This prospective observational cohort study was reviewed and formally approved by the Research and Ethics Committees of the Faculty of Medicine, Al-Azhar University, Damietta, Egypt (Institutional Review Board approval no. DFM-IRB 00012367-23-12-014). All clinical procedures, specimen collections, and data analyses involving human participants were conducted in strict accordance with the ethical tenets of the 1964 Declaration of Helsinki and its subsequent amendments. Written informed consent was obtained from all participants or their authorized legal representatives prior to study enrollment. Consent for specimen acquisition was verified by the attending surgical team.

A consecutive series of 40 adult patients who underwent LDLT at the Gastrointestinal Surgery Center, Faculty of Medicine, Mansoura University, Egypt, were enrolled between December 2023 and July 2024. All surgical procedures involved right-lobe living donor hepatectomies with standard vascular reconstruction and duct-to-duct or Roux-en-Y biliary anastomoses.

Recipients were stratified into two clinical cohorts—non-early allograft dysfunction (NEAD, n = 24) and early allograft dysfunction (EAD, n = 16)—according to the standardized, widely validated criteria formulated by Olthoff et al.11. EAD was defined by the presence of one or more of the following parameters: (1) Serum total bilirubin (TB) ≥ 10 mg/dL on postoperative day 7 (POD-7); (2) International normalized ratio (INR) ≥ 1.6 on POD-7; and/or (3) Serum alanine aminotransferase (ALT) or aspartate aminotransferase (AST) > 2,000 U/L within the first 7 postoperative days11.

Patients were excluded if they met any of the following predefined criteria: pregnancy or women of childbearing potential without verified contraception; advanced or end-stage cardiopulmonary or neurological disorders; severe or irreversible portopulmonary hypertension; active illicit substance abuse; active extrahepatic malignancy (excluding well-differentiated neuroendocrine tumors); active, uncontrolled systemic infection (with the exception of successfully treated spontaneous bacterial peritonitis); requirement for multi-organ transplantation (e.g., combined liver-kidney, liver-heart, liver-pancreas, or multivisceral grafting); severe active psychiatric disorders impairing adherence to immunosuppressive regimens; human immunodeficiency virus (HIV) positivity or acquired immunodeficiency syndrome (AIDS); complete, uncorrectable portomesenteric venous thrombosis; or moribund clinical status with severely restricted life expectancy.

Venous blood samples were collected from each recipient at two defined timepoints: pre-transplantation baseline (1 day prior to surgery; Pre) and post-transplantation day 7 (POD-7). Each blood sample was divided into two sterile aliquots: the first aliquot was drawn into a plain, additive-free vacuum tube, allowed to clot at room temperature for 30 minutes, and centrifuged at 3,000 rpm for 10 minutes to obtain clear serum for cytokine quantification and biochemical profiles. Serum was immediately separated and preserved at −80°C until batch analysis. The second aliquot was collected in a vacuum tube containing tripotassium ethylenediaminetetraacetic acid (K3-EDTA) for immediate hematological determination.

Serum IL-6 concentrations were measured in duplicate using a commercial solid-phase sandwich enzyme-linked immunosorbent assay (ELISA) kit (Bioassay Technology Laboratory, Human IL-6 ELISA Kit, Cat. No. E0090Hu, Shanghai, China) following the manufacturer's protocol, with absorbance read at 450 nm on an automated microplate reader. Serum activities of ALT and AST, as well as concentrations of direct bilirubin (DB) and albumin (Alb), were measured using automated spectrophotometric kits (Chemelex, S.A., Barcelona, Spain). Gamma-glutamyl transferase (GGT) activity was determined using the enzymatic rate method described by Szasz12. Complete blood count (CBC) parameters—including hemoglobin (Hb), total red blood cell (RBC) count, white blood cell (WBC) count, and platelet count—were analyzed on a calibrated automated hematology analyzer (Cell-Dyn 1800, Abbott Diagnostics, Abbott Park, IL, USA).

Serological markers for hepatitis C virus antibodies (HCV-Ab) and hepatitis B surface antigen (HBs-Ag) were detected using standardized third-generation ELISA diagnostic platforms based on solid-phase antibody-antigen binding13. Enzymatic conversion of specific chromogenic substrates was quantified photometrically, with absorbance levels directly proportional to analyte concentration.

Statistical analyses were performed using SPSS version 26.0 for Windows (SPSS Inc., Chicago, IL, USA) and the R statistical computing environment (version 4.6.1; R Foundation for Statistical Computing, Vienna, Austria). Continuous variables were assessed for distributional normality using the Shapiro-Wilk test. Normally distributed variables are reported as mean ± standard deviation (SD) and compared using the independent Student's t-test. Non-normally distributed variables are expressed as median with interquartile range (IQR; 25th–75th percentiles) and compared using the Mann-Whitney U test. Categorical variables are presented as absolute frequencies and percentages [n (%)] and compared between groups using Pearson's chi-square (χ²) test or Fisher's exact test when expected cell counts were less than 5.

Paired temporal comparisons between preoperative baseline and POD-7 measurements within the same cohort were analyzed using the paired Student's t-test or the Wilcoxon signed-rank test, as appropriate. Discriminatory performance was evaluated using receiver operating characteristic (ROC) curve analysis, determining the area under the curve (AUC), 95% confidence intervals (CI), sensitivity, and specificity. Optimal threshold values were established using the maximum Youden's index (J = sensitivity + specificity − 1).

Univariable binary logistic regression was performed to identify independent predictors associated with EAD. Variables reaching statistical significance or marginal clinical relevance (p < 0.10) were considered for multivariable binary logistic regression modeling using forward stepwise selection. To adjust for model optimism and prevent overfitting given the cohort size, internal validation was executed via non-parametric bootstrapping with 1,000 resamples using the rms package in R, generating optimism-corrected AUC/C-index values. Calibration between predicted probabilities and observed outcomes was evaluated using the Hosmer-Lemeshow goodness-of-fit test, with a p-value > 0.05 reflecting satisfactory goodness-of-fit. All statistical tests were two-tailed, and p < 0.05 was considered statistically significant.

A total of 40 patients undergoing LDLT were included, comprising 24 patients (60.0%) in the NEAD cohort and 16 patients (40.0%) in the EAD cohort. Male recipients predominated in both groups (79.2% in NEAD vs. 93.8% in EAD, p = 0.206). No statistically significant differences were observed between the cohorts regarding recipient age (median 49.50 [IQR: 41.2–57.7] vs. 45.0 [IQR: 28.0–56.0] years, p = 0.411) or body mass index (median 27.5 [IQR: 23.8–31.8] vs. 27.9 [IQR: 21.2–30.4] kg/m², p = 0.356).

All recipients in both cohorts presented with established liver cirrhosis and concurrent chronic cholecystitis. The primary etiologies of liver disease, including chronic HCV infection (45.8% vs. 37.5%, p = 0.601), chronic HBV infection (4.2% vs. 18.8%, p = 0.132), autoimmune hepatitis (16.7% vs. 12.5%, p = 0.718), and secondary hepatocellular carcinoma (16.7% vs. 12.5%, p = 0.718), showed balanced distributions between the NEAD and EAD groups. The prevalence of histological cholestasis (33.3% vs. 18.8%, p = 0.312) and steatosis (12.5% vs. 0.0%, p = 0.141) did not differ significantly. Primary sclerosing cholangitis was identified in two patients (8.3%) in the NEAD group and none in the EAD group (p = 0.236), whereas Budd-Chiari syndrome was documented in one patient (6.3%) in the EAD group and none in the NEAD group (p = 0.215) (Table 1).

Preoperative and postoperative laboratory parameters are summarized in Table 2. Preoperative serum IL-6 concentrations were significantly higher in recipients who subsequently developed EAD than in those who did not (10.67 ± 2.77 vs. 8.01 ± 2.98 ng/dL, p = 0.007). On POD-7, circulating IL-6 remained significantly elevated in the EAD group relative to the NEAD group (13.10 ± 4.45 vs. 10.28 ± 3.00 ng/dL, p = 0.034). Paired longitudinal analysis demonstrated significant postoperative surges in IL-6 from baseline to POD-7 in both the NEAD cohort (p < 0.0001) and the EAD cohort (p = 0.012).

Regarding hepatic excretory and synthetic markers, both total bilirubin and direct bilirubin increased significantly by POD-7 across all recipients (p < 0.05). However, EAD recipients exhibited significantly higher POD-7 total bilirubin (median 6.60 [IQR: 2.72–9.20] vs. 3.50 [IQR: 1.47–6.67] mg/dL, p = 0.036) and POD-7 direct bilirubin (median 5.05 [IQR: 2.00–7.75] vs. 2.50 [IQR: 0.95–4.97] mg/dL, p = 0.022) compared to NEAD recipients. In contrast, while preoperative serum albumin levels were comparable (median 3.05 g/dL in both groups, p = 0.561), POD-7 serum albumin was significantly lower in the EAD group than in the NEAD group (median 2.90 [IQR: 2.62–3.10] vs. 3.20 [IQR: 3.00–3.60] g/dL, p = 0.003).

Preoperative GGT activity was significantly lower in EAD recipients than in NEAD recipients (median 19.5 [IQR: 10.7–29.0] vs. 39.5 [IQR: 16.5–112.5] U/L, p = 0.045). Although both cohorts exhibited pronounced postoperative rises in GGT activity by POD-7, the compensatory surge was significantly blunted in the EAD cohort (median 67.5 [IQR: 40.0–130.0] vs. 124.5 [IQR: 68.5–234.2] U/L, p = 0.031). Transaminase activities (ALT and AST) did not show statistically significant differences between the two groups at either timepoint.

Among hematological indices, preoperative total leukocyte counts were significantly lower in the EAD cohort (median 2.45 [IQR: 2.00–3.77] vs. 3.40 [IQR: 2.25–4.30] × 10³/μL, p = 0.042). Postoperatively, EAD recipients had significantly lower hemoglobin concentrations on POD-7 (median 7.10 [IQR: 6.70–7.62] vs. 7.85 [IQR: 7.20–9.07] g/dL, p = 0.021). Both groups demonstrated expected declines in hemoglobin and RBC counts from baseline to POD-7 (p < 0.05) (Table 2).

The discriminatory accuracy of individual candidate biomarkers was assessed via ROC curve analysis (Table 3). Preoperative serum IL-6 achieved an AUC of 0.757 (95% CI: 0.596–0.917, p = 0.007). At an optimal cutoff threshold of 8.82 ng/dL, preoperative IL-6 yielded a sensitivity of 75.0% and a specificity of 75.0%. For POD-7 IL-6, the AUC was 0.718 (95% CI: 0.541–0.895, p = 0.033), with an optimal cutoff of 11.27 ng/dL providing 71.4% sensitivity and 75.0% specificity.

Among postoperative clinical chemistry indices, POD-7 albumin demonstrated the highest individual discriminatory capacity (AUC = 0.781, 95% CI: 0.640–0.922, p = 0.003; sensitivity 68.8%, specificity 66.7% at ≤ 3.05 g/dL). POD-7 direct bilirubin demonstrated an AUC of 0.716 (95% CI: 0.549–0.883, p = 0.022; sensitivity 62.5%, specificity 70.8% at > 4.40 mg/dL), and POD-7 hemoglobin yielded an AUC of 0.717 (95% CI: 0.555–0.880, p = 0.021; sensitivity 75.0%, specificity 66.7% at ≤ 7.50 g/dL). Preoperative indices including Pre-GGT (AUC = 0.689, p = 0.045) and Pre-WBC count (AUC = 0.691, p = 0.042) displayed modest predictive utility (Table 3).

Univariable binary logistic regression revealed significant associations between several candidate markers and EAD development (Table 4). Elevated preoperative IL-6 significantly increased EAD risk (odds ratio [OR] = 1.410, 95% CI: 1.058–1.879, p = 0.019). Elevated POD-7 direct bilirubin was likewise associated with increased risk (OR = 1.356, 95% CI: 1.026–1.793, p = 0.032). Conversely, higher POD-7 albumin conferred strong protection against EAD (OR = 0.038, 95% CI: 0.003–0.446, p = 0.009). Higher POD-7 GGT activity showed a weak, protective association with EAD (OR = 0.990, 95% CI: 0.980–0.999, p = 0.036).

Based on multivariable binary logistic regression analysis of significant independent variables, two distinct clinical prediction models were formulated (Table 5):

Model 1 (Preoperative Model): Formulated to enable risk stratification prior to surgical incision based on baseline circulating cytokine levels:

Logit(P) = 0.343 × (Pre-IL-6) − 3.575

Where Pre-IL-6 is expressed in ng/dL. Model 1 demonstrated an OR of 1.410 (95% CI: 1.058–1.879, p = 0.019), an apparent AUC of 0.7565 (95% CI: 0.596–0.917), and balanced sensitivity and specificity of 75.0% at a cutoff of 8.82 ng/dL.

Model 2 (Postoperative Model): Formulated to capture dynamic graft recovery and excretory function at the conclusion of the first postoperative week:

Logit(P) = 0.403 × (POD-7 DB) − 4.175 × (POD-7 Alb) + 10.680

Where POD-7 DB is expressed in mg/dL and POD-7 Alb is expressed in g/dL. Both variables remained independent predictors in multivariable modeling (POD-7 DB: OR = 1.496, 95% CI: 1.032–2.169, p = 0.034; POD-7 Alb: OR = 0.015, 95% CI: 0.001–0.396, p = 0.012). Model 2 achieved high discriminatory performance, with an apparent AUC of 0.8776 (95% CI: 0.762–0.993) and both sensitivity and specificity of 87.5% at an optimal predicted probability threshold of 0.419.

Internal validation of both models was conducted using 1,000 bootstrap resamples to assess model stability and quantify overfitting optimism. For Model 1, the apparent AUC was 0.7565, with an estimated optimism of only 0.0003, resulting in an optimism-corrected AUC of 0.7568 (p = 0.007). For Model 2, the apparent AUC was 0.8776, with an optimism of 0.0009, yielding an optimism-corrected AUC of 0.8767 (p < 0.0001). The negligible optimism values confirm strong internal consistency and minimal over-optimism. Calibration assessment using the Hosmer-Lemeshow goodness-of-fit test demonstrated excellent agreement between predicted and observed EAD events for Model 1 (χ² = 6.456, degrees of freedom [df] = 7, p = 0.488) and Model 2 (χ² = 11.374, df = 8, p = 0.181) (Table 5, Figure 1).

AIH: autoimmune hepatitis; Alb: albumin; ALF: acute liver failure; ALT: alanine aminotransferase; AST: aspartate aminotransferase; AUC: area under the receiver operating characteristic curve; B: unstandardized regression coefficient; BCS: Budd-Chiari syndrome; BMI: body mass index; CBC: complete blood count; CI: confidence interval; DB: direct bilirubin; DDLT: deceased donor liver transplantation; df: degrees of freedom; EAD: early allograft dysfunction; ELISA: enzyme-linked immunosorbent assay; GGT: gamma-glutamyl transferase; Hb: hemoglobin; HBs-Ag: hepatitis B surface antigen; HCC: hepatocellular carcinoma; HCV: hepatitis C virus; HCV-Ab: hepatitis C virus antibody; HIV: human immunodeficiency virus; INR: international normalized ratio; IQR: interquartile range; IRI: ischemia-reperfusion injury; LDLT: living donor liver transplantation; LT: liver transplantation; MELD: Model for End-Stage Liver Disease; NEAD: non-early allograft dysfunction; OR: odds ratio; PBC: primary biliary cirrhosis/cholangitis; POD: postoperative day; POD-7: postoperative day 7; Pre: preoperative baseline (day prior to transplantation); PSC: primary sclerosing cholangitis; RBC: red blood cell; ROC: receiver operating characteristic; SD: standard deviation; SE: standard error; SIRS: systemic inflammatory response syndrome; SPSS: Statistical Package for the Social Sciences; TB: total bilirubin; WBC: white blood cell.

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This research received no external grant or funding from public, commercial, or not-for-profit agencies. The study was entirely self-funded by the authors. No honorarium or other financial support was provided to produce this manuscript.

The datasets generated and analyzed during the current study are available from the corresponding author upon reasonable request.

The study protocol was reviewed and approved by the Research and Ethics Committees of the Faculty of Medicine, Al-Azhar University, Damietta, Egypt (Approval No. DFM-IRB 00012367-23-12-014). All clinical procedures and investigations were conducted in compliance with the ethical guidelines of the 1964 Declaration of Helsinki and its subsequent revisions. Written informed consent was obtained from all patients or their legal representatives prior to enrollment.

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The authors declare that they have no competing interests.