Improved assay for fecal calprotectin

Abstract

Fecal calprotectin is a marker of inflammatory and neoplastic disease in the lower gastrointestinal tract. A new fecal sample preparation procedure for the measurement of calprotectin has been developed, with higher calprotectin yield and lower contamination risk. Changes in the new method compared to the original [Røseth AG, Fagerhol MK, Aadland E, Schønsby H. Assessment of the neutrophil dominating protein calprotectin in feces. A methodologic study. Scand J Gastroenterol 1992;27(9):793–798] are smaller sample size, higher dilution of the sample, presence of dissociating agents in the extraction solution and procedure performed in closed disposable tubes. The extraction yield was 78% (41–100%) of total calprotectin, giving an overall five-fold increase compared to the original method. Samples with high calprotectin values were increased to a slightly higher degree, than low calprotectin samples, thus improving the separation between high and low calprotectin levels. Median calprotectin level in healthy subjects was 26 μg/g. Pathological samples with pancolitis showed levels up to 30 000 μg/g. The mean C.V. (coefficient of variation) in blended feces was lower than that of unblended, suggesting uneven distribution of calprotectin. However, no significant difference between spot measurements was found when five samples from each of 47 stools were measured. Thus measurements of calprotectin in fecal samples were accurate and reproducible. No interference with foods or relevant oral pharmaceuticals or nutraceuticals was found.

Introduction

Calprotectin is a 36.5-kD calcium binding protein consisting of three polypeptide chains. In the presence of Ca²⁺, calprotectin is remarkably resistant towards proteolysis [2], [10], [13], [14], [23]. Calprotectin is abundant in neutrophil granulocytes, monocytes, macrophages and submucosal epithelial cells [5], [8]. In neutrophils the protein may account for up to 60% of the protein in the cytosolic fraction [9]. Calprotectin exerts antimicrobial properties in vitro, both bactericidal and fungicidal [6], [32], [33], [35]. The protein also appears to have a regulatory function in the inflammatory process [34].

Several studies have described the clinical relevance of measuring calprotectin. Elevated concentrations of calprotectin in feces have been measured in patients with colorectal cancer, inflammatory bowel diseases and bacterial infections in the gastrointestinal tract (GI) [19], [28], [29]. The highest levels of calprotectin in plasma has been found in patients with rheumatoid arthritis, cystic fibrosis, Crohn’s disease and acute bacterial infections such as sepsis and meningococcal disease [3], [4], [11], [12], [26], [27], [28], [29].

Røseth et al. developed a method for extraction of calprotectin in feces and quantification by an enzyme linked immunoassay [26]. Several clinical studies suggest a diagnostic value of fecal calprotectin measured with this method [15], [18], [19], [22], [27]. The original method has, however, a low calprotectin yield and a high contamination risk with the procedure based on 5 g feces and homogenization with a mechanical rod mixer in an open tube. A new simplified feces sample preparation method has been developed, based on 50–100 mg feces, sample preparation in closed tubes with disposable equipment and dissociating extraction solution. In the present study the new method is compared with the original fecal calprotectin method.