Recently, Fusconi et al16
reported anti‐CCP positivity in a relatively high percentage of patients with AIH‐1. They performed an anti‐CCP test (second generation; DIASTAT anti‐CCP, Axis‐Shield, Dundee, UK) on these patients to investigate whether anti‐CCP autoantibodies are part of the large pool of autoantibodies that are present in sera from patients with AIH‐1. In their cohort of 133 patients with AIH‐1, they observed anti‐CCP positivity in 9% (n
12) of the patients, of which 58% had titres
20 U/ml (cut‐off
5 U/ml). Of the control rheumatoid arthritis sera, 60% (53 out of 89) were positive. Very low percentages of positivity were observed in primary biliary cirrhosis (2 out of 49) and hepatitis C virus‐related disease (1 out of 80) patients, in both cases with titres <20 U/ml. The anti‐CCP test can be performed on patients with chronic hepatitis with arthritic involvement to help in diagnosing a possible coexisting rheumatoid arthritis, for which the test has a reported specificity of 95%.4
Given the absolute requirement for the citrulline moiety of the epitope9
and the empirically established rheumatoid arthritis‐specificity, the diagnostic anti‐CCP kits have been optimised for low background of normal sera and only include citrulline‐containing antigen(s).
To check whether the reactivity of the anti‐CCP positive AIH‐1 sera was indeed citrulline‐dependent, we tested both CCP2 (IMMUNOSCAN RA kit) and its arginine‐control variant (plate supplied by Euro‐Diagnostica BV) and compared the results. In parallel, we also tested anti‐CCP positive sera from patients with other rheumatologic (non‐rheumatoid) and hepatologic conditions. Sera were considered CCP2 positive in accordance with manufacturer's instructions and the recommended 25 U/ml cut‐off. The CCP2 positive sera were divided into three groups: (a) citrulline‐preferred reactivity when, regardless of their reactivity with the arginine‐variant (positive or not), the cut‐off index (COI) for the citrulline‐variant was >1 and more than twice the COI for arginine; (b) completely citrulline‐specific when they reacted with CCP2, but not with its arginine‐variant (this group is obviously a subset of the first group); (c) non‐citrulline‐specific (citrulline‐independent positivity) when the sera reacted with the arginine‐variant and the COI for citrulline was less than twice the COI for arginine. Table 1 shows the results for CCP2. In total, we found 12 anti‐CCP2 positive patients with AIH‐1, with the IMMUNOSCAN RA test (for features of the anti‐CCP2 positive patients with AIH‐1 see Table 2, where also anti‐CCP1 positive cases (see below) are reported). Out of 12 Dutch patients with AIH‐1, never tested for CCP2, 3 (25%) were positive. Regarding the previously tested samples there was some discrepancy: 6 out of 9 DIASTAT positive Italian AIH‐1 sera were IMMUNOSCAN RA positive positive, whereas only 3 out of 26 EliA positive or borderline AIH‐1 sera scored positive in IMMUNOSCAN ELISA. None of the 10 previously reported anti‐CCP negative patients with AIH‐1 that were tested, reacted with CCP2 by IMMUNOSCAN RA. The CCP2 values measured in AIH‐1 sera were in most cases fairly low, which could explain some of the discrepancies between the various tests.
Table 1Percentages of citrulline‐specific reactivity and citrulline‐preferred reactivity in sera of different patients groups
Table 2Clinical and laboratory features of anti‐CCP2 and/or anti‐CCP1 positive patients with AIH‐1
With regard to citrulline‐dependency of AIH‐1 sera, out of 12 anti‐CCP2 positive patients, 7 showed a citrulline‐preferred reactivity (6 (50%) of them were completely citrulline‐specific) whereas 5 (42%) showed a citrulline‐independent positivity. Interestingly, 2 out of the 6 citrulline‐specific sera were from patients with an overlapping rheumatic disease (table 2). If we exclude these two cases, the percentage of non‐citrulline‐specific positivity in patients with AIH‐1 is 50% (5 out of 10 patients). Of the three cases with other liver diseases, two reacted as positive with IMMUNOSCAN, and both of them in a citrulline‐independent way.
As for the CCP positive rheumatic patients without rheumatoid arthritis, out of 63 tested cases 54 were positive with IMMUNOSCAN CCP2 in our hands; regarding citrulline‐dependence, 50 (93%) showed a citrulline‐preferred reactivity (47 of them (87%) completely citrulline‐specific); only 4 patients (7%) had a citrulline‐independent reactivity. Particularly, all the sera from patients with PsA, SjS and palindromic rheumatism displayed a citrulline‐preferred reactivity (almost all completely citrulline‐specific) (table 1). As for patients with rheumatoid arthritis, all the 41 tested cases reacted with the IMMUNOSCAN kit. As expected, all of them showed a citrulline‐preferred reactivity (36 (88%) of them were completely citrulline‐dependent) (see table 1 and fig 1). In figure 2 the observed reactivity patterns of individual AIH‐1 sera are depicted with connecting lines between the COIs for “citrulline” and “arginine”. For the non citrulline‐specific sera, most have equal COIs (almost horizontal lines). One serum is far more reactive in the arginine‐control ELISA.
Figure 1Reactivity of sera against CCP2 citrulline‐ and arginine‐variant. CCP2 (X) versus arginine‐control (R) reactivity of rheumatoid arthritis (RA), autoimmune hepatitis type 1 (AIH‐1), psoriatic arthritis (PsA), (more ...)
Figure 2Corresponding cut‐off index (COI) levels for cyclic citrullinated peptide‐2 positive individual type 1 autoimmune hepatitis (AIH‐1) sera.
Subsets of AIH‐1 sera (n
31, 9 anti‐CCP2 positive with IMMUNOSCAN and 22 negative) and RA sera (n
5) were also tested for both citrulline‐ and arginine‐variants of the anti‐CCP1 test to see if the AIH‐1 reactivity was dependent on the antigenic peptides used. The results (shown in Table 2) indicate that the majority of CCP1 reactive AIH‐1 sera (12 out of 14
86%) did not show a citrulline‐preferred reactivity. In contrast, all the five RA sera used as control showed a citrulline‐specific reactivity. Some differences in reactivity for CCP1 compared to CCP2 were observed: all the AIH‐1 sera that reacted with CCP2 (IMMUNOSCAN) reacted also with CCP1, except one (case 15, Table 2). Two of these sera changed from citrulline‐preferred or citrulline‐specific reactivity in CCP2 to citrulline‐independent reactivity in CCP1. Six sera that did not react with CCP2 reacted with CCP1, in all cases in a citrulline‐independent fashion.
To determine whether components of the ELISA system, independent from the peptides, are responsible for the non‐specific reactivity by AIH‐1 sera several control experiments (both citrulline‐ and arginine‐variants) in an anti‐CCP1 setting were performed (eg, streptavidine‐plates instead of DNA‐bind plate; with or without bovine serum albumin). Sera maintained their reactivity for CCP1 with these different approaches (results not shown) indicating that the reactivity of the AIH‐1 sera was not introduced by the method of testing and that the antibodies really target the presented antigen.