The role of urinary biomarkers in the early diagnosis of acute appendicitis in children

Authors

Abstract

Aim Acute appendicitis is a common surgical emergency in children that may cause serious complications if not diagnosed early. This study aimed to evaluate urinary levels of Neutrophil Gelatinase-Associated Lipocalin, L-Fatty Acid-Binding Protein, and I-Fatty Acid-Binding Protein in pediatric patients with acute appendicitis and to investigate their relationships with other biochemical parameters and their possible prognostic value.
Materials and Methods The study included fifty-four children diagnosed with acute appendicitis and sixty-four healthy controls. Complete blood count, high-sensitive C-reactive protein, procalcitonin, erythrocyte sedimentation rate, bilirubin, albumin, total protein, full urinalysis, and urinary levels of creatinine, Neutrophil Gelatinase-Associated Lipocalin, L-Fatty Acid-Binding Protein, and I-Fatty Acid-Binding Protein were measured and compared between the two groups.
Results There were no significant differences in urinary Neutrophil Gelatinase-Associated Lipocalin, L-Fatty Acid-Binding Protein, creatinine, urea, or uric acid levels between children with acute appendicitis and healthy controls (p > 0.05). However, I-Fatty Acid-Binding Protein, sodium, potassium, and albumin levels were significantly lower in patients, while high-sensitivity C-reactive protein and procalcitonin levels were significantly higher (p < 0.05). Each 1 milligram per liter increase in high-sensitivity C-reactive protein was associated with a 2.36-fold higher risk of appendicitis (p = 0.003), whereas each one milliequivalent per deciliter increase in potassium was related to a 0.001-fold lower risk (p = 0.002).
Discussion These biomarkers may serve as indicators of intestinal inflammation and may support the diagnosis of acute appendicitis; however, none appear adequate to be used alone as a definitive diagnostic test.

Keywords

Introduction

Acute appendicitis (AA) remains the most common surgical emergency in pediatric patients. Its diagnosis continues to be a significant challenge for clinicians due to the atypical clinical presentation in children [1, 2]. The lifetime risk of developing AA is between 7% and 9% [3]. Delays in diagnosis can lead to serious complications, including perforation and peritonitis, increasing morbidity and hospital stays [4]. While a combination of clinical scores, laboratory markers, and imaging modalities is used to guide diagnosis, a single, highly sensitive, and specific non-invasive test remains elusive [5, 6].
In recent years, the search for novel biomarkers has intensified to aid in the early and accurate diagnosis of AA. Neutrophil Gelatinase-Associated Lipocalin (NGAL), a protein secreted by activated neutrophils and epithelial cells, has been extensively investigated for its role in inflammation and tissue damage [7]. Studies have shown elevated NGAL levels in patients with appendicitis, suggesting its potential as a diagnostic marker [8, 9].
Similarly, Fatty Acid-Binding Proteins (FABPs) are being explored as indicators of organ-specific ischemia and inflammation. Intestinal-type FABP (I-FABP) and Liver-type FABP (L-FABP) are released from damaged intestinal and hepatic tissues, respectively [10, 11]. Previous research has demonstrated a strong correlation between elevated serum and urinary FABP levels and intestinal ischemia, which often occurs in advanced stages of appendicitis [12]. Given that urine is a non-invasive and easily collected sample, its potential for use in a point-of-care setting makes these biomarkers particularly appealing for pediatric emergency departments.
This study aims to evaluate the diagnostic utility of urinary NGAL, I-FABP, and L-FABP in pediatric patients with suspected AA. We investigated their levels and correlations with established biochemical parameters to determine their potential as reliable indicators for early diagnosis and their prognostic significance.

Materials and Methods

Study Design and Patients Study Population: This prospective study was conducted at the Pediatric Emergency Department of Sakarya University Faculty of Medicine Training and Research Hospital from February to September 2019. A total of 64 healthy controls and 63 pediatric patients with a clinical diagnosis of acute appendicitis were initially enrolled. Nine patients whose AA diagnosis was pathologically excluded post-operatively were removed, resulting in a final cohort of 64 healthy controls and 54 acute appendicitis patients.
Inclusion and Exclusion Criteria: The patient group included children aged 17 or younger who underwent surgery for a clinical diagnosis of AA. The control group consisted of healthy children with similar demographic profiles. Exclusion criteria were defined as any chronic disease, recent medication use, an infection within two weeks prior to enrollment, or a history of abdominal trauma. Patients with a non-confirmed pathological diagnosis of AA were also excluded.
Acute Appendicitis Definition: The diagnosis of AA was based on a comprehensive assessment including clinical signs and symptoms (e.g., Alvarado or Pediatric Appendicitis Scores), laboratory findings (WBC count, hs-CRP, procalcitonin), and abdominal ultrasonography (appendiceal diameter > 6 mm and wall thickness > 2 mm) [13, 14]. The final diagnosis for all patients was confirmed by histopathological examination of the surgical specimen.
Biochemical Parameters Sample collection: Peripheral venous blood and urine samples were collected upon admission from AA patients and from healthy controls. Samples were immediately centrifuged, and the serum and cell-free urine were stored at -80°C until analysis. Standard laboratory tests: Complete blood counts were analyzed with an automated analyzer. Serum hs-CRP and procalcitonin levels were measured using immunonephelometric and electrochemiluminescence methods, respectively [15]. Serum urea, creatinine, uric acid, and albumin levels were determined using an automated analyzer, and sodium and potassium were measured via the Ion-Selective Electrode (ISE) method. Urinary biomarker analysis: Urinary NGAL, I-FABP, and L-FABP levels were measured using commercially available ELISA kits. Urinary creatinine levels were also measured to adjust for hydration status.
Statistical Analysis Descriptive statistics were used for demographic data. The normality of variable distributions was assessed using the Kolmogorov-Smirnov test. Group comparisons were performed using Student’s t-test or the Mann-Whitney U test, and categorical variables were compared with the chi-squared or Fisher’s exact chi-squared test. Relationships between variables were analyzed using Pearson’s and Spearman’s correlation coefficients. A stepwise multivariable logistic regression model was used to identify independent predictors for AA risk. ROC curve analysis was performed to evaluate the diagnostic accuracy of the biomarkers. All statistical analyses were conducted using SPSS for Windows software (ver. 20.0), with a p-value of<0.05 considered statistically significant.
Ethical Approval This study was approved by the Ethics Committee of Sakarya University (Date: 2020-03-21, No: 16214662/050.01.04/29).

Results

There was no significant difference between the patient and control groups in terms of age and gender (p > 0.05). Similarly, there was no difference in NGAL, L-FABP, urinary creatinine, urea, creatinine, and uric acid levels between the patient and control groups (p > 0.05). It was determined that I-FABP, sodium, potassium, and albumin values were lower in the patient group compared to the control group, while hs-CRP and procalcitonin values were higher (p < 0.05) (Table 1). In the patient group, the PAS value was 7.04±1.37, the anterior-posterior diameter of the appendix was 10.32±3.13 mm, and the appendix wall thickness was 2.04 ± 0.47 mm.
While a negative correlation (r = -0.203, p = 0.039) was observed between NGAL and uric acid in the patient group, no correlation was found with other parameters. A negative correlation was observed between L-FABP and urine creatinine (r = -0.198, p = 0.031) and appendiceal anterior-posterior diameter (r = -0.343, p = 0.016), with no other significant correlations. A negative correlation was found between I-FABP and leukocyte count (r = -0.198, p = 0.032), hs-CRP (rho = -0.341, p < 0.001), and
procalcitonin (rho = -0.292, p = 0.001), while it was positively correlated with sodium (r = 0.249, p = 0.008) and albumin (r = 0.191, p = 0.039) (Figure 1). Correlation analyses are shown in Table 2.
In the multivariate logistic regression analysis performed to determine the risk of appendicitis, a 1 mg/L increase in hs-CRP levels was found to increase the risk of appendicitis by 2.36 times (p = 0.003), while a one mEq/dL increase in potassium levels decreased the risk of appendicitis by 0.001 times (p = 0.002) (Table 3). NGAL, L-FABP, I-FABP, leukocyte count, hs-CRP, procalcitonin, urine creatinine, urea, serum creatinine, uric acid, sodium, potassium, and albumin parameters were included in the logistic regression model, and the Backward Wald elimination method was applied.
The sensitivity and specificity of NGAL, L-FABP, and I-FABP parameters in the diagnosis of AA were evaluated with ROC curves (Figure 2). When the optimal cut-off value for I-FABP was set at 19.70 ng/mL, its sensitivity was 45.3% and specificity was 79.6% (AUC = 0.668, p = 0.002). AUC values of NGAL and L-FABP parameters were below 0.600, and p > 0.05.

Discussion

Our study confirms the established utility of conventional inflammatory markers, such as hs-CRP and procalcitonin, for diagnosing and prognosticating pediatric acute appendicitis [15, 16]. The significant elevation of these markers, along with altered electrolyte and albumin levels (Table 1), is consistent with the systemic inflammatory response and fluid shifts that characterize the disease [17]. The finding that a 1 mg/L increase in hs-CRP levels increases the risk of AA by 2.36-fold highlights its substantial predictive value, supporting its use as a core diagnostic tool.
However, a key finding of our study was the lack of a significant increase in urinary NGAL and L-FABP levels, and a surprising decrease in urinary I-FABP levels, in the patient group (Table 1). This result is in stark contrast to prior reports that suggested these biomarkers could be useful in AA diagnosis [8, 9]. We believe this discrepancy can be attributed to our study population, which consisted primarily of non-perforated and early-stage AA cases. The release of these biomarkers, particularly I-FABP, may be more pronounced in later stages of inflammation when there is more extensive intestinal epithelial damage, a common finding in complicated or perforated appendicitis [18, 19]. Therefore, the absence of elevated levels in our cohort may reflect the early nature of the disease in these patients rather than the ineffectiveness of the biomarkers themselves. This suggests that these markers may be more useful in differentiating between complicated and uncomplicated AA, a critical need in clinical practice [20, 21].
The low sensitivity and moderate specificity of I-FABP, as shown by the ROC curve analysis (Figure 2), further suggest that these urinary biomarkers should not be used as standalone diagnostic tools. Their values can also be affected by various other conditions, such as acute kidney injury and diabetic nephropathy, which limit their specificity for AA [22, 23]. While our findings reinforce the importance of established inflammatory markers, they highlight the limitations of relying on a single biomarker for definitive diagnosis. The negative correlations observed between I-FABP and established inflammatory markers like leukocyte count, hs-CRP, and procalcitonin (Table 2), while unexpected, further support the notion that I-FABP may not be a reliable early-stage marker.
Our study reinforces the established importance of hs-CRP, procalcitonin, and electrolyte balance in the diagnosis and prognosis of acute appendicitis. We found that while urinary NGAL and FABPs alone are not sufficient for a reliable early diagnosis of AA, they may have a more nuanced role in the context of disease staging. Our findings suggest that these markers may be more indicative of the severity of inflammation or widespread tissue damage that occurs in later stages of appendicitis, a hypothesis that warrants further investigation. A primary limitation of our study is the absence of corresponding serum data for these biomarkers, which would have provided a more comprehensive understanding of their diagnostic potential. Based on these findings, we recommend future research to focus on developing biomarker panels that combine novel and conventional markers for a more robust diagnostic tool, conducting longitudinal studies to track the kinetics of these markers, and comparing biomarker levels between perforated and non-perforated cases to predict disease severity. The role of other inflammatory markers, such as the neutrophil-to-lymphocyte ratio, can also be integrated into these panels in future studies to help determine diagnosis and disease severity [24, 25]. Further research addressing these limitations is essential to fully understand the potential of these biomarkers in pediatric acute appendicitis.

Limitations

The main limitation of this study is that, since it was conducted at a single center, the results may not fully represent different populations or clinical settings.

Conclusion

Our study contributes to the growing body of evidence emphasizing the necessity of a multifactorial approach in the diagnosis and treatment of pediatric acute appendicitis. By integrating both traditional and emerging biomarkers, future diagnostic strategies may achieve earlier and more accurate detection, ultimately improving clinical outcomes.

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