- Laboratory analysis
The samples were immediately centrifuged at 5,000 rpm and 4 °C for 10 min to remove cells and debris and stored in aliquots at –70 °C until analysis. The urinary N-methylhistamine, histamine, and tryptase levels were measured using test kits by the sandwich enzyme-linked immunosorbent assay (Genzyme Corporation, Cambridge, MA, USA). The specific biotinylated detection antibody and avidin horseradish peroxidase conjugate were added to the wells and incubated. After removing the non-washing parts, the blue color was created in the boxes by adding a substrate solution. The reaction was stopped by adding the stop solution, and the blue color was observed to turn yellow. Optical density (OD) was determined spectrophotometrically at a wavelength of 450 ± 2 nm. Since the OD value was proportional to mast cell mediator levels in the samples, the N-methylhistamine, histamine, and tryptase levels concentrations were calculated using standard curve graphics.