ABSTRACT
Background and Aims
Chronically elevated liver enzymes without a clear etiology remain a frequent diagnostic challenge. This study evaluated the diagnostic yield of liver biopsy in such cases and assessed if laboratory parameters predict histological clarification.
Methods
We retrospectively analyzed 71 patients with unexplained elevated liver enzymes who underwent percutaneous liver biopsy between 2015 and 2021 at a tertiary referral center in Germany. Clinical characteristics and lab values were compared between patients with clarified and unclarified diagnoses. ROC analysis was performed for ALT and AST. Histopathological characteristics and biopsy‐related complications were recorded.
Results
A definitive diagnosis was reached in 47.9% of cases. The most frequent findings were autoimmune hepatitis (35.3%), drug‐induced liver injury (23.5%), and NAFLD (23.5%). ALT and AST levels were significantly higher in patients with clarified diagnoses (ALT: p = 0.0079; AST: p = 0.0096). ROC analysis showed moderate performance (ALT AUC = 0.684; AST AUC = 0.679). Fibrosis stages ≥ F2 were found in 28.2% of patients. Biopsy complications occurred in 2.8%, all minor.
Conclusions
Liver biopsy clarified the etiology in nearly half of patients and revealed diagnoses not apparent through non‐invasive work‐up. While ALT and AST levels were associated with diagnostic yield, they lacked sufficient predictive value. Biopsy remains a valuable tool in selected patients with unexplained liver enzyme elevations.
1. Introduction
Persistently elevated liver enzymes—specifically alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (AP), and gamma‐glutamyltransferase (γ‐GT)—of unclear etiology represent a common and challenging scenario in clinical hepatology. Mild abnormalities in liver enzymes are frequently encountered—population studies report elevated aminotransferases in approximately 8%–15% of individuals [1, 2].
The first approach to chronically elevated liver enzymes is typically non‐invasive, including medical history, laboratory evaluation, and imaging studies. These non‐invasive methods usually detect the most common causes of elevated liver enzymes, such as metabolic dysfunction‐associated steatotic liver disease (MASLD; formerly known as nonalcoholic fatty liver disease, NAFLD), alcohol‐related liver disease, or viral hepatitis. However, despite comprehensive non‐invasive assessment, approximately 10% of cases remain unexplained [3, 4, 5]. Serologic panels and imaging studies can lack sensitivity or specificity for certain occult liver diseases. For example, ultrasound can detect moderate‐to‐severe steatosis but may miss mild fatty change [6, 7, 8], and serologies for autoimmune hepatitis (AIH) rely on the presence of autoantibodies that are in up to 20% of AIH cases not present [9, 10]. This diagnostic uncertainty poses a clinical dilemma: while many patients may harbor benign or non‐progressive conditions, a subset could have an early‐stage treatable liver disease that is simply not apparent on standard tests. Distinguishing these possibilities is crucial, as even asymptomatic elevations can portend clinically significant liver pathology or progression over time. In such cases, where non‐invasive diagnostics remain inconclusive, liver biopsy continues to play a pivotal role in establishing a definitive diagnosis.
The decision to biopsy is often guided by clinical context (e.g., degree of enzyme elevation, duration of abnormality, and index of suspicion for specific diseases). In clinical practice, liver biopsy is often performed when elevated liver enzymes persist for more than 6 months without a clear explanation, despite comprehensive laboratory and imaging work‐up. This approach is supported by international guidelines, which recommend biopsy as the next diagnostic step when non‐invasive methods are inconclusive—particularly to confirm diagnoses such as MASLD or AIH [11, 12, 13].
Only few studies have systematically examined the diagnostic role of liver biopsy in this setting, and the reported diagnostic yield has varied substantially across cohorts. Khalifa et al. reported on 383 patients who underwent biopsy for unexplained abnormal liver tests: remarkably, histopathologic diagnoses were established in 85% of cases, leading to a specific clinical diagnosis in 87% [14]. Importantly, this study underlined that routine lab‐based evaluation could not reliably discriminate among these diagnoses prior to biopsy. In an other study cohort of 87 patients with cryptogenic transaminase, Pietri et al. demonstrated the liver biopsy revealed minimal or no histologic changes in 48% of cases, effectively ruling out significant liver disease. However, about one in every seven patients (~15%) had biopsy findings that directly impacted clinical management, identifying conditions such as AIH, primary biliary cholangitis (PBC), or metabolic dysfunction–associated steatohepatitis (MASH) that were previously unsuspected and for which targeted treatments or interventions were available [15]. Although not every liver biopsy leads to a diagnosis that alters clinical management, a substantial proportion provide histological findings that are diagnostically or prognostically meaningful. This underscores the value of liver biopsy as a diagnostic tool, particularly when non‐invasive methods fail to determine the cause of chronically elevated liver enzymes.
Taken together—and considering that liver biopsy is an invasive procedure with inherent risks—its use should be carefully weighed against the expected diagnostic benefit in each individual case. This retrospective study aims to assess the value of liver biopsy in patients with persistently elevated liver enzymes of unclear origin despite thorough non‐invasive evaluation. In particular, it examines whether liver biopsy can clarify the underlying etiology and whether its diagnostic yield justifies its limitations and potential complications.
2. Methods
2.1. Study Design and Patient Selection
This retrospective study analyzed clinical data from all patients aged 18 years or older who underwent diagnostic liver biopsy at the University Medical Center Göttingen (UMG) between January 1, 2015, and December 31, 2022.
During this period, a total of 853 liver biopsies were performed. Patients were categorized based on the indication for biopsy.
Since this study focuses on unclear chronically elevated transaminases, this subgroup was identified based on the Criteria in Table 1.
TABLE 1.
Inclusion and exclusion criteria for study cohort selection.
| Inclusion criteria | Exclusion criteria (at least one applicable) |
|---|---|
|
Chronically elevated liver enzymes (> 6 months) Defined as:
|
Known etiology of elevated liver enzymes |
| History of alcohol abuse (> 30 g/day for men, > 20 g/day for women) | |
| History of liver transplantation | |
| Serologic evidence of viral hepatitis A–E | |
| Serologic evidence of hemochromatosis, Wilson's disease, or alpha‐1 antitrypsin deficiency | |
| Imaging evidence of NAFLD or hepatic steatosis | |
| AMA positivity suggestive of PBC | |
| Suspicion of AIH with Hennes score > 5 without histology | |
| Typical PSC findings on MRCP | |
| Use of known hepatotoxic drugs or agents within the last 6 months. | |
| Age < 17 years | |
| Clinical suspicion of cryptogenic cirrhosis when no etiology could be determined non‐invasively |
The data were collected from hospital records, sonography reports, pathology findings, microbiology results, and laboratory data, as well as external reports. To preserve patient anonymity, each case was assigned a pseudonymized ID for data analysis.
Ethical approval for the study was granted by the Ethics Committee of the University Medical Center Göttingen (Application Number 24/8/21) on August 9, 2021.
2.2. Pre‐Biopsy Non‐Invasive Evaluation
Prior to liver biopsy, all patients underwent a standardized non‐invasive work‐up including detailed clinical history and laboratory testing. This included assessment of liver enzymes, coagulation parameters, viral hepatitis serologies (including hepatitis A, B, C, and E), autoimmune markers (antinuclear antibodies [ANA], smooth muscle antibodies [SMA], liver‐kidney microsomal antibodies [LKM‐1], anti‐mitochondrial antibodies [AMA], and immunoglobulin G [IgG]), metabolic markers (ceruloplasmin, ferritin, transferrin saturation, alpha‐1‐antitrypsin, lipid profile), and extrahepatic parameters such as tissue transglutaminase antibodies and creatine kinase (CK). Additionally, protein electrophoresis was performed. All patients underwent abdominal ultrasound, and in selected cases, further imaging (CT, MRI) or ultrasound‐based elastography was performed.
2.3. Biopsy Procedure and Histological Assessment
Biopsies were performed percutaneously under ultrasound guidance using an 18‐gauge semiautomatic full core biopsy instrument (BioPince; Argon Medical Devices, Frisco, Texas, USA). Vital parameters were monitored, and blood counts were checked 3–4 h postprocedure to assess for potential complications. Biopsy tissue was fixed in formalin and sent to the pathology department for histological analysis according to current guidelines.
Standardized scoring systems were applied to ensure consistent interpretation and comparability of results. Grading of inflammatory activity and staging of fibrosis were assessed using the Desmet classification [16], while the NAFLD Activity Score was used for the evaluation of nonalcoholic steatohepatitis (NASH), and steatosis grading was performed according to the Kleiner classification [17].
2.4. Statistical Analysis
A Shapiro–Wilk test was applied to assess data normality. As most variables were not normally distributed, non‐parametric tests were used throughout the analysis. The Mann–Whitney U test was performed to compare laboratory values between the groups “Etiology clarified” and “Etiology unclarified.”
To further assess the diagnostic utility of ALT and AST, a receiver operating characteristic (ROC) analysis was performed, and optimal cut‐off values were identified by calculating the Youden Index (J = sensitivity + specificity−1), which was performed using Microsoft Excel (Version 16.78.3). The diagnostic performance was expressed as area under the curve (AUC), sensitivity, and specificity. To compare clinical and laboratory parameters across diagnostic subgroups, non‐parametric testing was performed due to non‐normal data distribution. Continuous variables were summarized as median with interquartile range (IQR). Differences between diagnostic categories were assessed using the Kruskal–Wallis test. A two‐sided p‐value < 0.05 was considered statistically significant.
To evaluate whether non‐invasive laboratory parameters were independently associated with a conclusive histological diagnosis, a multivariate logistic regression analysis was performed. The dependent variable was defined as “etiology clarified by liver biopsy” (yes = 1, no = 0). Independent variables included routinely assessed biochemical markers: ALT, AST, GGT, AP, total bilirubin, and Quick value. All variables were entered into the model simultaneously. A p‐value < 0.05 was considered statistically significant.
Statistical analysis was performed using SPSS (IBM Corp., Version 29.0.0.0) and Microsoft Excel (Version 16.78.3).
Literature searches were supported by Open Evidence, an AI‐assisted research platform used to identify relevant peer‐reviewed studies. All sources and citations were independently verified by the authors, who take full responsibility for data interpretation and content.
3. Results
3.1. Description of the Study Cohort
Between 2015 and 2021, a total of 853 liver biopsies were performed at the UMG. The indications and selection process leading to the study cohort are summarized in Figure 1. Indications included hepatic lesions (n = 503; 58.98%), suspected clinical diagnoses (n = 250; 29.27%), cryptogenic cirrhosis (n = 13; 1.5%), and post–liver transplantation evaluation (n = 16; 1.9%). A distinct subgroup of 71 patients (8.31%) presented with chronically elevated liver enzymes of unclear etiology and met the inclusion criteria defined in Table 1. This group formed the main cohort for the further analysis.
FIGURE 1.
Flowchart of patient selection for the study cohort. Flowchart illustrating the selection of the final study cohort. Between 2015 and 2021, a total of 853 liver biopsies were performed at the UMG. After excluding biopsies conducted for hepatic lesion evaluation (n = 503), 350 cases with elevated enzymes remained. Further exclusions were made for biopsies performed to confirm a suspected diagnosis (n = 250), post–liver transplantation cases (n = 16), and cryptogenic liver cirrhosis (n = 13). The final study cohort included 71 patients with persistently unexplained elevated liver enzymes.
The median age was 48 years (±16), and 53.5% of the patients were female. The median body mass index (BMI) was 25.3 kg/m2. Common comorbidities included type II diabetes mellitus (5.6%) and hyperlipoproteinemia (14.08%). An overview of clinical and laboratory parameters is provided in Table 2.
TABLE 2.
Clinical and laboratory parameters of the study cohort (n = 71).
| Characteristic | Unclear hepatopathy (n = 71) |
|---|---|
| Age, median (Q1–Q3) | 48 (35–60) |
| Female sex, n (%) | 38 (53.5%) |
| BMI, median (Q1–Q3) (kg/m2) | 25.3 (22.2–27.45) |
| Type 2 diabetes mellitus, n (%) | 5 (6.8%) |
| Hyperlipoproteinemia, n (%) | 13 (18.5%) |
| Celiac disease, n (%) | 2 (3.2%) |
| ALT, median (Q1–Q3) (U/L) | 86 (50–259) |
| AST, median (Q1–Q3) (U/L) | 56 (44–122) |
| γ‐GT, median (Q1–Q3) (U/L) | 150 (86–303) |
| AP, median (Q1–Q3) (U/L) | 106 (79–173) |
| Bilirubin, Median (Q1–Q3) (mg/dL) | 0.8 (0.5–1.4) |
| Quick value, median (Q1–Q3) (%) | 93 (84.5–99) |
| Hennes score, median (Q1–Q3) | 4 (2–5) |
Note: This table summarizes the demographic, clinical, and laboratory characteristics of the final study cohort (n = 71). Continuous variables are presented as median and interquartile range (Q1–Q3); categorical variables are reported as absolute numbers and percentages. Laboratory values reflect results closest to the time of liver biopsy.
Most patients were asymptomatic before biopsy, with only 40.8% reporting symptoms, the most common being fatigue (18.3%) and jaundice (9.9%). Less frequent complaints included nausea, weight loss, abdominal pain, loss of appetite, pruritus, and xanthelasma (each ≤ 2.8%).
3.2. Histopathological Findings in Patients With Unexplained Elevated Liver Enzymes
A total of 71 liver biopsy specimens were analyzed, with an average biopsy length of 2.62 cm. In 93% of cases, the biopsy was obtained from the right hepatic lobe; in two cases, the biopsy location was not recorded. The number of portal tracts was documented in 49.3% of the specimens, with a mean of 9.54 portal tracts per biopsy. Histological assessment included fibrosis staging and inflammatory activity grading according to Desmet [16], as well as steatosis grading according to the Kleiner classification [17].
The fibrosis stage distribution (Figure 2A) showed no fibrosis (F0) in 59.15% of cases, mild fibrosis (F1) in 12.68%, moderate fibrosis (F2) in 16.90%, advanced fibrosis (F3) in 9.86%, and cirrhosis (F4) in 1.41%. The inflammatory activity grade (Figure 2B) revealed no inflammation (G0) in 41.18% of cases, minimal activity (G1) in 26.47%, mild activity (G2) in 17.65%, moderate activity (G3) in 10.29%, and severe activity (G4) in 4.41%.
FIGURE 2.
Histopathological and diagnostic findings in the study cohort. (A) Shows the post‐biopsy Desmet fibrosis classification among 71 patients with persistently elevated liver enzymes, depicting the distribution across fibrosis stages F0–F4. (B) Illustrates the post‐biopsy Desmet inflammation grades in the same cohort, ranging from no inflammation (G0) to severe inflammation (G4). (C) Presents the steatosis grading according to Kleiner, highlighting the proportion of patients with no steatosis, mild, moderate, or severe steatosis. (D) Displays the distribution of diagnoses among the 34 patients in whom liver biopsy successfully clarified the etiology of liver enzyme elevation, including AIH, DILI, NAFLD, and other rarer conditions (small duct PSC, IgG4‐associated cholangitis, hemochromatosis, CH, AMA‐negative PBC, and AIH–PSC overlap). Bar heights represent the absolute number of cases (n), and the corresponding percentage relative to the analyzed subgroup is shown above each bar.
Regarding hepatic steatosis (Figure 2C), 73.24% of specimens showed no steatosis (< 5%), 25.35% had grade 1 steatosis (5%–33%), none had grade 2 steatosis (> 33%–66%), and 1.41% had grade 3 steatosis (> 66%).
3.3. Diagnostic Yield of Liver Biopsy
In the study cohort, a definitive diagnosis could be established in 47.89% of cases based on the histopathological findings and clinical correlation.
Among the 34 patients in whom liver biopsy clarified the etiology of liver enzyme elevation, the most frequent diagnoses were AIH (35.3%), NAFLD (23.53%), and drug‐induced liver injury (DILI) (23.53%). Representative histological images of these key diagnostic entities are shown in Figure 3.
FIGURE 3.
Representative liver histology of key diagnostic entities. Shown are examples of the three most frequent biopsy‐based diagnoses in the cohort, stained with hematoxylin and eosin. (A) AIH with dense portal and periportal lymphoplasmacytic infiltrates, interface activity, and hepatocellular rosette formation (Hennes score = 2). (B) DILI showing mixed portal and lobular inflammation with eosinophils and zone 3 hepatocellular necrosis consistent with a toxic pattern of injury. (C) NAFLD/NASH displaying macrovesicular steatosis involving approximately 25% of hepatocytes and minimal lobular inflammation. Scale bar = 50 μm.
In DILI cases, no clear temporal association between the initiation of a potentially hepatotoxic medication and the onset of liver test abnormalities was evident prior to biopsy. Histological review subsequently prompted a re‐evaluation of the medication history, which then revealed possible drug exposures. The predominant histological patterns included portal and lobular mixed inflammatory infiltrates with eosinophils, zone 3 hepatocellular necrosis, and cholestatic hepatitis. Drugs implicated were imatinib, amoxicillin‐clavulanate, atorvastatin, metamizole, gabapentin, and cotrimoxazole. In one case, the patient reported the use of plant‐based cough remedies that could not be further classified retrospectively. Another patient had multiple potentially hepatotoxic medications, including pantoprazole, amlodipine, and metoprolol.
The remaining six patients were diagnosed with one of the following: small duct primary sclerosing cholangitis (PSC), IgG4‐associated cholangitis, hemochromatosis, congestive hepatopathy (CH), AMA‐negative primary biliary cholangitis (PBC), or AIH–PSC overlap syndrome.
In 52.11% of cases, the underlying cause of the chronic hepatopathy remained unclear, or the histopathological examination revealed no relevant pathological findings (normal histology) in 11.27% of cases. No histological or clinical features suggestive of porto‐sinusoidal vascular disease (PSVD) were identified according to the VALDIG consensus definition [18].
3.4. Analysis of Non‐Invasive Predictors
To identify potential non‐invasive predictors of biopsy yield, laboratory parameters were compared between patients with clarified versus unresolved diagnoses. ALT and AST levels were significantly higher in the group in which biopsy led to a diagnosis (ALT: 193 U/L vs. 81 U/L, p = 0.0079; AST: 122 U/L vs. 51.5 U/L, p = 0.0096) (Figure 4A). In contrast, no significant differences were observed for AP, γ‐GT, total bilirubin, or Quick value.
FIGURE 4.
Diagnostic utility of transaminases in patients with unclear liver enzyme elevations. (A) Distribution of AST and ALT values stratified by diagnostic outcome. Enzyme levels were significantly higher in patients with a clarified etiology (green) (p < 0.05, Mann–Whitney U test). (B) Receiver operating characteristic (ROC) curves for AST and ALT in predicting a clarified diagnosis following liver biopsy. The area under the curve (AUC) was 0.68 for both enzymes, indicating moderate diagnostic accuracy. The optimal cut‐off values determined by the Youden index were 77 U/L for AST (sensitivity: 62.9%, specificity: 75.0%) and 102 U/L for ALT (sensitivity: 71.4%, specificity: 61.1%).
Receiver operating characteristic (ROC) analysis demonstrated moderate diagnostic accuracy for transaminases. For ALT, the area under the curve (AUC) was 0.684, with an optimal cut‐off value of 102 U/L (sensitivity 71%, specificity 61%). For AST, the AUC was 0.679, with a cut‐off of 77 U/L (sensitivity 63%, specificity 75%) (Figure 4B).
To explore whether biochemical parameters differed by histologically confirmed diagnosis, we compared laboratory values across the five most common diagnostic subgroups. A Kruskal–Wallis test revealed significant differences in ALT and AST levels between groups (both p < 0.001), while AP, γ‐GT, and bilirubin levels showed no significant variation (Table 3).
TABLE 3.
Comparison of clinical and biochemical parameters across diagnostic groups.
| Parameter | AIH n: 12 | DILI n: 8 | NAFLD n: 8 | Normal histology n: 8 | Unresolved n: 29 | Kruskal–Wallis H | p |
|---|---|---|---|---|---|---|---|
| ALT | 1041.0 (554.8–1264.5) | 526.0 (216.0–833.0) | 86.0 (56.2–118.8) | 110.0 (86.0–157.0) | 66.5 (48.2–143.5) | 19.98 | < 0.001 |
| AST | 851.0 (230.0–1016.2) | 294.0 (204.5–418.5) | 44.5 (37.5–56.5) | 70.0 (56.0–73.0) | 48.5 (39.0–79.2) | 25.56 | < 0.001 |
| γ‐GT | 94.0 (81.2–211.5) | 109.0 (81.5–342.5) | 91.0 (57.8–315.8) | 269.5 (179.5–475.2) | 149.0 (109.8–218.5) | 5.35 | 0.253 |
| AP | 133.0 (110.8–172.5) | 100.0 (86.0–155.5) | 106.0 (90.0–142.8) | 136.5 (91.0–194.8) | 117.0 (83.5–167.8) | 1.37 | 0.850 |
| Bilirubin | 5.1 (0.9–10.6) | 1.0 (0.6–1.5) | 0.7 (0.6–0.7) | 0.9 (0.7–1.4) | 0.8 (0.5–1.4) | 8.99 | 0.061 |
| Quick | 72.5 (66.2–96.5) | 93.0 (89.0–106.5) | 106.5 (97.0–112.2) | 103.0 (94.0–110.5) | 99.5 (91.0–107.2) | 7.79 | 0.100 |
| BMI | 24.9 (21.1–28.9) | 22.7 (21.9–27.7) | 28.4 (26.3–30.4) | 27.1 (23.2–28.0) | 24.3 (22.7–26.6) | 5.86 | 0.210 |
Note: Values are presented as median and interquartile range (IQR). Group differences were assessed using the Kruskal–Wallis test. Statistically significant p‐values (p < 0.05) indicate variables that differ significantly between diagnostic categories.
In the multivariate logistic regression model, none of the included laboratory parameters showed a statistically significant association with the likelihood of establishing a histological diagnosis. While ALT and AST had shown trends toward higher levels in the group with clarified etiology in univariate analyses, these associations did not remain significant after adjusting for other parameters (ALT: p = 0.63; AST: p = 0.97). Similarly, GGT, AP, bilirubin, and Quick value were not independently predictive. These findings suggest that no single laboratory parameter is sufficient to predict the diagnostic yield of liver biopsy in this patient population when considered in combination with others.
3.5. Complication Rates of Liver Biopsy
Procedure‐related complications were observed in 2.8% of cases (2 out of 71 patients). One patient experienced mild, self‐limited pain that resolved without intervention. In the second case, the patient reported more persistent and severe pain, prompting further diagnostic evaluation by abdominal CT, which revealed a localized bilioma. No major complications such as hemorrhage, infection, or biopsy‐related mortality occurred.
4. Discussion and Clinical Implications
The aim of this study was to evaluate the diagnostic utility of liver biopsy in patients with chronically elevated liver enzymes of unclear etiology. In addition, we assessed whether clinical predictors could help anticipate diagnostic yield. A total of 71 patients who underwent liver biopsy were retrospectively analyzed.
To assess the representativeness of our study population, clinical and laboratory characteristics were compared with previously published cohorts addressing similar clinical scenarios. The median age in our study was 48 years, with 53.5% female and a BMI of 25.3 kg/m2, closely matching data from a large retrospective cohort reporting a mean age of 51 years, 53% female, and similarly elevated transaminase levels [14]. Another study focusing on a more narrowly defined population with isolated transaminase elevation described comparable demographics [15]. These similarities suggest that our cohort is representative of patients commonly seen in hepatology practice with unexplained liver enzyme abnormalities, supporting the generalizability of our findings.
In this retrospective study, the underlying cause was clarified in 47.9% of cases. Compared to studies with more selective cohorts, such as one reporting a biopsy‐related diagnostic gain in approximately 30% of cases [15], our findings indicate a higher diagnostic yield. That cohort was evaluated by two hepatologists and relied primarily on subjective reassessment of pre‐biopsy diagnoses. In contrast, our approach was systematic and histology‐driven, based on comprehensive non‐invasive pre‐evaluation. Higher diagnostic rates have been reported elsewhere, such as 66.6% in one retrospective study [19], and up to 87% in a large cohort using both histological findings and clinical consensus for final diagnosis [14].
In our cohort, the most common histological diagnoses were AIH (35.3%), DILI (23.5%), and NAFLD (23.5%). These findings are consistent with previous reports, in which AIH accounted for 34%, NAFLD/NASH for 17%, and DILI for 15% [14], while another study identified NAFLD/NASH in 33%, AIH in 10%, and DILI in 8% of patients [14].
AIH emerged as the most frequent histological diagnosis (35.3%). Notably, several patients lacked classical serologic markers, highlighting the diagnostic value of liver biopsy in seronegative or atypical presentations. Although the simplified Hennes score is commonly used to support the diagnosis of AIH, it may fail to capture atypical cases. These findings support the role of biopsy in diagnostically uncertain settings, particularly when AIH is clinically suspected but not serologically confirmed. Importantly, the detection of AIH has immediate therapeutic implications, as timely immunosuppressive treatment can prevent fibrosis progression. Our findings are in line with previous reports that identified AIH in 10%–34% of similar cohorts [14, 15, 19], but suggest that autoimmune mechanisms may be underdiagnosed in the absence of histological evaluation.
Eight patients (23.5%) were diagnosed with NAFLD or NASH based on liver biopsy, despite the absence of steatosis on sonography. These patients had a BMI below the WHO‐defined threshold for class I obesity. Only one patient had type 2 diabetes mellitus and another had hyperlipoproteinemia. Thus, the classical risk constellation for NAFLD was not fully met. Histopathology revealed mild to moderate steatosis: in four cases, a 5% fat content was observed; in one case 15%; and in three cases 20%. Low‐grade steatosis is often difficult to detect via conventional ultrasound. These findings underscore the continued diagnostic relevance of liver biopsy, particularly in cases where mild steatosis is suspected but not visualized on conventional imaging. While liver biopsy remains the gold standard for detecting subtle histopathological changes, recent advancements in ultrasound‐based technologies offer promising non‐invasive alternatives. Quantitative ultrasound (QUS) techniques, which analyze radiofrequency backscatter to derive spectral and envelope‐based parameters, have shown promising results in accurately quantifying liver fat content, particularly in the range of 5% to 30% steatosis [7].
DILI accounted for 23.5% of all definitive diagnoses in our cohort, reflecting the frequent contribution of drug‐related injury to unexplained aminotransferase elevation. As a clinicopathologic diagnosis, DILI requires careful integration of histological features and clinical context. In our series, liver biopsy often played a pivotal role by revealing histological patterns that prompted a re‐evaluation of the medication history, even when drug exposure had been long‐standing or initially considered unrelated. The observed histological spectrum, including lobular disarray with eosinophil‐rich inflammation, zone 3 necrosis, and occasional cholestatic injury, corresponds well with previous descriptions [20, 21]. Importantly, biopsy often served not only for confirmation but also to exclude competing causes such as autoimmune or metabolic liver disease, supporting its continued diagnostic relevance in selected DILI cases.
Another relevant finding of this study was that 28.2% of patients with unexplained hepatopathy demonstrated advanced architectural remodeling of the liver parenchyma. Specifically, 18.3% showed moderate fibrosis, 8.45% had severe fibrosis, and 1.4% were diagnosed with cirrhosis. These findings align with the results reported in [22], who observed fibrosis in 20% and cirrhosis in 6% of patients undergoing liver biopsy for chronically elevated liver enzymes of unclear etiology. Similarly [15], described histological evidence of advanced liver remodeling in 12% of asymptomatic patients. While such histological changes do not always lead to immediate therapeutic consequences, they offer valuable insight for patient management, such as counseling on lifestyle modifications and scheduling of follow‐up evaluations. However, it should be noted that the information on fibrosis stage—although valuable—is increasingly accessible through non‐invasive techniques such as transient elastography, shear wave elastography, and magnetic resonance elastography. These methods have demonstrated good accuracy and are widely recommended for fibrosis assessment and risk stratification [12, 23]. In this light, the diagnostic value of liver biopsy regarding fibrosis staging may be considered limited, as comparable information can often be obtained non‐invasively.
Moreover, 11.3% of patients in our cohort with persistently elevated liver enzymes had biopsy specimens without any pathological findings. This is consistent with the literature, where Skelly et al. reported 6%, Schwake et al. 8%–11%, and Khalifa et al. 15% of patients with histologically normal liver tissue despite biochemical abnormalities [14, 19, 24]. The rates observed in our study cannot be solely explained by the expected 2.5% of physiologically elevated liver enzymes based on normal distribution. Beyond sampling error or technical limitations, such cases may reflect underlying conditions that elude detection by conventional pathology, particularly late‐onset genetic liver diseases. Advanced genetic testing, including targeted gene panels and exome sequencing, has shown clinical utility in uncovering pathogenic variants in transport proteins (e.g., ABCB4, ABCB11) and other rare cholestatic or metabolic disorders that often lack distinctive histological features. In selected cohorts with unexplained cholestasis, diagnostic yields of 27%–54% have been reported with targeted panels [25, 26, 27, 28], while broader next‐generation sequencing approaches can further increase yield, identifying actionable diagnoses in up to 25% of adults with idiopathic liver disease [27, 28, 29, 30]. Such findings can directly impact management, including targeted therapies, family screening, and avoidance of hepatotoxic agents.
Our study also assessed the diagnostic relevance of laboratory markers such as AST, ALT, γ‐GT, and AP. Although elevated transaminase levels were common in patients with biopsy‐confirmed pathology, no individual parameter demonstrated sufficient predictive power to independently guide clinical decision‐making. ROC analysis revealed cut‐off values of 81 U/L for AST and 101.5 U/L for ALT, with moderate sensitivity and specificity (AST: 58.8%/81.1%; ALT: 73.5%/62.0%), highlighting the limited diagnostic accuracy of these markers. Importantly, neither bilirubin levels nor cholestatic markers (γ‐GT, AP) were significantly associated with diagnostic yield. Furthermore, regression analyses did not identify any single laboratory value as a statistically significant predictor of biopsy outcome. In the multivariate analysis, none of the standard laboratory parameters—including ALT, AST, GGT, AP, bilirubin, or Quick—were independently associated with a diagnostic outcome. Although ALT and AST showed trends in univariate comparisons, these did not remain significant after adjustment. This indicates that routine liver tests alone are insufficient to predict the diagnostic yield of liver biopsy in patients with unexplained enzyme elevations. These findings align with prior studies reporting that transaminase elevations, while indicative of hepatocellular injury, are nonspecific and poorly correlated with histological severity or etiology [14, 22]. One important limitation of this study is the lack of systematic assessment of liver stiffness using ultrasound‐based elastography prior to biopsy. This may have affected the overall diagnostic yield and the decision‐making process for performing liver biopsy. Another limitation is that testing for telomere biology disorders was not routinely performed; however, at the time of liver biopsy, none of the patients showed clinical or hematologic features suggestive of such a disorder.
This study also assessed the complication rate of percutaneous liver biopsy. Complications occurred in 2.8% of cases (2 out of 71 patients), both of which were mild, self‐limiting, and classified as minor—transient pain being the predominant symptom. These findings are consistent with previously published safety data. Neuberger et al. reported a 2.4% rate of major complications in a large prospective cohort, supporting the general safety of ultrasound‐guided liver biopsy under standardized conditions [31]. While our rate was slightly higher than the 0.5% pooled estimate from the meta‐analysis by Tian et al., the absence of any major adverse events in our cohort further confirms the low‐risk nature of the procedure [32]. Overall, these data underscore that liver biopsy remains a safe diagnostic tool when appropriately performed.
In conclusion, liver biopsy proved to be a valuable diagnostic tool in patients with chronically elevated liver enzymes of unclear etiology, providing a definitive diagnosis in nearly 48% of cases. AIH was the most frequent finding, often undetectable by non‐invasive tests alone. Despite elevated transaminases, no single marker reliably predicted biopsy outcomes. The procedure also revealed advanced fibrosis in a significant subset and was safe with minimal complications. These findings support the continued role of liver biopsy when non‐invasive methods fail to clarify hepatic pathology.
Funding
The authors have nothing to report.
Conflicts of Interest
The authors declare no conflicts of interest.
Acknowledgments
Open Access funding enabled and organized by Projekt DEAL.
Jaawan S., Krämer A., Masri R., et al., “Diagnostic Utility of Liver Biopsy in Persistent Unexplained Liver Enzyme Elevation: A Retrospective Cohort Study,” JGH Open 9, no. 11 (2025): e70310, 10.1002/jgh3.70310.
Data Availability Statement
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.