European Journal of Cardiovascular Medicine

The vermiform appendix is a narrow, blind-ended tubular structure arising from the cecum of the large intestine. The term vermiform, meaning “worm-like,” reflects its slender anatomical configuration [1]. Its clinical significance in surgery primarily relates to its propensity for inflammation, leading to acute appendicitis, one of the most frequent surgical emergencies worldwide. Acute appendicitis most commonly results from obstruction of the appendiceal lumen by fecaliths, inspissated stool, lymphoid hyperplasia, or infectious agents, with subsequent polymicrobial infection contributing to disease progression. If left untreated, this condition may culminate in serious complications such as perforation, generalised peritonitis, or intra-abdominal abscess formation [2,3]. Acute appendicitis remains a major healthcare burden, accounting for more than 40,000 hospital admissions annually in England alone [4]. The lifetime risk is higher in males (8.6%) compared to females (6.7%), with the peak incidence observed during the second decade of life (10–19 years). Nearly 70% of cases occur in individuals younger than 30 years [5]. While uncomplicated appendicitis carries a relatively low mortality rate of 0.1–0.5%, the mortality associated with perforated appendicitis rises substantially, ranging from approximately 3% in the general population to as high as 15% among elderly patients [6]. Reported perforation rates vary between 16% and 40%, with notably higher rates in younger children (40–57%) and in patients older than 50 years (55–70%) [7,8]. Despite advances in diagnostic modalities, the diagnosis of acute appendicitis continues to be challenging. It is largely based on a combination of clinical history, physical examination findings, laboratory investigations, and imaging studies. Typical symptoms include periumbilical abdominal pain, anorexia, nausea, migration of pain to the right lower quadrant, and low-grade fever [9]. However, these classical features may be absent or attenuated in elderly patients, while paediatric presentations are often nonspecific due to limited communication abilities [10]. Furthermore, acute appendicitis may mimic a wide range of intra-abdominal and pelvic conditions, including diverticulitis, mesenteric adenitis, Crohn’s disease, endometriosis, ectopic pregnancy, omental torsion, pelvic inflammatory disease, ruptured ovarian cysts, and urinary tract infections. Consequently, diagnostic error rates remain substantial, reported to be 12–23% in men and 24–42% in women globally [11]. Delayed diagnosis and surgical intervention significantly increase the risk of complications, even in initially non-perforated cases, potentially leading to gangrene or perforation. Appendiceal perforation may further result in abscess formation, diffuse peritonitis, faecal fistulae, intestinal obstruction, portal pyaemia, sepsis, and infertility in women [4]. Conversely, reliance on clinical suspicion alone in diagnostically uncertain cases may lead to unnecessary appendectomies, with negative appendectomy rates reported to be as high as 20% [12]. Such unwarranted surgical interventions expose patients to avoidable complications, including wound infection, postoperative adhesions causing bowel obstruction, and incisional hernia formation. Although laboratory investigations support clinical decision-making, no single biomarker has been identified as both highly sensitive and specific for acute appendicitis. Elevated white cell count (WCC) is commonly observed but lacks diagnostic specificity, as it may rise in various inflammatory conditions. C-reactive protein (CRP) is frequently used in the diagnosis of suspected appendicitis; however, its diagnostic accuracy and specificity vary considerably among studies [13]. Clinical scoring systems such as the Alvarado and Modified Alvarado scores provide structured assessment tools, yet limitations persist. Moreover, advanced imaging modalities, including computed tomography (CT) and magnetic resonance imaging (MRI) are not consistently reliable in differentiating uncomplicated appendicitis from complicated or perforated disease [14]. Therefore, there is a continued need for additional, easily measurable biomarkers to complement existing diagnostic approaches and facilitate early identification of severe disease.

The present study aimed to evaluate the association between hyperbilirubinemia and the severity of acute appendicitis before surgical intervention. Specifically, we assessed the diagnostic utility of serum bilirubin levels in distinguishing inflamed appendices from normal appendices in patients undergoing emergency appendectomy. In addition, we examined the relationship between hyperbilirubinemia and appendiceal perforation and compared the diagnostic specificity of bilirubin with that of WCC and CRP in both uncomplicated and perforated appendicitis.

This retrospective observational study was conducted in the Department of General Surgery at the Indira Gandhi Institute of Medical Sciences (IGIMS), Patna, Bihar. The study included the patients who underwent emergency appendectomy for clinically suspected acute appendicitis over a period of one year. The study was conducted in full accordance with ethical standards and adhered to the principles outlined in the Declaration of Helsinki.

Sample Size Calculation:

The sample size for the present study was calculated using a power analysis performed with G*Power software, version 3.0.1 (Franz Faul, Universität Kiel, Germany). The calculation was based on an alpha error of 5% (α = 0.05) and a statistical power of 80% (1−β = 0.80). An anticipated moderate effect size of 0.6 was assumed, derived from previously published studies evaluating the diagnostic role of hyperbilirubinemia in acute appendicitis and appendiceal perforation. Based on these parameters, the minimum required sample size was estimated to be 65 patients. This sample size was considered adequate to detect a statistically significant association between serum bilirubin levels and the severity of appendicitis, while ensuring reliable and clinically meaningful results.

Patient data were retrieved from the computerised hospital information system. Collected variables included demographic details, preoperative laboratory parameters such as serum total bilirubin levels, total leukocyte count (WCC), and C-reactive protein (CRP) levels, operative findings, and histopathological examination reports. Histopathological evaluation of the resected appendix was considered the gold standard for confirming the diagnosis.

Patients of adult age groups and both genders with complete preoperative laboratory data and available histopathological reports were included. Patients with known liver disease, biliary tract pathology, viral hepatitis, chronic alcoholism, haemolytic disorders, gastrointestinal malignancy, or use of hepatotoxic drugs were excluded. Cases with incomplete medical records or those who underwent interval appendectomy or appendectomy for indications other than suspected acute appendicitis were also excluded.

Histopathological findings were used to classify cases into five categories: normal appendix, acute focal appendicitis, acute suppurative appendicitis, gangrenous appendicitis, and perforated appendicitis. For analytical purposes, three broader groups were formed: normal appendices, non-perforated appendicitis (including acute focal, suppurative, and gangrenous appendicitis), and perforated appendicitis. Mean serum bilirubin, WCC, and CRP levels were compared among these groups, and the proportion of patients with elevated biomarker levels was also analysed.

Hyperbilirubinemia was defined as a serum total bilirubin level greater than 20 μmol/L, leukocytosis as a WCC exceeding 11 × 10⁹/L, and elevated CRP as a value greater than 10 mg/L. Diagnostic accuracy parameters, including sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and odds ratios, were calculated to evaluate the diagnostic performance of serum bilirubin, WCC, and CRP in identifying non-perforated and perforated appendicitis.

Statistical Analysis:

All collected data were entered into Microsoft Excel and subsequently analysed using the Statistical Package for the Social Sciences (SPSS), version 25.0 (IBM Corp., Armonk, NY, USA). Continuous variables were assessed for normality using the Shapiro–Wilk test. Normally distributed data were expressed as mean ± standard deviation and compared using Student’s t-test (for two groups) or one-way analysis of variance (ANOVA) (for more than two groups). Non-normally distributed data were analysed using the Mann–Whitney U test or Kruskal–Wallis test, as appropriate. Categorical variables were presented as frequencies and percentages and analysed using the Chi-square test. Fisher’s exact test was applied when expected cell counts were less than five. Diagnostic accuracy parameters, including sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and odds ratios with 95% confidence intervals, were calculated to evaluate the diagnostic performance of serum bilirubin, white blood cell count (WCC), and C-reactive protein (CRP) in identifying non-perforated and perforated appendicitis.

A p-value of less than 0.05 was considered statistically significant

A total of 65 patients who underwent emergency appendectomy for clinically suspected acute appendicitis during the study period were included in the analysis. The age of the patients ranged from 24 to 76 years, with a mean age of 33.54 ± 14.59 years. Males constituted 61.5% of the study population, while females accounted for 38.5%.

Histopathological examination of the resected appendices revealed non-perforated appendicitis in 48 patients (73.8%), perforated appendicitis in 10 patients (15.4%), and a normal appendix in 7 patients (10.8%) (Table 1 and Figure 1). Thus, the negative appendectomy rate in the present study was 10.8%. Gender-wise analysis demonstrated a male predominance in both non-perforated and perforated appendicitis. Among patients with non-perforated appendicitis, 62.5% were male, and 37.5% were female, while perforated appendicitis was observed in 70.0% males and 30.0% females. In contrast, females constituted a higher proportion of patients with a normal appendix (57.1%) compared to males (42.9%) (Table 1). Although a male predominance was observed in both non-perforated and perforated appendicitis, the association between gender and histopathological diagnosis was not statistically significant (Fisher’s exact test, p = 0.63).

Table 1: Gender-wise distribution of appendicitis based on histopathological findings

Figure 1: Gender wise distribution of appendicitis

Comparison of laboratory parameters across histopathological groups showed a significant increase in mean serum bilirubin levels with increasing disease severity. Patients with perforated appendicitis had the highest mean bilirubin levels (24.8 ± 18.2 μmol/L), followed by those with non-perforated appendicitis (13.6 ± 7.9 μmol/L), while the lowest levels were observed in patients with a normal appendix (9.2 ± 4.8 μmol/L). This difference was statistically significant (p < 0.001). A similar trend was observed for total leukocyte count and C-reactive protein levels, with significantly higher mean values in perforated appendicitis compared to non-perforated appendicitis and normal appendices (p < 0.001 for all comparisons) (Table 2).

Table 2: Mean laboratory parameters according to histopathological groups

Analysis of elevated biomarker levels revealed that hyperbilirubinemia (serum bilirubin >20 μmol/L) was present in 70.0% of patients with perforated appendicitis, compared to 20.8% in non-perforated appendicitis and 14.3% in patients with a normal appendix. Raised WCC and elevated CRP levels were observed more frequently across both non-perforated and perforated appendicitis; however, these markers were also elevated in a substantial proportion of patients with a normal appendix (Table 3).

Table 3: Proportion of patients with elevated biomarkers

Diagnostic accuracy analysis demonstrated that serum bilirubin had a high specificity (92.9%) but low sensitivity (20.8%) in differentiating non-perforated appendicitis from a normal appendix. In comparison, WCC and CRP exhibited higher sensitivity but lower specificity. When perforated appendicitis was compared with non-perforated appendicitis, hyperbilirubinemia showed a sensitivity of 70.0% and a specificity of 79.2%, with an odds ratio of 8.6, indicating a strong association between elevated bilirubin levels and appendiceal perforation. Although WCC and CRP demonstrated higher sensitivity in predicting perforation, their specificity was considerably lower than that of serum bilirubin (Table 4). Odds ratios were calculated with corresponding 95% confidence intervals. Overall, serum bilirubin emerged as a highly specific marker for both non-perforated and perforated appendicitis, whereas WCC and CRP served as more sensitive but less specific inflammatory markers.

Table 4: Diagnostic performance of Serum Bilirubin, WCC, and CRP

1. Non-perforated Appendicitis vs Normal Appendix

1. Perforated Appendicitis vs Non-perforated Appendicitis

Acute appendicitis remains one of the most common surgical emergencies, with an estimated annual incidence of approximately 100 cases per 100,000 population [5]. Despite significant advancements in diagnostic imaging modalities, including graded compression ultrasonography, high-resolution computed tomography, and diagnostic laparoscopy, the rates of misdiagnosis and appendiceal rupture have not declined substantially over time [5]. Moreover, these diagnostic approaches are associated with several limitations, such as increased cost, radiation exposure, operator dependence, contrast-related adverse reactions, false-positive and false-negative results, and the need for anaesthesia in certain situations. Consequently, there is an ongoing need for reliable, inexpensive, and easily accessible laboratory markers that can aid in the early diagnosis and risk stratification of acute appendicitis.

The clinical course of uncomplicated acute appendicitis is usually benign following timely appendectomy; however, disease progression to gangrenous or perforated appendicitis markedly increases morbidity and mortality. Perforated appendicitis is associated with serious complications, including generalised peritonitis, intra-abdominal abscess formation, intestinal obstruction, and systemic sepsis [15,16]. Early identification of patients at risk for perforation is therefore crucial. Unfortunately, no single clinical feature or laboratory investigation has been shown to reliably predict complicated appendicitis at presentation. In this context, serum bilirubin estimation has emerged as a promising adjunctive marker, fulfilling several desirable criteria such as simplicity, affordability, widespread availability, and rapid turnaround time.

Traditionally, the role of hyperbilirubinemia in uncomplicated acute appendicitis has been underappreciated, with most earlier studies focusing primarily on its association with appendiceal perforation. However, the findings of the present study demonstrate that hyperbilirubinemia is not only associated with perforated appendicitis but also shows significant diagnostic value in uncomplicated acute appendicitis. Patients with elevated serum bilirubin levels in our study were more likely to have histologically confirmed appendicitis than those with normal bilirubin values, suggesting that bilirubin may serve as a useful diagnostic adjunct even in the early stages of the disease.

Importantly, our study identified a strong association between hyperbilirubinemia and appendiceal perforation. A significantly higher proportion of patients with perforated appendicitis exhibited elevated bilirubin levels compared to those with non-perforated appendicitis, underscoring its potential role as a predictor of disease severity. The observed prevalence of non-perforated appendicitis and perforated appendicitis in the present study was comparable to previously reported figures, further supporting the external validity of our findings. The high specificity of hyperbilirubinemia for both non-perforated appendicitis and appendiceal perforation suggests that elevated bilirubin levels should prompt heightened clinical suspicion and consideration of early surgical intervention.

Although serum bilirubin demonstrated superior specificity, traditional inflammatory markers such as WCC and CRP showed higher sensitivity in detecting appendicitis. This complementary pattern indicates that hyperbilirubinemia functions best as a rule-in test rather than a screening tool. When used in conjunction with WCC, CRP, and clinical assessment, serum bilirubin may enhance diagnostic accuracy and assist in identifying patients at greater risk for complications.

Our findings are consistent with those reported by Sand et al. [17], who demonstrated a higher specificity of serum bilirubin for perforated appendicitis compared to CRP. Similarly, the diagnostic performance of bilirubin observed in the present study aligns closely with that reported by Kar et al. [18], particularly in terms of its high specificity for complicated appendicitis. Minor variations in specificity values across studies may be attributable to differences in study design, patient demographics, sample size, and laboratory cut-off values.

Emmanuel et al. [19] also reported high specificity of hyperbilirubinemia for both uncomplicated and gangrenous or perforated appendicitis, with significantly elevated bilirubin levels in patients with advanced disease. The diagnostic accuracy parameters observed in our study, including sensitivity, specificity, positive predictive value, and negative predictive value, were comparable to those reported in earlier literature. Furthermore, the significantly elevated mean bilirubin levels observed in patients with appendiceal perforation in the present study corroborate the findings of Vaziri et al. [20], who similarly demonstrated higher bilirubin levels in complicated appendicitis.

Overall, the results of the present study reinforce the growing body of evidence supporting the diagnostic and prognostic utility of serum bilirubin in acute appendicitis. Incorporation of serum bilirubin estimation into the routine evaluation of patients with suspected appendicitis may aid clinicians in early identification of severe disease, facilitate timely surgical decision-making, and potentially reduce morbidity associated with delayed diagnosis.

Limitations of the study: This study has certain limitations. Its retrospective, single-centre design may limit the generalizability of the findings. The relatively small sample size, particularly in the perforated appendicitis and normal appendix groups, may have contributed to variability in some laboratory parameters. Nevertheless, the use of histopathological confirmation as the diagnostic gold standard and standardised laboratory cut-off values strengthens the reliability of the results. Larger, prospective multicenter studies are recommended to further validate these findings.

The findings of this study demonstrate a significant association between hyperbilirubinemia and the severity of acute appendicitis. Serum bilirubin showed high specificity in differentiating appendicitis from a normal appendix and in predicting appendiceal perforation, outperforming traditional inflammatory markers such as WCC and CRP in specificity. Although WCC and CRP were more sensitive, serum bilirubin emerged as a reliable rule-in marker for complicated appendicitis. Given its simplicity, cost-effectiveness, and widespread availability, routine assessment of serum bilirubin may enhance the diagnostic accuracy and early identification of severe appendicitis when used alongside clinical evaluation and other laboratory parameters.

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