Hwajeong Lee, MD, Robert T. Stapp, DO, Adrian H. Ormsby, MD, and Veena V. Shah, MD

Anatomic Pathology / Overlap Syndrome The Usefulness of IgG and IgM Immunostaining of Periportal Inflammatory Cells (Plasma Cells and Lymphocytes) for the Distinction of Autoimmune Hepatitis and Primary Biliary Cirrhosis and Their Staining Pattern in Autoimmune Hepatitis Primary Biliary Cirrhosis Overlap Syndrome Hwajeong Lee, MD, Robert T. Stapp, DO, Adrian H. Ormsby, MD, and Veena V. Shah, MD Key Words: Autoimmune liver disease; Overlap syndrome; Autoimmune hepatitis; Primary biliary cirrhosis; IgG; IgM Abstract Autoimmune hepatitis (AIH) primary biliary cirrhosis (PBC) overlap syndrome (OS) is a vaguely defined entity demonstrating features of AIH and PBC. We investigated the usefulness of IgG and IgM immunostaining for the distinction of AIH and PBC and their staining pattern in cases of possible OS. The approximate quantity of IgG+ and IgM+ periportal inflammatory cells in immunohistochemical analysis were compared in cases of AIH, PBC, and OS. AIH cases showed predominant IgG immunostaining of periportal inflammatory cells. A significant number of PBC cases also demonstrated IgG predominance rather than IgM. Six OS cases had IgG predominance, 4 had IgM predominance, and 1 was equivocal. The usefulness of IgG and IgM immunostaining is limited in PBC cases with IgM predominance for excluding AIH. IgG predominance is not specific for AIH. OS does not demonstrate either IgG or IgM predominance (P >.2) and does not help classify OS into either category. Autoimmune liver disease is divided into a hepatitic form, represented by autoimmune hepatitis (AIH), and a cholestatic form, including primary biliary cirrhosis (PBC) and primary sclerosing cholangitis. Overlap syndrome (OS) is a heterogeneous group of entities that demonstrate overlapping clinical, serologic, biochemical, and histologic features of more than 1 autoimmune liver disease, usually mixed hepatitic and cholestatic clinical features. AIH-PBC OS is the most commonly encountered, 1 with its reported incidence ranging from 5% to 20% of either AIH or PBC. 2-4 Still, AIH-PBC OS is a vaguely defined entity for which the criteria for diagnosis or terminology are not standardized, 5 and it is controversial whether AIH- PBC OS is a distinct entity, a variant of AIH or PBC, or simultaneous manifestations of AIH and PBC. 6-8 Despite its uncertainty, identifying and differentiating AIH-PBC OS from other autoimmune liver diseases has serious clinical implication because patients with AIH-PBC OS seem to benefit from combined ursodeoxycholic acid and immunosuppressive therapy. 2,9 In a recent article, Daniels et al 10 reported that IgG and IgM immunostaining can differentiate AIH from PBC; IgG+ plasma cells are predominant in liver biopsy specimens from AIH cases, whereas IgM+ cells are predominant in PBC. As an extension of their study, we investigated and compared the IgG and IgM staining patterns in established cases of AIH, PBC, and AIH-PBC OS. First, we aimed to validate usefulness of IgG and IgM immunostaining for the distinction of AIH and PBC. Second, we investigated the immunostaining pattern of AIH-PBC OS cases for IgG and IgM to evaluate whether the result of stains would push overlap cases into the AIH or PBC category. 430 Am J Clin Pathol 2010;133:430-437 Downloaded 430 from https://academic.oup.com/ajcp/article-abstract/133/3/430/1766134

Anatomic Pathology / Original Article Materials and Methods This study was approved by our institutional review board. Case Selection For the study, 28 cases of PBC, 36 cases of AIH, and 13 cases of possible AIH-PBC OS were retrieved from the surgical pathology files at Henry Ford Hospital (1998-2008) by a Systematized Nomenclature of Medicine Clinical Terminology (SNOMED) search. The search terms for AIH- PBC OS included immunocholangitis, autoimmune cholangitis, autoimmune cholangiopathy, variant syndrome, crossover syndrome, and overlap syndrome. Preliminary AIH-PBC OS cases had histologic overlapping features of both AIH and PBC. H&E-stained slides (1-20 slides per case) of the cases were reviewed to select tissue blocks for tissue microarray or recuts. Cases with missing blocks or slides and needle biopsy specimens showing very sparse portal inflammation were excluded. Clinical history and serologic data, including antimitochondrial antibody (AMA), anti smooth muscle antibody (ASMA), anti liver kidney microsome (LKM)-1 antibody, and antinuclear antibody (ANA) using indirect fluorescent antibody procedures, were reviewed. One case from the possible AIH-PBC OS group was reassigned to the PBC group after reviewing the clinical note. Another OS case in a 20-year-old man turned out to be OS of AIH and primary sclerosing cholangitis on follow-up and was excluded from the study. In addition, 1 AIH case was excluded owing to subsequent serologic detection of hepatitis C viral markers. Final selections consisted of 27 PBC cases, 27 AIH cases, and 11 AIH-PBC OS cases, of which 11 were hepatectomy specimens from liver transplants, 1 was from a wedge biopsy, and the rest were from needle biopsies. Furthermore, tissue samples from 1 PBC case and 3 AIH cases were lost or exhausted during process, leaving 26 PBC cases, 24 AIH cases, and 11 AIH-PBC OS cases for study. Tissue Microarray and Recuts Areas of portal triads with the most inflammation were selected for tissue microarray. Four microarray paraffin tissue blocks (Beecher Instruments, Sun Prairie, WI) were manually prepared from the blocks, consisting of 2 cores per case. Recuts were performed for 12 cases that were not deemed amenable to tissue microarray. In addition, 1 AIH-PBC OS case was selected for recuts for better appreciation of the staining pattern. Degree of Inflammation and Fibrosis The Ishak scoring system 11 was used to score the degree of inflammation (histologic activity index [HAI]) and fibrosis for each case with original slides. In some cases, Massontrichrome stained slides were available for evaluation of the degree of fibrosis. Stains H&E staining was performed on 4-μm-thick sections from the tissue microarray blocks. Immunostaining with antihuman IgG (polyclonal rabbit, dilution 1:10,000; DakoCytomation, Carpinteria, CA) and antihuman IgM (polyclonal rabbit, dilution 1:5000, DakoCytomation) was performed on 4-μm-thick sections from 4 tissue microarray blocks and recuts from 13 cases. Tissue sections were pretreated with Proteinase K for 10 minutes, followed by avidin-biotin blocking for another 10 minutes. Next, the slides were incubated in LINK (biotinylated antibody; a kit from DAKO, Carpinteria, CA) for 30 minutes at room temperature and then were incubated in LSAB+ (labeled streptavidin biotin; a kit from DAKO) before the application of the antibodies. AEC+ (DAKO) was used as the chromogen. Immunostain Interpretation The percentage of IgG+ or IgM+ inflammatory cells, including plasma cells and lymphocytes of portal triads, was approximated. The numbers of cases with more IgG+ cells, more IgM+ cells, and equivocal staining were recorded and counted for all 3 groups. We grouped cases with IgM predominance and equivocal staining together, as did an index study. 10 Results Age and Sex The mean age of the PBC group (57 years; range, 39-74 years) was higher than that of the AIH group (40 years; range, 15-66 years). The mean age of the AIH-PBC OS group was 48 years (range, 24-68 years). All 3 groups showed a female predominance, with 24 of 26 patients in the PBC group being female and all patients in the AIH-PBC OS group being female. The AIH group consisted of 20 patients; 16 were female. One of the patients had 2 liver biopsy specimens and another, 3 liver biopsy specimens and 1 hepatectomy specimen included in this group. Case Description and Histologic Features of AIH-PBC OS The laboratory results and clinical impression for the AIH-PBC OS group are given in Table 1. The cases are described in the following paragraphs. Case 1 Jaundice developed in a 42-year-old woman who was treated for AIH for 20 years. The biochemical profile revealed cholestasis with an increased alkaline phosphatase (ALP) level. She underwent liver transplantation due to cirrhosis. The explanted liver showed marked cholestasis and atypical ductular proliferation with a decreased number of bile ducts Downloaded from https://academic.oup.com/ajcp/article-abstract/133/3/430/1766134 Am J Clin Pathol 2010;133:430-437 431 431 431

Lee et al / Overlap Syndrome Table 1 Clinicopathologic Data * Increased Laboratory Values Serology Case No./ AST ALT ALP GGT TBil γ-glob IgG IgA IgM Clinical Age (y) (U/L) (U/L) (U/L) (U/L) (mg/dl) (g/dl) (mg/dl) (mg/dl) (mg/dl) ANA ASMA AMA Impression 1/42 636 369 508 191 7.8 1.12 1,550 344 182 AIH-PBC 2/24 86 127 259 NA 1.4 3.3 1,620 257 146 + OS AIH-PBC OS 3/66 74 111 359 NA NR 1.10 1,260 347 510 +? PBC 4/33 279 330 1,096 NA 2.3 5.5 1,450 333 1,080 w+ + +? PBC 5/42 158 204 992 1,174 4.5 3.6 1,540 438 533 + + AIH-PBC OS 6/41 141 203 220 393 1.7 5.9 3,350 233 211 +? AIH 7/42 95 129 216 NA NR 3.6 1,640 228 427 NA NA? PBC 8/68 91 90 NR NA 1.4 3.2 NA NA NA + + +? AIH 9/68 41 351 NR 374 NR 1.39 1,190 263 737 + AMA PBC 10/54 51 NR 266 NA NR 3.8 1,320 274 958 w+ AMA PBC 11/43 970 1,008 194 264 4.3 1.54 1,570 349 265 NA + AMA PBC AIH, autoimmune hepatitis; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AMA, antimitochondrial antibody; ANA, antinuclear antibody; ASMA, anti smooth muscle antibody; AST, aspartate aminotransferase; γ-glob, γ-globulin; GGT, γ-glutamyl transpeptidase (glutamyltransferase); Ig, immunoglobulin; NA, not available; NR, within normal range; OS, overlap syndrome; PBC, primary biliary cirrhosis; TBil, total bilirubin; w+, weak positive;?, questionable;, negative; +, positive. * All patients were women. Reference ranges are as follows: ALP, 0-120 U/L; ALT, 10-40 U/L; AST, 15-35 U/L; γ-glob, 0.58-1.50 g/dl; GGT, <50 U/L; IgA, 70-400 mg/dl; IgG, 700-1,600 mg/dl; IgM, 40-230 mg/dl;.and TBil, <1.2 mg/dl. in many portal triads and mild periportal lymphoplasmacytic infiltrates. The bile duct damage and the ductopenia are not compatible with pure AIH, nor are these changes typical of PBC. Hence, this case was reclassified as OS. Case 2 In a 24-year-old woman with acute hepatitis and cholestasis, serologic study showed increased transaminase, IgG, and ALP levels and a positive ASMA and negative AMA. The 2 initial biopsies showed features suggesting AIH with periportal and interface hepatitis composed of lymphocytes, plasma cells, and neutrophils. A subsequent liver biopsy revealed ductopenia without granulomas. The material used in the present study is from the second biopsy. This case showed overlapping features of AIH and AMA PBC. Case 3 A 66-year-old woman with a cholestatic serologic profile with increased ALP and IgM levels underwent liver biopsy with a clinical impression of probable PBC. AMA and ASMA tests were negative, and the ANA test was positive. A biopsy showed periportal plasmacytic infiltrate, interface hepatitis, and a few periportal noncaseating granulomas and bile duct damage. These microscopic features are a combination of AIH and PBC. Case 4 A 33-year-old woman with a history of autoimmune liver disease of uncertain type underwent liver biopsy with a clinical impression of PBC. The transaminase, ALP, and IgM levels were increased. Serologic studies were remarkable for positive AMA, ASMA, and ANA (weak reaction). The biopsy exhibited marked interface hepatitis, numerous plasma cells in portal triads, and a reduced number of interlobular bile ducts. Case 5 A 42-year-old woman with cholestatic autoimmune liver disease underwent liver biopsy. The ALP, γ-glutamyltransferase (GGT), and IgM levels were increased. In addition, AMA and ASMA results were positive. A biopsy showed marked periportal plasmacytic infiltrate and piecemeal necrosis along with ductular proliferation and periportal granulomas. Case 6 Cholestasis developed in a 41-year-old woman with a presumed diagnosis of AIH and a positive ASMA result. Transaminase, ALP, GGT, and IgG levels were increased. In the biopsy specimen, bile duct damage suggestive of PBC was noted along with extensive periportal plasmacytic infiltrate and marked interface hepatitis. Case 7 A 42-year-old woman with cholestasis and hepatitis underwent liver biopsy. Transaminase, ALP, IgM, and IgG levels were increased. Autoimmune antibodies were not detected. However, the patient had a history of AMA and ANA positivity determined outside of our institution. A biopsy 432 Am J Clin Pathol 2010;133:430-437 Downloaded 432 from https://academic.oup.com/ajcp/article-abstract/133/3/430/1766134

Anatomic Pathology / Original Article demonstrated marked periportal plasmacytic inflammation and a moderate degree of interface hepatitis. Occasional bile duct damage was seen, and 2 small granulomas unassociated with bile ducts were noted. Case 8 A 68-year-old woman with a clinical picture of AIH underwent liver biopsy owing to cholestasis. Previously, the patient had symptoms of gradual onset of fatigue, scleral icterus, and darkening of her urine. The AMA, ASMA, and ANA results were positive. A biopsy showed moderate periportal lymphoplasmacytic infiltrate with interface hepatitis and bile duct damage in some of the portal triads, overlapping features of AIH and PBC. Case 9 A 68-year-old woman was seen because of elevated ALP and GGT levels with mild elevation of the transaminase levels. An autoimmune screen and AMA test were negative. A liver biopsy showed a dense periportal lymphoplasmacytic infiltrate, moderate interface inflammation, and marked bile duct damage. Focal bridging fibrosis was noted. Case 10 A 54-year-old woman had long-standing history of autoimmune thyroiditis, an elevated ALP level, and a negative AMA result. The liver biopsy showed a prominent periportal mixed inflammatory infiltrate with many plasma cells. There was focal ductopenia and interlobular ductular damage. The microscopic features were a combination of AIH and PBC. Case 11 A 43-year-old woman with end-stage liver disease, ascites, and bleeding esophageal varices had a presumed diagnosis of PBC. However, her autoimmune screen and AMA test were negative. She underwent liver transplantation. The explanted liver showed cirrhosis with marked ductopenia. However, there were no diagnostic ductal lesions or significant ductular proliferation in keeping with the diagnosis of PBC. The microscopic features were consistent with OS. Serology Of 24 patients with PBC for whom AMA results were available by electronic medical record, 20 had positive results. Among the AMA+ patients, 6 also showed seropositivity for ANA and/or ASMA. Two patients had weakly positive results for ANA only, without AMA positivity. Of 19 patients with AIH for whom serologic data were available, 18 were positive for ANA or ASMA or both. One patient with AIH had seropositivity for AMA and for both ASMA and ANA. Serologic antibody profiles of 6 patients with AIH-PBC OS were similar to AIH with ANA and/or ASMA positivity. Three were positive for AMA and ASMA. Two patients were seronegative for all antibodies. No case was seropositive for anti-lkm-1 antibody. HAI and Fibrosis Data for the HAI and degree of fibrosis are given in Table 2 and Table 3. The mean HAI values for the PBC, AIH, and AIH-PBC OS groups was 4 (range, 1-7), 8 (range, 2-12), and 6 (range, 2-10), respectively. The mean fibrosis score for all 3 groups was 3. Half (13/26) of the PBC cases showed advanced fibrosis (stage 4-6), as did 10 of 25 AIH Table 3 Primary Biliary Cirrhosis Autoimmune Hepatitis Overlap Syndrome Histologic Fibrosis Dominant Activity Index Stage Immunostain Case No. 1 10 6 IgG 2 5 1 IgG 3 2 2 IgG 4 3 4 IgG 5 4 2 IgG 6 9 2 IgG 7 5 1 IgM 8 6 2 IgM 9 8 3 IgM 10 8 2 IgM 11 5 6 Equivocal Mean 5.9 2.8 Table 2 HAI, Fibrosis, and Immunostaining Pattern of PBC, AIH, and OS * IgG > IgM IgM IgG HAI Fibrosis Stage AMA (Serology) ANA ± ASMA (Serology) OS (n = 11) 6 5 6 (2-10) 3 (1-6) 3 6 AIH (n = 24) 24 0 8 (2-12) 3 (1-6) 1 22 PBC (n = 26) 12 14 4 (1-7) 3 (0-6) 20 8 AIH, autoimmune hepatitis; AMA, antimitochondrial antibody; ANA, antinuclear antibody; ASMA, anti smooth muscle antibody; HAI, histologic activity index; OS, overlap syndrome; PBC, primary biliary cirrhosis. * Data are given as number of cases or mean (range). Downloaded from https://academic.oup.com/ajcp/article-abstract/133/3/430/1766134 Am J Clin Pathol 2010;133:430-437 433 433 433

Lee et al / Overlap Syndrome cases. In AIH-PBC OS, 2 explant cases showed advanced fibrosis (stage 6). IgG and IgM Immunostaining Tables 2 and 3 also provide data on immunostaining. In the AIH group, all 24 cases showed predominantly IgG+ inflammatory cells in the portal area Image 1. In contrast, 14 of 26 PBC cases demonstrated equivocal (7 cases) or predominant IgM (7 cases) staining Image 2. There was no significant difference in HAI or fibrosis between the 7 cases with equivocal staining and the 7 with IgM predominance (P >.5; unpaired t test). The staining pattern in the PBC and AIH groups with IgG and IgM was significantly different (P <.0001; Fisher exact test). The difference was significant regardless of the degree of inflammation or fibrosis (P <.5; unpaired t test). In the AIH-PBC OS group, IgG+ portal inflammatory cells outnumbered the IgM+ cells in 6 cases. In 4 cases, IgM+ A B Image 1 Autoimmune hepatitis. Most of the inflammatory cells of the same portal triad stain positively with the IgG immunostain (A, IgG, 200), whereas the IgM immunostain of the same area shows sparse IgM+ inflammatory cells (B, IgM, 200). Inset, Scanning view of liver biopsy specimen demonstrates marked periportal inflammatory infiltrate (H&E, 100). A B Image 2 Comparison of IgG (A) and IgM (B) immunostains of a primary biliary cirrhosis case demonstrates more IgM+ inflammatory cells in the same portal triad despite a relatively heavy background stain in the IgG immunostain (A, IgG, 200; B, IgM, 200; inset, H&E, 200). 434 Am J Clin Pathol 2010;133:430-437 Downloaded 434 from https://academic.oup.com/ajcp/article-abstract/133/3/430/1766134

Anatomic Pathology / Original Article cells outnumbered the IgG+ cells Image 3. One case showed equivocal staining. AIH-PBC OS did not demonstrate IgG or IgM predominance (P >.2; Fisher exact test), regardless of the degree of inflammation or fibrosis (P >.5; unpaired t test). Discussion AIH and PBC are relatively well-defined entities; nevertheless, histologic distinction of AIH and PBC is sometimes challenging because marked interface hepatitis with many plasma cells is not uncommon in PBC; likewise, bile ducts with overflowing inflammatory cells in AIH may mimic bile duct lesions of PBC. In a recently published study, Daniels et al 10 demonstrated that immunophenotyping of periportal plasma cells using IgG and IgM immunohistochemical staining can help differentiate AIH from PBC cases. They reported that all AIH cases showed predominance of IgG+ cells and a majority (89%) of PBC cases demonstrated equivocal or more IgM+ cells. 10 Similar to their study, our results demonstrated that all AIH cases showed IgG predominance in the periportal inflammatory infiltrate. However, the staining pattern of the PBC group was different from that of Daniels et al 10 in that a significant number (12/26), although not the majority, of PBC cases also demonstrated IgG predominance. The IgG and IgM staining patterns of the 2 groups were significantly different using the Fisher exact test owing to the IgM predominance in greater numbers (14/26) of PBC cases. Also the difference remained statistically valid regardless of the degree of inflammation (HAI) or fibrosis. Our observation brings up a few limitations of this panel of immunostains as a diagnostic adjunct. First, we observed that 46% of PBC cases (12/26) demonstrated more IgG staining. To rephrase, of the 36 cases that showed IgG predominance, excluding AIH-PBC OS, one third were PBC cases. Therefore, predominance of IgG+ cells in a portal triad does not aid in differentiating AIH from PBC. Although IgM predominance seems specific for PBC, IgG predominance is not specific for AIH. Moreover, IgM staining has low sensitivity (54%) for the PBC group. The difference in immunostaining results in the PBC group with that of the reference study 10 (IgG predominance in 11% of PBC cases [2/18]) may be largely due to sampling. In the reference study, it appeared to be a routine practice to perform IgG and IgM immunostaining on any liver biopsy specimens with a clinical suspicion of AIH or PBC. Therefore, the authors were able to assess the entire length of stained biopsy specimens and select areas with the most inflammation for evaluation. In contrast, we built tissue microarray blocks from previous biopsy specimens, and, as a result, we had a much lower number of portal triads per case for evaluation. We acknowledge that sampling imposed some limitations on the interpretation of the stains, and the different results may be attributable to this issue. Second, it should be noted that equivocal staining was lumped with the IgM predominance category, thereby rendering a significant chance of interobserver variation. For example, 7 PBC cases showed equivocal staining, and those cases were added to 7 other IgM-predominant cases in our study. Some of the equivocally stained cases may well be interpreted as slight IgG predominance by another A B Image 3 (Case 7) A, A case of autoimmune hepatitis primary biliary cirrhosis overlap syndrome showing a few IgM+ inflammatory cells, including plasma cells (arrow) of a portal triad. B, Fewer IgG+ cells are seen in the same portal triad (A, IgM, 200; inset, H&E, 200; B, IgG, 200). Downloaded from https://academic.oup.com/ajcp/article-abstract/133/3/430/1766134 Am J Clin Pathol 2010;133:430-437 435 435 435

Lee et al / Overlap Syndrome pathologist. Therefore, practical usefulness of the stains in differentiating AIH from PBC seems to be limited to only PBC cases in which IgM+ cells indubitably outnumber IgG+ cells to rule out AIH. Stratifying cases according to degree of inflammation or fibrosis would not increase the sensitivity of detecting IgM-predominant cases, because neither the HAI nor the fibrosis score correlated with IgM predominance (P >.5; unpaired t test). Last, we had some difficulty in interpreting immunostaining owing to the presence of background staining, especially for IgG, even though the avidin-biotin blocking step was included in the pretreatment procedure and reagents were diluted according to the validated protocol in our immunology laboratory. We could roughly estimate the percentage of positively stained cells through background staining in most cases. In a few cases, however, only back-to-back comparisons of identical portal triads from identical cases allowed us to categorize them into IgG- or IgM-predominant groups. Again, background staining may well raise the possibility of interobserver variation in interpretation of the staining. It is not a routine practice to perform IgG or IgM immunostaining on liver tissue in our group. If both immunostains were to be performed routinely on liver tissue as in the setting of Daniels et al, 10 we might be able to overcome the background staining issue by practice and experience or improve the quality of stain by testing different dilutions of the polyclonal antibodies on liver tissue. Nevertheless, the practical usefulness of IgG and IgM immunostaining in distinguishing AIH from PBC is questionable for all of the aforementioned reasons. Since its first description by Guebel et al 12 in 1976, AIH- PBC OS has been in the midst of controversy about terminology, diagnostic criteria, pathophysiology, and treatment. There is no consensus on diagnostic criteria for AIH-PBC OS, and patients have mixed features of AIH and PBC, including increased serum aminotransferase levels, cholestatic markers, γ immunoglobulins (IgG and IgM), AMA seropositivity, and inconclusive histologic findings. 13 Because patients with AIH-PBC OS seem to benefit from combined immunosuppressive and ursodeoxycholic acid therapy, 1,2,7,9 some authors identified AIH-PBC OS by their response to combined treatment. 12,14 In our study, we used soft diagnostic criteria for AIH- PBC OS. We performed a SNOMED search from our pathology archives for initial screening of possible OS cases; hence, we selected cases that exhibited histologic overlapping features of AIH and PBC. These cases did not match with either serology or clinical impression based on biochemistry. We did not include response to treatment in the diagnostic criteria. If we were to apply strict criteria for all aspects histologic, serologic, biochemical, and clinical many of our AIH-PBC OS cases might not suffice. However, all patients in the AIH-PBC group did not have a solid diagnosis of AIH or PBC, despite the best efforts of pathologists and clinicians. We intended to study whether the panel of IgM and IgG immunostaining would help us categorize these problematic, possible AIH-PBC OS cases into one or the other. AIH-PBC OS consisted of 14% of initial cases retrieved (11/76; 1 case turned out to be seropositive for hepatitis C viral marker). The histologic features of AIH-PBC OS were not closer to either entity but were between AIH and PBC. For example, the HAI of OS was between that for AIH and PBC, and the degree of fibrosis was similar to both. Similarly, the IgG and IgM immunostaining pattern in AIH-PBC OS was between that in AIH and PBC; there was no predominant immunostaining of lymphocytes and plasma cells of portal triads with either IgG or IgM. Thus, AIH-PBC OS seems to be a heterogeneous group with no predictable IgG or IgM predominance in the periportal inflammatory infiltrate, and IgG or IgM staining does not aid in grouping this entity into AIH or PBC. Furthermore, serologic findings of AIH-PBC OS were not specific for any entity, as noted by presence or absence of all autoimmune markers in 5 cases. Some authors applied the exclusive term autoimmune cholangitis (AIC) for patients with a clinical picture of PBC without AMA 15,16 and categorized it as outlier (variant) syndrome, different from AIH-PBC OS. 17 Our AIH-PBC OS cases 9, 10, and 11 may well be considered as AIC AMAnegative PBC rather than AIH-PBC OS because they had clinical pictures of PBC, AMA negativity, ANA positivity, and histologic findings not specific for AIH. Although case 3 also demonstrated a PBC-like clinical picture with negative AMA, its histologic features were close to those of AIH. It is also worthy of mention that these 3 possible AIC cases showed IgM predominance (cases 9 and 10) or equivocal staining (case 11), which is a specific immunoprofile of PBC cases. If we exclude these 3 cases from AIH-PBC OS, the incidence of AIH-PBC OS in this report is 11% (8/76) during the 10-year period. Although there is a statistically significant difference in IgG and IgM immunostaining of periportal inflammatory cells in established AIH and PBC cases, the panel s usefulness in daily practice is doubtful owing to low specificity of IgG immunostaining for AIH and low sensitivity of IgM immunostaining for PBC. Also, our setting in which IgG and IgM immunostaining on liver tissue is not a standard procedure further limits the stains usefulness owing to background staining. There is no predictable IgG or IgM immunostaining of periportal lymphoplasmacytic inflammatory cells in AIH-PBC OS. Comprehensive analysis of serologic, biochemical, and histologic data in conjunction with clinical correlation is required for the correct diagnosis of autoimmune liver disease. From the Department of Pathology and Laboratory Medicine, Henry Ford Hospital, Detroit, MI. 436 Am J Clin Pathol 2010;133:430-437 Downloaded 436 from https://academic.oup.com/ajcp/article-abstract/133/3/430/1766134

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