The evolution of knowledge in the laboratory diagnostics of autoimmune hepatopathies, and in particular of primary biliary cholangitis, well represents the profound changes that have affected the field of autoimmune disease diagnostics in the last decade.
In recent years, in fact, molecular tests based on the use of purified or recombinant antigens and new diagnostic strategies and solutions, some of which are automated, have been added to the traditional methods in immunofluorescence and are currently the subject of different study protocols.
Autoimmune diseases of the liver are a heterogeneous group of immune-mediated pathologies characterized by an autoimmune aggression towards hepatocytes or cholangiocytes and can be classified into three major entities, primary biliary cholangitis, autoimmune hepatitis and primary sclerosing cholangitis, which contain within them some variants as well as overlapping syndromes, so-called Overlap, where both a cholestatic and an inflammatory com ponent is present to varying degrees. In this field, the distinction between hepatocellular damage and cholestatic damage is fundamental for the purposes of differential diagnosis, and consequent therapeutic choices and antibody diagnostics plays an essential role in the correct classification of the different nosological entities.
Autoimmune hepatitis (AIH) is a chronic inflammation of the liver of unknown cause, in which tolerance to the hepatocyte is lost. The mechanisms underlying liver damage are cell-mediated and direct cytotoxicity.
The disease mainly affects females in both childhood and adulthood and may be associated with other autoimmune conditions (ulcerative colitis, autoimmune thyroiditis, primary biliary cholangitis and primary sclerosing cholangitis).
It is usual to classify AIH on the basis of specific autoantibodies, which for type 1 are mainly ANA and anti-smooth muscle antibodies (ASMA) (Fig. 1); type 2, on the other hand, usually presents LKM1 (liver-kidney microsomal antibodies). Other antibodies found in AIH are anti-SLA (soluble liver antigens), anti-LP (liver-pancreas), anti-ASGPR (asialo-glycoprotein) and anti-LC1 (liver cytosol type 1). Positivity for ANA, ASMA, LKM1 and SLA is included in the diagnostic criteria for AIH.
Fig1. Fluoroscopic pictures relating to positivity for anti-nucleus (ANA +, left) and anti-smooth muscle (ASMA +, right) autoantibodies
Primary biliary cholangitis (CBP) is a relatively rare chronic cholestatic hepatopathy with slow evolution and autoimmune aetiology characterized by an aggression of T lymphocytes against the cells of the intrahepatic bile ducts; the consequent damage makes the drainage of bile from the liver to the intestine difficult, thus causing stagnation in the liver and resulting in liver failure in a variable time.
Patients with CBP are primarily female (90%), usually diagnosed in the fifth or sixth decade of life. It has been suggested that genetic susceptibility is a predisposing factor for CBP, while environmental factors such as infections, chemicals and smoking may play a causal role.
The course of the disease is variable, and an accurate diagnosis in the early stages is crucial as early drug treatment can slow the progression to liver failure and improve survival.
The diagnosis is based on a combination of clinical/histological features, abnormalities of the liver biochemical profile in a setting of persistent cholestasis for more than 6 months and the presence of AMA and/or specific ANA antibodies in the serum.
AMA antibodies are detected in about 90% of CBP patients, and their high sensitivity and specificity makes their presence one of the three diagnostic criteria for CBP. The AMA are actually a heterogeneous group of antibodies and are directed with various percentages of frequency against different autoantigens that are part of the structural complex of the enzyme 2-oxyacid dehydrogenase, namely the sub units E2 of the enzyme pyruvate dehydrogenase (PDC-E2 – frequency 90%), the complex oxyglutaric dehydrogenase (OGDC-E2 – frequency 50%) and branched-chain ketoacid dehydrogenase (BCOAD-E2 – frequency 50%).
AMAs can be detected in the serum of asymptomatic patients with normal liver enzyme balance; follow-up studies have shown that over time most of these subjects develop CBP.
For a long time, the indirect immunofluorescence test (IIF) on cryostat sections of the liver, kidney and stomach of rats was considered the gold standard for routine screening of AMA. In recent years, on one hand, the limitations presented by this test (complex as execution, difficult to standardize, not fully automated, operator-dependent interpretation) and, on the other hand, the identification of molecular targets of AMA have led to the development, vali dation and introduction in the laboratory diagnostics of molecular antigen-specific tests using the technique of ELISA immunoassays or Immunoblotting.
The promising results obtained with the latest generation of ELISA tests using the MIT3 antigen (PDC-E2, OGDC-E2 and BCOAD-E2), capable of detecting positivity in about half of AMA IIF negative patients, suggest the use of these tests in place of or alongside IIF for AMA testing, particularly when laboratories are unfamiliar with IIF use and interpretation.
Immunoblotting assays also represent an interesting diagnostic alternative to the AMA in IIF because this multiplex assay allows the simultaneous detection of different CBP- specific autoantibodies using recombinant antigens.
In addition to AMA, ANA, usually associated with auto immune rheumatic diseases, are frequently found in CBP patients. At present, the method considered the gold standard for the determination of ANA is IIF, using HEp-2 cells as substrate. CBP-specific ANA have been detected by several authors in 30–50% of patients, and two different fluorescence patterns have been described for these autoantibodies: rime-like/membranous (specificity for gp210 and Nucleoporin p62 proteins) and multiple nuclear dots (specificity for sp100, PML and small ubiquitin-like modifier proteins). The detection of these antibodies, characterized by a low sensitivity, allows to confirm the diagnosis of CBP in AMA-negative patients, frequently liable to misclassification. There is also some evidence that the presence of specific CBP ANA, particularly anti-gp210, is associated with a worse prognosis and a more aggressive disease. Finally, as for AMA, also for specific CBP ANA, the new solid phase tests (ELISA and Immunoblotting), especially those using recombinant antigens, such as MIT3, appear to be more sensitive and less subjective than IIF (Fig. 2).
Fig2. Molecular antigens and analogous CBP-associated fluoroscopic pictures
Other non CBP-specific ANA, mainly anti-Centromere and anti-SSA/Ro – 52 kDa antibodies, are often detected (10–30%) in CBP patients, conferring, according to some authors, unfavourable prognostic features to the disease course. This finding correlates with the observation of the association of CBP with other autoimmune diseases, such as scleroderma, Sјogren’s syndrome and CREST syndrome.
Finally, it is interesting to report new markers currently under study for the laboratory diagnosis of CBP, namely two new self-antigens (KLHL12 and HK1), genetic markers, particular metabolic profiles, miRNAs and epigenetic factors.
With regard to CBP, it is interesting to note, in summary, how the increased knowledge of the serological associations of the disease, together with the widespread use of non- invasive tests, such as laboratory tests, has significantly modified the initial clinical presentation in recent years, often allowing an early diagnosis to be made with respect to the detection of advanced liver disease.
Primary sclerosing cholangitis (PSC) is a chronic cholestatic syndrome characterized by inflammatory fibrosis of the intra- and extrahepatic bile ducts.
SPC typically affects young males and is commonly associated with inflammatory bowel disease, especially ulcerative colitis (CU).
From the point of view of laboratory diagnostics, most patients present an elevation of serum alkaline phosphatase and γGT that may be accompanied by a modest increase in transaminases; unlike CBP, in CSP, the search for AMA anti bodies is negative. The determination of anti-neutrophil cytoplasmic antibodies (ANCA) with a pANCA panel in ethanol, an autoantibody marker also present in a high per centage of patients with CU (approximately 80%), was found to be useful for diagnosis.
In summary, laboratory diagnostics of hepatic autoimmune diseases, both in terms of diagnosis differential and early diagnosis, has made important progress in recent years and, without doubt, the prospect is open to new developments. In this field, an issue that could be of great interest in the near future is the improvement in the standardization of methods and the definition of new diagnostic algorithms.
At present, the detection of specific autoantibody profiles is essential, among autoimmune liver diseases, for the diagnosis of AIH and CBP, while the diagnosis of CSP is made by means of the picture provided by endoscopic retrograde cholangiopancreatography. The crucial value of autoantibodies for the differential diagnosis of EAI and CBP is expressed in the consequent therapeutic choices.
Failure to detect autoantibodies does not exclude the presence of an autoimmune hepatopathy, for example CBP AMA negative.
In this diagnostic field, in perspective, the percentage of patients negative for AMAs, which still represent the key immunological marker for CBP, will be significantly reduced by the use of new antigen-specific laboratory tests (ELISA and Immunoblotting tests) based on the use of recombinant antigens.
In addition, the increased use of automated systems that include a full panel of related CBP self-antigens, including the nuclear antigenic targets gp210 and sp100, will contribute to the optimization of new diagnostic algorithms. Conducting multicentre studies evaluating newly diagnosed CBP patients may effectively allow the evaluation of these new analytical tools in the screening and differential diagnostics of CBP. The results of these studies could contribute to redesign the entire diagnostic process, starting from the formulation of a reasonable clinical suspicion to the request for tests, up to the use of the most appropriate autoantibody tests and ensuring through the shared implementation of common algorithms a harmonization among different laboratories.
In other words, clinical governance and standardization in the field of immunological laboratory diagnostics of autoimmune liver diseases, as for other autoimmune diseases, are two essential elements to enhance the potential of autoantibody biomarkers both in diagnostic and prognostic fields.
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