Clinical Information

Primary biliary cholangitis (PBC) is a chronic and progressive autoimmune liver disease characterized by the destruction of the small intrahepatic bile ducts and a variable clinical course, which may include fatigue and pruritus. Untreated patients with PBC have a high risk of liver cirrhosis and related complications, liver failure and death.(1,2) The serological hallmark of PBC is the presence of antimitochondrial antibody (AMA) characterized by cytoplasmic reticular/AMA (anti-cell 21 [AC-21] based on the International Consensus on Antinuclear Antibody Patterns [ICAP] nomenclature) staining pattern on HEp-2 substrate by indirect immunofluorescence assay (IFA).(3) In addition, autoantibodies associated with the HEp-2 IFA nuclear patterns have been reported in a subset of patients with PBC who are seronegative for AMA or may be positive for AMA but have uncertain clinical or phenotypic attributes.(1,2,4,5) The HEp-2 IFA nuclear patterns in PBC include multiple nuclear dots (MND or AC-6) and punctate nuclear envelope (AC-12), which are associated with anti-Sp100 and anti-gp210 antibodies, respectively. (3) The diagnosis of PBC can be established if 2 out of the 3 following criteria are met: sustained elevated levels of alkaline phosphatase (ALP), evidence AMA or specific antinuclear antibody (ANA) (anti-Sp100 and anti-gp210 antibodies), and diagnostic liver histology.(2) Based on these criteria, a biopsy can be avoided in case of high ALP levels and detection of these PBC-specific autoantibodies.(1,2) Therefore, reliable and accurate serologic determination of PBC-specific autoantibodies play a critical role in disease evaluation.

Positivity of AMA ranges from 90% to 95% in patients with PBC, while the PBC-specific ANA (anti-Sp100 and anti-gp210 antibodies) may occur in approximately 30% of all patients with PBC, and up to 50% of patients who are AMA-negative.(6) The M2-type AMA (AMA-M2) is the dominant target of the 9 subunits of the mitochondrial antigenic complex.(1,2) AMA-M2 target components of the 2-oxo-acid dehydrogenase complex: pyruvate dehydrogenase complex (PDC), 2-oxoglutarate dehydrogenase complex (OGDC) and branched-chain 2-oxoacid dehydrogenase complex (BCOADC). Specifically, autoantibodies mainly recognize the E2 subunits of these complexes: PDC-E2 (80%-90% of cases), BCOADC-E2 (50%-80% of cases) and OGDC-E2 (20%-60% of cases), and to a lesser extent, the E1 and E3 subunits.(2). Although the sensitivities of the anti-Sp100 and anti-gp210 antibodies are low, their specificities for PBC are excellent; therefore, both tests have been reported to be useful in confirming a diagnosis of PBC or predicting development of disease in nonestablished PBC cases with positive AMA.(4,5) In addition to the diagnostic relevance of anti-gp210 IgG antibody, a few studies have suggested a role for their use in the risk stratification and prognosis in PBC; however, the significance of these remain contentious. In one study, the presence of anti-gp210 antibodies was reported to pose a significant risk for hepatic failure type progression, more severe interface hepatitis, and lobular inflammation, compared to those with centromere antibodies who had relatively higher ductular reaction.(7) In addition to MND and punctate nuclear envelope, the anticentromere (AC-3) and the speckled (AC-4 and AC-5) patterns can be found in variable prevalence in patients with PBC with overlapping connective tissue diseases (systemic sclerosis [SSc] and Sjogren syndrome).(8) In the context of other liver diseases, the cytoplasmic fibrillar linear (AC-15) HEp-2 IFA pattern associated with autoimmune hepatitis (AIH) may also be seen when PBC overlaps in patients with AIH or other liver diseases, such as hepatitis B virus infection, hepatitis C virus infection, and hepatic carcinoma.(9) In general, a mixed pattern composed of at least two HEp-2 IFA patterns is mostly found in patients with PBC rather than in other liver diseases.(9)

Traditionally, the IFA method was used for the detection of AMA; however, antigen-specific solid-phase immunoassays (SPA), such as enzyme-linked immunosorbent assay (ELISA), line blot immunoassay (LIA), and dot immunoassay (DIA) have been developed and are increasingly being used in the laboratory evaluation of PBC.(4,5,7-10) The AMA SPA use a variety of M2 antigens, including fusion protein combining the three E2 subunits, a mixture of recombinant E2 subunits, or the three E2 recombinant subunits isolated, among others.(4,5,7,10) The anti-Sp100 and anti-gp210 antibodies can also be determined using analyte-specific ELISA, LIA and DIA. In addition to the SPA for detecting antibodies to AMA, Sp100, and gp210, the use HEp-2 substrate by IFA provides a simple and strategic approach for confirming the presence of AMA cytoplasmic staining if positive by enzyme immunoassay (EIA) with the possibility of identifying patients who may be AMA-negative but positive to nuclear antibodies. In PBC patients, the nuclear envelope pattern is associated with anti-gp210 antibody while the multiple nuclear dot pattern is specific for anti-Sp100 antibodies. However, expression of the MND and the nuclear envelope patterns may not be easily identified in the presence of other antibodies. Testing for these antibodies is indicated in patients who are AMA positive by EIA as well as patients at-risk for PBC but are AMA negative. In addition to providing additional support for PBC diagnosis in AMA-positive and AMA-negative patients, the use of HEp-2 substrate offers the possibility to identify patients at-risk for PBC who may present with coexisting systemic autoimmune rheumatic diseases (systemic lupus erythematosus, systemic sclerosis, and Sjogren syndrome) or autoimmune liver disease (autoimmune hepatitis) through additional pattern recognition. The use of SPA for ANA testing do not provide these additional diagnostic insights.