Detection and diagnostic value of urine leucine-rich α-2-glycoprotein in children with suspected acute appendicitis

Abstract

Study objective: Previously, we used a proteomics approach for the discovery of new diagnostic markers of acute appendicitis and identified leucine-rich α-2-glycoprotein (LRG) that was elevated in the urine of children with acute appendicitis and enriched in diseased appendices. Here, we sought to evaluate the diagnostic utility of enzyme-linked immunosorbent assay (ELISA) of urine LRG in a blinded, prospective, cohort study of children being evaluated for acute abdominal pain.

Methods: Urine LRG concentration was measured with a commercially available LRG ELISA and selected ion monitoring mass spectrometry. Urine LRG test performance was evaluated blindly against the pathologic diagnosis and histologic grade of appendicitis.

Results: Urine LRG was measured in 49 patients. Mean urine LRG concentration measured with commercial LRG ELISA was significantly elevated in patients with acute appendicitis but exhibited an interference effect. Direct measurements using selected ion monitoring mass spectrometry demonstrated that LRG was elevated more than 100-fold in patients with acute appendicitis compared with those without, with the receiver operating characteristic area under the curve of 0.98 (95% confidence interval 0.96 to 1.0). Among patients with acute appendicitis, elevations of urine LRG measured with ELISA and selected ion monitoring mass spectrometry correlated with the histologic severity of appendicitis.

Conclusion: Urine LRG ELISA allows for discrimination between patients with and without acute appendicitis but exhibits limited accuracy because of immunoassay interference. Direct measurements of urine LRG with selected ion monitoring mass spectrometry demonstrate superior diagnostic performance. Development of a clinical-grade urine LRG assay is needed to advance the diagnostic accuracy of clinical evaluations of appendicitis.

Figure 1

A. Urine LRG ELISA is limited by immunoassay interference that can be partially mitigated by serial dilution. Apparent urine LRG concentration measured using commercial LRG ELISA as a function of urine dilution of 49 specimens. Most specimens exhibit increases in apparent LRG concentration with increasing dilution of urine, indicating the presence of a substance that interferes with the antibody adsorption and/or detection of urine LRG. This effect is absent in some specimens, with relatively constant LRG concentrations among repeated measurements of different dilutions (green). The immunoassay interference can be partially overcome by increasing urine dilution, with relatively constant LRG concentrations in specimens diluted by more than 100-fold (red). B. Urine LRG ELISA discriminates patients with appendicitis and correlates with disease severity. Boxplots of ELISA interference-adjusted urine LRG concentrations in patients without appendicitis (black), as compared to those with appendicitis with increasing histologic severity. LRG ELISA values in some patients with appendicitis overlap with those in patients without appendicitis or histologically normal appendices. C. Direct measurement of urine LRG using SIM MS overcomes immunoassay interference effects. SIM MS urine LRG levels as measured using area under the curve (AUC) for the extracted ion currents of LRG peptide VAAGAFQGLR in patients with histologically normal appendices, as compared to those with appendicitis with increasing histologic severity. Box center and ranges represent median and standard error values, respectively. Stars denote mean values for the two groups. Whiskers represent 95% confidence intervals.