antibody serum concentrations and the degree of lymphocytic infiltration in salivary glands[29]. The notion that anti-alpha-fodrin antibodies may participate in early pathogenic processes is supported by finding that pSS patients with IgG-antibodies to alpha-fodrin had a shorter disease duration. In addition, antibodies in the sera of children with 7

ACCEPTED MANUSCRIPT primary SS or secondary SS were observed even before anti-Ro/SSA or anti-La/SSB antibodies became positive[30].

2.3 Autoantibodies associated with other autoimmune diseases Anti-centromere antibodies (ACAs) are commonly found among patients with limited

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cutaneous scleroderma. The prevalence of ACA in primary Sjögren’s syndrome ranges from 4%

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to 27.0% when detected by indirect immunofluorescence (IIF) [31-33]. Immunoblotting of the nuclear extracts revealed the 3 major polypeptide antigens recognized by ACA, designated

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CENP-A, CENP-B, and CENP-C. The pattern of CENP recognition differs markedly in primary SS

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and limited scleroderma. Patients with primary SS predominantly recognize CENP-C alone, whereas dual recognition of CENP-B and CENP-C is most frequently seen in scleroderma[34].

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Studies comparing primary SS patients positive for to those who are negative for ACA found

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that that positive ACA patients had a higher mean age at disease onset and a greater frequency of Raynaud’s phenomenon, keratoconjunctivitis sicca, peripheral neuropathy and concomitant

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autoimmune disorders, such as primary biliary cirrhosis when compared to the negative ACA

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group. [32, 33, 35]. Furthermore, ACA positive SS patients had a lower prevalence of anti-

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Ro/SSA and anti-La/SSB antibodies, lower rates of RF positivity, lower frequency of leukocytopenia and hypergammaglobulinaemia but a greater prevalence of autoantibodies other than anti-SSA/SSB or ACA[31-33, 35]. It was hypothesized that patients with pSS with serum antibodies to certain centromere proteins represent a distinct clinical subset of the SS population. Anti-mitochondrial antibodies (AMA) are considered the serological hallmark of primary biliary cirrhosis (PBC). Several studies have shown an association of SS to PBC, with a 8

ACCEPTED MANUSCRIPT prevalence of sicca manifestations or SS among PBC patients ranging from 47 to 81%[36]. AMA antibodies have been found positive in 1.7-13% of pSS patients when detected by IIF[16, 37-39]. In addition to having a higher frequency of liver involvement, AMA-positive patients also showed a higher prevalence of Raynaud phenomenon, peripheral neuropathy,

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hypergammaglobulinaemia and ESR >50 mm5[16]. AMA was proposed as a sensitive indicator of liver involvement in pSS patients that predispose them to develop autoimmune cholangitis

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similar to PBC[38].

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Anti-cyclic citrullinated peptide antibodies(Anti-CCP) are considered as specific markers for the diagnosis of rheumatoid arthritis (RA). The prevalence of anti-CCP antibodies in pSS ranges

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from 3 to 10%[40-43]. In one study, Anti-CCP positive pSS patients did not differ significantly

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from the anti-CCP negative group in terms of demographic features, synovitis, extraglandular

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involvement or other immunologic markers and in minor salivary gland focus scores [40, 41]. On the contrary, a study of 141 Italian pSSpatients found a positive association of anti-CCP

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antibodies with non-erosive synovitis in multivariate analysis[42]. A Japanese study also

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suggested anassociation of anti-CCP antibodies with the presence of symptoms from the joints in pSS, was also suggested by a Japanese study that detected anti-CCP positivity in 3 out of 46

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pSS patients with articular manifestations, but in none of 26 pSS patients without articular involvement[43]. Interestingly, some of the above studies recognized a group of anti-CCP positive patients, with SS and non-erosive arthritis, fulfilling the ACR classification criteria for RA[40, 43]. This group could be either classified as having RA with secondary SS, or as pSS with anti-CCP and non-erosive arthritis. Although one cannot rule out the possibility that pSS patients with anti-CCP antibodies may progress to RA, a common observation is that non9

ACCEPTED MANUSCRIPT erosive arthritis prevails in anti-CCP antibody-positive SS patients, whereas in RA anti-CCP is a marker of persistent, erosive disease[44].

3. Update on antibody detection methods Autoantibody immunoassays have been used extensively for over 50 years with

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continuous change in the technologies and antigens used. In principle, the detection of

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antibody/antigen recognition depends on the specific interaction between the antibody and its epitope, choice of laboratory assay relies on the specificity of the autoantibody/antigen

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interaction and it is important to be familiar with the limitations of the available techniques.

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Indirect immunofluorescence staining uses tissue sections or cultured cells as an antigenic source and detects the specific recognition of autoantibodies to native antigens. The

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presence of multiple antigenic targets on tissue/cell sections provides overall good sensitivity at

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appropriate sera dilution, it doesn't require sophisticated equipment and it is not expensive; IIF is one of the most commonly used techniques for antibody detection especially during the

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initial screening step[45]. The advances in identifying and cataloguing the molecular targets

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bound by autoantibodies led to the next generation of diagnostic technologies that included

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antigen-specific immunoassays using different platform such as enzyme linked immunoassay (ELISA), Line immunoblots Assay (LIA), multiplexed immunoassays such as addressable laser bead immunoassays (ALBIA) and chemiluminescence assay (CLA). In general, the increase of sensitivity and high throughput nature make these technologies ideal choice for autoantibodies confirmation assay[45]. The rapid proliferation of autoantibodies specificities and the emergence and adoption of novel technologies has created a dilemma for standardization of autoantibodies testing 10

ACCEPTED MANUSCRIPT protocols and the results that are reported to clinicians. The evolving standards of Anti-Ro/SSA autoantibodies system offer a perfect example. The target antigen was originally called “SjD” and follow by double name Ro and SS-A which was derived from the description of this autoantibody system by two research groups: one part of the nomenclature relating to the

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name of a SLE patient (“Ro”) and the other nomenclature related to its association with SjS (“SS”)[46-48]. Eventually, the nomenclature became SSA/Ro60 and SSA/Ro52 to include the

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molecular masses of the respective antigens. The primary target antigen for anti-Ro/SSA was

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first identified as a 60 kDa protein component of small cytoplasmic ribonucleoprotein complexes (hY-RNA complexes)[49]. Later, it was confirmed that the Ro52 and the Ro60

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antigens indeed consisted of two different proteins coded by different cDNAs[50]. Although

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initially suggested to be closely related, a direct interaction of the Ro52 and Ro60 proteins

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could never be conclusively proven. Recent studies indicate that Ro52 and Ro60 proteins are localized to different cell compartments and perform a slightly different function. Ro60 protein,

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having a configuration that resembles a doughnut, binds to misfolded, noncoding RNAs in

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vertebrate cells and acts as a quality checkpoint for RNA misfolding with molecular chaperones for defective RNAs. The misfolded RNAs are recognized and then tagged by Ro60 for

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degradation[51]. The 52 kDa Ro antigen was eventually identified as TRIM21, a family member of the RING/Bbox/coiled-coil (RBCC) tripartite motif proteins (TRIM), and as an ubiquitin-ligase that is over-expressed in peripheral blood mononuclear cells in SS and SLE patients[52, 53]. Ro52 is thought to modify the role or stability of its substrates through ubiquitination, and this modification might result in the Ro52-mediated biological events. Ro52 is biochemically and immunologically distinct from Ro60[53, 54]. 11

ACCEPTED MANUSCRIPT A study evaluating anti-Ro60 and anti-Ro52 autoantibodies in different autoimmune disease entities found differing prevalence distributions for anti-Ro60 and anti-Ro52 using three independent methods: line immunoassay (LIA), ALBIA and ELISA. [54]. The frequency of antiRo52 autoantibody was similar to the frequency of anti-Ro60 in cohorts of systemic lupus

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erythematosus (SLE), Sjögren's syndrome (SS), subacute cutaneous lupus and neonatal lupus syndrome using all three methods. However, this was not true for myositis and

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Scleroderma/ystemic Sclerosis (SSc) cohorts. The percentages of anti-Ro52 autoantibody that

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occur without anti-Ro60 antibody also varied from 5.4% in childhood SLE to 35.4% in the myositis group. In the SS group, 63.2% of anti-Ro52 sera had also antibody to Ro60[54].

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Compared to anti-Ro60, anti-Ro52 antibody seem to be merely associated with myositis and to

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a lesser extent with SSc, whereas reactivity against both antigens and to a lesser extent against

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Ro60 alone seemed to be associated with SS or SLE in the context of connective tissue diseases[53, 54].

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Due to the lack of highly purified and diagnostically reliable recombinant antigens, the

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different associations of anti-Ro/SSA autoantibodies have remained a matter of debate for over two decades until recombinant Ro60 antigen become available and validated[54]. Traditionally,

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anti-Ro/SSA autoantibodies were detected by indirect immunofluorescence (IIF) on HEp-2 cells and confirmed by immunodiffusion (ID), immunoblot or ELISA, mostly using a mixture of both Ro52 and Ro60 as the antigens. With advances in the expression and purification of recombinant protein, immunoassays such as ELISA, LIA, ALBIA or autoantigen arrays became available that allow the separate detection of anti-Ro52 and anti-Ro60 autoantibodies[54]. The importance of the separate detection of those two autoantibodies was analyzed in several 12

ACCEPTED MANUSCRIPT studies and became a matter of debate especially since commercial ELISA kits are currently often marketed as SSA ELISA that employ mixtures of both antigens together in a single assay. It was shown that Anti-Ro52 and anti-Ro60 reactivity can mask each other, thus more than 20% Ro positive samples can remain undetected in assays that utilize blended antigens[54]. These

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findings provide additional evidence and rationale for the recommendation that anti-Ro52 and anti-Ro60 should be detected separately with recombinant antigens [54]. However, in the

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clinical evaluation of different cohorts of SS patients, only few studies have adapted to this

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recommendation and the definition of SSA antibody positivity in the most current classification criteria of SS is still a mix of anti-SSA/Ro60 with anti-SSA/Ro52 autoantibodies[10, 55-57].

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Another example of knowledge gap between technology advance with clinical reporting

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is the recent issue in the United States regarding the standard antinuclear antibody (ANA)

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screening. The method of screening ANA was switched from conventional IIF assay using HEp-2 slide to multiplex beads assay without providing sufficient educational clarification for the

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clinicians. When both assays were labelled as “ANA screening test”, While the ANA multiplex

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assay is used to detect a few specific autoantibodies the ANA IF assay is used for general screening for many autoantibodies. But this causes confusion. In an effort to clarify ANA testing

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for the clinicians, the American College of Rheumatology released a position statement in 2009[58]. The following positions were stated in this statement: 1) The IF ANA assay is the gold standard for ANA testing with greater sensitivity than solidphase assays.

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ACCEPTED MANUSCRIPT 2) HEp-2 cells have approximately 100–150 possible autoantigens. These cells are used to detect ANAs by the IF method, in which both pattern and titer can be described, and to display a variety of autoantigens not present in multiplex ANA tests. 3) Many commercial laboratories and some hospital laboratories have switched their ANA

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screening test to solid-phase immunoassays, such as a multiplex platform. The latter technique can screen and process large volumes of clinical specimens more quickly and

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at less cost than the traditional IF ANA test using fixed HEp-2 cells as substrate.

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4) These multiplex assays can detect only the specific autoantibodies directed against the limited number (typically 8–10) autoantigens that are displayed.

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5) Laboratories should indicate the method used when reporting ANA results.

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A large number of autoantibodies have been identified in the serum of patients with SS. Depend on the methods used for antibody detection, the prevalence of these autoantibodies in

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Sjögren’s syndrome was summarized in table 1.

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4. Autoantibodies panel for the early diagnosis of Sjögren’s syndrome 4.1 Autoantibody production precedes the development of Sjögren’s syndrome The presence of autoantibodies is one of several hallmarks of the autoimmune disease. It is now becoming increasingly clear that autoantibodies may also play a pivotal role in the pathogenesis of many autoimmune diseases and that autoantibodies can mediate both systemic inflammation and tissue injury [59]. From a clinical standpoint, the detection of serum autoantibodies has a central role in the diagnosis and classification of autoimmune 14

ACCEPTED MANUSCRIPT disease. The finding that patients with RA and SLE produced autoantibodies several years before the onset of symptoms and diagnosis [60] s indicates that the autoimmune process in autoimmune disease often starts a very long time before clinical recognition[60]. In families with multiple cases of autoimmune disease, autoantibody profiling, along with

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assessment of genetic risk, enables identification of susceptible individuals in a pre-disease state[56].

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While mothers of babies with neonatal lupus syndrome can have anti-Ro/SSA antibodies

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and remain healthy, follow up studies shown that more than a quarter of such women develop primary SS within 10 years of giving birth. If both SSA and SSB autoantibodies are

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present, an even greater percentage of those women will develop SS[61, 62]. Recently, it

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was shown that autoantibodies profiling may identify individuals at risk of developing

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Sjögren’s syndrome many years before disease onset[55, 56]. ANAs, RF, and antibodies against Ro60/SSA and Ro52/SSA were detected in samples obtained as early as 19–20 years

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(median 4.3–5.1 years) before diagnosis, whereas antibodies against La/SSB were detected

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as early as 16 years (median 3.5 years) before diagnosis [56]. In 75% of the patients studied, serum samples obtained before diagnosis were found to contain autoantibodies (primarily

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ANAs, followed by RF, and anti-Ro60/SSA, anti-Ro52/SSA, and anti-La/SSB, in that order)[56]. In IL-14a transgenic mice, an animal model of SS, autoantibody deposition in salivary glands and decreased salivary flow were present at 6 months of age when lymphocytic infiltration of the salivary and lacrimal glands had not yet begun. Lymphocytic infiltration could be demonstrated at 9 months of age in the submandibular glands, at 12 months of age in the lacrimal glands, and at 15 months of age in the parotid glands. By 18 months of 15

ACCEPTED MANUSCRIPT age, malignant transformations were noted in submandibular and parotid glands, and salivary function had steadily declined throughout the whole process. These findings suggest that, in mouse models, and potentially in humans, a humoral stage of Sjögren’s disease, characterized by autoantibody production and deposition, precedes a cellular stage,

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characterized by progressive glandular inflammation, destruction, and malignant transformation[22, 23].

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4.2 Improvement in the sensitivity of autoantibodies for the diagnosis of Sjögren’s

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syndrome

While anti-SSA/SSB autoantibody has been the main serological markers for the diagnosis of

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SS, detection of additional autoantibodies is still necessary to overcome the limitation of

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SSA/SSB. In a recent study of a SICCA cohort, positive anti-SSA/SSB serology was present in

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1138 (76%) of those participants who met the ACR criteria and in 1067 (73%) of those who met the AECG criteria for SS[10]. While the existence of anti-SSA sero-negative patients can be

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overcome by further confirmed with the biopsy of minor salivary glands (MSG), there is a strong

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need to identify new serological biomarkers that can reflect the disease activity in response to treatment. The SSA antibody profile that is presented at the diagnosis of pSS seems to remain

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constant throughout the course of the disease[14], even after the administration of B-cell depletion therapy with rituximab[15]. Recent identification of organ-specific Sjö autoantibodies may expedite the diagnosis of SS in the future. Sjö autoantibodies included three different autoantibodies, each targeted a salivary gland related antigens. For Anti-SP1, the antigen salivary gland protein 1 is a protein highly expressed in the submandibular and lachrymal glands and expressed at lower levels in 16

ACCEPTED MANUSCRIPT the parotid and submandibular glands. For Anti-CA6, the antigen Carbonic Anhydrase 6 is a secreted protein highly expressed in the acinar cells of the submandibular and pa rotid glands, it may serve as a binding site for particular viruses. For Anti-PSP, the antigen Parotid secretory protein is a secreted protein expressed at high levels in the parotid glands but also in the

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submandibular and sublingual gland and it may also serve as a binding site for particular viruses. In a cohort analysis of 20 patients who met the 2002 ACR diagnostic criteria for SS (including

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positive salivary biopsy) but who were anti-Ro/SSA and anti-La/SSB negative, 45% were positive

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for anti-SP1 and 5% were positive for anti-CA6[23]. In a recent study of 61 confirmed SS patients, the sensitivity of serological markers was increased from 80% (by anti-Ro/SSA

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positivity alone) to 93% (by combination of anti-SSA positivity and Sjö markers positivity) with a

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specificity of 95% (Figure 1, unpublished data). In SS patients with disease duration less then 5

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years, 68.4% was anti-SSA positive, 26.4% was anti-SSA negative but Sjö markers positive and 5.2% was anti-SSA negative and Sjö markers negative. In SS patients with disease duration

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between 5 to 10 years, 80% was anti-SSA positive, 20% was anti-SSA negative but Sjö markers

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positive and 10% was anti-SSA negative and Sjö markers negative. In SS patients with disease duration longer than 10 years, 90.9% was anti-SSA positive, 4.5% was anti-SSA negative but Sjö

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markers positive and 4.5% was anti-SSA negative and Sjö markers negative (Table 2). Additional larger studies are needed to validate these observations. 4.3 Ideal autoantibody panel for the early detection of Sjögren’s syndrome The symptoms of SS can progress slowly and are often highly variable in presentation which making the diagnosis difficult and challenge[4]. There is a huge gap between the SS patient’s description of their symptoms and the objective findings. Unfortunately, the nature of sicca 17

ACCEPTED MANUSCRIPT symptoms is very non-specific, especially in the immediate absence of other SS-related complaints or finding. When SS is suspected in a patient with thoroughly evaluation of sicca symptoms, an objective serological test will be very useful to help the physician making the correct diagnosis of SS. For the balance of sensitivity and specificity, an ideal serology panel for

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patients with sicca symptoms should include initial screen using HEp-2 cells as IIF substrate which will be able to detect ANA, AMA and ACA autoantibodies (Figure 2), followed by

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confirmation assays for anti-SSA/Ro60, anti-SSA/Ro52, anti-SSB/la, RF, anti-SP1, anti-CA6, anti-

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PSP, anti-alpha-fodrin, and anti-CCP autoantibodies by one of the solid phase assays (ELISA, LIA or multiplex bead assay). Any positive serological result with clinical define sicca symptoms

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should raise the flag for more frequently follow up of patients for the development of SS.

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Table 1: Detection methods and Prevalence of autoantibodies in Sjögren syndrome

Rheumatoid Factors (RF)

36-74%

Antimitochondrial antibodies (AMA)

3-13%

Anticentromere antibodies (ACA) Sjö autoantibodies (Anti-CA6, Anti-PSP and Anti-SP1) Anti-alpha Fodrin Anti-CCP

3-27%

IIF (gold standard) ELISA: lower cost, less reliable Multiplex: Automation, limited autoantibodies detected ELISA (IgM) and Lattex agglutination test have comparable sensitivity (66-69%) and specificity (8691%) ELISA (M2) has a better sensitivity compared to IIF and CFT (99% versus 85-97%), but specificity was lower compared to IIF

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Good agreement between IIF and ELISA for detection of ACA.

ELISA

AC

30%-45%

T

85-90%

IP

Anti-Nuclear Antibodies (ANA)

Comments RNA precipitation (gold standard) ELISA: sensitivity 72%-specificity 95% CIE: sensitivity 89% -specificity 100% Multiplex: sensitivity 80% -specificity 98%

CR

25-40%

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Anti-La/SSB

Detection Methods RNA precipitation assay Counter-immunoelectrophoresis (CIE) Immunoblotting ELISA Multiplex Beads Assay Indirect immunofluorescence (IIF) on HEp-2 cells Solid phase immunoassays, (bead based multiplex platforms, ELISA) ELISA Particle agglutination tests Waaler-Rose hemagglutination Laser nephelometry Indirect immunofluorescence (IIF), Complement fixation test (CFT), Immunodiffusion, Radioimmunoassay (RIA), ELISA, Immunoblotting (IB) and functional assays Indirect immunofluorescence (IIF) on HEp-2 cells ELISA for the detection of CENPs

AN

Prevalence 50-70%

M

Autoantibodies Anti-Ro/SSA

38%-42%

ELISA

7%-10%

ELISA

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Table 2. Prevalence of autoantibodies in primary SS at different disease duration

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Figure 1. Sensitivity of SSA and Sjö autoantibodies in Chinese primary SS patients

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Figure 2. Antibody patterns in IIF on HEp-2 cells Anti-mitochondrial antibodies (AMA)

Anti-centromere antibodies (ACA)

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Anti-Nuclear Antibodies (ANA) with a fine speckled pattern

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The presence of autoantibodies is one of several hallmarks of Sjögren’s Syndrome, the detection of serum autoantibodies has a central role in the diagnosis and classification of Sjögren’s syndrome. In this review, we will discuss autoantibodies that are helpful in the diagnosis of Sjögren’s syndrome. This includes the traditional autoantibodies for disease classification (ANA, Anti-Ro/SSA, Anti-La/SSB, RF), autoantibodies identified from mouse models (Anti-SP1, Anti- PSP, Anti-CA6, and anti-alpha fodrin) and autoantibodies associated with other autoimmune disease (ACA, AMA, and Anti-CCP). We will also review the methods for the detection of autoantibodies and associated challenges for clinical results reporting. The significance of using an autoantibody panel for the diagnosis of SS will be also be reviewed.

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