Detection of autoantibodies assists in the diagnosis and classification of autoimmune liver diseases, namely autoimmune hepatitis (AIH), primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC). Routine autoantibody testing by indirect immunofluorescence [IFL] has been recently complemented by molecular assays such as ELISA, blots, dot/line immunoassays or bead assays based on purified or recombinant antigens. This article critically discusses the current understanding of the diagnostic and clinical significance of autoantibodies specifically detected in patients with autoimmune liver diseases.
by Dr D. P. Bogdanos
Autoantibodies are important tools for the correct diagnosis and classification of autoimmune liver diseases (AiLD), a broad range of disorders that can affect liver cells (hepatocytes) in autoimmune hepatitis (AIH), and biliary duct cells (cholangiocytes) in primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC) [1, 2].
Autoimmune hepatitis (AIH)
AIH is a chronic autoimmune liver disease of unknown origin characterised by hyperglobulinemia, high-titre autoantibodies and histological features of interface hepatitis [3]. The disease has a female bias, occurs in both children and adults, and affects different ethnic groups. AIH patients can present as asymptomatic or with symptoms indistinguishable from those of acute viral hepatitis or fulminant hepatic failure. Extrahepatic diseases such as autoimmune thyroiditis, type 1 diabetes mellitus and rheumatological conditions are relatively common in AIH. A prompt diagnosis of the disease enables timely implementation of immunosuppression and drug-induced remission is achieved in approximately 90% AIH cases [3].
Primary biliary cirrhosis (PBC)
PBC is a chronic progressive cholestatic liver disease affecting middle-aged women. The clinical spectrum of PBC varies from asymptomatic cholestasis to liver failure. Extrahepatic diseases are common in patients with PBC [4]. The disease is suspected in all cases of unexplained chronic cholestasis and presence of disease-specific autoantibodies. A liver biopsy can confirm the diagnosis, if needed. While ursodeoxycholic (UDCA) may slow progression, there is not yet a curative therapy and end-stage disease patients require liver transplantation [4].
Primary Sclerosing Cholangitis (PSC)
PSC is characterised by strictures of the intra- and/or extrahepatic bile ducts leading to cirrhosis and liver failure [5]. The aetiology of the disease is unknown. PSC usually affects young and middle-aged men and has a 2:1 male predominance. Some 15-50% of cases remain asymptomatic at presentation. The disease is characterised by periodic remissions and exacerbations. UDCA treatment improves liver cholestatic biochemistry but has no effect on disease progression. Patients with PSC do not respond well to immunosuppressive treatment. One third of patients will develop hepatocellular carcinoma [5]. In the last two decades more cases with AIH, PBC or PSC are diagnosed at early stages. It is not yet clear whether this reflects changes in the natural history of these diseases or greater awareness of the disease and better application of improved diagnostic tools [1, 2, 6, 7].
Autoantibody serology
Antibodies to nuclei (ANA) and/or to smooth muscle (SMA) characterise type 1 AIH (AIH-1) and antibodies to liver kidney microsomal-1 (anti-LKM) or liver cytosol-1 (anti-LC1) define patients with type 2 AIH (AIH-2) [2, 6, 7]. Anti-mitochondrial antibody (AMA) and ANA reactive with specific nuclear body and nuclear pore complex antigens are the serological markers of PBC [1]. Perinuclear anti-neutrophil cytoplasmic antibody (p-ANCA) is the most frequent antibody reactivity in PSC 1. Autoimmune sclerosing cholangitis (ASC), a paediatric form of sclerosing cholangitis, has an autoantibody profile indistinguishable from that of AIH-1 [1].
Currently, IFL is the main technique for routine autoantibody testing [8]. It is based on the use of rodent substrate that usually includes kidney, liver and stomach, a combination that allows the detection of ANA, SMA, anti-LKM-1, anti-LC1 - if anti-LKM1 is absent – and AMA. IFL using ethanol-fixed human neutrophils permits detection of ANCA. Positive sera should be titrated to extinction. In adults, significant titres equal or exceed 1:40 dilution with IFL. In children, titres of 1:20 for ANA or SMA and 1:10 for anti-LKM1 are diagnostically relevant and are used in combination with other laboratory and clinical findings to confirm the diagnosis [2, 8]. AMA or PBC-specific ANA exceeding 1:40 are diagnostic for PBC. Atypical p-ANCA titres equal or greater than 1:20 are considered positive and can act as an additional pointer towards the diagnosis of PSC or AIH, particularly in the absence of other
autoantibodies [1].
Anti-nuclear antibodies (ANA)
ANA positive AIH-1 cases react with centromere, histones, double-stranded DNA, chromatin, and ribonucleoprotein complexes but there is no individual reactivity or combination of specificities typical of the disease [Table 1], [2]. Thus, the ANA profile test systems developed for the diagnosis of ANA in rheumatological diseases, as replacement for IFL, are not suitable for the detection of ANA in AIH. ANA are readily detectable as nuclear staining in all three tissues of the composite substrate. A better ANA pattern can be characterised using human larynx epithelioma cancer cells (HEp-2), which have larger nuclei than those of rodent tissues [2]. HEp-2 cells, however, are not suitable for screening, because ANA are frequently present at 1:40 dilution in the normal adult population. Moreover, HEp-2 cells are not as sensitive in detecting SMA and AMA compared to the rodent tissue and do not allow the detection of anti-LKM1 and LC1. The homogenous ANA pattern is the most characteristic of AIH-1 [2]. HEp-2 cells are very useful for the detection of the PBC-specific ANA giving multiple nuclear dot (MND) or rim-like membranous (RLM) patterns [1, 6]. These autoantibody patterns are present in 25-70% cases with PBC. Anti-MND antibodies recognise sp100 and promyelocytic leukaemia (PML) proteins. Antibodies giving an RLM pattern react with nuclear envelope proteins and in particular gp210, lamin B receptor (LBR), and nucleoporin p62.
Smooth muscle antibodies (SMA)
SMA is the serological marker of AIH-1. SMA can be detected, by IFL, using rodent kidney and stomach tissue sections, fibroblasts, HEp-2 cells and VSM 47 cells derived from rat embryonic thoracic aorta [Figure 1], [2]. The SMA staining of arterial vessels (V), glomerular mesangium (G) and fibers surrounding the kidney tubules (T), (and therefore responsible for the VG and VGT patterns) is confined to AIH-1 cases [2]. Most routine laboratories report the presence or absence of SMA reactivity and not the patterns of SMA. At the molecular level, AIH-1 specific SMA react with actin, mainly in its polymerised filamentous (F)-actin form. However, some 20% of the AIH-specific SMA positive cases do not react with purified F-actin while anti-F-actin positivity, usually at low titres, can be present in diseases unrelated to AIH-1 [1, 2].
Liver kidney microsomal type 1
antibody (anti-LKM1) and liver cytosol antibody (anti-LC1)
Anti-LKM1 antibodies define a second serological type of AIH, i.e. AIH type 2 [1-3]. Their target antigen is cytochrome P450 IID6 (CYP2D6). Anti-LC1 antibodies recognise formiminotransferase cyclodeaminase (FTCD), an enzyme highly-expressed in the liver that catalyses the conversion of histidine to glutamic acid. These autoantibodies are detected in isolation or in association with anti-LKM1
antibodies in patients with AIH-2.
Anti-mitochondrial antibody (AMA)
AMA is so strongly associated with PBC, being present in 90-97% of PBC cases, that in its absence, the diagnosis of the disease should be questioned [6]. These autoantibodies are easily detected by IFL [Figure 2]. They recognise components of the 2-oxo-acid dehydrogenase complexes, and the predominant targets are the E2 subunits of pyruvate dehydrogenase complex (PDC), branched chain oxoacid dehydrogenase complex (BCOADC) and oxoglutarate dehydrogenase complex (OGDC), PDC-E3 binding protein and to a lesser extent the PDC-E1α and PDC-E1β subunits [Figure 3], [6].
Antineutrophil cytoplasmic antibodies (ANCA)
ANCA encompass a variety of heterogeneous antibodies targeting diverse components of human neutrophilic granulocytes, and with IFL a cytoplasmic (c-ANCA) or perinuclear (p-ANCA) staining can result. Most classical p-ANCA are present in microscopic polyangiitis and react with myeloperoxidase, whilst c-ANCA against proteinase 3 is detected in patients with Wegener granulomatosis. An atypical p-ANCA staining, which gives a perinuclear staining subtly different from the classical p-ANCA and recognises a 50 kDa nuclear envelope antigen has been described in patients with PSC (90%), AIH-1 (up to 70%) and
inflammatory bowel diseases [8].
Antibodies against soluble liver antigen/liver-pancreas antigen (SLA/LP)
The characterisation of other autoantibodies frequently present in AIH but undetectable by IFL has led to the identification of antibodies against SLA/LP. These autoantibodies are present in isolation or in association with ANA and SMA in patients with AIH-1 and recognise a selenocysteine synthase [2]. ELISA and dot/line immunoassays based on the recombinant antigen have been developed to facilitate
detection of anti-SLA/LP antibodies.
Appropriate detection of autoantibodies is essential for the diagnosis of autoimmune liver disease
Autoantibody testing should be performed in all patients with abnormal liver function tests and/or liver disease of unknown aetiology [1, 8]. The diagnostic importance of these autoantibodies is reflected in the recent, simplified diagnostic scoring system of AIH whereby the presence of disease-related autoantibodies is one of the four criteria required for a definitive diagnosis of this disease [9].
The presence of high-titre anti-LKM1 and/or anti-LC1 antibodies in peadiatric cases of otherwise unexplained hepatitis is highly indicative of AIH-2; these cases require early commencement of immunosuppressive treatment to avoid life-threatening complications from end-stage liver disease [2, 10].
Disease-specific AMA and ANA are so relevant to the diagnosis of PBC that in the presence of these antibodies and unexplained cholestasis, a liver biopsy is not needed to confirm the diagnosis [1]. ANA patterns in PBC can be obscured by the confounding simultaneous presence of AMA in PBC cases. If there is a doubt, ELISA or similarly observer-independent commercial assays allow an accurate estimation of PBC-specific ANA against gp210, LBR, sp100 and PML [1].
Interpretative problems may also arise when laboratories base their IFL screening on rodent kidney tissue sections alone [1, 8]. Such testing does not allow detection of anti-LC1 antibodies and additional tests using FTCD- (LC1) based assays are needed to exclude the presence of these antibodies. Another diagnostic problem of IFL using kidney tissue as substrate relates to the resemblance of anti-LKM1 antibodies with AMA, which can create some confusion [8]. The fact that AMA stains the mitochondria-rich small distal tubules, whereas anti-LKM-1 stains the third portion of the proximal tubules, is only appreciated by a very well trained eye. Assays based on recombinant human CYP2D6 for anti-LKM-1 and the conventional M2 antigen or three major oxo-acid dehydrogenase complex E2 subunits (MIT3) for AMA can easily discriminate between the two autoantibody IFL specificities [4, 8, 11, 12].
Diagnostic and clinical significance of autoantibodies
Lack of autoantibodies at presentation cannot exclude AIH and repeat testing is needed to confirm the autoantibody status and to allow correct disease classification [3]. The presence or absence of the antibody, rather than the titre or concentration of autoantibody, is most important. At times a sharp fall or loss of autoantibody is a valid indicator of response to immunosuppressive treatment [2].
ANA and SMA may be present in other liver diseases but their frequency, titre and pattern are different from those seen in AIH and the clinician needs to be aware of this. Thus, if a patient fulfils the other diagnostic criteria and has high titre ANA of the homogenous pattern and/or SMA of the VGT or F-actin pattern the diagnosis will almost certainly be of AIH-1 [2]. Conversely, AIH patients will always be negative for the PBC-specific ANA except in the case of co-occurrence of both diseases [2].
Anti-LKM1 and anti-LC1 are reported in a proportion (up to 11%) of patients with a chronic hepatitis C virus infection. The necessity to differentiate between HCV and AIH-2 amongst those who are anti-LKM1 antibody positive stems from the fact that immunosuppression, the first line treatment for AIH-2, may be harmful in HCV infected patients while, interferon-α can exacerbate autoimmune manifestations and can lead to flares of liver damage in anti-LKM1 antibody positive HCV infected cases [13].
Anti-SLA antibody positive cases have a worse outcome and do not respond as well as anti-SLA negative AIH cases to immunosuppression [14]. Based on these findings, anti-SLA antibody has been considered as a marker of disease severity and all cases with AIH are screened for the presence of this autoantibody [2, 15].
AMA titres do not relate to the stage of PBC and their fluctuation over time does not seem to have clinical significance [1, 4]. Thus, most clinicians only order AMA at presentation to help establish the diagnosis. Repeat testing is normally requested only in cases that are seronegative for AMA at presentation but with clinical or laboratory findings compatible with PBC [4].
AMA titres do not seem to be associated with disease severity but those of the IgG3 subclass may identify patients prone to develop more severe disease compared to those without AMA-IgG3 [16]. PBC-specific ANA, and in particular those showing the RLM antibody pattern, have been found more frequently in patients with advanced disease [6, 7, 17]. These findings have obvious implications for the clinical management of PBC as testing for these autoantibodies may be important in identifying asymptomatic patients with a likely unfavourable disease course.
This review article has covered the major developments in the autoantibody serology of autoimmune liver diseases and addressed the pros and cons of various tests which may create problems with their clinical interpretation. It is clear that an ideal molecular-based test system for the detection of autoantibodies in patients with autoimmune liver diseases would be one that encompasses all the diagnostically and clinically-relevant autoantibody specificities. We are currently working towards the development of such a test system in a project known as the ‘LIVERpool’ project (www.bogdanoslab.com). It aims to identify all the molecular targets of ANA, SMA, anti-LKM1, anti-LC1, AMA, PBC-specific ANA and p-ANCA, with high specificity and diagnostic and clinical utility for liver autoimmunity. Our ultimate goal is to develop an assay that is able to detect all these autoantibody specificities simultaneously. As we have highlighted in this article, we have much work to do before this methodological approach yields a fruitful result.
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The author
Dimitrios P. Bogdanos, M.D., Ph.D.
Liver Immunodiagnostics,
Liver Unit and Institute of Liver Studies,
King’s College London School of Medicine at King’s College Hospital,
London, UK
Correspondence to:
Dimitrios P. Bogdanos, MD, PhD
Clinical Senior Lecturer & Consultant
Institute of Liver Studies,
King’s College London School of Medicine at King’s College Hospital,
Denmark Hill Campus, London SE5 9RS, UK
e-mail:
dimitrios.bogdanos@kcl.ac.uk;
dimitrios.bogdanos@kch.nhs.uk
Website: www.bogdanoslab.com
Tel/Fax: +44 203299 3397
