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Autoimmune serology testing in clinical practice: An updated roadmap for the diagnosis of autoimmune hepatitis

  • George N. Dalekos
    Correspondence
    Corresponding author: Head of the Department of Medicine and the Research Laboratory of Internal Medicine, National Expertise Center of Greece in Autoimmune Liver Diseases, Full Member of the European Reference Network on Hepatological Diseases (ERN RARE-LIVER), General University Hospital of Larissa, Mezourlo area, Larissa 41110, Greece
    Affiliations
    Department of Medicine and Research Laboratory of Internal Medicine, National Expertise Center of Greece in Autoimmune Liver Diseases, General University Hospital of Larissa, Larissa, Greece

    European Reference Network on Hepatological Diseases (ERN RARE-LIVER), General University Hospital of Larissa, Larissa, Greece
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  • Nikolaos K. Gatselis
    Affiliations
    Department of Medicine and Research Laboratory of Internal Medicine, National Expertise Center of Greece in Autoimmune Liver Diseases, General University Hospital of Larissa, Larissa, Greece

    European Reference Network on Hepatological Diseases (ERN RARE-LIVER), General University Hospital of Larissa, Larissa, Greece
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Published:November 16, 2022DOI:https://doi.org/10.1016/j.ejim.2022.11.013

      Highlights

      • There is not a single specific laboratory marker to diagnose or exclude AIH.
      • Therefore, diagnosis of AIH is in most cases challenging for physicians.
      • Antibodies detection is mandatory even though not pathognomonic for AIH diagnosis.
      • Assessment of autoimmune serology in strict adherence to guidelines is essential.
      • IgG increase and autoimmunity background support an in-depth serological assessment.

      Abstract

      Diagnosis of autoimmune hepatitis (AIH) is in most cases challenging for clinicians as there is not a single specific laboratory or histological marker to diagnose or exclude the presence of the disease. The clinical spectrum of AIH varies from completely asymptomatic to acute-severe or even rarely fulminant hepatic failure, while everybody can be affected irrespective of age, gender, and ethnicity. The old revised and the newer simplified diagnostic scores have been established by the International Autoimmune Hepatitis Group (IAIHG) in 1999 and 2008, respectively, which are based on several clinical, laboratory and histological parameters. Additionally, a thorough differential diagnosis from other diseases mimicking AIH is absolutely indicated. In this context, autoantibodies detection in patients with suspected AIH is mandatory -even though not pathognomonic- not only for AIH diagnosis but furthermore, for AIH classification (AIH-type 1 and AIH-type 2). Although autoimmune serology can be supportive of AIH diagnosis in ≥95% of cases if testing has been performed according to the IAIHG guidelines, this is not the case under real-life circumstances in routine clinical laboratories. Clinicians should be careful both for the importance of the required testing and how to interpret the results and therefore, they should communicate and discuss with the laboratory personnel to achieve the maximum benefit for the patient. Herein, a detailed and updated review of the diagnostic work-up for AIH diagnosis under real-life conditions is given to minimize the underestimation and misdiagnosis of AIH which can result in progression of the disease and unfavourable outcomes.

      Keywords

      Abbreviations:

      AIH (autoimmune hepatitis), IAIHG (International AIH Group), IgG (immunoglobulin G), AIH-1 (autoimmune hepatitis type 1), AIH-2 (autoimmune hepatitis type 2), ANA (antinuclear antibodies), SMA (smooth muscle antibodies), anti-LKM1 (liver kidney microsomal type-1 antibodies), anti-LKM3 (liver kidney microsomal type-3 antibodies), anti-LC1 (liver cytosol type-1 antibodies), anti-SLA/LP (antibodies against soluble liver antigens/liver pancreas), IIF (indirect immunofluorescence), HEp-2 (human larynx epithelioma cancer cell lines), dsDNA (double-stranded DNA), DILI (drug induced liver injury), ELISA (enzyme-linked immunosorbent assay), V-pattern (arterial vessels), VG-pattern (arterial vessels and mesangium of renal glomeruli), VGT-pattern (arterial vessels, glomeruli and intracellular fibrils in renal tubules), F-actin (filamentous actin), anti-α-actinin (antibodies against alpha-actinin), anti-Ro52 (antibodies against ribonucleoprotein/Sjogren's syndrome 52kDa antigen), ANCA (anti-neutrophil cytoplasmic antibodies), cANCA (cytoplasmic ANCA), pANCA (perinuclear ANCA), pANNA (perinuclear antineutrophil nuclear antibody), PSC (primary sclerosing cholangitis), PBC (primary biliary cholangitis), anti-LKM2 (liver kidney microsomal type-2 antibodies), AMA (anti-mitochondrial antibodies), SLE (systemic lupus erythematosus), pIgG (polyreactive IgG)

      1. Introduction

      The diagnosis of autoimmune hepatitis (AIH) is based on a combination of clinical, laboratory and histological findings in patients with unexplained acute or chronic hepatitis [
      European Association for the Study of the Liver
      EASL clinical practice guidelines: autoimmune hepatitis.
      ,
      • Dalekos G.N.
      • Koskinas J.
      • Papatheodoridis G.V.
      Hellenic association for the study of the liver clinical practice guidelines: autoimmune hepatitis.
      ,
      • Mack C.L.
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      • Manns M.P.
      • Mayo M.J.
      • et al.
      Diagnosis and management of autoimmune hepatitis in adults and children: 2019 practice guidance and guidelines from the american association for the study of liver diseases.
      ]. The disease is considered rare but recent population-based studies from England and New Zealand have shown that AIH incidence is on the rise [
      • Grønbaek L.
      • Otete H.
      • Ban L.
      • Crooks C.
      • Card T.
      • Jepsen P.
      • et al.
      Incidence, prevalence and mortality of autoimmune hepatitis in England 1997-2015. A population-based cohort study.
      ,
      • Lamba M.
      • Ngu J.H.
      • Stedman C.A.M.
      Trends in incidence of autoimmune liver diseases and increasing incidence of autoimmune hepatitis.
      ]. As in most autoimmune diseases, AIH is characterized by female predominance while every subject can be affected irrespective of age, gender, and ethnicity [
      • Gatselis N.K.
      • Zachou K.
      • Koukoulis G.K.
      • Dalekos G.N.
      Autoimmune hepatitis, one disease with many faces: etiopathogenetic, clinico-laboratory and histological characteristics.
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      • Dalekos G.N.
      • Azariadis K.
      • Lygoura V.
      • Arvaniti P.
      • Gampeta S.
      • Gatselis N.K.
      Autoimmune hepatitis in patients aged 70 years or older: disease characteristics, treatment response and outcome.
      ]. Differential diagnosis includes viral, toxic, genetic, and metabolic liver diseases [
      European Association for the Study of the Liver
      EASL clinical practice guidelines: autoimmune hepatitis.
      ,
      • Dalekos G.N.
      • Koskinas J.
      • Papatheodoridis G.V.
      Hellenic association for the study of the liver clinical practice guidelines: autoimmune hepatitis.
      ,
      • Mack C.L.
      • Adams D.
      • Assis D.N.
      • Kerkar N.
      • Manns M.P.
      • Mayo M.J.
      • et al.
      Diagnosis and management of autoimmune hepatitis in adults and children: 2019 practice guidance and guidelines from the american association for the study of liver diseases.
      ,
      • Gatselis N.K.
      • Zachou K.
      • Koukoulis G.K.
      • Dalekos G.N.
      Autoimmune hepatitis, one disease with many faces: etiopathogenetic, clinico-laboratory and histological characteristics.
      ]. However, not rarely, AIH can be concurrent with other hepatic and non-hepatic diseases making its diagnosis difficult and challenging [
      • Rigopoulou E.I.
      • Zachou K.
      • Gatselis N.
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      Autoimmune hepatitis in patients with chronic HBV and HCV infections: patterns of clinical characteristics, disease progression and outcome.
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      • Beisel C.
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      • Teufel A.
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      Association of autoimmune hepatitis and systemic lupus erythematodes: a case series and review of the literature.
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      • Rigopoulou E.I.
      • Gyftaki S.
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      • Tsimourtou V.
      • Koukoulis G.K.
      • Hadjigeorgiou G.
      • et al.
      Autoimmune hepatitis in patients with multiple sclerosis: the role of immunomodulatory treatment.
      ,
      • Dalekos G.N.
      • Gatselis N.K.
      • Zachou K.
      • Koukoulis G.K.
      NAFLD and autoimmune hepatitis: do not judge a book by its cover.
      ,
      • Rigopoulou E.I.
      • Gatselis N.
      • Arvaniti P.
      • Koukoulis G.K.
      • Dalekos G.N.
      Alcoholic liver disease and autoimmune hepatitis: sometimes a closer look under the surface is needed.
      ].
      The international autoimmune hepatitis group (IAIHG) has established diagnostic scores to help for a timely and prompt diagnosis of the disease namely, the revised and the simplified score in 1999 and 2008, respectively [
      • Alvarez F.
      • Berg P.A.
      • Bianchi F.B.
      • Bianchi L.
      • Burroughs A.K.
      • Cancado E.L.
      • et al.
      International Autoimmune Hepatitis Group Report: review of criteria for diagnosis of autoimmune hepatitis.
      ,
      • Hennes E.M.
      • Zeniya M.
      • Czaja A.J.
      • Parés A.
      • Dalekos G.N.
      • Krawitt E.L.
      • et al.
      Simplified criteria for the diagnosis of autoimmune hepatitis.
      ]. The latter seems very easy and friendly for use in everyday clinical practice as only four parameters have been included namely, autoantibodies detection, gamma-globulins or immunoglobulin G (IgG) serum levels, histopathology of the liver, and seronegativity for viral hepatitis markers (Supplementary Table 1) [
      • Hennes E.M.
      • Zeniya M.
      • Czaja A.J.
      • Parés A.
      • Dalekos G.N.
      • Krawitt E.L.
      • et al.
      Simplified criteria for the diagnosis of autoimmune hepatitis.
      ]. However, even though the abovementioned simplified criteria represent a good tool for daily clinical practice still, there is not a diagnostic ''gold standard'' and therefore, the clinicians must regard any diagnostic score only as an aid to AIH diagnosis and the criteria should be used alongside clinical judgement [
      European Association for the Study of the Liver
      EASL clinical practice guidelines: autoimmune hepatitis.
      ,
      • Dalekos G.N.
      • Koskinas J.
      • Papatheodoridis G.V.
      Hellenic association for the study of the liver clinical practice guidelines: autoimmune hepatitis.
      ,
      • Mack C.L.
      • Adams D.
      • Assis D.N.
      • Kerkar N.
      • Manns M.P.
      • Mayo M.J.
      • et al.
      Diagnosis and management of autoimmune hepatitis in adults and children: 2019 practice guidance and guidelines from the american association for the study of liver diseases.
      ].
      It should be noted also, that recently many doubts have been raised concerning the significance of emperipolesis and hepatocyte rosettes as typical characteristics of AIH at the histological level [
      • de Boer Y.S.
      • van Nieuwkerk C.M.
      • Witte B.I.
      • Mulder C.J.
      • Bouma G.
      • Bloemena E.
      Assessment of the histopathological key features in autoimmune hepatitis.
      ,
      • Gurung A.
      • Assis D.N.
      • McCarty T.R.
      • Mitchell K.A.
      • Boyer J.L.
      • Jain D.
      Histologic features of autoimmune hepatitis: a critical appraisal.
      ]. Indeed, it seems that these lesions are indicative of hepatocytes damage from many causes and therefore, are rather unspecific for AIH diagnosis [
      • de Boer Y.S.
      • van Nieuwkerk C.M.
      • Witte B.I.
      • Mulder C.J.
      • Bouma G.
      • Bloemena E.
      Assessment of the histopathological key features in autoimmune hepatitis.
      ,
      • Gurung A.
      • Assis D.N.
      • McCarty T.R.
      • Mitchell K.A.
      • Boyer J.L.
      • Jain D.
      Histologic features of autoimmune hepatitis: a critical appraisal.
      ]. In this regard, the International AIH Pathology Group very recently published a consensus report which proposed a homogenous approach for AIH diagnosis based on liver histology [
      • Lohse A.W.
      • Sebode M.
      • Bhathal P.S.
      • Clouston A.D.
      • Dienes H.P.
      • Jain D.
      • et al.
      Consensus recommendations for histological criteria of autoimmune hepatitis from the International AIH Pathology Group: results of a workshop on AIH histology hosted by the European Reference Network on Hepatological Diseases and the European Society of Pathology: results of a workshop on AIH histology hosted by the European Reference Network on Hepatological Diseases and the European Society of Pathology.
      ]. However, up to the present, this consensus lacks external validation and therefore, cannot be used safely in everyday clinical practice by replacing that of the simplified criteria. Besides, the consensus opinion is that even though hepatocyte rosettes and emperipolesis are not specific histological characteristics of AIH, they can be reported as surrogate markers of AIH severity [
      • Lohse A.W.
      • Sebode M.
      • Bhathal P.S.
      • Clouston A.D.
      • Dienes H.P.
      • Jain D.
      • et al.
      Consensus recommendations for histological criteria of autoimmune hepatitis from the International AIH Pathology Group: results of a workshop on AIH histology hosted by the European Reference Network on Hepatological Diseases and the European Society of Pathology: results of a workshop on AIH histology hosted by the European Reference Network on Hepatological Diseases and the European Society of Pathology.
      ]. Detailed description of the new proposed recommendations for the histological criteria of AIH is beyond the scope of this review (for further reading see ref. [
      • Lohse A.W.
      • Sebode M.
      • Bhathal P.S.
      • Clouston A.D.
      • Dienes H.P.
      • Jain D.
      • et al.
      Consensus recommendations for histological criteria of autoimmune hepatitis from the International AIH Pathology Group: results of a workshop on AIH histology hosted by the European Reference Network on Hepatological Diseases and the European Society of Pathology: results of a workshop on AIH histology hosted by the European Reference Network on Hepatological Diseases and the European Society of Pathology.
      ] and Supplementary Table 2).
      According to the autoantibodies detected, AIH is classified as AIH-type 1 (AIH-1) or AIH-type 2 (AIH-2). Patients with AIH-1 have detectable anti–nuclear autoantibodies (ANA) and/or smooth muscle autoantibodies (SMA). Patients with AIH-2 have detectable anti–liver kidney microsomal type-1 (anti-LKM1) or rarely anti–liver kidney microsomal type-3 (anti-LKM3), and/or anti–liver cytosol type-1 (anti-LC1) antibodies [
      European Association for the Study of the Liver
      EASL clinical practice guidelines: autoimmune hepatitis.
      ,
      • Dalekos G.N.
      • Koskinas J.
      • Papatheodoridis G.V.
      Hellenic association for the study of the liver clinical practice guidelines: autoimmune hepatitis.
      ,
      • Mack C.L.
      • Adams D.
      • Assis D.N.
      • Kerkar N.
      • Manns M.P.
      • Mayo M.J.
      • et al.
      Diagnosis and management of autoimmune hepatitis in adults and children: 2019 practice guidance and guidelines from the american association for the study of liver diseases.
      ,
      • Gatselis N.K.
      • Zachou K.
      • Koukoulis G.K.
      • Dalekos G.N.
      Autoimmune hepatitis, one disease with many faces: etiopathogenetic, clinico-laboratory and histological characteristics.
      ,
      • Dalekos G.N.
      • Samakidou A.
      • Lyberopoulou A.
      • Banakou E.
      • Gatselis N.K.
      Recent advances in the diagnosis and management of autoimmune hepatitis.
      ]. Antibodies against soluble liver antigens/liver pancreas autoantigen (anti-SLA/LP) can also be detected, mainly in AIH-1 patients [
      • Dalekos G.N.
      • Samakidou A.
      • Lyberopoulou A.
      • Banakou E.
      • Gatselis N.K.
      Recent advances in the diagnosis and management of autoimmune hepatitis.
      ,
      • Wies I.
      • Brunner S.
      • Henninger J.
      • Herkel J.
      • Kanzler S.
      • Meyer zum Büschenfelde K.H.
      • et al.
      Identification of target antigen for SLA/LP autoantibodies in autoimmune hepatitis.
      ,
      • Herkel J.
      • Heidrich B.
      • Nieraad N.
      • Wies I.
      • Rother M.
      • Lohse A.W.
      Fine specificity of autoantibodies to soluble liver antigen and liver/pancreas.
      ]. The clinical significance of the subclassification of AIH is shown in Table 1.
      Table 1Clinical, demographic, and serological differences between AIH-1 and AIH-2.
      CharacteristicAIH-1AIH-2
      Age at onsetAny ageCommonly in childhood and young adulthood
      SeverityVariableUsually acute or acute/severe
      Genetic susceptibilityHLA DR3, DR4, DR13HLA DR3, DR7
      AutoantibodiesANA, SMA, anti-alpha actinin, anti-F actin, anti-SLA/LPanti-LKM1, anti-LC1, anti-LKM3 (rarely)
      Liver histologyMild to severe disease

      and/or cirrhosis
      Usually, advanced disease
      No response or insufficient responseRareCommon
      Relapse during steroids tapering or complete cessation of immunosuppressionVariableCommon
      Long-term maintenance treatmentVariableVery common
      Abbreviations are same as in the text.
      Autoantibodies are mandatory to achieve a prompt diagnosis of AIH if they are determined according to the guidelines [
      • Gatselis N.K.
      • Zachou K.
      • Koukoulis G.K.
      • Dalekos G.N.
      Autoimmune hepatitis, one disease with many faces: etiopathogenetic, clinico-laboratory and histological characteristics.
      ,
      • Dalekos G.N.
      • Samakidou A.
      • Lyberopoulou A.
      • Banakou E.
      • Gatselis N.K.
      Recent advances in the diagnosis and management of autoimmune hepatitis.
      ,
      • Vergani D.
      • Alvarez F.
      • Bianchi F.B.
      • Cançado E.L.
      • Mackay I.R.
      • Manns M.P.
      • et al.
      Liver autoimmune serology: a consensus statement from the committee for autoimmune serology of the International Autoimmune Hepatitis Group.
      ,
      • Terziroli Beretta-Piccoli B.
      • Mieli-Vergani G.
      • Vergani D.
      Serology in autoimmune hepatitis: a clinical-practice approach.
      ]. This review aims to provide an updated roadmap for the performance of autoimmune serology to facilitate physicians both to be aware of the importance of this testing but also to be efficient to interpretate the results.

      2. Autoantibodies detection

      2.1 Anti-nuclear (ANA) and smooth muscle antibodies (SMA)

      According to the current guidelines, ANA should be detected in first-line screening by indirect immunofluorescence (IIF) on fresh-frozen rodent stomach, liver, and kidney tissue substrates (Fig. 1, Fig. 2) [
      European Association for the Study of the Liver
      EASL clinical practice guidelines: autoimmune hepatitis.
      ,
      • Dalekos G.N.
      • Koskinas J.
      • Papatheodoridis G.V.
      Hellenic association for the study of the liver clinical practice guidelines: autoimmune hepatitis.
      ,
      • Gatselis N.K.
      • Zachou K.
      • Koukoulis G.K.
      • Dalekos G.N.
      Autoimmune hepatitis, one disease with many faces: etiopathogenetic, clinico-laboratory and histological characteristics.
      ,
      • Dalekos G.N.
      • Samakidou A.
      • Lyberopoulou A.
      • Banakou E.
      • Gatselis N.K.
      Recent advances in the diagnosis and management of autoimmune hepatitis.
      ,
      • Vergani D.
      • Alvarez F.
      • Bianchi F.B.
      • Cançado E.L.
      • Mackay I.R.
      • Manns M.P.
      • et al.
      Liver autoimmune serology: a consensus statement from the committee for autoimmune serology of the International Autoimmune Hepatitis Group.
      ,
      • Terziroli Beretta-Piccoli B.
      • Mieli-Vergani G.
      • Vergani D.
      Serology in autoimmune hepatitis: a clinical-practice approach.
      ]. Up to the present, several target-autoantigens of ANA have been identified in AIH patients, including single- and double-stranded DNA (dsDNA), chromatin, histones, centromere, ribonucleoproteins, and cyclin A (Table 2). However, neither the staining pattern by using the human larynx epithelioma cancer (HEp-2) cell lines as substrate, nor the identification of the target-autoantigens have any known specific clinical implication in patients with AIH.
      Fig 1
      Fig. 1Antinuclear antibodies by indirect immunofluorescence on (A) HEp2 cells showing a homogeneous nuclear staining and (B) fresh rodent liver section with staining of the nuclei of hepatocytes. Original magnification × 40.
      Fig 2
      Fig. 2Abbreviations are same as in the text. Diagnostic algorithm of investigation in patients with acute or chronic hepatitis of unknown cause. Autoantibodies can be detected in more than 95% of patients if testing performed according to the guidelines. Under real life conditions, in cases with acute severe AIH (jaundice, international normalized ratio≥1.5, no hepatic encephalopathy, no previously recognized liver disease) a diagnostic trial with oral or intravenous corticosteroids is rather justified before obtaining the results of autoimmune serology and liver biopsy, as the abovementioned diagnostic algorithm is somehow time consuming.
      *IgG levels can also be within normal limits in about 39% of acute severe cases. **ANA and SMA can also be evaluated by IIF on HEp2 cells or by ELISAs (for details and rules see text and ) [
      • Galaski J.
      • Weiler-Normann C.
      • Schakat M.
      • Zachou K.
      • Muratori P.
      • Lampalzer S.
      • et al.
      Update of the simplified criteria for autoimmune hepatitis: Evaluation of the methodology for immunoserological testing.
      ]. The laboratories should comply to the guidelines regarding the assays they use along with the cut-offs considered for reactivity.
      Table 2Significance of antibodies in autoimmune hepatitis (AIH).
      AntibodyTarget-autoantigens/Detection MethodsClinical significance
      ANAHistones, chromatin, ribonucleoproteins; single- and double-stranded DNA, cyclin A, centromere; undefined antigens 20–30%; IIF on multi-organ rodent tissue substrates*AIH-1 but not specific; Rare in AIH-2
      SMAFilamentous actin, desmin, vimentin; Undefined antigens in 20%; IIF on multi-organ rodent tissue substrates*AIH-1 particularly in combi-nation with ANA; VG/VGT patterns highly specific; Rare in AIH-2
      Anti-LKM1Cytochrome P450 2D6 (molecular weight: 50 kDa); IIF on multi-organ rodent tissue substrates; also, by ELISA or western blotHighly specific for AIH-2 (absent in AIH-1); detectable in HCV infection (10%)
      Anti-LKM3UGT1 (molecular weight: 55 kDa); IIF on multi-organ rodent tissue substrates or by western blotSpecific antibody for AIH-2 but rare; present in up to 13% of HDV infection
      Anti-LC1FTCD (molecular weight: 58–62 kDa); IIF on multi-organ rodent tissue substrates; also, by immunodiffusion, ELISA, or western blot (very important in patients with coexistence of anti-LKM1 by IIF)Liver specific antibody for AIH-2 (absent in AIH-1 and rare in HCV); usually coexists with anti-LKM1; can be the only marker (10% of AIH-2 cases)
      Anti-SLA/LPSynthase (S) converting O-phosphoseryl-tRNA (Sep) to selenocysteinyl-tRNA (Sec) (molecular weight: 50 kDa); ELISA or western blotHighly specific for AIH-1 (15–30% of patients; specifi-city: 99%); rare in AIH-2; coexists with anti-Ro52 anti-bodies (77–98% of cases); need for permanent treatment
      pANCA/ pANNAUnknown target-autoantigen; IIF on fixed granulocytesExclusively in AIH-1 (60–96%); very few cases with isolated detection; also in PSC, IBD and ASC patients
      PBC-specific ANANuclear body speckled 100 kDa (sp100) and 210 kDa glycoprotein (gp210) as main target autoantigens of MND and RLM patterns by IIF on HEp2 cells; also, by ELISAs or western blotVery specific for PBC (up to 65% of PBC patients by using specific IgG subclasses as secondary antibody on HEp2 vs. 15–20% by the common anti-total IgG use); associate with PBC severity
      Anti-α-actininα-actinin (F-actin cross-linking protein); investigational serologic marker; ELISA or western blotCommon in SLE and AIH-1; 42% of AIH-1 and in 2/3 of anti-F-actin positive (double reactivity is associated with severe AIH, insufficient res-ponse, and relapse episodes)
      Anti-dsDNAdsDNA usually by ELISAs or IIF on Crithidia luciliae (higher specificity than molecular-based assays)Highly specific for SLE and AIH (30% of AIH and up to 60% of AIH/PBC variant); SLE misdiagnosis instead of concurrent AIH or AIH alone is not uncommon with po-tential catastrophic consequ-ences for the patients
      AMAE2 subunits of the 2-OADC; IIF on multi-organ rodent tissue substrates; also, by specific ELISAs or western blotLaboratory hallmark of PBC but also in 5–10% of typical AIH cases (significance unknown); similar manage-ment as in AMA-negative AIH patients
      Abbreviations are same as in the text. *For updates regarding their detection by IIF on HEp2-cells or ELISAs see Table 3 and text; CYP2D6, cytochrome P450 2D6; HCV, hepatitis C virus; UGT1, family 1 of uridine diphosphate glucuronosyl-transferases; HDV, hepatitis D virus; FTCD, formiminotransferase cyclodeaminase; IBD, inflammatory bowel disease; ASC, autoimmune sclerosing cholangitis; PBC, primary biliary cholangitis; MND, multiple nuclear dots; RLM, rim like membranous; 2-OADC, 2-oxo-acid dehydrogenase complexes
      Even though ANA are currently the most sensitive marker of AIH, they lack specificity as they are present in a variety of liver diseases, including fatty liver disease, drug-induced liver injury (DILI), viral hepatitis, Wilson's disease, and alcoholic liver disease [
      • Dalekos G.N.
      • Gatselis N.K.
      • Zachou K.
      • Koukoulis G.K.
      NAFLD and autoimmune hepatitis: do not judge a book by its cover.
      ,
      • Gatselis N.K.
      • Georgiadou S.P.
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      • Zachou K.
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      Clinical significance of organ- and non-organ-specific autoantibodies on the response to anti-viral treatment of patients with chronic hepatitis C.
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      ]. They may also be detected both in healthy adults and diseases which may coexist with AIH (e.g., Hashimoto thyroiditis, celiac disease) [
      • Tan E.M.
      • Feltkamp T.E.
      • Smolen J.S.
      • Butcher B.
      • Dawkins R.
      • Fritzler M.J.
      • et al.
      Range of antinuclear antibodies in “healthy” individuals.
      ].
      As in ANA, the detection of SMA is recommended by IIF on fresh-frozen triple rodent substrates. Τhey stain the lamina propria and muscularis mucosae of the stomach (Fig. 3A) and arterial walls of the liver, while three different IIF patterns characterize SMA on rodent kidney substrate: (1) V-pattern (arterial vessels), (2) VG-pattern (arterial vessels and mesangium of renal glomeruli), and (3) VGT-pattern (arterial vessels, glomeruli and intracellular fibrils in renal tubule) (Fig. 3B) [
      • Bottazzo G.F.
      • Florin-Christensen A.
      • Fairfax A.
      • Swana G.
      • Doniach D.
      • Groeschel-Stewart U.
      Classification of smooth muscle autoantibodies detected by immunofluorescence.
      ]. SMA target-autoantigens include structures of the cytoskeleton, mainly the filamentous actin (anti-F-actin which is present in 80% of AIH patients with SMA of VGT-pattern), but also vimentin, tubulin desmin, and troponin (Table 2) [
      • Dalekos G.N.
      • Samakidou A.
      • Lyberopoulou A.
      • Banakou E.
      • Gatselis N.K.
      Recent advances in the diagnosis and management of autoimmune hepatitis.
      ,
      • Vergani D.
      • Alvarez F.
      • Bianchi F.B.
      • Cançado E.L.
      • Mackay I.R.
      • Manns M.P.
      • et al.
      Liver autoimmune serology: a consensus statement from the committee for autoimmune serology of the International Autoimmune Hepatitis Group.
      ,
      • Terziroli Beretta-Piccoli B.
      • Mieli-Vergani G.
      • Vergani D.
      Serology in autoimmune hepatitis: a clinical-practice approach.
      ,
      • Muratori P.
      • Muratori L.
      • Agostinelli D.
      • Pappas G.
      • Veronesi L.
      • Granito A.
      • et al.
      Smooth muscle antibodies and type 1 autoimmune hepatitis.
      ]. SMA, especially of V-pattern, are not disease specific and could be identified in different liver diseases [
      • Dalekos G.N.
      • Samakidou A.
      • Lyberopoulou A.
      • Banakou E.
      • Gatselis N.K.
      Recent advances in the diagnosis and management of autoimmune hepatitis.
      ,
      • Bottazzo G.F.
      • Florin-Christensen A.
      • Fairfax A.
      • Swana G.
      • Doniach D.
      • Groeschel-Stewart U.
      Classification of smooth muscle autoantibodies detected by immunofluorescence.
      ,
      • Muratori P.
      • Muratori L.
      • Agostinelli D.
      • Pappas G.
      • Veronesi L.
      • Granito A.
      • et al.
      Smooth muscle antibodies and type 1 autoimmune hepatitis.
      ], while SMA-VG or -VGT patterns are more specific for AIH and correlate with F-actin reactivity (Table 2) [
      • Bottazzo G.F.
      • Florin-Christensen A.
      • Fairfax A.
      • Swana G.
      • Doniach D.
      • Groeschel-Stewart U.
      Classification of smooth muscle autoantibodies detected by immunofluorescence.
      ,
      • Granito A.
      • Muratori L.
      • Muratori P.
      • Pappas G.
      • Guidi M.
      • Cassani F.
      • et al.
      Antibodies to filamentous actin (F-actin) in type 1 autoimmune hepatitis.
      ].
      Fig 3
      Fig. 3Smooth muscle antibodies on fresh rodent tissue sections by indirect immunofluorescence. (A) Staining of the smooth muscle fibers of lamina propria and muscularis mucosae of the stomach and (B) Staining of the smooth muscle fibers within the arterial vessels and glomeruli of the kidney. Original magnification × 40.
      According to the current criteria for AIH, IIF titers ≥1:40 in adults and ≥1:20 in children are considered positive for ANA and SMA during the investigation of liver diseases [
      European Association for the Study of the Liver
      EASL clinical practice guidelines: autoimmune hepatitis.
      ,
      • Dalekos G.N.
      • Koskinas J.
      • Papatheodoridis G.V.
      Hellenic association for the study of the liver clinical practice guidelines: autoimmune hepatitis.
      ,
      • Hennes E.M.
      • Zeniya M.
      • Czaja A.J.
      • Parés A.
      • Dalekos G.N.
      • Krawitt E.L.
      • et al.
      Simplified criteria for the diagnosis of autoimmune hepatitis.
      ,
      • Mieli-Vergani G.
      • Vergani D.
      • Baumann U.
      • Czubkowski P.
      • Debray D.
      • Dezsofi A.
      • et al.
      Diagnosis and management of pediatric autoimmune liver disease: ESPGHAN hepatology committee position statement.
      ]. Of interest, SMA and ANA are only useful for diagnosing AIH, whereas they are not associated with prognosis. SMA are typically found in conjunction with ANA in about 50% of patients, whereas isolated SMA are detected in approximately 35% and ANA alone in 15% of cases [
      • Dalekos G.N.
      • Samakidou A.
      • Lyberopoulou A.
      • Banakou E.
      • Gatselis N.K.
      Recent advances in the diagnosis and management of autoimmune hepatitis.
      ,
      • Vergani D.
      • Alvarez F.
      • Bianchi F.B.
      • Cançado E.L.
      • Mackay I.R.
      • Manns M.P.
      • et al.
      Liver autoimmune serology: a consensus statement from the committee for autoimmune serology of the International Autoimmune Hepatitis Group.
      ,
      • Terziroli Beretta-Piccoli B.
      • Mieli-Vergani G.
      • Vergani D.
      Serology in autoimmune hepatitis: a clinical-practice approach.
      ].

      2.2 Antibodies against soluble liver antigens/liver pancreas (anti-SLA/LP)

      Anti‐SLA/LP are detected by enzyme-linked immunosorbent assay (ELISA), immunoblot or radioligand assays but not with conventional IIF in 15%‐30% of AIH patients and according to the guidelines should be investigated during the first-line screening (Fig. 2) [
      European Association for the Study of the Liver
      EASL clinical practice guidelines: autoimmune hepatitis.
      ,
      • Dalekos G.N.
      • Koskinas J.
      • Papatheodoridis G.V.
      Hellenic association for the study of the liver clinical practice guidelines: autoimmune hepatitis.
      ,
      • Gatselis N.K.
      • Zachou K.
      • Koukoulis G.K.
      • Dalekos G.N.
      Autoimmune hepatitis, one disease with many faces: etiopathogenetic, clinico-laboratory and histological characteristics.
      ,
      • Hennes E.M.
      • Zeniya M.
      • Czaja A.J.
      • Parés A.
      • Dalekos G.N.
      • Krawitt E.L.
      • et al.
      Simplified criteria for the diagnosis of autoimmune hepatitis.
      ,
      • Dalekos G.N.
      • Samakidou A.
      • Lyberopoulou A.
      • Banakou E.
      • Gatselis N.K.
      Recent advances in the diagnosis and management of autoimmune hepatitis.
      ,
      • Vergani D.
      • Alvarez F.
      • Bianchi F.B.
      • Cançado E.L.
      • Mackay I.R.
      • Manns M.P.
      • et al.
      Liver autoimmune serology: a consensus statement from the committee for autoimmune serology of the International Autoimmune Hepatitis Group.
      ,
      • Terziroli Beretta-Piccoli B.
      • Mieli-Vergani G.
      • Vergani D.
      Serology in autoimmune hepatitis: a clinical-practice approach.
      ]. The target-autoantigen of anti-SLA/LP has been identified as a synthase (S) converting O-phosphoseryl-tRNA (Sep) to selenocysteinyl-tRNA (Sec) (Table 2) [
      • Palioura S.
      • Sherrer R.L.
      • Steitz T.A.
      • Söll D.
      • Simonovic M.
      The human SepSecS-tRNASec complex reveals the mechanism of selenocysteine formation.
      ]. These antibodies have very high diagnostic accuracy for the diagnosis of AIH [
      • Dalekos G.N.
      • Samakidou A.
      • Lyberopoulou A.
      • Banakou E.
      • Gatselis N.K.
      Recent advances in the diagnosis and management of autoimmune hepatitis.
      ,
      • Herkel J.
      • Heidrich B.
      • Nieraad N.
      • Wies I.
      • Rother M.
      • Lohse A.W.
      Fine specificity of autoantibodies to soluble liver antigen and liver/pancreas.
      ,
      • Ballot E.
      • Homberg J.C.
      • Johanet C.
      Antibodies to soluble liver antigen: an additional marker in type 1 autoimmune hepatitis.
      ,
      • Baeres M.
      • Herkel J.
      • Czaja A.J.
      • Wies I.
      • Kanzler S.
      • Cancado E.L.
      • et al.
      Establishment of standardised SLA/LP immunoassays: specificity for autoimmune hepatitis, worldwide occurrence, and clinical characteristics.
      ,
      • Zhang W.C.
      • Zhao F.R.
      • Chen J.
      • Chen W.X.
      Meta-analysis: diagnostic accuracy of antinuclear antibodies, smooth muscle antibodies and antibodies to a soluble liver antigen/liver pancreas in autoimmune hepatitis.
      ]. Recent studies in large cohorts of patients have shown that seropositivity for anti-SLA/LP was not associated with the clinical, serological, biochemical, or histological features of AIH patients [
      • Zachou K.
      • Gampeta S.
      • Gatselis N.K.
      • Oikonomou K.
      • Goulis J.
      • Manoussakis M.N.
      • et al.
      Anti-SLA/LP alone or in combination with anti-Ro52 and fine specificity of anti-Ro52 antibodies in patients with autoimmune hepatitis.
      ,
      • Kirstein M.M.
      • Metzler F.
      • Geiger E.
      • Heinrich E.
      • Hallensleben M.
      • Manns M.P.
      • et al.
      Prediction of short- and long-term outcome in patients with autoimmune hepatitis.
      ,
      • Zachou K.
      • Weiler-Normann C.
      • Muratori L.
      • Muratori P.
      • Lohse A.W.
      • Dalekos G.N.
      Permanent immunosuppression in SLA/LP-positive autoimmune hepatitis is required although overall response and survival are similar.
      ]. In addition, overall treatment response and survival were identical between anti-SLA/LP-positive and anti-SLA-negative AIH patients [
      • Zachou K.
      • Gampeta S.
      • Gatselis N.K.
      • Oikonomou K.
      • Goulis J.
      • Manoussakis M.N.
      • et al.
      Anti-SLA/LP alone or in combination with anti-Ro52 and fine specificity of anti-Ro52 antibodies in patients with autoimmune hepatitis.
      ,
      • Zachou K.
      • Weiler-Normann C.
      • Muratori L.
      • Muratori P.
      • Lohse A.W.
      • Dalekos G.N.
      Permanent immunosuppression in SLA/LP-positive autoimmune hepatitis is required although overall response and survival are similar.
      ]. However, anti-SLA/LP-positive patients have lower sustained rates of biochemical response after complete cessation of treatment suggesting that rather permanent immunosuppression is needed in this subgroup of AIH patients [
      • Zachou K.
      • Weiler-Normann C.
      • Muratori L.
      • Muratori P.
      • Lohse A.W.
      • Dalekos G.N.
      Permanent immunosuppression in SLA/LP-positive autoimmune hepatitis is required although overall response and survival are similar.
      ].
      Another important point is that anti-SLA/LP is associated with the concurrent detection of autoantibodies against to the ribonucleoprotein/Sjogren's syndrome A antigen (anti-Ro/SSA) [
      • Zachou K.
      • Gampeta S.
      • Gatselis N.K.
      • Oikonomou K.
      • Goulis J.
      • Manoussakis M.N.
      • et al.
      Anti-SLA/LP alone or in combination with anti-Ro52 and fine specificity of anti-Ro52 antibodies in patients with autoimmune hepatitis.
      ,
      • Liaskos C.
      • Bogdanos D.P.
      • Rigopoulou E.I.
      • Norman G.L.
      • Shurns Z.
      • Al-Chalabi T.
      • et al.
      Antibody responses specific for soluble liver antigen co-occur with Ro-52 autoantibodies in patients with autoimmune hepatitis.
      ,
      • Eyraud V.
      • Chazouilleres O.
      • Ballot E.
      • Corpechot C.
      • Poupon R.
      • Johanet C.
      Significance of antibodies to soluble liver antigen/liver pancreas: a large French study.
      ,
      • Montano-Loza A.J.
      • Shums Z.
      • Norman G.L.
      • Czaja A.J.
      Prognostic implications of antibodies to Ro/SSA and soluble liver antigen in type 1 autoimmune hepatitis.
      ,
      • Van der Woude F.J.
      • Rasmussen N.
      • Lobatto S.
      • Wiik A.
      • Permin H.
      • van Es L.A.
      • et al.
      Autoantibodies against neutrophils and monocytes: tool for diagnosis and marker of disease activity in Wegener's granulomatosis.
      ]. Indeed 77–98% of anti-SLA/LP-positive European and North American AIH patients have also anti-Ro52 antibodies which is not a result of cross-reactivity [
      • Liaskos C.
      • Bogdanos D.P.
      • Rigopoulou E.I.
      • Norman G.L.
      • Shurns Z.
      • Al-Chalabi T.
      • et al.
      Antibody responses specific for soluble liver antigen co-occur with Ro-52 autoantibodies in patients with autoimmune hepatitis.
      ]. From the clinical and diagnostic points of view, the abovementioned findings seem very important as the clinician could suspect the presence of underlying AIH in a patient with unexplained hepatitis and anti-Ro52 antibodies by searching for the potential concurrent presence of anti-SLA/LP in case this testing has not been done (Table 2).

      2.3 Anti-neutrophil cytoplasmic antibodies (ANCA)

      These autoantibodies have been originally described by IIF on fixed granulocytes in patients with granulomatosis with polyangiitis (typically associated with diffuse cytoplasmic granular fluorescence; cANCA; major target-autoantigen: proteinase 3; Fig. 4A) or microscopic polyangiitis and eosinophilic granulomatosis (typically associated with perinuclear staining pattern; pANCA; major target-autoantigen: myeloperoxidase) [
      • Salvador F.
      ANCA associated vasculitis.
      ,
      • Lindgren S.
      • Nilsson S.
      • Nassberger L.
      • Verbaan H.
      • Wieslander J.
      Anti-neutrophil cytoplasmic antibodies in patients with chronic liver diseases: prevalence, antigen specificity and predictive value for diagnosis of autoimmune liver disease. Swedish Internal Medicine Liver Club (SILK).
      ]. The latter staining pattern is an artifact because of the ethanol fixation of granulocytes, which results in the migration of positive cytoplasmic proteins to the negative nuclear cell membrane. For these reasons, both formalin and ethanol fixed granulocytes should be used in ANCA investigation. If the pattern of staining is not affected by ethanol fixation, the result should be given as “atypical” pANCA (Fig. 4B), or with the more appropriate term “perinuclear antineutrophil nuclear antibody” (pANNA; positive cut-off titre 1:20; Fig. 4C; Table 3).
      Fig 4
      Fig. 4Anti-neutrophil cytoplasmic antibodies (ANCA) by indirect immunofluorescence on ethanol fixed human granulocytes: (A) Granular cytoplasmic staining (cANCA); (B) “Atypical” perinuclear staining (pANCA); (C) Anti-neutrophil nuclear antibody staining (ANNA). Original magnification x 40.
      Table 3Update of the simplified diagnostic criteria of the International AIH Group [adapted from 60].
      FeatureCut-offPoints1
      ANA or SMA/anti-F-actinPositive21
      ANA or SMA/anti-F-actinStrongly positive3
      or anti-LKM≥1:402
      or anti-SLA/LPPositive
      IgG>Upper limit of normal1
      >1.1x upper limit of normal2
      Liver histology (with evidence of hepatitis)Compatible with AIH1
      Typical AIH2
      Absence of viral hepatitisYes2
      ≥6: probable AIH
      ≥7: definite AIH
      Abbreviations are same as in the text. 1Addition of points achieved (maximum 2 points for autoantibodies). 2Indirect immunofluorescence: ≥1:40 when assessed on tissue sections; ≥1:80 or 1:160 for ANA when assessed on HEp-2 cells, depending on local standards. ELISA with locally established cut-offs. 3Indirect immunofluorescence: ≥1:80 when assessed on tissue sections; ≥1:160 or 1:320 for ANA when assessed on HEp-2 cells. ELISA with cut-offs established locally; Important note: If ELISA-based autoantibody assessment is negative despite clinical suspicion of autoimmune hepatitis, indirect immunofluorescence should be performed in addition.
      Searching for specific autoantibodies against proteinase 3 and myeloperoxidase by molecular-based assays has not any sense in the investigation of an index patient with suspected AIH and detectable pANCA/ANNA. Neither the determination of antigenic specificities of pANCA/ANNA seems to be important as the clinical relevance of such investigation in AIH patients is obscure [
      • Lindgren S.
      • Nilsson S.
      • Nassberger L.
      • Verbaan H.
      • Wieslander J.
      Anti-neutrophil cytoplasmic antibodies in patients with chronic liver diseases: prevalence, antigen specificity and predictive value for diagnosis of autoimmune liver disease. Swedish Internal Medicine Liver Club (SILK).
      ]. The prevalence of pANCA/ANNA in AIH cases (mostly of AIH-1) is 60–96% [
      • Vergani D.
      • Alvarez F.
      • Bianchi F.B.
      • Cançado E.L.
      • Mackay I.R.
      • Manns M.P.
      • et al.
      Liver autoimmune serology: a consensus statement from the committee for autoimmune serology of the International Autoimmune Hepatitis Group.
      ,
      • Terziroli Beretta-Piccoli B.
      • Mieli-Vergani G.
      • Vergani D.
      Serology in autoimmune hepatitis: a clinical-practice approach.
      ,
      • Zauli D.
      • Ghetti S.
      • Grassi A.
      • Descovich C.
      • Cassani F.
      • Ballardini G.
      • et al.
      Anti-neutrophil cytoplasmic antibodies in type 1 and 2 autoimmune hepatitis.
      ]. However, very few AIH-1 patients have isolated pANCA/ANNA and therefore, this autoantibody should be tested only in patients who are negative for ANA, SMA, and anti-SLA/LP. pANCA/ANNA has also been reported frequently in other immune-mediated diseases such as, primary sclerosing cholangitis (PSC), inflammatory bowel disease and autoimmune sclerosing cholangitis a specific AIH/PSC variant in children (Table 2) [
      • Dalekos G.N.
      • Manoussakis M.N.
      • Goussia A.C.
      • Tsianos E.V.
      • Moutsopoulos H.M.
      Soluble interleukin-2 receptors, antineutrophil cytoplasmic antibodies and other autoantibodies in patients with ulcerative colitis.
      ,
      • Hov J.R.
      • Boberg K.M.
      • Taraldsrud E.
      • Vesterhus M.
      • Boyadzhieva M.
      • Solberg I.C.
      • et al.
      Antineutrophil antibodies define clinical and genetic subgroups in primary sclerosing cholangitis.
      ,
      • Di Giorgio A.
      • Hadzic N.
      • Dhawan A.
      • Deheragoda M.
      • Heneghan M.A.
      • Vergani D.
      • et al.
      Seamless management of juvenile autoimmune liver disease: long-term medical and social outcome.
      ].

      2.4 Liver kidney microsomal (anti-LKM) and liver cytosol type 1 antibodies (anti-LC1)

      Anti-LKM antibodies include three isoforms: anti-LKM1, anti–liver kidney microsomal type2 (anti-LKM2), and anti-LKM3 [
      • Gatselis N.K.
      • Zachou K.
      • Koukoulis G.K.
      • Dalekos G.N.
      Autoimmune hepatitis, one disease with many faces: etiopathogenetic, clinico-laboratory and histological characteristics.
      ,
      • Dalekos G.N.
      • Samakidou A.
      • Lyberopoulou A.
      • Banakou E.
      • Gatselis N.K.
      Recent advances in the diagnosis and management of autoimmune hepatitis.
      ,
      • Vergani D.
      • Alvarez F.
      • Bianchi F.B.
      • Cançado E.L.
      • Mackay I.R.
      • Manns M.P.
      • et al.
      Liver autoimmune serology: a consensus statement from the committee for autoimmune serology of the International Autoimmune Hepatitis Group.
      ,
      • Terziroli Beretta-Piccoli B.
      • Mieli-Vergani G.
      • Vergani D.
      Serology in autoimmune hepatitis: a clinical-practice approach.
      ,
      • Czaja A.J.
      Autoantibodies in autoimmune liver disease.
      ]. Anti-LKM1 and less commonly anti-LKM3 define AIH-2, whereas anti-LKM2 antibodies are associated with DILI due to tienilic acid and never with AIH (Table 2) [
      • Gatselis N.K.
      • Zachou K.
      • Koukoulis G.K.
      • Dalekos G.N.
      Autoimmune hepatitis, one disease with many faces: etiopathogenetic, clinico-laboratory and histological characteristics.
      ,
      • Dalekos G.N.
      • Samakidou A.
      • Lyberopoulou A.
      • Banakou E.
      • Gatselis N.K.
      Recent advances in the diagnosis and management of autoimmune hepatitis.
      ,
      • Vergani D.
      • Alvarez F.
      • Bianchi F.B.
      • Cançado E.L.
      • Mackay I.R.
      • Manns M.P.
      • et al.
      Liver autoimmune serology: a consensus statement from the committee for autoimmune serology of the International Autoimmune Hepatitis Group.
      ,
      • Terziroli Beretta-Piccoli B.
      • Mieli-Vergani G.
      • Vergani D.
      Serology in autoimmune hepatitis: a clinical-practice approach.
      ,
      • Czaja A.J.
      Autoantibodies in autoimmune liver disease.
      ,
      • Homberg J.C.
      • Andre C.
      • Abuaf N.
      A new anti-liver-kidney microsome antibody (anti-LKM2) in tienilic acid-induced hepatitis.
      ]. On fresh rodent tissue sections, anti-LKM1 shows by IIF a restricted staining of the third portion of the proximal renal tubules (Fig. 5A). Besides, anti-LKM1 stain the cytoplasm of the complete liver lobule (Fig. 5B). Anti-LKM1 are easily distinguished from anti-mitochondrial antibodies (AMA) as the latter antibodies stain both the proximal and distal renal tubules. In any case, if there are problems regarding the discrimination between AMA and anti-LKM by IIF, these should be resolved by using complementary molecular-based assays.
      Fig 5
      Fig. 5Liver kidney microsomal antibodies type 1 (anti-LKM1) and liver cytosol type 1 antibodies (anti-LC1) on fresh rodent tissue sections by indirect immunofluorescence. Anti-LKM1 shows (A) a restricted staining of the P3 segment of the proximal tubules sparing the distal tubules and (B) a diffuse staining of the cytoplasm of hepatocytes in the entire liver lobule. (C) Anti-LC1 exhibits a pattern of cytoplasmic staining of the periportal hepatocytes while sparing the area around the central veins. Original magnification × 40.
      The main target-autoantigen of anti-LKM1 is the cytochrome P450 2D6, which allowed the development of specific molecular-based assays for anti-LKM1 detection, such as ELISA and immunoblot. Interestingly, about 10% of patients with chronic hepatitis C but not with hepatitis B, have detectable anti-LKM1 [
      • Dalekos G.N.
      • Samakidou A.
      • Lyberopoulou A.
      • Banakou E.
      • Gatselis N.K.
      Recent advances in the diagnosis and management of autoimmune hepatitis.
      ,
      • Vergani D.
      • Alvarez F.
      • Bianchi F.B.
      • Cançado E.L.
      • Mackay I.R.
      • Manns M.P.
      • et al.
      Liver autoimmune serology: a consensus statement from the committee for autoimmune serology of the International Autoimmune Hepatitis Group.
      ,
      • Terziroli Beretta-Piccoli B.
      • Mieli-Vergani G.
      • Vergani D.
      Serology in autoimmune hepatitis: a clinical-practice approach.
      ,
      • Dalekos G.N.
      • Wedemeyer H.
      • Obermayer-Straub P.
      • Kayser A.
      • Barut A.
      • Frank H.
      • et al.
      Epitope mapping of cytochrome P4502D6 autoantigen in patients with chronic hepatitis C during alpha-interferon treatment.
      ,
      • Dalekos G.N.
      • Makri E.
      • Loges S.
      • Obermayer-Straub P.
      • Zachou K.
      • Tsikrikas T.
      • et al.
      Increased incidence of anti-LKM autoantibodies in a consecutive cohort of hepatitis C patients from central Greece.
      ].
      Although anti-LKM3 antibodies have been initially described in up to 13% of patients with chronic hepatitis delta [
      • Crivelli O.
      • Lavarini C.
      • Chiaberge E.
      • Amoroso A.
      • Farci P.
      • Negro F.
      • et al.
      Microsomal autoantibodies in chronic infection with the HBsAg associated delta (delta) agent.
      ], they have also been reported in about 5–10% of AIH-2 patients [
      • Czaja A.J.
      Autoantibodies in autoimmune liver disease.
      ,
      • Strassburg C.P.
      • Obermayer-Straub P.
      • Alex B.
      • Durazzo M.
      • Rizzetto M.
      • Tukey R.H.
      • et al.
      Autoantibodies against glucuronosyltransferases differ between viral hepatitis and autoimmune hepatitis.
      ]. The major target-autoantigens of anti-LKM3 are members of the family 1 uridine diphosphate glucuronosyl-transferases (Table 2) [
      • Strassburg C.P.
      • Obermayer-Straub P.
      • Alex B.
      • Durazzo M.
      • Rizzetto M.
      • Tukey R.H.
      • et al.
      Autoantibodies against glucuronosyltransferases differ between viral hepatitis and autoimmune hepatitis.
      ].
      Anti-LC1 antibodies are detected in about one-third of AIH-2 patients (coexistence with anti-LKM1 in approximately 50% of cases) [
      • Dalekos G.N.
      • Samakidou A.
      • Lyberopoulou A.
      • Banakou E.
      • Gatselis N.K.
      Recent advances in the diagnosis and management of autoimmune hepatitis.
      ,
      • Vergani D.
      • Alvarez F.
      • Bianchi F.B.
      • Cançado E.L.
      • Mackay I.R.
      • Manns M.P.
      • et al.
      Liver autoimmune serology: a consensus statement from the committee for autoimmune serology of the International Autoimmune Hepatitis Group.
      ,
      • Terziroli Beretta-Piccoli B.
      • Mieli-Vergani G.
      • Vergani D.
      Serology in autoimmune hepatitis: a clinical-practice approach.
      ,
      • Martini E.
      • Abuaf N.
      • Cavalli F.
      • Durand V.
      • Johanet C.
      • Homberg J.C.
      Antibody to liver cytosol (anti-LC1) in patients with autoimmune chronic active hepatitis type 2.
      ,
      • Abuaf N.
      • Johanet C.
      • Chretien P.
      • Martini E.
      • Soulier E.
      • Laperche S.
      • et al.
      Characterization of the liver cytosol antigen type 1 reacting with autoantibodies in chronic active hepatitis.
      ]. In about 10% of AIH-2 patients, anti-LC1 autoantibodies are detected solely, so it is mandatory to be included at first-line screening in the diagnostic algorithm of suspected cases (Fig. 2) [
      • Abuaf N.
      • Johanet C.
      • Chretien P.
      • Martini E.
      • Soulier E.
      • Laperche S.
      • et al.
      Characterization of the liver cytosol antigen type 1 reacting with autoantibodies in chronic active hepatitis.
      ]. Anti-LC1 exhibits by IIF a cytoplasmic staining pattern of the periportal hepatocytes on fresh rodent tissue sections, sparing typically the area around the central veins (Fig. 5C) [
      • Martini E.
      • Abuaf N.
      • Cavalli F.
      • Durand V.
      • Johanet C.
      • Homberg J.C.
      Antibody to liver cytosol (anti-LC1) in patients with autoimmune chronic active hepatitis type 2.
      ,
      • Abuaf N.
      • Johanet C.
      • Chretien P.
      • Martini E.
      • Soulier E.
      • Laperche S.
      • et al.
      Characterization of the liver cytosol antigen type 1 reacting with autoantibodies in chronic active hepatitis.
      ]. As a result, in cases of anti-LC1 coexistence with anti-LKM1 antibodies, IIF fails to detect them because anti-LKM1 stains the cytoplasm of hepatocytes in the entire liver lobule (Fig. 5B). Therefore, additional methods like immunodiffusion, ELISA or immunoblot are required [
      • Martini E.
      • Abuaf N.
      • Cavalli F.
      • Durand V.
      • Johanet C.
      • Homberg J.C.
      Antibody to liver cytosol (anti-LC1) in patients with autoimmune chronic active hepatitis type 2.
      ,
      • Abuaf N.
      • Johanet C.
      • Chretien P.
      • Martini E.
      • Soulier E.
      • Laperche S.
      • et al.
      Characterization of the liver cytosol antigen type 1 reacting with autoantibodies in chronic active hepatitis.
      ,
      • Muratori L.
      • Cataleta M.
      • Muratori P.
      • Manotti P.
      • Lenzi M.
      • Cassani F.
      • et al.
      Detection of anti-liver cytosol antibody type 1 (anti-LC1) by immunodiffusion, counterimmunoelectrophoresis and immunoblotting: comparison of different techniques.
      ]. The formiminotransferase cyclodeaminase, a liver enzyme involved in folate metabolism, has already been documented as the molecular target of anti-LC1 [
      • Lapierre P.
      • Hajoui O.
      • Homberg J.C.
      • Alvarez F.
      Formiminotransferase cyclodeaminase is an organ-specific autoantigen recognized by sera of patients with autoimmune hepatitis.
      ]. According to the current guidelines, IIF titers ≥1:40 in adults and ≥1:10 in children are considered positive for both anti-LKM1 and anti-LC1 [
      European Association for the Study of the Liver
      EASL clinical practice guidelines: autoimmune hepatitis.
      ,
      • Dalekos G.N.
      • Koskinas J.
      • Papatheodoridis G.V.
      Hellenic association for the study of the liver clinical practice guidelines: autoimmune hepatitis.
      ,
      • Hennes E.M.
      • Zeniya M.
      • Czaja A.J.
      • Parés A.
      • Dalekos G.N.
      • Krawitt E.L.
      • et al.
      Simplified criteria for the diagnosis of autoimmune hepatitis.
      ,
      • Mieli-Vergani G.
      • Vergani D.
      • Baumann U.
      • Czubkowski P.
      • Debray D.
      • Dezsofi A.
      • et al.
      Diagnosis and management of pediatric autoimmune liver disease: ESPGHAN hepatology committee position statement.
      ].

      3. Diagnostic application in patients with deranged liver enzymes of unknown aetiology

      The investigation of autoantibodies remains the hallmark for AIH diagnosis, even though they are not pathognomonic and cannot support a definite diagnosis on their own. A step-by-step serological investigation which should be applied in patients with deranged liver enzymes of unexplained cause is shown in Fig. 2. It should be noted however, that in cases with acute severe form of the disease (jaundice, international normalized ratio≥1.5, no hepatic encephalopathy, no previously recognized liver disease) a diagnostic trial with oral or intravenous corticosteroids is rather justified before obtaining the results of autoimmune serology and liver biopsy [
      • Zachou K.
      • Arvaniti P.
      • Azariadis K.
      • Lygoura V.
      • Gatselis N.K.
      • Lyberopoulou A.
      • et al.
      Prompt initiation of high-dose i.v. corticosteroids seem to prevent progression to liver failure in patients with original acute severe autoimmune hepatitis.
      ,
      • Dalekos G.N.
      • Gatselis N.K.
      • Zachou K.
      Acute severe autoimmune hepatitis: corticosteroids or liver transplantation?.
      ]. Furthermore, it should be clear that in cases of suspected AIH with acute liver failure (which includes by definition the presence of hepatic encephalopathy), the patients should be immediately listed for liver transplantation even though an autoimmune serology testing could be in process [
      European Association for the Study of the Liver
      EASL clinical practice guidelines: autoimmune hepatitis.
      ,
      • Dalekos G.N.
      • Koskinas J.
      • Papatheodoridis G.V.
      Hellenic association for the study of the liver clinical practice guidelines: autoimmune hepatitis.
      ].
      ANA, SMA, anti-LKM-1 and anti-LC1 antibodies must be investigated first by IIF on fresh-frozen substrates from combined rodent liver, stomach, and kidney sections ideally before treatment initiation as immunosuppression may affect the results. In parallel, investigation for anti-SLA/LP antibodies by molecular-based assays (ELISAs and/or western blot) should be done (Fig. 2). Reactivity for one or more of these autoantibodies, in conjunction with the appropriate clinical background, suggests proceeding to liver biopsy, as AIH is highly likely [
      • Lohse A.W.
      • Sebode M.
      • Bhathal P.S.
      • Clouston A.D.
      • Dienes H.P.
      • Jain D.
      • et al.
      Consensus recommendations for histological criteria of autoimmune hepatitis from the International AIH Pathology Group: results of a workshop on AIH histology hosted by the European Reference Network on Hepatological Diseases and the European Society of Pathology: results of a workshop on AIH histology hosted by the European Reference Network on Hepatological Diseases and the European Society of Pathology.
      ]. Absence of reactivity besides clinical suspicion, raises the justification for additional specific searching and repeated investigation for ANA, SMA, anti-LKM-1, anti-LC1 and anti-SLA/LP and non-standard autoantibodies in a reference laboratory (Fig. 2). Insistence for second testing is further supported if there are concurrent extra-hepatic autoimmune diseases in the patient or her/his first-degree relatives [
      • Fogel R.
      • Comerford M.
      • Chilukuri P.
      • Orman E.
      • Chalasani N.
      • Lammert C.
      Extrahepatic autoimmune diseases are prevalent in autoimmune hepatitis patients and their first-degree relatives: survey study.
      ]. Positivity during second investigation suggests proceeding to liver biopsy, as AIH is highly likely or possible [
      • Lohse A.W.
      • Sebode M.
      • Bhathal P.S.
      • Clouston A.D.
      • Dienes H.P.
      • Jain D.
      • et al.
      Consensus recommendations for histological criteria of autoimmune hepatitis from the International AIH Pathology Group: results of a workshop on AIH histology hosted by the European Reference Network on Hepatological Diseases and the European Society of Pathology: results of a workshop on AIH histology hosted by the European Reference Network on Hepatological Diseases and the European Society of Pathology.
      ]. Absence of reactivity after the second investigation means that AIH is very unlikely and other diagnosis should be considered or seronegative AIH is present. It is worthy to state here, that the above thorough serological investigation could be engaged even for patients with other liver disorders if there is distinct IgG elevation and an autoimmune extra-hepatic background because coexistence of AIH with another liver condition cannot be excluded [
      • Rigopoulou E.I.
      • Zachou K.
      • Gatselis N.
      • Koukoulis G.K.
      • Dalekos G.N.
      Autoimmune hepatitis in patients with chronic HBV and HCV infections: patterns of clinical characteristics, disease progression and outcome.
      ,
      • Beisel C.
      • Weiler-Normann C.
      • Teufel A.
      • Lohse A.W.
      Association of autoimmune hepatitis and systemic lupus erythematodes: a case series and review of the literature.
      ,
      • Rigopoulou E.I.
      • Gyftaki S.
      • Arvaniti P.
      • Tsimourtou V.
      • Koukoulis G.K.
      • Hadjigeorgiou G.
      • et al.
      Autoimmune hepatitis in patients with multiple sclerosis: the role of immunomodulatory treatment.
      ,
      • Dalekos G.N.
      • Gatselis N.K.
      • Zachou K.
      • Koukoulis G.K.
      NAFLD and autoimmune hepatitis: do not judge a book by its cover.
      ,
      • Rigopoulou E.I.
      • Gatselis N.
      • Arvaniti P.
      • Koukoulis G.K.
      • Dalekos G.N.
      Alcoholic liver disease and autoimmune hepatitis: sometimes a closer look under the surface is needed.
      ].
      The routine clinical laboratories should adhere to the guidelines both regarding the assays they use as well as the cut-off they consider for positivity and this information should be provided clearly to the clinicians to assist them achieving the appropriate interpretation of the results [
      European Association for the Study of the Liver
      EASL clinical practice guidelines: autoimmune hepatitis.
      ,
      • Dalekos G.N.
      • Koskinas J.
      • Papatheodoridis G.V.
      Hellenic association for the study of the liver clinical practice guidelines: autoimmune hepatitis.
      ,
      • Gatselis N.K.
      • Zachou K.
      • Koukoulis G.K.
      • Dalekos G.N.
      Autoimmune hepatitis, one disease with many faces: etiopathogenetic, clinico-laboratory and histological characteristics.
      ,
      • Hennes E.M.
      • Zeniya M.
      • Czaja A.J.
      • Parés A.
      • Dalekos G.N.
      • Krawitt E.L.
      • et al.
      Simplified criteria for the diagnosis of autoimmune hepatitis.
      ,
      • Dalekos G.N.
      • Samakidou A.
      • Lyberopoulou A.
      • Banakou E.
      • Gatselis N.K.
      Recent advances in the diagnosis and management of autoimmune hepatitis.
      ,
      • Vergani D.
      • Alvarez F.
      • Bianchi F.B.
      • Cançado E.L.
      • Mackay I.R.
      • Manns M.P.
      • et al.
      Liver autoimmune serology: a consensus statement from the committee for autoimmune serology of the International Autoimmune Hepatitis Group.
      ,
      • Terziroli Beretta-Piccoli B.
      • Mieli-Vergani G.
      • Vergani D.
      Serology in autoimmune hepatitis: a clinical-practice approach.
      ,
      • Galaski J.
      • Weiler-Normann C.
      • Schakat M.
      • Zachou K.
      • Muratori P.
      • Lampalzer S.
      • et al.
      Update of the simplified criteria for autoimmune hepatitis: Evaluation of the methodology for immunoserological testing.
      ]. In this context, it is pivotal the laboratory to report all renal reactivity patterns of SMA, as SMA-VG/VGT patterns have the highest specificity for AIH diagnosis.

      3.1 Common difficult clinical questions

      My lab does not perform IIF testing on triple rodent substrates but relies on IIF on HEp-2 cells and/or ELISAs for ANA and SMA detection. How shall I manage this difficulty?
      It should be emphasized that the original simplified score (Supplementary Table 1) does not account for ANA or SMA detection by IIF on the HEp-2 cell lines or by using ELISAs even though, these assays are indeed the preferable ways of screening in many routine laboratories because they are much easier and more friendly in everyday use. However, it is not clear how the results by using HEp2 cells and ELISAs may be converted into the simplified diagnostic score [
      • Gatselis N.K.
      • Zachou K.
      • Koukoulis G.K.
      • Dalekos G.N.
      Autoimmune hepatitis, one disease with many faces: etiopathogenetic, clinico-laboratory and histological characteristics.
      ,
      • Dalekos G.N.
      • Samakidou A.
      • Lyberopoulou A.
      • Banakou E.
      • Gatselis N.K.
      Recent advances in the diagnosis and management of autoimmune hepatitis.
      ,
      • Vergani D.
      • Alvarez F.
      • Bianchi F.B.
      • Cançado E.L.
      • Mackay I.R.
      • Manns M.P.
      • et al.
      Liver autoimmune serology: a consensus statement from the committee for autoimmune serology of the International Autoimmune Hepatitis Group.
      ]. In addition, investigation for ANA detection by ELISAs is not recommended as may result in false negative results in up to 20–30% of AIH patients [
      • Dalekos G.N.
      • Samakidou A.
      • Lyberopoulou A.
      • Banakou E.
      • Gatselis N.K.
      Recent advances in the diagnosis and management of autoimmune hepatitis.
      ,
      • Vergani D.
      • Alvarez F.
      • Bianchi F.B.
      • Cançado E.L.
      • Mackay I.R.
      • Manns M.P.
      • et al.
      Liver autoimmune serology: a consensus statement from the committee for autoimmune serology of the International Autoimmune Hepatitis Group.
      ,
      • Terziroli Beretta-Piccoli B.
      • Mieli-Vergani G.
      • Vergani D.
      Serology in autoimmune hepatitis: a clinical-practice approach.
      ]. This happens because most of these assays have been developed mainly for the investigation of patients with autoimmune rheumatic diseases and therefore, they utilize molecularly defined antigens only as substrate and not whole nuclear extracts which is very important for the investigation of suspected cases of AIH [
      • Galaski J.
      • Weiler-Normann C.
      • Schakat M.
      • Zachou K.
      • Muratori P.
      • Lampalzer S.
      • et al.
      Update of the simplified criteria for autoimmune hepatitis: Evaluation of the methodology for immunoserological testing.
      ,
      • Andrade L.E.C.
      • Klotz W.
      • Herold M.
      • Conrad K.
      • Ronnelid J.
      • Fritzler M.J.
      • et al.
      International consensus on antinuclear antibody patterns: definition of the AC-29 pattern associated with antibodies to DNA topoisomerase I.
      ].
      Moreover, the use of F-actin ELISA for SMA detection can lose about 20% of cases, as SMA do not target F-actin exclusively, and therefore, anti-F-actin molecular assays should only be used in conjunction with IIF [
      • Dalekos G.N.
      • Samakidou A.
      • Lyberopoulou A.
      • Banakou E.
      • Gatselis N.K.
      Recent advances in the diagnosis and management of autoimmune hepatitis.
      ,
      • Vergani D.
      • Alvarez F.
      • Bianchi F.B.
      • Cançado E.L.
      • Mackay I.R.
      • Manns M.P.
      • et al.
      Liver autoimmune serology: a consensus statement from the committee for autoimmune serology of the International Autoimmune Hepatitis Group.
      ,
      • Czaja A.J.
      • Cassani F.
      • Cataleta M.
      • Valentini P.
      • Bianchi F.B.
      Frequency and significance of antibodies to actin in type 1 autoimmune hepatitis.
      ,
      • Liaskos C.
      • Bogdanos D.P.
      • Davies E.T.
      • Dalekos G.N.
      Diagnostic relevance of anti-filamentous actin antibodies in autoimmune hepatitis.
      ]. To sum up, clinicians should be very careful and aware of these arbitrary assumptions of the laboratories as they may have dramatic consequences for the patients with suspected AIH.
      Of note, the IAIHG recently aimed to address these issues by comparing the performance of IIF on HEp-2 cells and tissue sections for the detection of ANA and SMA in AIH patients and controls from three European centres [
      • Galaski J.
      • Weiler-Normann C.
      • Schakat M.
      • Zachou K.
      • Muratori P.
      • Lampalzer S.
      • et al.
      Update of the simplified criteria for autoimmune hepatitis: Evaluation of the methodology for immunoserological testing.
      ]. The group also evaluated one commercial ELISA for SMA testing (anti-F-actin ELISA) and three different commercial ELISAs for ANA testing [
      • Galaski J.
      • Weiler-Normann C.
      • Schakat M.
      • Zachou K.
      • Muratori P.
      • Lampalzer S.
      • et al.
      Update of the simplified criteria for autoimmune hepatitis: Evaluation of the methodology for immunoserological testing.
      ]. It was concluded that, if ANA titres on HEp-2 cells adjust to higher thresholds (1/160 for positivity and 1/320 for strong positivity; Table 3), then this substrate could be used as a reliable alternative assay for ANA detection [
      • Galaski J.
      • Weiler-Normann C.
      • Schakat M.
      • Zachou K.
      • Muratori P.
      • Lampalzer S.
      • et al.
      Update of the simplified criteria for autoimmune hepatitis: Evaluation of the methodology for immunoserological testing.
      ].
      Regarding the various ELISA kits, it was shown that they can also be used as alternative assays for ANA and SMA but for ANA the use of total HEp-2 nuclear extracts should be applied in order to account for unrecognised nuclear autoantigens in addition to molecularly defined antigens, whereas for both ANA and SMA ELISAs there is a need for locally established cut-offs in each laboratory as the cut-offs of these commercial ELISAs have not been validated globally [
      • Galaski J.
      • Weiler-Normann C.
      • Schakat M.
      • Zachou K.
      • Muratori P.
      • Lampalzer S.
      • et al.
      Update of the simplified criteria for autoimmune hepatitis: Evaluation of the methodology for immunoserological testing.
      ]. The proposed updated simplified score which, however, needs validation is shown in Table 3.
      A special mention on screening by HEp-2 cells is needed for two specific ANA patterns namely, the multiple-nuclear dot and rim-like membranous patterns which are commonly seen in PBC patients (Fig. 6; Table 2) [
      • Invernizzi P.
      • Selmi C.
      • Ranftler C.
      • Podda M.
      • Wesierska-Gadek J.
      Antinuclear antibodies in primary biliary cirrhosis.
      ,
      European Association for the Study of the Liver. EASL Clinical Practice Guidelines: the diagnosis and management of patients with primary biliary cholangitis.
      ,
      • Gatselis N.K.
      • Dalekos G.N.
      Molecular diagnostic testing for primary biliary cholangitis.
      ]. These autoantibodies are considered specific for the diagnosis of PBC and have been associated with more severe disease [
      European Association for the Study of the Liver. EASL Clinical Practice Guidelines: the diagnosis and management of patients with primary biliary cholangitis.
      ,
      • Rigopoulou E.I.
      • Davies E.T.
      • Pares A.
      • Zachou K.
      • Liaskos C.
      • Bogdanos D.P.
      • et al.
      Prevalence and clinical significance of isotype specific antinuclear antibodies in primary biliary cirrhosis.
      ,
      • Gatselis N.K.
      • Zachou K.
      • Norman G.L.
      • Gabeta S.
      • Papamichalis P.
      • Koukoulis G.K.
      • et al.
      Clinical significance of the fluctuation of primary biliary cirrhosis-related autoantibodies during the course of the disease.
      ,
      • Nakamura M.
      • Kondo H.
      • Tanaka A.
      • Komori A.
      • Ito M.
      • Yamamoto K.
      • et al.
      Autoantibody status and histological variables influence biochemical response to treatment and long-term outcomes in Japanese patients with primary biliary cirrhosis.
      ]. The presence of PBC-specific ANA in AIH patients in conjunction with a cholestatic biochemical profile may suggest the diagnosis of AIH/PBC variant and therefore, liver biopsy should be carefully evaluated in these cases [
      European Association for the Study of the Liver
      EASL clinical practice guidelines: autoimmune hepatitis.
      ,
      • Dalekos G.N.
      • Koskinas J.
      • Papatheodoridis G.V.
      Hellenic association for the study of the liver clinical practice guidelines: autoimmune hepatitis.
      ,
      • Mack C.L.
      • Adams D.
      • Assis D.N.
      • Kerkar N.
      • Manns M.P.
      • Mayo M.J.
      • et al.
      Diagnosis and management of autoimmune hepatitis in adults and children: 2019 practice guidance and guidelines from the american association for the study of liver diseases.
      ].
      Fig 6
      Fig. 6Antinuclear antibodies specific for primary biliary cholangitis. Detection by indirect immunofluorescence on HEp-2 cells. (A) Multiple-nuclear dot staining pattern of 3–20 dots of variable size in the nucleus; (B) Rim-like membranous staining pattern of the nuclear membrane. Granular cytoplasmic staining is due to the presence of antimitochondrial antibodies. Original magnification x 40.
      My patient has high titre SMA but normal IgG, while ANA testing is also positive. What should I do?
      As it is shown in Fig. 2, serum IgG levels should be determined first as it is one of the four diagnostic criteria of AIH [
      • Hennes E.M.
      • Zeniya M.
      • Czaja A.J.
      • Parés A.
      • Dalekos G.N.
      • Krawitt E.L.
      • et al.
      Simplified criteria for the diagnosis of autoimmune hepatitis.
      ]. However, although a distinct increase of IgG characterizes most AIH patients, about 10–15% of chronic and up to 39% of the acute AIH cases may have indeed normal IgG at baseline [
      • Hartl J.
      • Miquel R.
      • Zachou K.
      • Wong G.W.
      • Asghar A.
      • Pape S.
      • et al.
      Features and outcome of AIH patients without elevation of IgG.
      ]. It should be emphasized however, that the “normal” range of IgG in routine laboratories is wide, as it is not practical to establish the “real normal ranges” of the respective population where an index subject with AIH is living. As a result, physicians may mistakenly disregard AIH from the differential diagnosis leading to delay of diagnosis with potential catastrophic consequences for the patients. Therefore, additional testing for autoantibodies is advised even in cases with “normal” IgG levels and if positive, liver biopsy should be performed to confirm or exclude AIH.
      It should be noted that even in patients with “normal” IgG, alterations of IgG could be used to monitor response to treatment, as a recent study has shown a considerable fall of IgG after immunosuppressive therapy, and a decline of IgG, sometimes below the lower limit of normal, in almost all AIH patients with “normal” IgG at diagnosis [
      • Hartl J.
      • Miquel R.
      • Zachou K.
      • Wong G.W.
      • Asghar A.
      • Pape S.
      • et al.
      Features and outcome of AIH patients without elevation of IgG.
      ].
      My patient has high titre ANA and anti-dsDNA antibodies but also moderate increase of transaminases for a long time with negative investigation for other liver disorders including viral hepatitis markers. Should I consider systemic lupus erythematosus (SLE) as the underlying diagnosis?
      It should be emphasized that seropositivity for ANA and anti-dsDNA antibodies should not lead the physicians to the “superficial diagnosis” of SLE if other criteria for its diagnosis are missing, because anti-dsDNA antibodies can also be detected in about 30% of AIH patients and up to 60% of patients with AIH/PBC variant [
      • Czaja A.J.
      • Morshed S.A.
      • Parveen S.
      • Nishioka M.
      Antibodies to single-stranded and double-stranded DNA in antinuclear antibody positive type 1-autoimmune hepatitis.
      ,
      • Pisetsky D.S.
      • Lipsky P.E.
      New insights into the role of antinuclear antibodies in systemic lupus erythematosus.
      ,
      • Granito A.
      • Muratori L.
      • Tovoli F.
      • Muratori P.
      Diagnostic role of anti-dsDNA antibodies: do not forget autoimmune hepatitis.
      ,
      • Muratori P.
      • Granito A.
      • Pappas G.
      • Pendino G.M.
      • Quarneti C.
      • Cicola R.
      • et al.
      The serological profile of the autoimmune hepatitis/primary biliary cirrhosis overlap syndrome.
      ]. Anti-dsDNA antibodies are usually detected by ELISAs or IIF on Crithidia luciliae substrate which incorporates high quantities of dsDNA and therefore, it permits a simple and easy way to detect them with higher specificity than the molecular-based ELISAs. As many AIH patients suffer from polyarthralgia syndrome without arthritis usually involving the small joints for years, it is reasonable for physicians to consider the diagnosis of SLE instead of AIH in an index patient with ANA and anti-dsDNA positivity (Table 2). However, involvement of the liver is not normally part of the clinical spectrum of SLE. Indeed, coincidence of viral hepatitis, non-alcoholic liver disease commonly induced by corticosteroids and DILI because of SLE-related therapies are the most frequent causes of deranged liver enzymes in patients with SLE [
      • Beisel C.
      • Weiler-Norman C.
      • Teufel A.
      • Lohse A.W.
      Association of autoimmune hepatitis and systemic lupus erythematodes: A case series and review of the literature.
      ].
      The key point for the suspicion of AIH in such cases is if the patients have abnormal transaminases along with high IgG serum levels and SMA with F-actin reactivity which direct towards AIH rather than SLE diagnosis. In this regard, liver biopsy will distinguish AIH from SLE as in the latter only non-specific findings are present [
      • Beisel C.
      • Weiler-Norman C.
      • Teufel A.
      • Lohse A.W.
      Association of autoimmune hepatitis and systemic lupus erythematodes: A case series and review of the literature.
      ]. From the clinical perspective, this misdiagnosis may have catastrophic consequences for the patients as we have seen a fair number of such cases who at initial diagnosis of AIH presented with advanced disease needing rescue therapies because of delayed diagnosis [
      • Dalekos G.N.
      • Azariadis K.
      • Lygoura V.
      • Arvaniti P.
      • Gampeta S.
      • Gatselis N.K.
      Autoimmune hepatitis in patients aged 70 years or older: disease characteristics, treatment response and outcome.
      ,
      • Rigopoulou E.I.
      • Dalekos G.
      • Bogdanos D.P.
      How common are connective tissue disorders in patients with autoimmune hepatitis?.
      ,
      • Dalekos G.N.
      • Arvaniti P.
      • Gatselis N.K.
      • et al.
      First results from a propensity matching trial of mycophenolate mofetil vs. azathioprine in treatment-naive AIH patients.
      ,
      • Arvaniti P.
      • Giannoulis G.
      • Gabeta S.
      • Zachou K.
      • Koukoulis G.K.
      • Dalekos G.N.
      Belimumab is a promising third-line treatment option for refractory autoimmune hepatitis.
      ,
      • Dalekos G.N.
      • Arvaniti P.
      • Gatselis N.K.
      • Gabeta S.
      • Samakidou A.
      • Giannoulis G.
      • et al.
      Long-term results of mycophenolate mofetil vs. azathioprine use in patients with autoimmune hepatitis.
      ].
      I performed all the serological tests at my patient. She/he has AIH with SMA but also AMA. What should I do?
      AMA, the laboratory hallmark for the diagnosis of PBC [
      • Gatselis N.K.
      • Dalekos G.N.
      Molecular diagnostic testing for primary biliary cholangitis.
      ], have been occasionally found in patients with other liver diseases including AIH (Table 2). Indeed, AMA detection has been reported in about 5–10% (up to 35% in some Japanese studies) of typical cases of AIH without any evidence of cholestatic disease. The clinical significance of this finding is currently enigmatic. Up to the present, most studies agree that AMA presence in patients with AIH is not corresponded to a specific subgroup of AIH patients requiring different therapeutic options or to a rapid development of PBC characteristics during follow-up [
      • Nezu S.
      • Tanaka A.
      • Yasui H.
      • Imamura M.
      • Nakajima H.
      • Ishida H.
      • et al.
      Presence of antimitochondrial autoantibodies in patients with autoimmune hepatitis.
      ,
      • O'Brien C.
      • Joshi S.
      • Feld J.J.
      • Guindi M.
      • Dienes H.P.
      • Heathcote E.J.
      Long-term follow-up of antimitochondrial antibody-positive autoimmune hepatitis.
      ,
      • Montano-Loza A.J.
      • Carpenter H.A.
      • Czaja A.J.
      Frequency, behavior, and prognostic implications of antimitochondrial antibodies in type 1 autoimmune hepatitis.
      ,
      • Liaskos C.
      • Bogdanos D.P.
      • Rigopoulou E.I.
      • Dalekos G.N.
      Development of antimitochondrial antibodies in patients with autoimmune hepatitis: art of facts or an artifact?.
      ,
      • Muratori P.
      • Efe C.
      • Muratori L.
      • Ozaslan E.
      • Schiano T.
      • Yoshida E.M.
      • et al.
      Clinical implications of antimitochondrial antibody seropositivity in autoimmune hepatitis: a multicentre study.
      ,
      • Dinani A.M.
      • Fischer S.E.
      • Mosko J.
      • Guindi M.
      • Hirschfield G.M.
      Patients with autoimmune hepatitis who have antimitochondrial antibodies need long-term follow-up to detect late development of primary biliary cirrhosis.
      ]. The limitations of short follow-up, infrequent sequential histologic examination and the small numbers of patients included in these studies may have led to the abovementioned inconclusive results. From the clinical point of view, clinicians should be aware of this phenomenon and manage their AIH patients with AMA reactivity as in those without if cholestatic indices are not present both at the biochemical and histological level.

      4. Concluding remarks and future perspectives

      AIH diagnosis is still challenging for physicians, as there is not a single test to diagnose or exclude the disease while its presentation is highly heterogeneous both at clinical and the serological level. Therefore, AIH should be absolutely considered in the differential diagnosis of all subjects with acute or chronic hepatitis of any severity irrespective of sex, age, or ethnicity. The precise guidelines for autoantibodies investigation [
      European Association for the Study of the Liver
      EASL clinical practice guidelines: autoimmune hepatitis.
      ,
      • Dalekos G.N.
      • Koskinas J.
      • Papatheodoridis G.V.
      Hellenic association for the study of the liver clinical practice guidelines: autoimmune hepatitis.
      ,
      • Gatselis N.K.
      • Zachou K.
      • Koukoulis G.K.
      • Dalekos G.N.
      Autoimmune hepatitis, one disease with many faces: etiopathogenetic, clinico-laboratory and histological characteristics.
      ,
      • Dalekos G.N.
      • Azariadis K.
      • Lygoura V.
      • Arvaniti P.
      • Gampeta S.
      • Gatselis N.K.
      Autoimmune hepatitis in patients aged 70 years or older: disease characteristics, treatment response and outcome.
      ,
      • Hennes E.M.
      • Zeniya M.
      • Czaja A.J.
      • Parés A.
      • Dalekos G.N.
      • Krawitt E.L.
      • et al.
      Simplified criteria for the diagnosis of autoimmune hepatitis.
      ,
      • Dalekos G.N.
      • Samakidou A.
      • Lyberopoulou A.
      • Banakou E.
      • Gatselis N.K.
      Recent advances in the diagnosis and management of autoimmune hepatitis.
      ,
      • Vergani D.
      • Alvarez F.
      • Bianchi F.B.
      • Cançado E.L.
      • Mackay I.R.
      • Manns M.P.
      • et al.
      Liver autoimmune serology: a consensus statement from the committee for autoimmune serology of the International Autoimmune Hepatitis Group.
      ,
      • Terziroli Beretta-Piccoli B.
      • Mieli-Vergani G.
      • Vergani D.
      Serology in autoimmune hepatitis: a clinical-practice approach.
      ] and the updated simplified criteria for the diagnosis of AIH [
      • Galaski J.
      • Weiler-Normann C.
      • Schakat M.
      • Zachou K.
      • Muratori P.
      • Lampalzer S.
      • et al.
      Update of the simplified criteria for autoimmune hepatitis: Evaluation of the methodology for immunoserological testing.
      ] all point directly to facilitate prompt recognition of the disease, which is of utmost importance to stop the disease progression and achieve favourable outcomes after treatment initiation. In this regard, physicians should be aware for the significance of the autoantibodies testing they order and should also be familiar with the interpretation of the results.
      It is clear that for a definite diagnosis of AIH, new biomarker(s) with both higher sensitivity and specificity than conventional autoantibodies are needed. Metabolomics and proteomics could be good candidates to identify in the future such specific molecules in AIH [
      • Wang J.B.
      • Pu S.B.
      • Sun Y.
      • Li Z.F.
      • Niu M.
      • Yan X.Z.
      • et al.
      Metabolomic profiling of autoimmune hepatitis: the diagnostic utility of nuclear magnetic resonance spectroscopy.
      ]. For instance, recently Taubert et al. [
      • Taubert R.
      • Engel B.
      • Diestelhorst J.
      • et al.
      Quantification of polyreactive immunoglobulin G facilitates the diagnosis of autoimmune hepatitis.
      ] discovered the presence of IgG antibodies with high capacity to bind to several human and non-human proteins by using a protein microarray. This polyreactive IgG (pIgG) showed greater specificity for the diagnosis of AIH than standard ANA and SMA tests, and a significantly higher sensitivity than anti-LKM and anti-SLA/LP [
      • Taubert R.
      • Engel B.
      • Diestelhorst J.
      • et al.
      Quantification of polyreactive immunoglobulin G facilitates the diagnosis of autoimmune hepatitis.
      ]. Of note, pIgG identified most patients with seronegative AIH along with most of patients with normal IgG [
      • Dinani A.M.
      • Fischer S.E.
      • Mosko J.
      • Guindi M.
      • Hirschfield G.M.
      Patients with autoimmune hepatitis who have antimitochondrial antibodies need long-term follow-up to detect late development of primary biliary cirrhosis.
      ]. Nevertheless, this new test needs external validation before general use at the screening phase of patients.

      Funding

      None

      Declaration of Competing Interest

      The authors declare they have no conflict of interest

      Appendix. Supplementary materials

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