Urinary 5-HIAA for Diagnosis of Acute Appendicitis in Children, Does it Help?

1.1- Study design: Patients were randomly categorized into two groups A and B. Randomization was achieved using closed envelop method.

Group A (n= 95) included patients that were evaluated using our institutional protocol. This protocol consists of assessment of children using PAS, ultrasound of abdomen and pelvis and serum levels of C-reactive protein.

Group B (n= 96) included children that were evaluated by our institutional protocol. In addition, all children gave a mid- stream urine sample for spot urinary 5-HIAA detection. Detailed history was taken. This mainly was to exclude cases with history of foods or drugs that may affect level of serotonin in urine such as mono amino oxidase inhibitors.

All related clinical data and laboratory investigations were recorded. 1.2- Quantitation of urinary 5-HIAA level: From each participant, a random urine sample was obtained to assess the urinary 5-HIAA level using a solid phase competitive enzyme-linked immunosorbent assay (ELISA) technique. The commercial kit was purchased and provided by LDN, DN Labor Diagnosika Nord GmbH \& Co. Nordhorn, Germany; Catalog #: BA E-1900. As stated by the manufacturer, the urine samples were methylated to derivatize the 5-HIAA. On the provided microtitre plate, the methylated standards, controls and samples are added. 5-HIAA antiserum was then added and incubated for 1 hour. After equilibration, excess free antigen and antigen-antibody complexes are washed out. The affixed bounded antibodies to the solid phase were detected by an anti-rabbit IgG-peroxidase conjugate using TMB as a substrate. The reaction is measured on a microplate reader (Tecan Spectra II, Switzerland), at 450 nm. Quantification of unknown sample concentration is processed by drawing a standard curve with known standard concentrations. The results of 5-HIAA were divided by urinary creatinine and expressed as mg/g creatinine. The inter-assay and intra-assay coefficient of variation were 10.8% and 8.6% respectively.

1.3- Clinical outcome: Diagnosis of acute appendicitis was confirmed by histopathological examination of resected appendix. According to operative findings and pathological reports, the condition ranged from negative appendix to different types of acute appendicitis (catarrhal, suppurative, perforated or gangrenous).

Children did not full filling the requirements for diagnosis as acute appendicitis were discharged on symptomatic treatment or after referral to pediatric emergency department.

Readmitted cases were recorded. 1.4- Statistical analysis: Statistical analysis was done using IBM SPSS V. 24 (IBM, NY, USA). A descriptive analysis was obtained for patients included in the study. Shapiro-Wilk test was conducted to check for normal distribution of dependent variables, such as urinary 5-HIAA, CRP and histopathological findings. X2 test was used to compare the categorical data. Student T-test was used to compare the normally distributed variable between two groups. Mann-Whitney U test and Kruskal-Wallis test were used for comparing the non-normally distributed variables of two and more than two groups, respectively. Binary logistic regression was performed to determine the independent predictors for acute appendicitis and to combine 5-HIAA and PAS results in both groups. Pearson correlation was used for correlating the urinary 5-HIAA with other acute appendicitis predictors. Receiver operating characteristic (ROC) curve was constructed to assess the diagnostic efficacy of acute appendicitis predictors. The optimal cutoff value for each predictor was assessed via the Youden index. The value of AUC ranged from 0.5 to 1.0. The ability of a diagnostic test to identify patients with appendicitis was considered optimal as AUC value reached closer to 1.0. P-value <0.05 was considered significant