Application of Histopathology, Immunohistochemistry, and Antibody Titers in the Diagnosis and Treatment Response Assessment of Autoimmune Liver Diseases

Application of Histopathology, Immunohistochemistry, and Antibody Titers in the Diagnosis and Treatment Response Assessment of Autoimmune Liver Diseases | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Application of Histopathology, Immunohistochemistry, and Antibody Titers in the Diagnosis and Treatment Response Assessment of Autoimmune Liver Diseases Su Jing, Liang Shi-yi, Chen yu-qing, Luo rui This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8776539/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Objective To investigate the differences in histopathological characteristics, immunohistochemical marker expression, and serum antibody titers among autoimmune hepatitis (AIH), primary biliary cholangitis (PBC), AIH-PBC overlap syndrome (OS)[ 39 ], and drug-induced autoimmune-like hepatitis (DI-AILH), providing basis to enhance the accuracy of differential diagnosis and rational prediction of treatment response. Methods A retrospective collection of liver biopsy specimens from 40 patients at the Department of Gastroenterology, Leshan People's Hospital, between January 2022 and August 2025 was conducted, including 11 AIH cases, 11 PBC cases, 10 AIH-PBC OS cases, and 8 DI-AILH cases. A comprehensive approach utilizing HE staining (assessing inflammation grade, fibrosis stage, and characteristic pathological changes), immunohistochemistry (detecting markers such as Mum-1, CD38, CD3, CD68), serum antibody titers (anti-nuclear antibody, ANA), and liver biochemical indices (ALT, ALP, IgG) was employed to quantitatively compare histological and immunohistochemical differences among the four groups. Patients with AIH, PBC, and OS were followed up for 6 months to analyze the correlation between treatment response and antibody titers, biochemical indices, and pathological changes. Results Both the AIH and OS groups exhibited significant interface hepatitis, hepatocyte rosette formation, and emperipolesis. The scores for portal plasma cell + lymphocyte infiltration [AIH: (2.00 ± 1.61), (3.91 ± 0.30); OS: (1.90 ± 1.20), (3.70 ± 0.48)] were significantly higher than those in the PBC group [(1.00 ± 0.63), (2.55 ± 1.13)] and the DI-AILH group [(0.38 ± 0.52), (2.38 ± 1.30)] (all P < 0.05). Inflammation grade (Scheuer grade) [AIH: (3.64 ± 0.50), OS: (3.00 ± 0.67)] and fibrosis stage (Scheuer stage) [AIH: (3.09 ± 0.83), OS: (2.80 ± 0.79)] were also higher in the AIH and OS groups compared to the other two groups (all P < 0.05). Serologically, the proportion of high-titer ANA (≥ 1:640) in the OS group was 80.0% (8/10), significantly higher than in other groups (P < 0.05). IgG levels in the AIH group [2520.00 (2190.00, 3565.00)] were significantly higher than in the other three groups (P < 0.05), while ALT levels in the DI-AILH group (360.12 ± 420.84) were significantly higher than in the other three groups (all P 0.05). Non-responders in the PBC group had significantly higher ALP and ALT levels [ALP: 917.00 ± NA; ALT: 215.00 ± NA] compared to responders [ALP: 195.50 ± 95.68 U/L; ALT: 64.40 ± 47.68 U/L] (both P < 0.05). Multivariate logistic regression analysis showed that elevated IgG was an independent diagnostic factor for AIH (OR = 1.02, 95% CI: 1.00-1.04, P < 0.05), and high-titer ANA was an independent predictive factor for OS (OR = 3.41, 95% CI: 1.12–10.38, P < 0.05). Immunohistochemical features: OS showed the most prominent CD3 and CD38 expression, reflecting dual "cellular immunity + immune activation" overlay. AIH exhibited active CD68 and Mum-1 expression, suggesting a combined "cellular immunity (macrophages) + humoral immunity" drive. PBC had Mum-1 levels comparable to AIH but lower T-cell and immune activation involvement, leaning towards "humoral immunity + bile duct-specific injury". DI-AILH generally showed lower involvement of all immune markers, reflecting the fundamental difference of its "drug-induced" immune injury mechanism compared to traditional autoimmune liver diseases. Conclusion Combining histopathological features (interface hepatitis, rosette formation), immunohistochemical markers (Mum-1, CD3, CD38, CD68), and serological indices (IgG, ANA titer, ALT) can effectively differentiate AIH, OS, PBC, and DI-AILH, improving diagnostic accuracy. Early identification of OS and selection of targeted immunosuppressive regimens can improve treatment response. Pre-treatment ALP and ALT levels in PBC patients can serve as important reference indicators for second-line treatment decisions. Autoimmune Liver Diseases Drug-Induced Autoimmune-Like Hepatitis (DI-AILH) Histopathology Immunohistochemistry Antibody Titers 6-Month Treatment Response Figures Figure 1 Figure 2 Figure 3 Introduction Autoimmune liver diseases (AILD) are a group of chronic inflammatory hepatobiliary diseases mediated by aberrant autoimmune responses, primarily including autoimmune hepatitis (AIH), primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), and overlap syndromes (OS). Among these, AIH and PBC are clinically most common and may present concurrently or sequentially in the same patient, known as AIH-PBC (OS). Diagnosis currently follows the internationally "Paris Criteria" updated 2011 [3、49]. Furthermore, drug-induced autoimmune-like hepatitis (DI-AILH), a special type of drug-induced liver injury, exhibits high similarity in clinical symptoms, serology, and histology to AIH[ 21 – 31 ], but responds well to corticosteroid therapy with a low relapse rate after discontinuation. According to the 2023 guidelines for the diagnosis and treatment of drug-induced liver injury(DILI) [ 32 ], differential diagnosis between DI-AILH and AIH requires combining liver biopsy pathological features with long-term follow-up (≥ 6 months). This study systematically analyzes the differences in clinical characteristics, histopathological morphology, immunohistochemical marker expression, serum antibody titers, and biochemical response among patients with AIH, PBC, AIH-PBC (OS), and DI-AILH, aiming to identify key indicators for differential diagnosis and explore predictive factors for treatment response, providing references for precise clinical diagnosis and treatment. 1. Materials and Methods 1.1 Study Subjects Forty patients who underwent ultrasound-guided liver biopsy at the Department of Gastroenterology, Leshan People's Hospital, from January 2022 to August 2025 were retrospectively enrolled and divided into four groups based on diagnostic criteria: AIH group (11 cases), PBC group (11 cases), AIH-PBC (OS) group (10 cases), and DI-AILH group (8 cases). Baseline clinical data (gender, age, comorbidities of autoimmune diseases), laboratory indices, liver histology data, and treatment outcomes were collected, with a follow-up duration of 6 months and an endpoint of August 2026. Inclusion Criteria: AIH: Meeting the 2021 revised diagnostic scoring system [49] (≥ 15 points, definitive diagnosis) by the International Autoimmune Hepatitis Group (IAIHG) [5]. PBC: Meeting at least 2 of the following: ① Elevated alkaline phosphatase (ALP) and γ-glutamyl transferase (γ-GT) persisting for ≥ 6 months; ② Positive anti-mitochondrial antibody (AMA); ③ Histological evidence of lymphocytic interlobular cholangitis. AIH-PBC (OS): Meeting the 2011 "Paris Criteria" [6] (simultaneously satisfying the AIH simplified diagnostic criteria ≥ 6 points and the main PBC diagnostic criteria). DI-AILH: Based on the RUCAM (Roussel Uclaf Causality Assessment Method) scale for drug and liver injury causality [7] (score ≥ 6) and clinically presenting with "autoimmune-like features" (positive anti-nuclear antibody(ANA)or elevated IgG, accompanied by interface hepatitis). Exclusion Criteria: Coexisting viral hepatitis (hepatitis B, C, etc.), metabolic liver diseases (Wilson's disease, hemochromatosis); alcoholic liver disease, non-alcoholic fatty liver disease, or other causes of liver dysfunction; coexisting liver malignancies (hepatocellular carcinoma, cholangiocarcinoma) or alpha-fetoprotein (AFP) > 100 ng/mL; liver biopsy specimen length < 10 mm or containing less than 6 lobules (insufficient reliability for pathological assessment). 1.2 Research Methods 1.2.1 Collection of Clinical and Laboratory Indices Patient gender, age, and comorbidities of rheumatic immune diseases (e.g., rheumatoid arthritis, Sjögren's syndrome) were recorded, along with biochemical response status after 6 months of treatment (complete response: ALT, ALP return to normal range; partial response: ALT, ALP decrease ≥ 50% from baseline but not normal; no response: not meeting partial response criteria). Peripheral venous blood was collected at enrollment. ALT, total bilirubin (TBil), γ-GT, and ALP levels were measured using an automatic biochemical analyzer. Immunoglobulin G (IgG) and immunoglobulin M (IgM) levels were measured by immunoturbidimetry. ANA titers were detected by indirect immunofluorescence (categorized as low titer < 1:320, medium titer 1:320-1:640, high titer ≥ 1:640). 1.2.2 Liver Histology and Immunohistochemistry Assessment Liver tissue specimens were fixed in 4% formaldehyde, embedded in paraffin, and sectioned consecutively at 4 µm for HE staining. Two experienced liver pathologists performed blinded evaluations, using the Scheuer scoring system(Table 1)[8] for inflammation grading (0–4) and fibrosis staging (0–4). A semi-quantitative scoring system [9–10] was used for infiltrating inflammatory cells (lymphocytes, plasma cells, neutrophils, eosinophils): 0 points (no infiltration), 1 point (mild infiltration, involving 50% of field). Immunohistochemistry Detection: The EnVision two-step method was used with primary antibodies: Mum-1 (rabbit anti-human, 1:100), CD38 (mouse anti-human, 1:200), CD3 (rabbit anti-human, 1:150), CD68 (mouse anti-human, 1:200). Positive expression was defined by the presence of brown-yellow granules in the cytoplasm or cell membrane. The percentage of positive cells was calculated, with ≥ 10% considered positive expression. Table 1 Criteria for histological evaluation Histological Characteristics Scoring System Scoring Criteria InflammatoryCell (TypePortal Tract / Lobule) 0/1/2/3/4 0 point: Absent Lymphocyte 0/1/2/3/4 1 point: ≤5% Plasma Cell 0/1/2/3/4 2 points: 5%ཞ20% Neutrophil 0/1/2/3/4 3 points: 20%ཞ50% Eosinophil 0/1/2/3/4 4 points: >50% Rosette Formation Absent/Present Hepatocyte arrangement around central lumen; mononuclear lymphocytes in hepatocyte cytoplasm Emperipolesis Absent/Present Inflammation Grade 0/1/2/3/4 Schruer Score Fibrosis Stage 0/1/2/3/4 Schruer Score Inflammatory Cell Cluster Portal Tract Absent/Present Cluster: ≥5 plasma cells aggregated Lobule Absent/Present Cholestasis Absent/Present Yellow material deposition in bile ducts or hepatocytes Bile Duct Injury Absent/Present Inflammatory reactive change of bile duct epithelial cells Steatosis 0/1/2/3/4 NASH Steatosis Scoring Hepatocyte Necrosis 0/1/2/3/4 0 point: Absent1 point: Focal necrosis (punctate) 2 points: Focal necrosis (patchy) 3 points:Bridging confluent necrosis 4 points: Bridging massive necrosis ANATiter Classification Negative:<320Low:640 InflammatoryCell (TypePortal Tract / Lobule) 0/1/2/3/4 0 point: Absent 1.3 Statistical Methods Data analysis was performed using SPSS 27.0 statistical software. Normally distributed measurement data were expressed as mean ± standard deviation (x̄ ± s), and comparisons among multiple groups were conducted using one-way analysis of variance (ANOVA), with pairwise comparisons using the LSD-t test. Non-normally distributed data were expressed as median (interquartile range) [M (Q1, Q3)], with comparisons among multiple groups using the Kruskal-Wallis H test and pairwise comparisons using the Nemenyi test. Count data were expressed as number (percentage) [n (%)], with intergroup comparisons using the χ² test or Fisher's exact probability method. Univariate and multivariate logistic regression analyses were used to identify independent predictive factors for diseases. Pearson/Spearman correlation analysis was used to assess the association between ANA titer and various indices. A P-value < 0.05 was considered statistically significant. 2. Results 2.1 General Data Among the 40 patients, 6 were male (15.0%) and 34 were female (85.0%), with a male-to-female ratio of 1:5.7. The mean age was (55.77 ± 12.34) years, predominantly middle-aged and elderly patients (≥ 50 years old accounted for 67.5%, 27/40). Five patients (12.5%) had other autoimmune diseases (2 Sjögren's syndrome, 2 rheumatoid arthritis, 1 systemic lupus erythematosus). Age and gender distribution across the four groups: The mean age of the DI-AILH group (48.75 ± 4.46 years) was significantly lower than the other three groups (AIH: 64.55 ± 10.68 years; PBC: 53.09 ± 13.68 years; OS: 54.70 ± 12.71 years) (P < 0.05). 2.2 Histological Features 2.2.1 Inflammation and Fibrosis Degree Both inflammation grade and fibrosis stage were higher in the AIH and OS groups compared to the DI-AILH and PBC groups. Inflammation grade: [AIH: (3.64 ± 0.50), PBC: (2.36 ± 0.67), OS: (3.00 ± 0.67), DI-AILH: (2.25 ± 1.16)]. Fibrosis stage: [AIH: (3.09 ± 0.83), PBC: (2.36 ± 1.29), OS: (2.80 ± 0.79), DI-AILH: (1.38 ± 0.74)]. The differences were statistically significant (all P < 0.05). 2.2.2 Characteristic Pathological Changes Rosette formation: AIH group 8 cases (72.7%), AIH-PBC (OS) group 7 cases (70.0%), significantly more than DI-AILH group 1 case (12.5%) and PBC group 2 cases (18.2%) (all P < 0.05). Portal inflammatory cell cluster infiltration: AIH group 9 cases (81.8%), AIH-PBC (OS) group 8 cases (80.0%), significantly more than PBC group 4 cases (36.4%) and DI-AILH group 3 cases (37.5%) (all P < 0.05). Severe interface hepatitis (Ishak grade 3): AIH group 3 cases (27.3%), AIH-PBC (OS) group 2 cases (20.0%), not present in the PBC and DI-AILH groups (P < 0.05). 2.3 ANA Titer and Disease Correlation Low-titer ANA ( 1:640)] was mainly distributed in the PBC group (9/11, 81.8%) and AIH-PBC (OS) group (8/10, 80.0%). The intergroup difference was statistically significant (P < 0.05). 2.4 Biochemical and Immunoglobulin Levels IgG: AIH group [2520.00 (2190.00, 3565.00)] was significantly higher than PBC group [1890.00 (1580.00, 2125.00)], AIH-PBC (OS) group [1705.00 (1612.50, 2180.00)], and DI-AILH group [1760.00 (1210.00, 2202.50)] (all P < 0.05). ALT: DI-AILH group (360.12 ± 420.84) was significantly higher than AIH group (295.36 ± 247.30), AIH-PBC (OS) group (116.50 ± 63.97), and PBC group (78.09 ± 64.10) (all P < 0.05). 2.5 Treatment Response Analysis Six-month complete biochemical response rate: PBC group 90.9% (10/11), AIH group 72.7% (8/11), AIH-PBC (OS) group 60.0% (6/10). The difference in complete response rate between the AIH and OS groups was not statistically significant (P > 0.05). In the PBC group, the single non-responder had significantly higher ALP and ALT levels compared to responders (both P < 0.05). In the AIH group, ANA titer showed a borderline negative correlation with treatment response (r=-0.58, P = 0.055), suggesting a tendency for higher response rates in patients with lower ANA titers. 2.6 Multivariate Analysis Using "whether it is AIH" as the dependent variable (1 = yes, 0 = no), indicators with P < 0.1 in univariate analysis (IgG, ANA titer, plasma cell infiltration score, etc.) were included in multivariate logistic regression. Results showed that elevated IgG was an independent diagnostic factor for AIH (OR = 1.01, 95% CI: 1.01 ~ 1.01, P < 0.05). Using "whether it is AIH-PBC (OS) " as the dependent variable (1 = yes, 0 = no), indicators with P < 0.1 in univariate analysis (ANA titer, IgM, ALP, etc.)[20、21] were included in multivariate logistic regression. Results showed that high-titer ANA (≥ 1:640) was an independent predictive factor for AIH-PBC (OS) (OR = 15.75, 95% CI: 2.30 ~ 107.93, P < 0.05). 2.7 Immunohistochemical Expression Comparison of positive rates of immunohistochemical markers among the four groups: (Figure 2、Figure 3 ) shows the comparison of positive rates for CD38, CD3, CD68, and Mum-1 immunohistochemical staining in AIH, PBC, DI-AILH, and AIH-PBC (OS) patients. AIH-PBC (OS): Showed the most prominent T-cell infiltration and immune activation (CD3, CD38), reflecting dual "cellular immunity + immune activation" overlay. AIH: Exhibited active macrophage infiltration (CD68) and plasma cell function (Mum-1), representing a combined "cellular immunity (macrophages) + humoral immunity" drive. PBC: Had plasma cell function (Mum-1) comparable to AIH, but lower T-cell and immune activation involvement, leaning towards "humoral immunity + bile duct-specific injury". DI-AILH: Generally showed lower involvement of all immune markers, reflecting the fundamental difference of its "drug-induced" immune injury mechanism compared to traditional autoimmune liver diseases. 3. Discussion "differential diagnosis among AILD (AIH, PBC, AIH-PBC(OS)) and DI-AILH）" and "factors influencing 6-month treatment response." It analyzes the specific manifestations and clinical significance of each disease group from three dimensions: pathological features, biochemical indices (e.g., IgG, ALP), and immunohistochemical mechanisms (e.g., CD3, CD38, CD68, Mum-1). Ultimately, it proposes diagnostic optimization suggestions and points out research limitations, providing references for clinical practice. Diagnostic Dilemmas and Multidimensional Evaluation Strategies of Autoimmune Hepatitis (AIH) Autoimmune hepatitis (AIH), a classic autoimmune liver disease, has a prognosis that is directly determined by treatment response.,A 2024 multicenter retrospective study by the International Autoimmune Hepatitis Group involving 2559 AIH patients showed that failure to achieve complete biochemical response (normalization of transaminases and IgG) at 6 months of treatment was an independent risk factor for liver-related death and liver transplantation [19、20、21、33]; furthermore, patients achieving response within 6 months had a significantly higher 20-year survival rate (91%) compared to non-responders (61%). This conclusion highlights the critical role of early accurate diagnosis and timely treatment in AIH prognosis. However, current AIH diagnosis faces significant bottlenecks—lack of specific biomarkers, reliance on scoring systems [1], low positivity rate and specificity of autoantibodies; some antibody-negative patients may even have normal IgG levels [19、41、42、43], further increasing diagnostic difficulty, leading to delayed immunosuppressive therapy for some patients and ultimately affecting treatment response. This study provides new insights for AIH diagnosis from pathological and immunological perspectives. Pathologically, AIH patients exhibit significant interface hepatitis, Rosette formation, emperipolesis, and clustered plasma cell infiltration at the limiting plate; portal lymphocyte infiltration is also pronounced [38]. Inflammation grade and fibrosis degree are higher than in other disease groups, likely due to cumulative chronic immune responses. Biochemically, elevated immunoglobulin IgG is an important factor influencing AIH diagnosis [19、20、21、37]. Immunohistochemistry further shows high expression of macrophage infiltration marker CD68 and plasma cell differentiation marker Mum-1 [40], suggesting AIH is a disease driven by both "macrophage-mediated cellular immunity and plasma cell-dominated humoral immunity" [16, 17, 18]. Based on this, characteristic pathological manifestations, immunohistochemical markers, and immunoglobulin levels can be integrated clinically to construct a multidimensional assessment system, breaking through traditional diagnostic limitations and improving AIH diagnostic efficiency. Differentiation between DI-AILH and AIH: From Morphological Similarity to Mechanistic Distinction DI-AILH [44], as a special type of DILI [50], shares histological similarities with AIH—such as lymphoplasmacytic infiltration in portal stroma and interface hepatitis, and varying degrees of confluent necrosis in liver parenchyma. However, different drugs can induce heterogeneous histological phenotypes, and there are fundamental differences in pathogenesis and clinical management between DI-AILH and AIH. Precise differentiation is crucial to avoid overtreatment or undertreatment. Serological characteristics provide important clues for differentiation. Low-titer autoantibodies are often associated with chronic DILI and idiosyncratic DILI, while higher IgG levels are closely linked to DI-AILH. Hisamochi et al. [45] found in a series study of 62 patients that the serum IgG-to-normal ratio could serve as a predictive indicator for DI-AILH, and serum IgG >1800 mg/dL had high sensitivity for DI-AILH. In this study, the average IgG level in the DI-AILH group was 1760.00 mg/dL, second only to the AIH group, further validating the clinical value of this association. Differences in immune mechanisms are the core basis for differentiation. This study found that the expression levels of various immune markers (CD3, CD38, CD68, Mum-1) in DI-AILH were generally lower than in traditional AILD like AIH, reflecting the fundamental difference between its "drug-induced" immune injury mechanism and autoimmune-driven liver disease—the immune abnormalities in DI-AILH are more likely a "secondary" reaction triggered directly or indirectly by drugs, rather than a "primary" lesion caused by autoimmune system dysregulation. This mechanistic difference not only clarifies the pathological boundary between the two but also provides theoretical support for formulating differential treatment strategies (e.g., priority to drug withdrawal and observation for DI-AILH, prompt immunosuppressive therapy for AIH). Clinical Challenges and Exploration of Mechanistic Features in PBC and AIH-PBC OS Treatment Response Challenges in PBC PBC, as an AILD centered on bile duct injury, sees approximately 40% of patients showing poor response to first-line UDCA, posing a significant clinical challenge. Previous studies suggest that alkaline phosphatase (ALP) is significantly associated with PBC protein components, and OS. Degree of liver inflammation and fibrosis are also key factors influencing response [18]. This study further confirms that elevated ALP and ALT are independent risk factors for non-response to treatment in PBC. More notably, some patients who do not fully meet diagnostic criteria for OS exhibit AIH-like features, termed "PBC with AIH-like features" in the literature [33, 34]. Failure to promptly add immunosuppressive therapy for such patients may lead to uncontrolled disease progression. Therefore, exploring "whether PBC patients with elevated ALP and ALT should initiate second-line drugs [35, 36] or immunosuppressants earlier" is necessary to improve treatment outcomes. Immunologically, plasma cell function (Mum-1 expression) in PBC is comparable to AIH, but T-cell infiltration (CD3) and immune activation (CD38) involvement are lower, suggesting its pathological mechanism leans more towards "humoral immunity dominance + bile duct-specific injury" [46, 47]. This contrasts sharply with the AIH model of "synergistic drive of cellular + humoral immunity," explaining why PBC treatment primarily focuses on bile duct protection, while AIH requires suppression of systemic immune responses. Recognition and Immune Overlay Mechanism of [14、48] is the most common overlap form in AILD, with a prevalence of about 5%~15% among PBC patients. The diagnosis is mainly based on the Paris criteria [3], and treatment recommends UDCA combined with immunosuppressants. However, due to complex clinical presentations, early recognition is challenging. This study found many commonalities in histopathology between OS and AIH [39]—such as interface hepatitis, Rosette formation, emperipolesis, clustered plasma cell infiltration at the limiting plate, and portal lymphocyte enrichment, with higher inflammation grade and fibrosis degree. High-titer ANA is an independent influencing factor for OS, and whether it can serve as a marker predicting progression to OS warrants further exploration in larger-scale studies. Immunohistochemical features further reveal the pathogenesis of OS: OS shows the most prominent T-cell infiltration (CD3) and immune activation (CD38) levels among the four disease groups, embodying a dual mechanism of "cellular immune activation + immune response overlay"—inheriting the T-cell-mediated immune response from AIH while retaining the bile duct injury-related immune characteristics of PBC. This also leads to more complex conditions and higher treatment difficulty in OS, necessitating combination therapies to balance different dimensions of immune abnormalities. 4.Conclusion Combining histopathological features (interface hepatitis, rosette formation)[12、13], immunohistochemical markers (Mum-1, CD38, CD3, CD68)[15], and serological indices (IgG, ANA titer, ALP) can significantly improve the differential diagnostic accuracy among AIH, AIH-PBC (OS), PBC, and DI-AILH. Elevated IgG is an independent diagnostic factor for AIH, and high-titer ANA is an independent predictive factor for AIH-PBC (OS), serving as core serum markers for disease classification. Pre-treatment ALP and ALT levels in PBC patients can predict response to ursodeoxycholic acid (UDCA) therapy; high levels indicate a risk of non-response, suggesting the need for early assessment for second-line therapy. Early precise classification and individualized treatment (e.g., combined "corticosteroids + UDCA" regimen for AIH-PBC (OS)) are key to improving the prognosis of patients with AILD. Study Limitations and Future Perspectives This study has certain limitations. Firstly, the sample size in each group is limited, and conclusions are based on small-sample statistics, potentially introducing selection bias. Secondly, differences in patient compliance may affect timely assessment of treatment efficacy, leading to potential bias in data interpretation. Future research could advance in three directions: First, conducting large-scale, multicenter cohort studies to validate findings such as the "AIH multidimensional diagnostic model," "DI-AILH IgG threshold," and "PBC response risk factors." Second, further exploring the predictive value of high-titer ANA for OS and developing more precise disease progression assessment tools. Third, integrating immunohistochemical markers with molecular detection to identify potential specific biomarkers, further enhancing diagnostic and therapeutic precision. With the deepening of research on AILD, more underlying pathogenic mechanisms will be elucidated, and more efficient assessment methods will be applied in clinical practice, ultimately addressing the core issues of "difficult differentiation and poor response," and providing patients with better diagnostic and therapeutic services. Declarations Standard Approval Statement:This study was approved by the Ethics Committee of the Leshan People's Hospital (Approval No. LYLL【2026】KY 075) on February 24, 2026. All procedures performed in this study involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8776539","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":593581426,"identity":"279ccd7d-53fb-4c0b-a474-463d4aad6478","order_by":0,"name":"Su Jing","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwUlEQVRIiWNgGAWjYBACfmbmAwcSeCTk+InWItnOlnjgg4yNsWQDsVoMzvMYH5xhk5a44QDR1hxmMDjMk3PY2Ph48gaGHxXbCOtgbGZIOMxz5rCc2ZlnBYw9Z24T1sLMzHDgMG/PYWOzGzkGzIxtRGhhY2ZsOMz773Di5hnEauEBWnNwBg/Q+xLEapFgZmM48IHHxlgC6JeDRPnF/vz5zx/AUdmevPHBjwoitCCBBIMDJKkHayFVxygYBaNgFIwQAACV9T72s4WSFwAAAABJRU5ErkJggg==","orcid":"","institution":"The People's Hospital of LeShan","correspondingAuthor":true,"prefix":"","firstName":"Su","middleName":"","lastName":"Jing","suffix":""},{"id":593581429,"identity":"86764697-5309-434f-85f3-940b8ac042ce","order_by":1,"name":"Liang Shi-yi","email":"","orcid":"","institution":"The People's Hospital of LeShan","correspondingAuthor":false,"prefix":"","firstName":"Liang","middleName":"","lastName":"Shi-yi","suffix":""},{"id":593581430,"identity":"24de142e-fa41-40ad-9f87-614044500dd1","order_by":2,"name":"Chen yu-qing","email":"","orcid":"","institution":"The People's Hospital of LeShan","correspondingAuthor":false,"prefix":"","firstName":"Chen","middleName":"","lastName":"yu-qing","suffix":""},{"id":593581431,"identity":"32177da3-6ce7-4fb3-a141-1b454a3e509e","order_by":3,"name":"Luo rui","email":"","orcid":"","institution":"The People's Hospital of LeShan","correspondingAuthor":false,"prefix":"","firstName":"Luo","middleName":"","lastName":"rui","suffix":""}],"badges":[],"createdAt":"2026-02-03 13:38:46","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8776539/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8776539/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103806108,"identity":"6b94492b-9b0d-4861-b3bc-4f27557e86db","added_by":"auto","created_at":"2026-03-03 07:22:51","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":224501,"visible":true,"origin":"","legend":"\u003cp\u003eFlow chart of the study design\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8776539/v1/15cfe397214ba49c0306b19c.jpeg"},{"id":103806107,"identity":"c9dffd33-9449-49a8-9554-91a156e03e9b","added_by":"auto","created_at":"2026-03-03 07:22:51","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":141949,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of the number of CD38, MUM1, CD4 and CD8 positive cells in AIH,PBC,DI-AILH and OS patients\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8776539/v1/9ef74fff2bb9ff7618dfea37.png"},{"id":104400414,"identity":"7aecdc73-62c5-4d37-8848-8b3405edffb8","added_by":"auto","created_at":"2026-03-11 12:09:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":576361,"visible":true,"origin":"","legend":"\u003cp\u003ePathological findings of CD38, MUM1, CD3, and CD68-positive staining in patients with autoimmune hepatitis (AIH), primary biliary cholangitis (PBC), drug-induced autoimmune-like hepatitis (DI-ALH), and AIH-PBC overlap syndrome (OS)\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8776539/v1/d792d6e1fc075984a392a1e5.png"},{"id":104408088,"identity":"644ea55d-2954-4ddc-a74d-d173402f15f1","added_by":"auto","created_at":"2026-03-11 12:41:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1574971,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8776539/v1/ff1bce4a-77f8-4307-aa4c-3235ae453bd9.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Application of Histopathology, Immunohistochemistry, and Antibody Titers in the Diagnosis and Treatment Response Assessment of Autoimmune Liver Diseases","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAutoimmune liver diseases (AILD) are a group of chronic inflammatory hepatobiliary diseases mediated by aberrant autoimmune responses, primarily including autoimmune hepatitis (AIH), primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), and overlap syndromes (OS). Among these, AIH and PBC are clinically most common and may present concurrently or sequentially in the same patient, known as AIH-PBC (OS). Diagnosis currently follows the internationally \"Paris Criteria\" updated 2011 [3、49]. Furthermore, drug-induced autoimmune-like hepatitis (DI-AILH), a special type of drug-induced liver injury, exhibits high similarity in clinical symptoms, serology, and histology to AIH[\u003cspan additionalcitationids=\"CR22 CR23 CR24 CR25 CR26 CR27 CR28 CR29 CR30\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], but responds well to corticosteroid therapy with a low relapse rate after discontinuation. According to the 2023 guidelines for the diagnosis and treatment of drug-induced liver injury(DILI) [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], differential diagnosis between DI-AILH and AIH requires combining liver biopsy pathological features with long-term follow-up (\u0026ge;\u0026thinsp;6 months). This study systematically analyzes the differences in clinical characteristics, histopathological morphology, immunohistochemical marker expression, serum antibody titers, and biochemical response among patients with AIH, PBC, AIH-PBC (OS), and DI-AILH, aiming to identify key indicators for differential diagnosis and explore predictive factors for treatment response, providing references for precise clinical diagnosis and treatment.\u003c/p\u003e"},{"header":"1. Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003e1.1\u003c/strong\u003e Study Subjects Forty patients who underwent ultrasound-guided liver biopsy at the Department of Gastroenterology, Leshan People\u0026apos;s Hospital, from January 2022 to August 2025 were retrospectively enrolled and divided into four groups based on diagnostic criteria: AIH group (11 cases), PBC group (11 cases), AIH-PBC (OS) group (10 cases), and DI-AILH group (8 cases). Baseline clinical data (gender, age, comorbidities of autoimmune diseases), laboratory indices, liver histology data, and treatment outcomes were collected, with a follow-up duration of 6 months and an endpoint of August 2026. Inclusion Criteria: AIH: Meeting the 2021 revised diagnostic scoring system [49] (\u0026ge;\u0026thinsp;15 points, definitive diagnosis) by the International Autoimmune Hepatitis Group (IAIHG) [5]. PBC: Meeting at least 2 of the following: ① Elevated alkaline phosphatase (ALP) and \u0026gamma;-glutamyl transferase (\u0026gamma;-GT) persisting for \u0026ge;\u0026thinsp;6 months; ② Positive anti-mitochondrial antibody (AMA); ③ Histological evidence of lymphocytic interlobular cholangitis. AIH-PBC (OS): Meeting the 2011 \u0026quot;Paris Criteria\u0026quot; [6] (simultaneously satisfying the AIH simplified diagnostic criteria\u0026thinsp;\u0026ge;\u0026thinsp;6 points and the main PBC diagnostic criteria). DI-AILH: Based on the RUCAM (Roussel Uclaf Causality Assessment Method) scale for drug and liver injury causality [7] (score\u0026thinsp;\u0026ge;\u0026thinsp;6) and clinically presenting with \u0026quot;autoimmune-like features\u0026quot; (positive anti-nuclear antibody(ANA)or elevated IgG, accompanied by interface hepatitis). Exclusion Criteria: Coexisting viral hepatitis (hepatitis B, C, etc.), metabolic liver diseases (Wilson\u0026apos;s disease, hemochromatosis); alcoholic liver disease, non-alcoholic fatty liver disease, or other causes of liver dysfunction; coexisting liver malignancies (hepatocellular carcinoma, cholangiocarcinoma) or alpha-fetoprotein (AFP)\u0026thinsp;\u0026gt;\u0026thinsp;100 ng/mL; liver biopsy specimen length\u0026thinsp;\u0026lt;\u0026thinsp;10 mm or containing less than 6 lobules (insufficient reliability for pathological assessment).\u003c/p\u003e\n\u003ch3\u003e1.2 Research Methods\u003c/h3\u003e\n\u003cp\u003e\u003cstrong\u003e1.2.1\u003c/strong\u003e Collection of Clinical and Laboratory Indices Patient gender, age, and comorbidities of rheumatic immune diseases (e.g., rheumatoid arthritis, Sj\u0026ouml;gren\u0026apos;s syndrome) were recorded, along with biochemical response status after 6 months of treatment (complete response: ALT, ALP return to normal range; partial response: ALT, ALP decrease\u0026thinsp;\u0026ge;\u0026thinsp;50% from baseline but not normal; no response: not meeting partial response criteria). Peripheral venous blood was collected at enrollment. ALT, total bilirubin (TBil), \u0026gamma;-GT, and ALP levels were measured using an automatic biochemical analyzer. Immunoglobulin G (IgG) and immunoglobulin M (IgM) levels were measured by immunoturbidimetry. ANA titers were detected by indirect immunofluorescence (categorized as low titer\u0026thinsp;\u0026lt;\u0026thinsp;1:320, medium titer 1:320-1:640, high titer\u0026thinsp;\u0026ge;\u0026thinsp;1:640).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e1.2.2\u003c/strong\u003e Liver Histology and Immunohistochemistry Assessment Liver tissue specimens were fixed in 4% formaldehyde, embedded in paraffin, and sectioned consecutively at 4 \u0026micro;m for HE staining. Two experienced liver pathologists performed blinded evaluations, using the Scheuer scoring system(Table 1)[8] for inflammation grading (0\u0026ndash;4) and fibrosis staging (0\u0026ndash;4). A semi-quantitative scoring system [9\u0026ndash;10] was used for infiltrating inflammatory cells (lymphocytes, plasma cells, neutrophils, eosinophils): 0 points (no infiltration), 1 point (mild infiltration, involving\u0026thinsp;\u0026lt;\u0026thinsp;10% of field), 2 points (moderate infiltration, involving 10%-30% of field), 3 points (marked infiltration, involving 30%-50% of field), 4 points (severe infiltration, involving\u0026thinsp;\u0026gt;\u0026thinsp;50% of field). Immunohistochemistry Detection: The EnVision two-step method was used with primary antibodies: Mum-1 (rabbit anti-human, 1:100), CD38 (mouse anti-human, 1:200), CD3 (rabbit anti-human, 1:150), CD68 (mouse anti-human, 1:200). Positive expression was defined by the presence of brown-yellow granules in the cytoplasm or cell membrane. The percentage of positive cells was calculated, with \u0026ge;\u0026thinsp;10% considered positive expression.\u003c/p\u003e\n\u003cdiv\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 1\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eCriteria for histological evaluation\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eHistological Characteristics\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eScoring System\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eScoring Criteria\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eInflammatoryCell (TypePortal Tract / Lobule)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0/1/2/3/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 point: Absent\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLymphocyte\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0/1/2/3/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1 point: \u0026le;5%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePlasma Cell\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0/1/2/3/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2 points: 5%ཞ20%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNeutrophil\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0/1/2/3/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3 points: 20%ཞ50%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEosinophil\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0/1/2/3/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4 points: \u0026gt;50%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRosette Formation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAbsent/Present\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHepatocyte arrangement around central lumen; mononuclear lymphocytes in hepatocyte cytoplasm\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEmperipolesis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAbsent/Present\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eInflammation Grade\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0/1/2/3/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSchruer Score\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFibrosis Stage\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0/1/2/3/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSchruer Score\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eInflammatory Cell Cluster\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePortal Tract\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAbsent/Present\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCluster: \u0026ge;5 plasma cells aggregated\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLobule\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAbsent/Present\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCholestasis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAbsent/Present\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eYellow material deposition in bile ducts or hepatocytes\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBile Duct Injury\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAbsent/Present\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eInflammatory reactive change of bile duct epithelial cells\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSteatosis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0/1/2/3/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNASH Steatosis Scoring\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHepatocyte Necrosis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0/1/2/3/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 point: Absent1 point: Focal necrosis (punctate)\u003c/p\u003e\n \u003cp\u003e2 points: Focal necrosis (patchy)\u003c/p\u003e\n \u003cp\u003e3 points:Bridging confluent necrosis\u003c/p\u003e\n \u003cp\u003e4 points: Bridging massive necrosis\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eANATiter Classification\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNegative:\u0026lt;320Low:\u0026lt;320Moderate: 320-640High: \u0026gt;640\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eInflammatoryCell (TypePortal Tract / Lobule)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0/1/2/3/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 point: Absent\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003e1.3\u003c/strong\u003e Statistical Methods Data analysis was performed using SPSS 27.0 statistical software. Normally distributed measurement data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (x̄ \u0026plusmn; s), and comparisons among multiple groups were conducted using one-way analysis of variance (ANOVA), with pairwise comparisons using the LSD-t test. Non-normally distributed data were expressed as median (interquartile range) [M (Q1, Q3)], with comparisons among multiple groups using the Kruskal-Wallis H test and pairwise comparisons using the Nemenyi test. Count data were expressed as number (percentage) [n (%)], with intergroup comparisons using the \u0026chi;\u0026sup2; test or Fisher\u0026apos;s exact probability method. Univariate and multivariate logistic regression analyses were used to identify independent predictive factors for diseases. Pearson/Spearman correlation analysis was used to assess the association between ANA titer and various indices. A P-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"2. Results","content":"\u003cp\u003e\u003cstrong\u003e2.1\u003c/strong\u003e General Data Among the 40 patients, 6 were male (15.0%) and 34 were female (85.0%), with a male-to-female ratio of 1:5.7. The mean age was (55.77\u0026thinsp;\u0026plusmn;\u0026thinsp;12.34) years, predominantly middle-aged and elderly patients (\u0026ge;\u0026thinsp;50 years old accounted for 67.5%, 27/40). Five patients (12.5%) had other autoimmune diseases (2 Sj\u0026ouml;gren\u0026apos;s syndrome, 2 rheumatoid arthritis, 1 systemic lupus erythematosus). Age and gender distribution across the four groups: The mean age of the DI-AILH group (48.75\u0026thinsp;\u0026plusmn;\u0026thinsp;4.46 years) was significantly lower than the other three groups (AIH: 64.55\u0026thinsp;\u0026plusmn;\u0026thinsp;10.68 years; PBC: 53.09\u0026thinsp;\u0026plusmn;\u0026thinsp;13.68 years; OS: 54.70\u0026thinsp;\u0026plusmn;\u0026thinsp;12.71 years) (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2\u003c/strong\u003e Histological Features\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2.1\u003c/strong\u003e Inflammation and Fibrosis Degree Both inflammation grade and fibrosis stage were higher in the AIH and OS groups compared to the DI-AILH and PBC groups. Inflammation grade: [AIH: (3.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50), PBC: (2.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.67), OS: (3.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.67), DI-AILH: (2.25\u0026thinsp;\u0026plusmn;\u0026thinsp;1.16)]. Fibrosis stage: [AIH: (3.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.83), PBC: (2.36\u0026thinsp;\u0026plusmn;\u0026thinsp;1.29), OS: (2.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.79), DI-AILH: (1.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.74)]. The differences were statistically significant (all P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2.2\u003c/strong\u003e Characteristic Pathological Changes Rosette formation: AIH group 8 cases (72.7%), AIH-PBC (OS) group 7 cases (70.0%), significantly more than DI-AILH group 1 case (12.5%) and PBC group 2 cases (18.2%) (all P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Portal inflammatory cell cluster infiltration: AIH group 9 cases (81.8%), AIH-PBC (OS) group 8 cases (80.0%), significantly more than PBC group 4 cases (36.4%) and DI-AILH group 3 cases (37.5%) (all P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Severe interface hepatitis (Ishak grade 3): AIH group 3 cases (27.3%), AIH-PBC (OS) group 2 cases (20.0%), not present in the PBC and DI-AILH groups (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3\u003c/strong\u003e ANA Titer and Disease Correlation Low-titer ANA (\u0026lt;\u0026thinsp;1:320) was mainly distributed in the AIH group (8/11, 72.7%) and DI-AILH group (7/8, 87.5%). Medium/high-titer ANA [(1:320-1:640), (\u0026gt;\u0026thinsp;1:640)] was mainly distributed in the PBC group (9/11, 81.8%) and AIH-PBC (OS) group (8/10, 80.0%). The intergroup difference was statistically significant (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4\u003c/strong\u003e Biochemical and Immunoglobulin Levels IgG: AIH group [2520.00 (2190.00, 3565.00)] was significantly higher than PBC group [1890.00 (1580.00, 2125.00)], AIH-PBC (OS) group [1705.00 (1612.50, 2180.00)], and DI-AILH group [1760.00 (1210.00, 2202.50)] (all P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). ALT: DI-AILH group (360.12\u0026thinsp;\u0026plusmn;\u0026thinsp;420.84) was significantly higher than AIH group (295.36\u0026thinsp;\u0026plusmn;\u0026thinsp;247.30), AIH-PBC (OS) group (116.50\u0026thinsp;\u0026plusmn;\u0026thinsp;63.97), and PBC group (78.09\u0026thinsp;\u0026plusmn;\u0026thinsp;64.10) (all P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5\u003c/strong\u003e Treatment Response Analysis Six-month complete biochemical response rate: PBC group 90.9% (10/11), AIH group 72.7% (8/11), AIH-PBC (OS) group 60.0% (6/10). The difference in complete response rate between the AIH and OS groups was not statistically significant (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05). In the PBC group, the single non-responder had significantly higher ALP and ALT levels compared to responders (both P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In the AIH group, ANA titer showed a borderline negative correlation with treatment response (r=-0.58, P\u0026thinsp;=\u0026thinsp;0.055), suggesting a tendency for higher response rates in patients with lower ANA titers.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6\u003c/strong\u003e Multivariate Analysis Using \u0026quot;whether it is AIH\u0026quot; as the dependent variable (1\u0026thinsp;=\u0026thinsp;yes, 0\u0026thinsp;=\u0026thinsp;no), indicators with P\u0026thinsp;\u0026lt;\u0026thinsp;0.1 in univariate analysis (IgG, ANA titer, plasma cell infiltration score, etc.) were included in multivariate logistic regression. Results showed that elevated IgG was an independent diagnostic factor for AIH (OR\u0026thinsp;=\u0026thinsp;1.01, 95% CI: 1.01\u0026thinsp;~\u0026thinsp;1.01, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Using \u0026quot;whether it is AIH-PBC (OS) \u0026quot; as the dependent variable (1\u0026thinsp;=\u0026thinsp;yes, 0\u0026thinsp;=\u0026thinsp;no), indicators with P\u0026thinsp;\u0026lt;\u0026thinsp;0.1 in univariate analysis (ANA titer, IgM, ALP, etc.)[20、21] were included in multivariate logistic regression. Results showed that high-titer ANA (\u0026ge;\u0026thinsp;1:640) was an independent predictive factor for AIH-PBC (OS) (OR\u0026thinsp;=\u0026thinsp;15.75, 95% CI: 2.30\u0026thinsp;~\u0026thinsp;107.93, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.7\u003c/strong\u003e Immunohistochemical Expression Comparison of positive rates of immunohistochemical markers among the four groups: (Figure 2、Figure 3 ) shows the comparison of positive rates for CD38, CD3, CD68, and Mum-1 immunohistochemical staining in AIH, PBC, DI-AILH, and AIH-PBC (OS) patients. AIH-PBC (OS): Showed the most prominent T-cell infiltration and immune activation (CD3, CD38), reflecting dual \u0026quot;cellular immunity\u0026thinsp;+\u0026thinsp;immune activation\u0026quot; overlay. AIH: Exhibited active macrophage infiltration (CD68) and plasma cell function (Mum-1), representing a combined \u0026quot;cellular immunity (macrophages) + humoral immunity\u0026quot; drive. PBC: Had plasma cell function (Mum-1) comparable to AIH, but lower T-cell and immune activation involvement, leaning towards \u0026quot;humoral immunity\u0026thinsp;+\u0026thinsp;bile duct-specific injury\u0026quot;. DI-AILH: Generally showed lower involvement of all immune markers, reflecting the fundamental difference of its \u0026quot;drug-induced\u0026quot; immune injury mechanism compared to traditional autoimmune liver diseases.\u003c/p\u003e"},{"header":"3. Discussion","content":"\u003cp\u003e\u0026quot;differential diagnosis among AILD (AIH, PBC, AIH-PBC(OS)) and DI-AILH）\u0026quot; and \u0026quot;factors influencing 6-month treatment response.\u0026quot; It analyzes the specific manifestations and clinical significance of each disease group from three dimensions: pathological features, biochemical indices (e.g., IgG, ALP), and immunohistochemical mechanisms (e.g., CD3, CD38, CD68, Mum-1). Ultimately, it proposes diagnostic optimization suggestions and points out research limitations, providing references for clinical practice.\u003c/p\u003e\n\u003col\u003e\n \u003cli\u003e\u0026nbsp;Diagnostic Dilemmas and Multidimensional Evaluation Strategies of Autoimmune Hepatitis (AIH)\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eAutoimmune hepatitis (AIH), a classic autoimmune liver disease, has a prognosis that is directly determined by treatment response.,A 2024 multicenter retrospective study by the International Autoimmune Hepatitis Group involving 2559 AIH patients showed that failure to achieve complete biochemical response (normalization of transaminases and IgG) at 6 months of treatment was an independent risk factor for liver-related death and liver transplantation [19、20、21、33]; furthermore, patients achieving response within 6 months had a significantly higher 20-year survival rate (91%) compared to non-responders (61%). This conclusion highlights the critical role of early accurate diagnosis and timely treatment in AIH prognosis. However, current AIH diagnosis faces significant bottlenecks\u0026mdash;lack of specific biomarkers, reliance on scoring systems [1], low positivity rate and specificity of autoantibodies; some antibody-negative patients may even have normal IgG levels [19、41、42、43], further increasing diagnostic difficulty, leading to delayed immunosuppressive therapy for some patients and ultimately affecting treatment response. This study provides new insights for AIH diagnosis from pathological and immunological perspectives. Pathologically, AIH patients exhibit significant interface hepatitis, Rosette formation, emperipolesis, and clustered plasma cell infiltration at the limiting plate; portal lymphocyte infiltration is also pronounced [38]. Inflammation grade and fibrosis degree are higher than in other disease groups, likely due to cumulative chronic immune responses. Biochemically, elevated immunoglobulin IgG is an important factor influencing AIH diagnosis [19、20、21、37]. Immunohistochemistry further shows high expression of macrophage infiltration marker CD68 and plasma cell differentiation marker Mum-1 [40], suggesting AIH is a disease driven by both \u0026quot;macrophage-mediated cellular immunity and plasma cell-dominated humoral immunity\u0026quot; [16, 17, 18]. Based on this, characteristic pathological manifestations, immunohistochemical markers, and immunoglobulin levels can be integrated clinically to construct a multidimensional assessment system, breaking through traditional diagnostic limitations and improving AIH diagnostic efficiency.\u0026nbsp;\u003c/p\u003e\n\u003col start=\"2\"\u003e\n \u003cli\u003eDifferentiation between DI-AILH and AIH: From Morphological Similarity to Mechanistic Distinction DI-AILH [44], as a special type of DILI [50], shares histological similarities with AIH\u0026mdash;such as lymphoplasmacytic infiltration in portal stroma and interface hepatitis, and varying degrees of confluent necrosis in liver parenchyma. However, different drugs can induce heterogeneous histological phenotypes, and there are fundamental differences in pathogenesis and clinical management between DI-AILH and AIH. Precise differentiation is crucial to avoid overtreatment or undertreatment. Serological characteristics provide important clues for differentiation. Low-titer autoantibodies are often associated with chronic DILI and idiosyncratic DILI, while higher IgG levels are closely linked to DI-AILH. Hisamochi et al. [45] found in a series study of 62 patients that the serum IgG-to-normal ratio could serve as a predictive indicator for DI-AILH, and serum IgG \u0026gt;1800 mg/dL had high sensitivity for DI-AILH. In this study, the average IgG level in the DI-AILH group was 1760.00 mg/dL, second only to the AIH group, further validating the clinical value of this association. Differences in immune mechanisms are the core basis for differentiation. This study found that the expression levels of various immune markers (CD3, CD38, CD68, Mum-1) in DI-AILH were generally lower than in traditional AILD like AIH, reflecting the fundamental difference between its \u0026quot;drug-induced\u0026quot; immune injury mechanism and autoimmune-driven liver disease\u0026mdash;the immune abnormalities in DI-AILH are more likely a \u0026quot;secondary\u0026quot; reaction triggered directly or indirectly by drugs, rather than a \u0026quot;primary\u0026quot; lesion caused by autoimmune system dysregulation. This mechanistic difference not only clarifies the pathological boundary between the two but also provides theoretical support for formulating differential treatment strategies (e.g., priority to drug withdrawal and observation for DI-AILH, prompt immunosuppressive therapy for AIH).\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eClinical Challenges and Exploration of Mechanistic Features in PBC and AIH-PBC OS\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eTreatment Response Challenges in PBC PBC, as an AILD centered on bile duct injury, sees approximately 40% of patients showing poor response to first-line UDCA, posing a significant clinical challenge. Previous studies suggest that alkaline phosphatase (ALP) is significantly associated with PBC protein components, and OS.\u0026nbsp;Degree of liver inflammation and fibrosis are also key factors influencing response\u0026nbsp;[18]. This study further confirms that elevated ALP and ALT are independent risk factors for non-response to treatment in PBC. More notably, some patients who do not fully meet diagnostic criteria for\u0026nbsp;OS\u0026nbsp;exhibit AIH-like features, termed \u0026quot;PBC with AIH-like features\u0026quot; in the literature [33, 34]. Failure to promptly add immunosuppressive therapy for such patients may lead to uncontrolled disease progression. Therefore, exploring \u0026quot;whether PBC patients with elevated ALP and ALT should initiate second-line drugs [35, 36] or immunosuppressants earlier\u0026quot; is necessary to improve treatment outcomes. Immunologically, plasma cell function (Mum-1 expression) in PBC is comparable to AIH, but T-cell infiltration (CD3) and immune activation (CD38) involvement are lower, suggesting its pathological mechanism leans more towards \u0026quot;humoral immunity dominance + bile duct-specific injury\u0026quot; [46, 47]. This contrasts sharply with the AIH model of \u0026quot;synergistic drive of cellular + humoral immunity,\u0026quot; explaining why PBC treatment primarily focuses on bile duct protection, while AIH requires suppression of systemic immune responses.\u003c/li\u003e\n \u003cli\u003eRecognition and Immune Overlay Mechanism of \u0026nbsp;[14、48] is the most common overlap form in AILD, with a prevalence of about 5%~15% among PBC patients. The diagnosis is mainly based on the Paris criteria [3], and treatment recommends UDCA combined with immunosuppressants. However, due to complex clinical presentations, early recognition is challenging. This study found many commonalities in histopathology between OS and AIH [39]\u0026mdash;such as interface hepatitis, Rosette formation, emperipolesis, clustered plasma cell infiltration at the limiting plate, and portal lymphocyte enrichment, with higher inflammation grade and fibrosis degree. High-titer ANA is an independent influencing factor for OS, and whether it can serve as a marker predicting progression to OS warrants further exploration in larger-scale studies. Immunohistochemical features further reveal the pathogenesis of OS: OS shows the most prominent T-cell infiltration (CD3) and immune activation (CD38) levels among the four disease groups, embodying a dual mechanism of \u0026quot;cellular immune activation + immune response overlay\u0026quot;\u0026mdash;inheriting the T-cell-mediated immune response from AIH while retaining the bile duct injury-related immune characteristics of PBC. This also leads to more complex conditions and higher treatment difficulty in OS, necessitating combination therapies to balance different dimensions of immune abnormalities.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"4.Conclusion","content":"\u003cp\u003eCombining histopathological features (interface hepatitis, rosette formation)[12、13], immunohistochemical markers (Mum-1, CD38, CD3, CD68)[15], and serological indices (IgG, ANA titer, ALP) can significantly improve the differential diagnostic accuracy among AIH, AIH-PBC (OS), PBC, and DI-AILH. Elevated IgG is an independent diagnostic factor for AIH, and high-titer ANA is an independent predictive factor for AIH-PBC (OS), serving as core serum markers for disease classification. Pre-treatment ALP and ALT levels in PBC patients can predict response to ursodeoxycholic acid (UDCA) therapy; high levels indicate a risk of non-response, suggesting the need for early assessment for second-line therapy. Early precise classification and individualized treatment (e.g., combined \"corticosteroids + UDCA\" regimen for AIH-PBC (OS)) are key to improving the prognosis of patients with AILD.\u003c/p\u003e\n\u003cp\u003eStudy Limitations and Future Perspectives\u003c/p\u003e\n\u003cp\u003eThis study has certain limitations. Firstly, the sample size in each group is limited, and conclusions are based on small-sample statistics, potentially introducing selection bias. Secondly, differences in patient compliance may affect timely assessment of treatment efficacy, leading to potential bias in data interpretation.\u003c/p\u003e\n\u003cp\u003eFuture research could advance in three directions: First, conducting large-scale, multicenter cohort studies to validate findings such as the \"AIH multidimensional diagnostic model,\" \"DI-AILH IgG threshold,\" and \"PBC response risk factors.\" Second, further exploring the predictive value of high-titer ANA for OS and developing more precise disease progression assessment tools. Third, integrating immunohistochemical markers with molecular detection to identify potential specific biomarkers, further enhancing diagnostic and therapeutic precision.\u003c/p\u003e\n\u003cp\u003eWith the deepening of research on AILD, more underlying pathogenic mechanisms will be elucidated, and more efficient assessment methods will be applied in clinical practice, ultimately addressing the core issues of \"difficult differentiation and poor response,\" and providing patients with better diagnostic and therapeutic services.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eStandard Approval Statement:This study was approved by the Ethics Committee of the Leshan People\u0026apos;s Hospital (Approval No. LYLL【2026】KY 075) on February 24, 2026. All procedures performed in this study involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Informed consent was obtained from all individual participants included in the study\u003c/p\u003e\u003ch2\u003eFunding Information:\u003c/h2\u003e \u003cp\u003eThis research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.\u003c/p\u003e \u003cp\u003eFor Figures:\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eResearch conception and design: Su Jing; Paper writing: Su Jing; Data collection and major revisions of the paper: Chen Yuqing, Luo Rui; Paper revision and final review: Liang Shiyi.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eShen Y, Lu C, Men R et al. 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J Clin Hepatol. 2023;39(8):1952\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Autoimmune Liver Diseases, Drug-Induced Autoimmune-Like Hepatitis (DI-AILH), Histopathology, Immunohistochemistry, Antibody Titers, 6-Month Treatment Response","lastPublishedDoi":"10.21203/rs.3.rs-8776539/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8776539/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjective\u003c/h2\u003e \u003cp\u003eTo investigate the differences in histopathological characteristics, immunohistochemical marker expression, and serum antibody titers among autoimmune hepatitis (AIH), primary biliary cholangitis (PBC), AIH-PBC overlap syndrome (OS)[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], and drug-induced autoimmune-like hepatitis (DI-AILH), providing basis to enhance the accuracy of differential diagnosis and rational prediction of treatment response.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eA retrospective collection of liver biopsy specimens from 40 patients at the Department of Gastroenterology, Leshan People's Hospital, between January 2022 and August 2025 was conducted, including 11 AIH cases, 11 PBC cases, 10 AIH-PBC OS cases, and 8 DI-AILH cases. A comprehensive approach utilizing HE staining (assessing inflammation grade, fibrosis stage, and characteristic pathological changes), immunohistochemistry (detecting markers such as Mum-1, CD38, CD3, CD68), serum antibody titers (anti-nuclear antibody, ANA), and liver biochemical indices (ALT, ALP, IgG) was employed to quantitatively compare histological and immunohistochemical differences among the four groups. Patients with AIH, PBC, and OS were followed up for 6 months to analyze the correlation between treatment response and antibody titers, biochemical indices, and pathological changes.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eBoth the AIH and OS groups exhibited significant interface hepatitis, hepatocyte rosette formation, and emperipolesis. The scores for portal plasma cell\u0026thinsp;+\u0026thinsp;lymphocyte infiltration [AIH: (2.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.61), (3.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30); OS: (1.90\u0026thinsp;\u0026plusmn;\u0026thinsp;1.20), (3.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48)] were significantly higher than those in the PBC group [(1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.63), (2.55\u0026thinsp;\u0026plusmn;\u0026thinsp;1.13)] and the DI-AILH group [(0.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52), (2.38\u0026thinsp;\u0026plusmn;\u0026thinsp;1.30)] (all P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Inflammation grade (Scheuer grade) [AIH: (3.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50), OS: (3.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.67)] and fibrosis stage (Scheuer stage) [AIH: (3.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.83), OS: (2.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.79)] were also higher in the AIH and OS groups compared to the other two groups (all P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Serologically, the proportion of high-titer ANA (\u0026ge;\u0026thinsp;1:640) in the OS group was 80.0% (8/10), significantly higher than in other groups (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). IgG levels in the AIH group [2520.00 (2190.00, 3565.00)] were significantly higher than in the other three groups (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while ALT levels in the DI-AILH group (360.12\u0026thinsp;\u0026plusmn;\u0026thinsp;420.84) were significantly higher than in the other three groups (all P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The 6-month follow-up showed no significant difference in the complete biochemical response rate between the AIH and OS groups (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Non-responders in the PBC group had significantly higher ALP and ALT levels [ALP: 917.00\u0026thinsp;\u0026plusmn;\u0026thinsp;NA; ALT: 215.00\u0026thinsp;\u0026plusmn;\u0026thinsp;NA] compared to responders [ALP: 195.50\u0026thinsp;\u0026plusmn;\u0026thinsp;95.68 U/L; ALT: 64.40\u0026thinsp;\u0026plusmn;\u0026thinsp;47.68 U/L] (both P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Multivariate logistic regression analysis showed that elevated IgG was an independent diagnostic factor for AIH (OR\u0026thinsp;=\u0026thinsp;1.02, 95% CI: 1.00-1.04, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and high-titer ANA was an independent predictive factor for OS (OR\u0026thinsp;=\u0026thinsp;3.41, 95% CI: 1.12\u0026ndash;10.38, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Immunohistochemical features: OS showed the most prominent CD3 and CD38 expression, reflecting dual \"cellular immunity\u0026thinsp;+\u0026thinsp;immune activation\" overlay. AIH exhibited active CD68 and Mum-1 expression, suggesting a combined \"cellular immunity (macrophages) + humoral immunity\" drive. PBC had Mum-1 levels comparable to AIH but lower T-cell and immune activation involvement, leaning towards \"humoral immunity\u0026thinsp;+\u0026thinsp;bile duct-specific injury\". DI-AILH generally showed lower involvement of all immune markers, reflecting the fundamental difference of its \"drug-induced\" immune injury mechanism compared to traditional autoimmune liver diseases.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eCombining histopathological features (interface hepatitis, rosette formation), immunohistochemical markers (Mum-1, CD3, CD38, CD68), and serological indices (IgG, ANA titer, ALT) can effectively differentiate AIH, OS, PBC, and DI-AILH, improving diagnostic accuracy. Early identification of OS and selection of targeted immunosuppressive regimens can improve treatment response. Pre-treatment ALP and ALT levels in PBC patients can serve as important reference indicators for second-line treatment decisions.\u003c/p\u003e","manuscriptTitle":"Application of Histopathology, Immunohistochemistry, and Antibody Titers in the Diagnosis and Treatment Response Assessment of Autoimmune Liver Diseases","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-03 07:22:46","doi":"10.21203/rs.3.rs-8776539/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b3ae0588-38b4-404f-acaa-0e45640a97ce","owner":[],"postedDate":"March 3rd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-29T15:53:25+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-03 07:22:46","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8776539","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8776539","identity":"rs-8776539","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}