Introduction

Liver transplantation (LT) is the definitive treatment for irreversible acute and chronic liver diseases 1. Graft rejection (GR) remains a major obstacle to graft survival and overall patient mortality in liver transplant recipients (LTRs) 2. In LTRs, GR is traditionally diagnosed by invasive liver biopsy 3. Current post-transplant survival rates are 89.6 % and 91.8 % at 1 year, 80.8 % and 83.8 % at 3 years, and 72.8 % and 76.1 % at 5 years for grafts and patients, respectively 4.

Compared with the standard method of care, namely liver biopsy, biomarkers provide a safer and contemporary means to identify and detect transplant rejection 5,6. These biomarkers offer a novel strategy for early risk stratification of GR 7. Biomarkers and molecular mechanisms of injury have been investigated in numerous studies to predict GR; however, there is still no global consensus on the early detection of graft rejection in clinical practice 8,9. A readily available biomarker capable of predicting short-term outcomes early in the postoperative course could permit prompt intervention and thereby decrease morbidity and mortality 10. Therefore, early non-invasive diagnosis of rejection may enable proactive management and improved outcomes 8. Nevertheless, no investigation has developed a clinically applicable protocol that would enable routinely accessible post-transplant serum analytes to be employed for graft rejection and patient survival prediction 11,12.

Through the analysis of routine postoperative clinical tests from postoperative day (POD) 1 to POD 4 in patients undergoing living-donor LT, this study aims to identify clinically practicable serum biomarkers that can discriminate patients at high risk of graft rejection and mortality within three months of LT. Furthermore, receiver-operating-characteristic (ROC) analysis was used to assess the diagnostic accuracy and cut-off values of these clinical parameters as non-invasive markers, underscoring their potential utility as indicators of post-transplant graft rejection and patient survival.

MethodsPatients

This retrospective study was conducted in 120 adult recipients who underwent living donor liver transplantation (LDLT) at the Gastrointestinal Surgery Center, Faculty of Medicine, Mansoura University. The attending physician collected clinical samples from the patients. All living donors and recipients provided written informed consent prior to enrolment, and written consent was also obtained from the attending physician. In accordance with the Declaration of Helsinki, the study protocol was approved by the Ethical Committee of Damietta Faculty of Medicine, Al-Azhar University (Egypt) (DFM-IRB 00012367-25-04-007). As this was a retrospective analysis, the study was not registered as a clinical trial. Patients with advanced chronic liver disease or acute fulminant liver failure refractory to medical or surgical treatment were included. Under these conditions, life expectancy is markedly reduced because of end-stage liver disease. Potential living donors aged 18–60 years were required to have ABO-compatible blood type and suitable hepatic anatomy. Donors with significant comorbidities, including malignancy, diabetes mellitus, cardiovascular disease, or intrinsic liver disease, were excluded. The eligibility criteria for transplantation complied with the practice recommendations of the American Association for the Study of Liver Diseases (AASLD) 13. All recipients were managed according to the standardized postoperative care protocol of the Gastrointestinal Surgery Center, Faculty of Medicine, Mansoura University. No surveillance liver biopsies were performed during postoperative days (POD) 1–4; biopsy was undertaken only when graft rejection was suspected. A uniform induction immunosuppressive protocol was administered to all recipients.

Exclusion criteria

Patients were excluded if they exhibited any of the following conditions: pregnancy; women of childbearing potential without effective contraception; end-stage cardiac, pulmonary, or neurologic disease; severe or irreversible pulmonary hypertension; active, untreated infection (excluding spontaneous bacterial peritonitis); active extrahepatic malignancy (except neuroendocrine tumors); active substance abuse; requirement for combined liver and heart, lung, pancreas, bone marrow/stem-cell, or intestinal transplantation; psychiatric illness with active symptoms or behavioral patterns that could impair adherence to post-transplant therapy; immunodeficiency, including HIV-positive serology or AIDS; inadequate social support; complete portal and mesenteric venous thrombosis; moribund status with a limited life expectancy.

Graft rejection

Graft rejection in the present study—defined as acute cellular rejection—was assessed histopathologically within 90 days after liver transplantation (LT). The diagnosis and grading were established according to the Banff criteria 14. Among the 25 rejection cases, 15 were classified as mild (rejection activity index [RAI] 4–5) and 10 as moderate (RAI 6–7). Chronic or severe rejection was not observed. Overall patient survival was monitored until death or the end of the study period. Graft-related mortality excluded deaths attributable to causes other than liver failure. No patients were lost to follow-up during the study period.

Biochemical analysis

On the preoperative day and on postoperative days 1–4, 5 mL of peripheral blood was collected from each patient. Each sample was divided into two aliquots: one aliquot was placed in a plain, sterile tube for serum separation, whereas the second aliquot was transferred to a tube containing anticoagulant for hematological evaluation. The following parameters were determined in freshly obtained specimens by routine biochemical and immunochemical methods: aspartate aminotransferase (AST), alanine aminotransferase (ALT), uric acid (UA), C-reactive protein (CRP), and the complete blood count (CBC). Alpha-fetoprotein was quantified by immunofluorescence assay (IFA), and hepatitis C virus (HCV) infection was confirmed by real-time polymerase chain reaction (RT-PCR).

Statistical analysis

The sample size was calculated using the Steven K. Thompson equation 15 based on the entire population of eligible cases, a pre-specified confidence interval, and an acceptable margin of error to ensure adequate statistical power. Descriptive data are presented as median (interquartile range, IQR) and were compared with the Mann–Whitney U-test. Normality was assessed with the Kolmogorov–Smirnov test. Categorical variables are expressed as proportions and were compared with the chi-square (χ) test or Fisher’s exact test, as appropriate. To evaluate the ability of independent variables to discriminate between the rejection and non-rejection groups and to stratify 3-month survival, receiver-operating characteristic (ROC) curves were generated and the area under the curve (AUC) was calculated. Optimal cut-off values were determined using the Youden index. Patient survival was estimated with the Kaplan–Meier method, and curves were compared using the log-rank test. Associations between independent variables and rejection were explored with univariate logistic regression, and the predictive value of variables at their optimal cut-offs for survival was assessed with Cox proportional hazards regression. Regression results are reported as odds ratios (ORs) or hazard ratios (HRs) with 95% confidence intervals (CIs). All tests were two-tailed, and a P-value < 0.05 was considered statistically significant. Statistical analyses were performed using SPSS, version 26.0 for Windows (SPSS Inc., Chicago, IL, USA).

Results

A total of 120 patients who underwent living donor liver transplantation (LDLT) were enrolled; 95 showed no evidence of rejection, whereas 25 developed acute rejection. The baseline characteristics of both groups and the distribution of liver disease etiologies are summarized in Supplementary Table S1. Among the non-rejection cohort, 18 (18.9 %) patients were female and 77 (81.1 %) were male, and the median age was 52 years (IQR, 43–57). In the rejection cohort, 7 (28 %) patients were female and 18 (72 %) were male, with a median age of 46 years (IQR, 31–55). The principal indications for transplantation were hepatitis C virus (HCV)-related cirrhosis, documented in 45 non-rejection cases (47.4 %) and 11 rejection cases (44 %), and alcoholic cirrhosis, present in 89 non-rejection cases (93.7 %) and all 25 rejection cases (100 %). Hepatocellular carcinoma was identified in 24 patients without rejection (25.3 %) and in 2 patients with rejection (8 %). Percentages of underlying etiologies are non-exclusive because some recipients harbored multiple causes of liver disease. The frequencies of cholecystitis, cholestasis, steatosis, autoimmune hepatitis, primary sclerosing cholangitis, Budd–Chiari syndrome, and portal vein thrombosis did not differ significantly between the two groups (P > 0.05).

The most significant biochemical variables distinguishing patients with and without rejection during postoperative days (POD) 1–4 at 3 months after living-donor liver transplantation (LDLT) were AST on POD4 (AST4), total bilirubin on POD1–3 (TB1–3), direct bilirubin on POD2–3 (DB2–3), γ-glutamyl transferase on POD2 (GGT2), and international normalised ratio on POD1 (INR1) (P < 0.05), whereas the most highly significant variables were TB4 and DB4 (both P < 0.0001; Table 1). Receiver-operating-characteristic (ROC) analysis (Table 2) yielded optimal cut-off values for predicting rejection at 3 months: AST4, 53.5 U/L; TB1–4, 3.85, 3.65, 4.35, and 4.35 mg/dL; DB2–4, 1.95, 3.10, and 3.35 mg/dL; GGT2, 23.5 U/L; and INR1, 2.15. The corresponding diagnostic accuracies were 67.15 %, 68.75 %, 71.90 %, 72.30 %, 73.80 %, 66.10 %, 69.80 %, 72.30 %, 61.90 %, and 62.55 %, respectively (all P < 0.05). Again, TB4 and DB4 remained highly significant (P < 0.0001). Univariate logistic regression performed for graft rejection prediction showed that TB1–4 and DB2–4 were significantly associated (P < 0.05), with strongest associations for TB4 and DB4 (P < 0.001). In contrast, AST4, GGT2, and INR1 were not statistically significant (P = 0.098, 0.41, and 0.136, respectively; Table 3).

Supplementary Figure S1 illustrates overall survival in 120 patients at 3 months post–living-donor liver transplantation (LDLT). Eleven patients died, corresponding to a 9.2 % mortality rate, while 109 survived (90.8 %). The mean ± SE survival time was 84.8 ± 1.65 days (95 % CI, 81.6–88.0 days). Supplementary Figure S2 compares survival between recipients with and without biopsy-proven rejection. Patients without rejection (n = 95) achieved a 93.7 % survival rate, whereas those with rejection (n = 25) exhibited an 80.0 % survival rate (log-rank χ = 4.32, P = 0.038). During the 3-month postoperative interval, 11 deaths were recorded: five were graft-related and six were attributable to non-graft-related causes. Consistent with these findings, no significant association was observed between graft rejection and mortality (Supplementary Table 3).

Receiver operating characteristic (ROC) analysis of the significant biochemical variables determined cut-off values that maximized sensitivity and specificity for predicting 3-month post-LDLT survival. Using these thresholds, patients were stratified into two groups; the findings are presented in Tables S2 and S3 of the Supplementary Material and depicted in Figure 1. The optimal cut-off values were ALT1 481 U/L; AST1 421.5 U/L; LDH1 463 U/L; INR2 1.85; CRP3 47.5 mg/L; UA1 5.25 mg/dL; Neu2 84.85 %; Lym 4.75 %; and NLR 15.83 (P < 0.05). The corresponding accuracy values for these predictors were 80.75 %, 77.6 %, 83.2 %, 71.9 %, 68.6 %, 75.2 %, 69.1 %, 76.7 %, and 74.3 % (Table S2 of the Supplementary Material).

Patient survival according to significant biochemical predictors within 3 months after LDLT is summarized in Table S3 of the Supplementary Material. For ALT, 80 patients (98.8 %) with a postoperative day (POD) 1 value <481 IU/L survived, whereas only 29 patients (74.4 %) with ALT ≥481 IU/L survived (log-rank = 19.77; P < 0.001; Figure 1A). Similarly, 85 patients (97.7 %) with AST <421.5 IU/L and 24 patients (72.7 %) with AST ≥421.5 IU/L survived (log-rank = 19.59; P < 0.001; Figure 1B). For LDH on POD1, survival rates were 97.6 % for values <463 IU/L and 77.1 % for values ≥463 IU/L (log-rank = 13.36; P < 0.001; Figure 1C). With respect to INR on POD2, survival was 95.9 % for INR <1.85 and 82.6 % for INR ≥1.85 (log-rank = 6.23; P = 0.013; Figure 2A). Higher survival was also observed in patients with CRP on POD3 <47.5 mg/L (97.0 %; log-rank = 6.96; P = 0.008) and serum uric acid on POD1 <5.25 mg/dL (97.5 %; log-rank = 13.19; P < 0.001) (Figure 2B,C). Figure 3A shows a 97.1 % survival rate for neutrophil count on POD2 <84.85 × 10/L (log-rank = 7.34; P = 0.007), whereas Figure 3B demonstrates a 96.7 % survival rate for lymphocyte count on POD2 >4.75 × 10/L (log-rank = 16.64; P < 0.001). Finally, patients with an NLR on POD2 <15.83 had a 97.4 % survival rate compared with 78.6 % for NLR ≥15.83 (log-rank = 11.96; P < 0.001; Figure 3C).

Table 4 presents the Cox regression analysis of the categorical variables used to predict patient survival three months after LDLT. The analysis confirmed that ALT1 (P = 0.003), AST1 (P = 0.001), LDH1 (P = 0.003), INR2 (P = 0.023), CRP3 (P = 0.021), UA1 (P = 0.003), Neu2 (P = 0.018), Lym2 (P = 0.001), and NLR2 (P = 0.005) were statistically significant predictors.

Table 1

Serum levels of different biochemical parameters on postoperative days from day one to day four (1-4) in patients with and without rejection after 3 months of living donor liver transplantation

VariableNo rejection (n = 95)Rejection (n = 25)P valueAST440(30–64)66(44.5–95)0.016TB13.1(2.1-4.7)4.5(2.55-5.85)0.026TB22.4(1.5-3.6)4.2(2.35-6.2)0.004TB32.6(1.8-4.6)5.7(3.15-8)0.001TB43(1.8-4.9)7.5(4.3-9)<0.0001DB21.4(0.9-2.6)2.7(1.2-4.7)0.013DB31.7(1-3.5)4(1.65-6.2)0.003DB42.1(1.2-3.4)6.3(2.55-7.65)<0.0001GGT223(17-33)31(19-57)0.043INR12(1.6-2.6)2.3(2-2.8)0.039Table 2

Area under curve (AUC), sensitivity, and specificity of statistically significant variables for prediction of rejection after 3 months in patients underwent living donor liver transplantation

VariableAUCCutoffSensitivity%Specificity%95% CIP valueAST40.65753.56866.30.538– 0.7760.016TB10.6453.856869.50.516 – 0.7750.026TB20.6893.656875.80.565 – 0.8140.004TB30.7124.357272.60.593 – 0.8310.001TB40.7744.357671.60.672-0.877<0.0001DB20.6611.956864.20.527-0.7960.013DB30.6963.16871.60.577-0.8150.003DB40.7623.357272.60.658-0.867<0.0001GGT20.63223.56855.80.501-0.7630.043INR10.6352.156461.10.523-0.7450.039Table 3

Simple logistic regression of some variables for prediction of rejection

VariableBSEP valueOR95% CI (OR)AST40.0050.0030.0981.0050.999– 1.012TB10.1930.0850.0231.2131.028 – 1.431TB20.2630.0950.0061.3011.079 – 1.568TB30.2010.0650.0021.2221.075– 1.390TB40.2450.069<0.00011.2771.117 – 1.461DB20.3510.1250.0051.4211.111-1.817DB30.2200.0770.0041.2461.071-1.449DB40.2920.081<0.00011.3391.141-1.570GGT20.0040.0050.411.0040.994-1.014INR10.4410.2960.1361.5550.87-2.778Table 4

Simple Cox regression analysis of statistically significant variables for prediction of survival after 3 months in patients underwent living donor liver transplantation

VariableBSEP valueHR95% CI (HR)

ALT1

ALT1 > 481

0.001

3.167

<0.0001

1.049

<0.0001

0.003

1.001

23.742

1.001– 1.002

3.037– 185.576

AST1

AST1 > 421.5

0.001

2.635

<0.0001

0.782

<0.0001

0.001

1.001

13.945

1.001– 1.002

3.009 – 64.617

LDH1

LDH1 > 463

0.001

2.328

<0.0001

0.791

0.01

0.003

1.001

10.260

1.000– 1.002

2.177 – 48.348

INR2

INR2 > 1.85

1.464

1.535

0.435

0.677

0.001

0.023

4.323

4.642

1.842– 10.145

1.231– 17.506

CRP3

CRP3 > 47.5

0.012

1.805

0.004

0.782

0.001

0.021

1.012

6.082

1.005– 1.02

1.314– 28.157

UA1

UA1 > 5.25

0.547

2.299

0.185

0.782

0.003

0.003

1.728

9.962

1.202-2.485

2.151-46.128

Neu2

Neu2 > 84.85

0.152

1.845

0.059

0.782

0.01

0.018

1.164

6.326

1.037-1.307

1.366-29.287