Abstract
IntroductionAntimitochondrial antibodies (AMAs) are the hallmark of primary biliary cholangitis (PBC) but can be identified also in patients with connective tissue disease, namely, systemic sclerosis (SSc). Protein immunoprecipitation (IP) and IP-Western blot (WB) can be used to confirm AMA positivity directed at the pyruvate dehydrogenase complex (PDC) subunits E1α, E1β, E2/E3, and E3BP in patients showing a cytoplasmic reticular pattern at indirect immunofluorescence when performed in a screening setting before the onset of overt cholestasis in rheumatic patients.Patients and MethodsWe studied sera from 285 patients affected by connective tissue disease [SSc, n = 144; dermato/polymyositis (DM/PM), n = 56; and undifferentiated connective tissue disease (UCTD), n = 85] by indirect immunofluorescence (IIF), protein-IP, and IP-WB to identify specific PDC subunits recognized by AMA.ResultsTwenty percent (57/285) of sera from patients with connective tissue disease had a cytoplasmic reticular pattern at IIF, and in 77% (44/57, including 20 SSc, 12 PM/DM, and 12 UCTD) of these, we detected different titers of autoantibodies against the PDC subunits, specifically against PDC-E2. Among these sera, 4 (9%) tested positive for anti-E1α, 15 (34%) for anti-E1β, and 16 (36%) for anti-E3BP. Four of the 20 AMA-positive SSc cases (20%) had been already diagnosed with PBC, and all were positive for autoantibodies against the subunits PDC-E2, E3, and E3BP.ConclusionsUsing IIF and IP, we confirm that autoantibodies against the PDC components are detected in rheumatic patients with PBC or without liver dysfunction. In view of the strong predictive value of AMA for PBC, a strict follow-up of these latter patients is warranted for an early diagnosis of the disease.
Highlights
Antimitochondrial antibodies (AMAs) are the hallmark of primary biliary cholangitis (PBC) but can be identified in patients with connective tissue disease, namely, systemic sclerosis (SSc)
As for controls used in the described experiments, in detail, we used (i) cytoplasmic-reticular pattern by indirect immunofluorescence (IIF) (AC-21) and the speckled cytoplasmic pattern by IIF (AC-20) both provided by ICAP as positive controls; (ii) 10 negative controls for each IIF, IP, and IP-Western blot (WB) analysis, as described in the Materials and Methods section; (iii) the positive and negative controls for AMA tissue analysis were included in the used kit (Astra Formedic, Milan, Italy)
20% (57/285) of sera had a cytoplasmic reticular pattern at IIF as described both on tissue slides and on HEp-2 cells (Figure 1) that we define possibly related to AMA positivity as specified by the ICAP website (AC-21 cytoplasmic pattern, www.anapatterns.org)
Summary
Antimitochondrial antibodies (AMAs) are the hallmark of primary biliary cholangitis (PBC) but can be identified in patients with connective tissue disease, namely, systemic sclerosis (SSc). AMA positivity has been reported by the International Consensus on Antinuclear Antibody (ANA) Patterns (ICAP; www.autoab.org) as a coarse granular filamentous staining extending throughout the cytoplasm by IIF on cellular substrates such as HEp-2 cells (AC-21), usually performed as a screening test for rheumatic diseases. Routine commercial tests used for AMA identification, including ELISA and immunoblot, and AMA analysis on triple tissue slides are largely used worldwide for PBC diagnosis but usually when a clinical suspect of PBC is already present [5] while being not routinely used as screening methods for autoantibody (autoAb) analysis as for IIF on HEp-2 slides and immunoprecipitation (IP)
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