Acute appendicitis is the most common acute surgical emergency of the abdomen, occurring in approximately 7–10% of the general population. It most commonly affects individuals between 10 and 30 years of age. Since its first documented surgical treatment in 1735 by Claudius Amyand and subsequent characterization by Reginald Fitz in 1886, appendicitis remains a significant diagnostic and management challenge. Despite advances in imaging and laboratory diagnostics, 15–50% of appendectomies still yield a normal or non-inflamed appendix, while delayed diagnosis remains a major contributor to morbidity and mortality.

The clinical diagnosis of acute appendicitis is based on history, physical examination, and the Alvarado scoring system. Standard investigations include complete blood picture (leucocytosis), imaging (ultrasound, CT scan), and C-reactive protein (CRP). However, predicting the severity of appendicitis—distinguishing between uncomplicated acute appendicitis and complicated forms such as perforated or gangrenous appendicitis—prior to surgery remains a clinical challenge. Complicated appendicitis is associated with significantly higher morbidity, prolonged hospital stay, and increased risk of postoperative complications including wound infection, intra-abdominal abscess, and sepsis.

Hyperbilirubinemia has been proposed as an additional laboratory marker for assessing appendicitis severity. The pathophysiological basis involves bacterial translocation of enteric organisms (primarily Escherichia coli and Bacteroides fragilis) through the appendiceal wall into the portal circulation via the superior mesenteric vein, resulting in interference with hepatic microcirculation and cause sinusoidal damage, finally leading to impaired bile secretion and hyperbilirubinemia . This mechanism is exacerbated with increasing severity of appendicitis, making hyperbilirubinemia potentially more pronounced in perforated or gangrenous disease.

Acute Appendicitis/Appendicular perforation

(Inflammatory response causes appendix to become more oedematous

and ischemic) ^[1]

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Causes transmigration/translocation of bacteria /toxins/ cytokines ^[1]

                                                  ↓

                Leading to endotoxemia / bacteraemia ^[1]

                                                  ↓

Invasion of Bacteria into the hepatic parenchyma interferes with the

physiology of excretion of bile.   ^[1]

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                               Hyperbilirubinemia ^[1] 

Multiple published studies by Estrada et al., Sand et al., Giordano et al., Biswal et al., Bali et al., and others have explored this association with varying degrees of diagnostic accuracy. Direct bilirubin has been specifically highlighted as a superior predictor compared to total serum bilirubin by some investigators (Sevinc et al., Eren et al.).

The present prospective observational study was undertaken with the objectives of: (1) evaluating preoperative serum total and direct bilirubin levels in patients with clinically suspected acute appendicitis, (2) correlating these bilirubin levels with the intraoperative and histopathological findings, (3) determining ROC curve-derived cut-off values with sensitivity and specificity for predicting complicated appendicitis, and (4) comparing preoperative and postoperative (POD-7) bilirubin levels to validate the hepatic mechanism.

Study Design and Setting This was a prospective observational study conducted in the Department of General Surgery, Bhaskar General Hospital & Medical College, Hyderabad, Telangana, India, over a period of three years (2021–2024). The study was approved by the Institutional Ethics Committee and registered with KNR University of Health Sciences, Warangal. Informed written consent was obtained from all participants. Study Population A total of 100 patients with clinically suspected acute appendicitis, fulfilling an Alvarado score of ≥5, who underwent emergency appendectomy were enrolled. Cases were recruited consecutively as they presented to the surgical emergency. Inclusion Criteria • Age > 10 years presenting with clinical features of acute appendicitis • Alvarado score ≥ 5 • Willingness to participate and provide informed written consent Exclusion Criteria • Known pre-existing liver disease (hepatitis, cirrhosis, obstructive jaundice) • Biliary tract disease • Pre-existing haemolytic disorders • Patients on hepatotoxic drugs • Paediatric patients under 10 years of age • Pregnancy • Patients refusing consent or lost to follow-up Data Collection and Laboratory Investigations All enrolled patients underwent a standardized preoperative evaluation including complete blood picture, serum total bilirubin (TSB), serum direct (conjugated) bilirubin, indirect bilirubin, and alkaline phosphatase (ALP). Blood samples were collected on admission prior to surgery. Bilirubin levels were estimated using the Jendrassik-Grof diazo method with colorimetric analysis. Postoperative serum bilirubin levels were re-measured on postoperative day 7 (POD-7) to assess the trend following appendectomy. Intraoperative findings were documented by the operating surgeon, classifying the appendix as: (1) acutely inflamed (acute appendicitis), (2) perforated, or (3) gangrenous. All resected specimens were sent for histopathological examination (HPE), which served as the definitive gold standard for diagnosis and classification. Statistical Analysis Data were entered in Microsoft Excel and analyzed using SPSS version 22. Quantitative variables were expressed as mean ± standard deviation. One-way ANOVA (F-test) was used to compare mean bilirubin levels across the three appendicitis groups. ROC (Receiver Operating Characteristic) curve analysis was employed to determine the optimal cut-off values of TSB and direct bilirubin for predicting perforated and gangrenous appendicitis, with calculation of area under the curve (AUC), sensitivity, and specificity. The pre- and post-operative bilirubin comparison was performed using paired t-test. A p-value of <0.05 was considered statistically significant.

Demographic Profile

Table 1. Age-wise distribution of study population (n=100)

The age of patients ranged from 10 to 60 years, with a mean age of 24.7 years. The majority of patients (69%) were in the 11–30 years age group. There was no statistically significant difference in age distribution between genders (p = 0.876).

Table 2. Gender distribution of study population (n=100)

Of the 100 patients, 64 were male and 36 were female, yielding a male-to-female ratio of 1.7:1.

Table 3. Distribution by Alvarado Score (n=100)

Intraoperative and Histopathological Findings

Table 4. Intraoperative findings and histopathological examination (HPE) results (n=100)

Intraoperative findings were consistent with HPE results in all 100 patients. Acute appendicitis was the most common finding (84%), followed by perforated appendix (9%) and gangrenous appendix (7%). Together, complicated appendicitis (perforation + gangrene) accounted for 16% of cases.

Preoperative Serum Bilirubin Levels

Table 5. Distribution of preoperative serum bilirubin levels (n=100)

Elevated total serum bilirubin (>1.2 mg%) was observed in 70% of study participants, with a mean of 1.53 ± 0.50 mg/dl (range: 0.4–4.1 mg/dl). Elevated direct bilirubin (>0.3 mg%) was present in 79% of patients, with a mean of 0.89 ± 0.58 mg/dl.

Correlation of Serum Bilirubin with Histopathological Severity

Table 6. Preoperative mean serum bilirubin levels by histopathological diagnosis (ANOVA)

Mean preoperative TSB was significantly higher in perforated appendix (2.3 mg%) compared to gangrenous appendix (1.883 mg%) and acute appendicitis (1.427 mg%) (F = 11.88, p < 0.001). Similarly, mean direct bilirubin was highest in the perforated group (1.633 mg%) compared to gangrenous (1.2 mg%) and acute appendicitis (0.795 mg%) (F = 15.09, p < 0.001).

Table 7. Percentage of patients with hyperbilirubinemia by appendicitis type

Hyperbilirubinemia (elevated TSB) was present in 89% of patients with perforated appendix, compared to 71.4% with gangrenous appendix and 66.6% with acute appendicitis. All patients with complicated appendicitis (perforated and gangrenous) had elevated direct bilirubin levels (100%), while 75% of uncomplicated AA patients had elevated direct bilirubin.

ROC Curve Analysis

Table 8. ROC curve analysis: diagnostic performance of serum bilirubin for complicated appendicitis

For predicting perforated appendicitis, TSB achieved an AUC of 0.866 with a cut-off value of 1.75 mg% (sensitivity 88.9%, specificity 80.2%), and direct bilirubin achieved an AUC of 0.883 with a cut-off of 1.15 mg% (sensitivity 88.9%, specificity 71.4%). For gangrenous appendicitis, TSB AUC was 0.71 (cut-off 1.85 mg%, sensitivity 71.7%, specificity 85.7%) and direct bilirubin AUC was 0.739 (cut-off 0.65 mg%, sensitivity 84.9%, specificity 77.6%). Total serum bilirubin >1.5 mg% was found to be highly predictive of complicated appendicitis.

Preoperative versus Postoperative Bilirubin Comparison (POD-7)

Table 9. Pre- and post-operative (POD-7) comparison of bilirubin and ALP levels by appendicitis type

A statistically significant decrease in serum total bilirubin, direct bilirubin, and ALP was observed at postoperative day 7 across all three groups (p < 0.005), confirming the reversible hepatic cholestatic mechanism related to appendiceal inflammation. The magnitude of decrease was greatest in the perforated appendix group.

        In this study, 50 This prospective study of 100 patients with acute appendicitis evaluated the diagnostic role of serum bilirubin levels in assessing severity and correlation with histopathological findings. The study population had a mean age of 24.7 years with a peak incidence in the 21–30 year age group (38%), consistent with published literature. Bali RS et al. reported a mean age of 29 years, Khan S reported 27.2 years, and Elmetwally AMS reported 21.98 years, all broadly similar to the present study. The male predominance (M:F = 1.7:1) is also consistent with the reported ratio of 1.5–1.9:1 in the literature (Marudanayagam: 1.5:1; Bali RS: 1.9:1).

       Intraoperative and HPE findings revealed 84% acute appendicitis, 9% perforated, and 7% gangrenous appendicitis—a complication rate of 16%. This is within the range reported in comparable studies: Ramasamy Ramu et al. (18% complicated), Mohammad Reza Motie (23.6%), Bali RS (33%), Estrada et al. (26%), and Poras C (16%). The consistency of intraoperative and HPE findings in all 100 cases supports the accuracy of intraoperative diagnosis.

       Pre-operative TSB was elevated (>1.2 mg%) in 70% of study participants, with a mean of 1.53 mg/dl. This is comparable with the series of Biswal et al. (50 patients, mean 1.49 mg/dl), Dalai P et al. (147 patients, 1.62 mg/dl), Hatata et al. (100 patients, 1.46 mg/dl), Sadhan Kumar Cheekuri et al. (110 patients, 1.21 mg/dl), and Sibabrata Kar et al. (128 patients, 1.35 mg/dl). The mean direct bilirubin of 0.89 mg/dl (SD 0.58) is comparable with Veeresh Kumar (1.0 mg/dl) and Bakshi Mandal (0.71 mg/dl).

       The key finding of the present study is the statistically significant association between serum bilirubin levels and histopathological severity of appendicitis. Mean TSB was 2.3 mg% in perforated appendix versus 1.883 mg% in gangrenous appendix and 1.427 mg% in acute appendicitis (F = 11.88, p < 0.001). This is consistent with findings of Biswal et al. (prospective study: 1.66 vs 1.07 mg%; sensitivity 57%, specificity 86%), Bakshi Mandal (complicated mean direct bilirubin 0.9 mg%, uncomplicated 0.53 mg%), and Bali RS (74% hyperbilirubinemia in perforated vs 30% in uncomplicated). In the present study, 89% of patients with perforated appendix had hyperbilirubinemia compared to 66.6% with acute appendicitis, demonstrating higher prevalence in complicated disease.

      Particularly important is the finding that 100% of both perforated and gangrenous appendicitis cases had elevated direct bilirubin, compared to 75% of acute appendicitis cases. This aligns with Eren et al., who described a 36-fold greater risk for perforated appendix or gangrene with elevated direct bilirubin, and Sevinc et al., who noted that direct bilirubin was more accurate than total bilirubin for diagnosis.

       ROC curve analysis demonstrated excellent diagnostic performance for both bilirubin fractions. For predicting perforated appendicitis, TSB AUC = 0.866 (sensitivity 88.9%, specificity 80.2%, cut-off 1.75 mg%) and direct bilirubin AUC = 0.883 (sensitivity 88.9%, specificity 71.4%, cut-off 1.15 mg%). These values compare favourably with other reported series: Bakshi and Mandal (TSB sensitivity 91.43%, specificity 88%, PPV 78.03%, NPV 95.65%), Sand et al. (sensitivity 70%, specificity 86%, cut-off 1.3 mg%), Khan et al. (sensitivity 100%, specificity 80%), and Elmetwally AMS (sensitivity 58%, specificity 82% for TSB ≥1.0 mg/dl). For gangrenous appendicitis, AUC values were lower (TSB 0.71, direct bilirubin 0.739), reflecting the inherently more variable bilirubin response in gangrenous (non-perforated) disease.

       The postoperative decline in bilirubin levels (p < 0.005 across all groups at POD-7) confirms the pathophysiological mechanism: elevation is secondary to bacterial translocation and transient hepatic cholestasis caused by the inflamed/perforated appendix. Resolution of the inflammatory focus following appendectomy normalizes bilirubin metabolism. This finding is consistent with Hassan A Saad et al. and supports the clinical validity of using bilirubin as a dynamic surgical biomarker.

       ALP was also elevated in complicated appendicitis (uncomplicated: 100.2 IU/L; complicated: 170 IU/L; p < 0.005), consistent with Mallikarjuna M et al. (uncomplicated 88.2 vs complicated 155 IU/L). Supplementing bilirubin with ALP, CRP, and total leucocyte count may further improve predictive accuracy, as highlighted in recommendations for future research.

       Limitations of the present study include the single-centre design with a relatively modest sample size of 100 patients from a specific geographical area, restricting generalizability. Additionally, only serum bilirubin levels (total and direct) were assessed; inclusion of additional LFT parameters such as indirect bilirubin and other inflammatory markers may provide a more comprehensive predictive model. Multi-centre prospective trials with larger cohorts are warranted to validate these findings.

Serum bilirubin—both total and direct—is a simple, inexpensive, and readily available laboratory marker that correlates significantly with the severity of acute appendicitis and histopathological findings. Serum bilirubin was elevated in 70% of patients with acute appendicitis, with higher prevalence and magnitude in complicated (perforated and gangrenous) disease. A total serum bilirubin >1.5 mg/dl and/or a direct bilirubin >0.7 mg/dl in a patient with clinical features of acute appendicitis should raise concern for complicated disease and guide timely surgical intervention. An increase in preoperative serum bilirubin should be regarded as a potential predictor of complicated appendicitis. Its routine inclusion in the preoperative work-up of patients with suspected acute appendicitis is recommended. Further multi-centre studies supplementing serum bilirubin with other LFT parameters (ALP, indirect bilirubin) and inflammatory markers (CRP, leucocyte count) may further refine the predictive model for complicated appendicitis.

Bakshi S, Mandal N. Evaluation of role of hyperbilirubinemia as a new diagnostic marker of complicated appendicitis. BMC Gastroenterol 21, 42 (2021). https://doi.org/10.1186/s12876-021-01614

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