Gastrointestinal
Serum Markers in Acute Appendicitis

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Background

We have recently profiled inflammatory gene expression in acute appendicitis (AA) and identified a set of genes that are overexpressed in AA. The purpose of this study was to determine whether serum levels of a subset of proteins coded for by these overexpressed genes could differentiate patients with AA from those with other causes of abdominal pain and whether the serum levels of these proteins correlate with the histologic severity of appendicitis.

Materials and Methods

Serum samples were obtained from a convenience sample of children between 1 and 21 y of age who presented to the emergency department (ED) with symptoms/signs of AA. Patients were assigned to the proven appendicitis (AA) or nonappendicitis (control) group based on histologic findings, chart review, and follow-up phone calls. The serum levels of haptoglobin, granulocyte colony stimulating factor (GCSF), interleukin 8 (IL-8), and C-reactive protein (CRP) were compared between groups. For patients with histologically proven appendicitis, a histologic severity score was assigned and correlated with the levels of potential serum biomarkers.

Results

thirty-two patients were enrolled; 23 patients (72%) had AA. Serum levels of CRP and GCSF were significantly different between AA and control groups (7.0 versus 0.8, P = 0.01 and 104.2 versus 58.7, P = 0.03, respectively.) In patients with AA, there was significant correlation between GCSF serum levels and the appendicitis severity score [r = 0.537, P = 0.02].

Conclusions

GCSF serum levels can distinguish between patients with AA and controls. GCSF may prove to be a useful adjunctive test in the diagnosis and staging of acute appendicitis.

Introduction

Acute appendicitis (AA) is the most common cause of acute abdomen requiring surgical intervention during childhood [1]. It accounts for 10% to 30% of pediatric presentations to emergency departments with abdominal pain 2, 3. Acute appendicitis is commonly diagnosed with a combination of clinical information including symptoms and physical examination findings; traditional biomarkers, such as white blood cell count (WBC), absolute neutrophil count (ANC), and C-reactive protein (CRP), as well as radiographic imaging (ultrasound and CT scan) 2, 3, 4. However, this diagnosis in children is problematic based on the patient's difficulty in communicating symptoms and the large overlap in presentation with other common pediatric diseases (i.e., gastroenteritis). Recent advances in computerized tomography have led to only modest improvements in the negative appendectomy rate and in the diagnosis of appendicitis 2, 5.

One potentially promising approach is to identify novel serum biomarkers that might more accurately predict the presence of disease than conventional diagnostics. Further, it is possible that serum biomarkers may not only indicate that appendicitis is present but may also correlate with the severity of appendicitis (i.e., early or late, perforated or not perforated.) This raises the idea that not only could biomarkers lead to more accurate diagnosis, but could be useful in disease staging, which in turn could potentially influence management options. A rational strategy to identify such markers could lead to important diagnostic advances.

It has been hypothesized that acute appendicitis is characterized by a sequence of events starting with an obstruction of the appendiceal lumen and decreased blood flow, destruction of the local epithelial barrier, bacterial invasion, infiltration by granulocytes, monocytes, T cells, and natural killer cells, subsequent tissue hypoxia, necrosis, and eventual perforation [6]. To further understand the gene expression events that characterize AA, Murphy et al. profiled gene expression within the appendices of pediatric patients who had undergone an appendectomy [7]. This study demonstrated that AA is characterized by an intense but relatively focal pattern of inflammatory gene expression. Further, this study identified a number of genes whose expression was elevated in both patients with mild and severe inflammation of the appendix. The observation of increased expression of select inflammatory related genes in the appendices of patients with both mild and severe appendicitis raised the question of whether the gene products of any of the genes whose expression was increased within the appendix of patients with AA could also be observed at elevated levels within the serum of patients with AA, and if so, could they serve as potential biomarkers to help improve diagnostic accuracy.

To evaluate this hypothesis, we chose to determine the serum levels of three proteins whose mRNA was elevated within the appendix of patients with AA. These were granulocyte colony stimulating factor (GCSF), haptoglobin, and interleukin-8 (IL-8). While levels of IL-8 have previously been evaluated in the serum of patients with appendicitis 8, 9, 10 there is little data regarding serum levels of GCSF 8, 11 and haptoglobin in patients with appendicitis. We also included in our analysis three well-studied serum biomarkers of acute appendicitis: WBC 3, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, ANC 3, 14, 16, 20, 23, and CRP 4, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 23, 24, 25, 26, 27. as comparative markers for the performance of potential new biomarkers. Here we report the results of these studies.

Section snippets

Study Design and Setting

This prospective observational study of a convenience sample was conducted from September 2006 to January 2008 at an urban, academic pediatric medical center with approximately 56,000 emergency department (ED) patient visits per year.

The study was approved by the Institutional Review Board of Children's Hospital, Boston. The study was compliant with the Health Insurance Portability and Accountability Act of 1996.

Participants and Enrollment

Children between 1 and 21 years of age who presented to the ED with possible

Characteristics of Subjects

A total of 32 patients were collected during the study period. Twenty-three patients were assigned to the appendicitis group. Nineteen of these had appendectomy during acute disease and had pathologic findings consistent with acute appendicitis (AA), and four who had CT scans demonstrating perforated appendicitis and went on to have interval appendectomies. Nine patients did not have appendicitis and were assigned to the control group as determined by an alternative diagnosis and a negative

Discussion

In this study, we compared plasma concentration of GCSF, haptoglobin, and IL-8, as potential biomarkers of acute appendicitis based on the recent finding that expression of the mRNA coding for these proteins is elevated within the inflamed appendix [7]. In addition, we compared the performance of these potential biomarkers with widely used biomarkers including WBC, ANC, and CRP. As expected, we found that WBC and CRP levels were significantly different between AA and control groups, while ANC

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