Value of Autoantibody Analysis in the Differential Diagnosis of Chronic Cholestatic Liver Disease

Transcription

1 CLINICAL GASTROENTEROLOGY AND HEPATOLOGY 2009;7: Value of Autoantibody Analysis in the Differential Diagnosis of Chronic Cholestatic Liver Disease PIOTR MILKIEWICZ,*, HAYMAN BUWANESWARAN,* CATALINA COLTESCU,* ZAKERA SHUMS, GARY L. NORMAN, and E. JENNY HEATHCOTE* *Department of Medicine, Toronto Western Hospital, University Health Network, University of Toronto, Toronto, Ontario, Canada; Liver Unit, Pomeranian Medical School, Szczecin, Poland; and INOVA Diagnostics, San Diego, California BACKGROUND & AIMS: It is challenging to diagnose patients with chronic cholestatic liver diseases when all the classic criteria are not fulfilled. We evaluated the performance of the recently developed MIT3-based enzyme-linked immunosorbent assay (ELISA), which detects antimitochondrial autoantibodies (AMAs), together with ELISAs for other autoimmune liver disease related antibodies in patients with chronic cholestatic liver disease. METHODS: Sera from 281 patients with chronic cholestatic conditions, including primary biliary cirrhosis (PBC), primary sclerosing cholangitis, AMA-positive autoimmune hepatitis, and undetermined cholangiopathy were tested for the following PBC-associated autoantibodies: antigp210, anti-sp100, conventional anti-m2, anti-m2 (MIT3) IgG, anti-m2 (MIT3) IgA, as well as anti-centromere, anti soluble liver antigen, and anti-chromatin. RESULTS: Of 57 patients with PBC who were AMA-negative by conventional M2 ELISA, 14 were found to be AMA-positive by the MIT3-IgG assay. Furthermore, on the basis of the data from 3 tests (MIT3-IgG, gp210, and sp100), PBC was confirmed in 20 of 57 (35%) patients diagnosed with AMA-negative PBC. We confirmed that sp100 and gp210 antibodies were detected only in patients with PBC and AMA-positive autoimmune hepatitis, whereas gp210 was detected more frequently in patients known to have had a poor outcome. Of the 11 patients with an undetermined cholangiopathy, 3 (27%) tested positive for PBC with the MIT3- IgG assay. In contrast to previous findings, anti-centromere antibodies were not found to be associated with poor outcome in PBC. CONCLUSIONS: ELISAs for AMAs and antinuclear antibodies are useful in diagnosis and prognosis of patients with features of PBC who lack conventional AMA and in patients with a cholangiopathy of undetermined etiology. In chronic cholestatic liver disease the presence of autoantibodies suggests an autoimmune basis for the disease, although they are considered to be only surrogate markers of an ongoing autoimmune process. 1 The autoantibodies most commonly used to facilitate a specific diagnosis in chronic cholestatic liver disease are antimitochondrial antibodies (AMAs) and antinuclear antibodies (ANAs). 2 Unfortunately, in clinical practice a proportion of patients with a chronic cholestatic liver disease characterized by bile duct loss and/or damage do not have the classic hallmarks of either primary sclerosing cholangitis (PSC) or primary biliary cirrhosis (PBC) and are frequently described as having vanishing bile duct syndrome or idiopathic ductopenia. These patients pose a diagnostic and a therapeutic dilemma. AMAs directed against the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2) remain a serologic signature of PBC and are considered the most highly disease-specific autoantibodies in humans. 3 On the basis of previous discoveries in this field, a series of new autoantibody enzyme-linked immunosorbent assay (ELISA) tests have been developed and evaluated in a broad range of presumed autoimmune liver diseases. 4 A novel ELISA test based on the patented recombinant molecule (MIT3) developed by Gershwin and Leung, containing the 3 major immunodominant epitopes targeted by AMAs present in the sera of patients with PBC, has recently been developed. 5 Studies evaluating this antigen have shown it to be more specific for detection of AMA than immunofluorescence or routine M2 (which primarily targets PDC-E2) ELISAs. 5,6 In addition, ELISAs based on the original discoveries of anti-sp100 (nuclear dot antibody) 7 and anti-gp210 (nuclear pore complex antibody), 8 10 as well as ELISAs for anti soluble liver antigen (SLA), anti-chromatin antibody, and anti-centromere antibody have been developed and are now widely available In this study we determined the frequency of 5 PBC-specific autoantibodies, as well as anti-centromere, anti-sla, and antichromatin in a large cohort of patients with chronic autoimmune and cholestatic liver conditions. Materials and Methods The study protocol was approved by the local ethics committee. Patient groups included AMA-positive autoimmune hepatitis (AIH) (IAH-G criteria fulfilled, no histologic features of PBC, and AMA-positive by routine ELISA; stable over many years); PSC (endoscopic retrograde cholangiopancreatography [ERCP]/ magnetic resonance cholangiopancreatography [MRCP] diagnostic for PSC, except in 3 patients who fulfilled histologic criteria for small duct PSC); undetermined cholangiopathy (elevated alkaline phosphatase [ALP] and/or -glutamyltransferase [GGT]; bile duct injury or ductopenia, and lack of diagnostic features for PBC, PSC, or AIH; normal ERCP/MRCP and no inflammatory bowel Abbreviations used in this paper: AIH, autoimmune hepatitis; ALP, alkaline phosphatase; AMA, antimitochondrial antibody; ANA, antinuclear antibody; ELISA, enzyme-linked immunosorbent assay; ERCP, endoscopic retrograde cholangiopancreatography; GGT, -glutamyltransferase; MRCP, magnetic resonance cholangiopancreatography; PBC, primary biliary cirrhosis; PDC-E2, E2 subunit of the pyruvate dehydrogenase complex; PSC, primary sclerosing cholangitis; SLA, soluble liver antigen by the AGA Institute /09/$36.00 doi: /j.cgh

2 1356 MILKIEWICZ ET AL CLINICAL GASTROENTEROLOGY AND HEPATOLOGY Vol. 7, No. 12 disease and history excluding other potential causes such as drugs or herbs; AMA-positive PBC and AMA-negative PBC (elevated ALP and/or GGT; histology compatible with PBC and positive or negative AMA by routine ELISA [M2]; PBC with bad outcome group [elevated ALP and/or GGT, histology compatible with PBC]; negative or positive AMA by routine ELISA [either at Toronto Western Hospital or INOVA]; required liver transplant or died as a result of liver disease). Serum samples were collected prospectively from consecutive patients seen at our Liver Clinic or were obtained from our PBC serum bank. The latter were from patients with PBC who were known to have had a bad outcome (see above). Only in these serum bank samples were AMA determinations tested by ELISA solely in the laboratory of INOVA Diagnostics, whereas in all other patients routine AMA antibodies were analyzed by ELISA in the laboratory of the University Health Network Toronto, Canada within 1 year of taking a serum sample for the current study. All serum samples were coded and sent to the laboratory of INOVA Diagnostics, where they were tested blinded to the diagnosis for M2 (MIT3) IgG, M2 (MIT3) IgA, gp210 IgG, sp100 IgG, SLA/ liver pancreas IgG, chromatin IgG, and centromere IgG antibodies. Routine biochemical tests and routine ANAs and anti smooth muscle antibodies were analyzed in the laboratory of Toronto Western Hospital. Enzyme-linked Immunosorbent Assays Antimitochondrial antibodies. Conventional M2 enzyme-linked immunosorbent assay. Two conventional M2 ELISA tests were used in this study. The Varelisa M2 ELISA (Pharmacia Diagnostics, Uppsala, Sweden) was used to test samples at University Health Network, Toronto. The QUANTA Lite M2 ELISA (INOVA Diagnostics, San Diego, CA) was used in the INOVA Diagnostics laboratory. These assays typically use antigen isolated from bovine tissue enriched for PDC-E2 for the detection of anti-m2 IgG antibodies. Enhanced M2 (MIT3) enzyme-linked immunosorbent assay. This assay (QUANTA Lite M2 EP (MIT3) IgG ELISA; INOVA Diagnostics, Inc) uses the patented Gershwin-Leung MIT3 antigen, which consists of a recombinant protein containing the immunodominant epitopes of the 3 major AMA targets. These are the E2 subunits of PDC-E2, the branched chain oxoacid dehydrogenase complex, and the oxoacid dehydrogenase complex. 15 A research use only version of this assay, modified to detect IgA MIT3 antibodies, was also used in the present study. Sp100 enzyme-linked immunosorbent assay. The QUANTA Lite sp100 ELISA (INOVA Diagnostics, Inc) uses a peptide incorporating immunodominant portions of the sp100 protein. This assay was developed following the original description of the immunodominant targets of these autoantibodies by Szostecki et al. 7 gp210 enzyme-linked immunosorbent assay. The QUANTA Lite gp210 ELISA (INOVA Diagnostics, Inc) uses a peptide incorporating immunodominant portions of the gp210 protein. This assay was developed on the basis of the discovery and description and immunodominant targets of these antibodies by Nickowitz and Worman. 10 Chromatin enzyme-linked immunosorbent assay. The QUANTA Lite Chromatin ELISA (INOVA Diagnostics, Inc) uses highly purified calf thymus chromatin, which has been depleted of histone H1 and non-histone proteins. Centromere enzyme-linked immunosorbent assay. The QUANTA Lite Centromere ELISA (INOVA Diagnostics, Inc) uses recombinant CENP-A and CENP-B proteins. Detailed performance data and instructions for the QUANTA Lite ELISA tests can be found online ( All QUANTA Lite ELISA tests were run according to the manufacturer s instructions. All kits used in this study have been cleared by the United States Food and Drug Administration for in vitro diagnostic use, with the exception of the QUANTA Lite M2 (MIT3) IgA ELISA, which is a research use only test. Statistical Analysis Statistical analysis was performed with 2, 2 with Yates modification, and Fisher exact test where appropriate by using Stat-View Program. P values less than.05 were considered significant. Results The patient population comprised 281 individuals for whom clinical and demographic data are summarized in Table 1. MIT3 Immunoglobulin G Antibodies Fourteen of 57 PBC patients (24.5%) identified as AMAnegative by the routine M2 ELISA were positive for MIT3 IgG antibodies. This test was also positive in 3 of 11 (27%) patients with an undetermined cholangiopathy. One of the 46 (2%) patients with PSC tested positive for MIT3 IgG. This test was positive in all patients with AMA-positive AIH. MIT3 Immunoglobulin A Antibodies MIT3 IgA antibodies were detected less frequently than MIT3 IgG antibodies. Eight of 57 (14%) patients with PBC and Table 1. Demographics of Study Population at Time of Test Diagnosis/no. of patients (n 285) Age (y) (mean SE) Gender (M/F) ALT (U/L) (mean SE) ALP(U/L) (mean SE) ANA, % positive SLA, % positive AIH (AMA ) (n 10) / PSC (n 46) / Undetermined cholangiopathy (n 11) / PBC (AMA ) (n 49) / PBC (AMA ) (n 127) / PBC (bad outcome) (n 38) (AMA, 30; AMA, 8) / Data presented as mean SE. Normal values for ALT are 3 30 U/L and for ALP are U/L. SE, standard error.

3 December 2009 AUTOANTIBODY ANALYSIS IN AUTOIMMUNE CONDITIONS 1357 negative by the routine AMA ELISA tested positive for MIT3 IgA antibodies. All of these patients were also positive for MIT3 IgG antibodies. The MIT3 IgA ELISA was positive in only one specimen that was negative by the routine M2 ELISA (INOVA) and borderline equivocal MIT3 IgG (20.2 units) (equivocal range, units). MIT3 IgA antibodies were found in 18.2% (2/11) of patients with undetermined cholangiopathy. In addition, MIT3 IgA was positive in 1 patient with PSC, in whom both routine AMA ELISA and MIT3 IgG ELISA were negative. This test was positive in 6 (60%) of patients with AMA-positive AIH. gp210 Antibodies Anti-gp210 antibodies were found in 3 of 49 (6%) patients with PBC who were AMA-negative with the routine ELISA assay. When the AMA-negative patients with bad outcome were included in this analysis, anti-gp210 antibodies were found in 10% (6/57) of AMA-negative patients. Regardless of AMA status, gp210 was detected in 14 of 38 (36.8%) cases of PBC with bad outcome. Anti-gp210 could be detected in 26 of 157 (17%) patients with PBC who tested positive for routine AMA or MIT3 IgG ELISA. Antibodies to gp210 were detected in no patient with PSC or an undetermined cholangiopathy. They were positive in 1 patient with AMA-positive AIH. Sp100 Antibodies Sp100 antibodies were found in 6 of 57 (10.5%) patients with PBC who were AMA-negative with the routine ELISA assay and in 35 (22%) PBC patients who were AMA-positive by the routine AMA ELISA. Sp100 antibodies were not detected in patients with PSC or an undetermined cholangiopathy. They were also positive in 4 (40%) patients with AMA-positive AIH. diagnosis of these patient populations. The overall frequency and magnitude of the various autoantibodies in the complete cohort of patients with PBC are shown in Figure 1. Examination of the 176 patients with PBC (excluding the 38 bad outcome PBC patients, because their sera had not undergone recent testing for AMA) with the 3 AMA ELISAs showed a sensitivity of 75% (132/176 [ ]) for the new MIT3 IgG ELISA, compared with 72.2% (127/176) and 68.2% (120/ 176) for the 2 routine M2 ELISAs we had used. The sensitivity increased to 77.1% (165/214) for the MIT3 IgG ELISA, compared with a sensitivity of 70.1% (150/214) for the conventional M2 ELISA when the data obtained on the 38 patients with a bad outcome were also included in the analysis. Examination of the 57 AMA-negative sera found that 24.5% (14/57) (Table 2) were positive by the MIT3 IgG assay, compared with 12% (7/57) by the M2 ELISA. In addition to the PBC-specific AMA antibodies detected by the MIT3 assay, 19% (41/214) of the patients with PBC tested positive for sp100, and 15% (32/214) had gp210 antibodies. Figures 2 and 3 illustrate the overlap of specificities observed in the complete PBC cohort (Figure 2) and on AMA-negative specimens only (Figure 3). Six specimens from AMA-negative PBC that did not have MIT3 IgG antibodies were found to have gp210 and/or sp100 antibodies. Combining the results of the gp210 and the sp100 tests with the MIT3 IgG test resulted in 79.4% (170/214) of the total cohort (including the AMA-negative PBC patients) being identified as positive for 1 or more PBC-specific markers (Figure 2). Excluding the AMA-negative specimens, the combined sensitivity on the PBC cohort was 97.5% (153/157). Anti-centromere Antibodies Anti-centromere antibodies were positive in 16 of 157 (10%) patients with PBC who tested positive with the routine AMA ELISA and in 11 of 57 (19.3%) patients with PBC who were negative for AMA. They were also positive in 1 patient with undetermined cholangiopathy, 1 patient with PSC, and 2 (20%) patients with AMA-positive AIH. Soluble Liver Antigen Antibodies SLA antibodies were positive in 1 patient with AMApositive PBC, 1 patient with PSC, and 2 (20%) patients with AMA-positive AIH. They were negative in all the other analyzed samples. Anti-chromatin Antibody Anti-chromatin antibodies were detected in 3 of 11 (27%) patients with undetermined cholangiopathy and in 8 of 46 (17%) patients with PSC. They were also positive in 8 of 57 (14%) patients who tested negative with the routine AMA and in 11 of 157 (7%) patients with PBC who tested positive with the routine AMA. They were positive in 3 (30%) patients with AMA-positive AIH. Frequency and Overlap of Autoantibodies After the assessment of the various autoantibodies in our patients, we proceeded to analyze the utility of individual and combined autoantibody measurements in the differential Figure 1. Autoantibodies in complete PBC cohort. Cutoff A (20 units) for CENP (anti-centromere antibodies) and chromatin ELISA; cutoff B (25 units) for all other ELISAs.

4 1358 MILKIEWICZ ET AL CLINICAL GASTROENTEROLOGY AND HEPATOLOGY Vol. 7, No. 12 Table 2. Summary of Autoantibody Prevalence in Analyzed Groups Diagnosis (no. of patients) AMA M2 conventional, a AMA M2 EP (MIT3) IgG, AMA M2 EP (MIT3) IgA, gp210, sp100, CENP, SLA, Chromatin, All analyzed groups AIH (AMA ) (n 10) 10 (100) 10 (100) 6 (60) 1 (10) 4 (40) 2 (20) 2 (20) 3 (30) PBC AMA (n 157) b 143 (91) 151 (96) 117 (75) 26 (17) 35 (22) 16 (10) 1 (0.6) 11 (7) PBC AMA (n 57) b 7 (12) 14 (24.5) 8 (14) 6 (10) 6 (11) 11 (19) 0 (0) 8 (14) PSC (n 46) 0 (0) 1 (2) 1 (2) 0 (0) 0 (0) 1 (2) 1 (2) 8 (17) Undetermined cholangiopathy (n 11) 2 (18) 3 (27) 2 (18) 0 (0) 0 (0) 1 (9) 0 (0) 3 (27) PBC patients with bad outcome (n 38) a 30 (78.9) 33 (86.8) 24 (63.2) 14 (36.8) 8 (21) 6 (15.8) 0 (0) 6 (15.8) PBC AMA (n 30) a 30 (100) 30 (100) 24 (80) 11 (36.7) 6 (20) 5 (16.7) 0 (0) 3 (10) PBC AMA (n 8) a 0 (0) 3 (37.5) 0 (0) 3 (37.5) 2 (25) 1 (12.5) 0 (0) 3 (37.5) The total number of patients with PBC was 214 (ie, 157 patients who were AMA and 57 who were AMA ). This total number includes PBC patients with bad outcome. CENP, anti-centromere antibodies. a AMA determined by conventional M2 ELISA assay (INOVA). b AMA determined by conventional M2 ELISA assay (Varelisa). In the AMA-negative group, the combined sensitivity was 35.1% (20/57) (Figure 3). The magnitude of the various antibodies detected in the AMA negative group is depicted in Figure 4. MIT3 IgG supported the diagnosis of PBC in 27% of patients with undetermined cholangiopathy, the most challenging group of patients diagnostically. Are MIT3 Immunoglobulin G Antibodies Associated With a Different Autoantibody Profile? Because we found that the MIT3 IgG ELISA is more sensitive for the detection of AMA than the routine conventional ELISA, we examined whether the overall antibody profile of MIT3-positive or MIT3-negative PBC patients was different. This analysis showed that although gp210 and sp100 antibodies appeared to be more frequent in subjects who were AMA MIT3 positive compared with the subjects who were AMA MIT3 negative, the difference was not statistically significant for either these or the other autoantibodies analyzed. Association of Autoantibodies With Bad Outcome The prevalence of gp210 was significantly higher in patients known to have had bad outcome. Antibodies to gp210 occurred in 37% of patients who had either died or needed a liver transplant as compared with 10% of the other patients with PBC who were currently being followed in clinic (P.0001). The prevalence of other autoantibodies was not statistically different when patients with or without bad outcome were compared. It should be noted that both gp210 and sp100 antibodies were never detected in the sera of patients with PSC or an undetermined cholangiopathy. Figure 2. Overlap of specificities in patients with PBC. Figure 3. Overlap of specificities in patients with AMA-negative PBC.

5 December 2009 AUTOANTIBODY ANALYSIS IN AUTOIMMUNE CONDITIONS 1359 Figure 4. Overlap of specificities in patients with AMA-negative PBC. Discussion In the present study, which included a large cohort of patients with various chronic, presumed autoimmune cholestatic liver conditions, we analyzed the prevalence of 5 PBCspecific (conventional anti-m2 IgG, anti-mit3 IgG and IgA, anti-gp210, anti-sp100) and 3 autoimmune liver disease associated autoantibodies (anti-sla, anti-chromatin, and anti-centromere) and assessed their potential role in the differential diagnosis of patients with chronic cholestasis caused by presumed autoimmune mediated duct injury and ductopenia. Although the immunofluorescence assay on rodent tissue sections or HEp-2 cells is the classic method for determination of AMA and is still used as the primary test by many laboratories, it is generally accepted that ELISA methodologies are more sensitive and less prone to operator-dependent errors in interpretation. A recent study reported that only 72% of patients thought to have PBC had detectable AMAs by immunofluoresence on HEp-2 cells or rodent tissue sections, but that 32% of these AMA-negative PBC patients were positive when tested by ELISA or immunoblot. 6 A more recent study that used an MIT3-based ELISA for 30 patients thought to have PBC, but who tested AMA-negative by immunofluorescence, immunoblot, and ELISA with recombinant antigen, found 20% tested positive for AMA with a bead assay. 15 Conventional ELISA tests for AMA use mitochondrial antigen isolated from animal tissue. These antigens are enriched for the E2 subunit of the PDC-E2. Although PDC-E2 is the major autoantigen recognized by patients with PBC, 2 other autoantigens, namely the E2 subunit of the branched chain 2-oxo-acid dehydrogenase complex and the E2 subunit of the 2-oxoglutarate dehydrogenase complex, are recognized by some patients. A recombinant antigen (MIT3) containing the immunodominant epitopes of these 3 autoantigens was constructed and shown to have increased sensitivity compared with conventional ELISAs. 5,6 Our study, which included a large cohort of patients who were AMA-negative but thought to have PBC, has confirmed the usefulness of this novel ELISA with the recombinant MIT3 molecule for the assessment of AMA status. We showed that one fourth of patients negative for AMA by M2 ELISA were AMA-positive with the new MIT3 IgG ELISA assay. Our assessment of the potential utility of MIT3 IgA antibodies found that although up to 75% of patients with PBC in the present study had anti MIT3 IgA antibodies, these antibodies were always present together with anti MIT3 IgG antibodies and consequently did not prove to be of particular help in increasing the chance of serologic confirmation of PBC in our cohorts. This finding is similar to that reported by Gabeta et al. 4 We have also shown that in chronic, presumed autoimmune cholestatic liver disease, both gp210 and sp100 are highly specific for PBC and might also be detected in some patients thought to have PBC, but who were negative for conventional AMA antibodies. These 2 antibodies were not detected in patients with proven PSC or those with an undetermined cholangiopathy. In a group of 13 AMA-negative patients, 7 (54%) were found to be either gp210 or sp100 positive. Autoantibodies to gp210 have been reported to be associated with worse outcome and probably more rapid progression of PBC. 11,16 Our current results concur with this observation in that we detected gp210 antibodies in 37% of those patients with PBC who had a known bad outcome (progressed sufficiently to require a liver transplant or who died), compared with 10% in a cohort of patients whose outcome is yet to be defined. Our study showed that a panel of 3 autoantibodies, MIT3 IgG, gp210, and sp100, allowed a serologic confirmation of PBC in more than one third of our patients who were thought to have PBC, but who tested negative for AMA by conventional assays. We also found that 27% (3/11) of patients with an undetermined cholangiopathy tested positive for MIT3 IgG antibodies, thus assisting the diagnosis of PBC in these subjects. We introduced the term undetermined cholangiopathy to characterize subjects with chronic cholestasis and liver biopsy evidence of duct loss and/or injury without diagnostic features of PBC or PSC (normal bile ducts on radiologic examination) and a negative history for drug-induced liver damage. Because some of these subjects have histologic features of bile duct injury but no obvious ductopenia in the liver tissue sample, we believed that this term describes their condition more appropriately than idiopathic ductopenia. Our study also included a group of patients with AIH who also tested positive for AMA. We recently provided a detailed description of this subgroup, because we believe that AMApositive AIH might be a separate clinical entity. 17 Certainly some might argue that these patients represent a true PBC/AIH overlap or will develop PBC in future. We found that all these patients consistently tested positive over many years with conventional and MIT3 IgG assays. In addition, 4 had sp100 and 1 had gp210 antibodies. Because all these patients were treated

6 1360 MILKIEWICZ ET AL CLINICAL GASTROENTEROLOGY AND HEPATOLOGY Vol. 7, No. 12 early with steroids, it is possible that the clinical and biochemical features of PBC were suppressed at an early stage. Indeed, a positive effect of steroids on the early PBC has been shown. 18,19 Thus the presence of these other PBC-specific antibodies in these patients confirms the specificity of MIT3 IgG assays in these case samples. Centromere antibodies, often with very high values, were detected in approximately 10% 19% of the various PBC subgroups examined. Centromere autoantibodies have been reported to be more common in those with a worse outcome (liver failure) in PBC in one study. 20 Our results did not confirm this observation and concur with findings of Rigamonti et al, 21 who showed no difference in liver-related deaths (bad outcome) between centromere-positive and centromere-negative PBC patients. The study of Nakamura et al 22 also found anti-centromere antibodies were not risk factors for a bad outcome (hepatic failure/liver transplant or death) in PBC. Anti-chromatin antibodies have been reported to be markers of disease activity in patients with AIH. 23 In our patient population reported here these antibodies were detected in approximately 10% of patients with PBC and in 17% of patients with PSC. The significance of anti-chromatin antibodies in these patients has not yet been studied and remains to be investigated. The availability of new assays for a variety of autoantibodies might play a significant role in confirming/establishing clinical diagnosis in patients with chronic autoimmune liver disorders. As we have shown, in patients with no obvious cause for a cholangiopathy up to one third have markers for PBC. These assays can strengthen the diagnosis of PBC. In addition, autoantibodies such as anti-gp210 might have prognostic value and prove useful in the management of patients with PBC. The utilization of these new assays will result in a decrease in the number of patients falling into the AMA-negative group and should result in patients receiving earlier treatment and better management. References 1. Czaja AJ, Norman GL. Autoantibodies in the diagnosis and management of liver disease. J Clin Gastroenterol 2003;37: Worman HJ, Courvalin JC. Antinuclear antibodies specific for primary biliary cirrhosis. Autoimmun Rev 2003;2: Kita H, Nalbandian G, Keeffe EB, et al. Pathogenesis of primary biliary cirrhosis. Clin Liver Dis 2003;7: Gabeta S, Norman GL, Liaskos C, et al. Diagnostic relevance and clinical significance of the new enhanced performance M2 (MIT3) ELISA for the detection of IgA and IgG antimitochondrial antibodies in primary biliary cirrhosis. J Clin Immunol 2007;27: Miyakawa H, Tanaka A, Kikuchi K, et al. Detection of antimitochondrial autoantibodies in immunofluorescent AMA-negative patients with primary biliary cirrhosis using recombinant autoantigens. Hepatology 2001;34: Muratori P, Muratori L, Gershwin ME, et al. True antimitochondrial antibody-negative primary biliary cirrhosis, low sensitivity of the routine assays, or both? Clin Exp Immunol 2004;135: Szostecki C, Will H, Netter HJ, et al. Autoantibodies to the nuclear Sp100 protein in primary biliary cirrhosis and associated diseases: epitope specificity and immunoglobulin class distribution. Scand J Immunol 1992;36: Bandin O, Courvalin JC, Poupon R, et al. Specificity and sensitivity of gp210 autoantibodies detected using an enzyme-linked immunosorbent assay and a synthetic polypeptide in the diagnosis of primary biliary cirrhosis. Hepatology 1996;23: Tartakovsky F, Worman HJ. Detection of Gp210 autoantibodies in primary biliary cirrhosis using a recombinant protein containing the predominant autoepitope. Hepatology 1995;21: Nickowitz RE, Worman HJ. Autoantibodies from patients with primary biliary cirrhosis recognize a restricted region within the cytoplasmic tail of nuclear pore membrane glycoprotein Gp210. J Exp Med 1993;178: Nakamura M, Shimizu-Yoshida Y, Takii Y, et al. Antibody titer to gp210-c terminal peptide as a clinical parameter for monitoring primary biliary cirrhosis. J Hepatol 2005;42: Leung PS, Rossaro L, Davis PA, et al. Antimitochondrial antibodies in acute liver failure: implications for primary biliary cirrhosis. Hepatology 2007;46: Manns M, Gerken G, Kyriatsoulis A, et al. Characterisation of a new subgroup of autoimmune chronic active hepatitis by autoantibodies against a soluble liver antigen. Lancet 1987;1: Wies I, Brunner S, Henninger J, et al. Identification of target antigen for SLA/LP autoantibodies in autoimmune hepatitis. Lancet 2000;355: Oertelt S, Rieger R, Selmi C, et al. A sensitive bead assay for antimitochondrial antibodies: chipping away at AMA-negative primary biliary cirrhosis. Hepatology 2007;45: Muratori P, Muratori L, Ferrari R, et al. Characterization and clinical impact of antinuclear antibodies in primary biliary cirrhosis. Am J Gastroenterol 2003;98: O Brien C, Joshi S, Feld JJ, et al. Long-term follow-up of antimitochondrial antibody-positive autoimmune hepatitis. Hepatology 2008;48: Mitchison HC, Bassendine MF, Malcolm AJ, et al. A pilot, doubleblind, controlled 1-year trial of prednisolone treatment in primary biliary cirrhosis: hepatic improvement but greater bone loss. Hepatology 1989;10: Rautiainen H, Karkkainen P, Karvonen AL, et al. Budesonide combined with UDCA to improve liver histology in primary biliary cirrhosis: a three-year randomized trial. Hepatology 2005;41: Yang WH, Yu JH, Nakajima A, et al. Do antinuclear antibodies in primary biliary cirrhosis patients identify increased risk for liver failure? Clin Gastroenterol Hepatol 2004;2: Rigamonti C, Shand LM, Feudjo M, et al. Clinical features and prognosis of primary biliary cirrhosis associated with systemic sclerosis. Gut 2006;55: Nakamura M, Komori A, Ito M, et al. Predictive role of anti-gp210 and anticentromere antibodies in long-term outcome of primary biliary cirrhosis. Hepatol Res 2007;37(Suppl 3):S412 S Czaja AJ, Shums Z, Binder WL, et al. Frequency and significance of antibodies to chromatin in autoimmune hepatitis. Dig Dis Sci 2003;48: Reprint requests Address requests for reprints to: Dr E. J. Heathcote, Toronto Western Hospital, 6B fell, Room 154, University Health Network, 399 Bathurst Street, Toronto, Ontario, M5T 2S8, Canada. jenny. heathcote@utoronto.ca; fax: (416) Conflicts of interest These authors disclose the following: Gary L. Norman and Zakera Shums are employees of INOVA Diagnostics, Inc. The remaining authors disclose no conflicts. Funding E. Jenny Heathcote has received an unrestricted grant from Axcan- Pharma.