Calprotectin is a small calcium-binding protein belonging to the S100 family (S100 A8/A9) with a molecular weight of 36.5 kDa (1,2). The protein is abundant predominantly in neutrophils but also in monocytes (3). Calprotectin accounts for 5% of the total proteins and 60% of the cytosol proteins in the neutrophils (2,4) and is considered to have both bactericidal and fungicidal properties (5). Elevated concentrations of calprotectin can be measured in plasma (6), synovial fluid (7), urine (8), and feces (9) when there is an ongoing inflammation with recruitment of neutrophils. In plasma, the normal calprotectin values of healthy adults are significantly higher among males than females (10). The fecal calprotectin concentration is approximately six times that of normal plasma (9), but no corresponding difference between the genders has been reported in fecal calprotectin concentrations in healthy adults.
Fecal calprotectin is a potentially important clinical test as a marker of inflammation in the gastrointestinal tract (11,12,13). Increased fecal calprotectin concentrations have been found in gastric cancer, colorectal cancer, and colonic polyps (14,15), as well as in chronic inflammatory bowel disease (9,16,17,18). In inflammatory bowel disease, high fecal calprotectin concentrations can be explained with increased turnover of leukocytes in the gut wall and increased migration of neutrophils into the gut lumen (16). An improved enzyme-linked immunosorbent assay (ELISA) for fecal calprotectin has been developed in which the cutoff is suggested to be <50 μg calprotectin/g feces for adults (19). The test is simple, noninvasive, and inexpensive and possibly a valuable tool in pediatric care. However, reference values for fecal calprotectin in children have not been defined. The aim of our study was to establish reference values for fecal calprotectin in healthy children between 4 and 17 years.
MATERIALS AND METHODS
Fecal samples were obtained from 117 healthy children (52 girls and 65 boys) ranging in ages from 4 to 17 years. The children were recruited from day nursery and school health services in Västerås (a city situated 110 km west of Stockholm, Sweden) and also from the families of the hospital staff. We categorized the children into four age groups: 4 to 6 years (12 girls and 15 boys), 7 to 10 years (16 girls and 14 boys), 11 to 14 years (10 girls and 17 boys), and 15 to 17 years (14 girls and 19 boys). To ensure that only eligible children were included, parents were asked to fill in a brief health questionnaire. All of the 117 children fulfilled the inclusion and exclusion criteria and were healthy without any known underlying chronic inflammatory disease. Children with treatment for allergic rhinoconjunctivitis (n = 4) or diet for lactose intolerance (n = 2) were accepted into the study. The exclusion criteria were any of the following: abdominal pain or diarrhea during the last month, acute upper respiratory tract infection or tonsillitis during the last 2 weeks, any intake of nonsteroidal anti-inflammatory drugs during the last 2 weeks, and menstrual or nasal bleeding during the last week. Twenty-two additional children (9 girls and 13 boys) delivered a fecal sample even though they did not comply with these criteria. Consequently, they were excluded from the study and classified as nonhealthy based on the following reasons: ongoing upper respiratory tract infection (n = 12), abdominal pain or diarrhea (n = 5), menstrual or nasal bleeding (n = 4), and ongoing medication with trimethoprim-sulfamethoxazole (n = 1).
The stool samples were prepared and analyzed according to the manufacturer's instructions (Calprest; Eurospital SpA, Trieste, Italy). Stool was collected in screw-capped plastic containers and sent the same or next day by mail to the laboratory. The weight of each sample (40–120 mg) was measured and an extraction buffer containing citrate and urea was added in a weight per volume ratio of 1:50. The samples were mixed for 30 seconds by a vortex method and homogenized for 25 minutes. One milliliter of the homogenate was transferred to a tube and centrifuged for 20 minutes. Finally, the supernatant was collected and frozen at −20°C. In most cases, time from sampling to preparation and freezing was estimated to be 1 to 3 days, except for a few samples that took 4 to 6 days before handling. The supernatants were thawed and analyzed later with Calprest, a quantitative calprotectin ELISA, for determination of calprotectin in stools. Calprotectin was expressed as micrograms per gram of feces.
Statistical analyses were performed using the SPSS version 10.1 for Windows (SPSS Inc., Chicago, IL, USA). The Kruskal-Wallis and the Mann-Whitney tests were used for comparison between groups. Statistically significant differences were assumed when P < 0.05. Simple regression analysis was used to assess the correlation between fecal calprotectin concentration and age.
Ethical approval was obtained from the Regional Research Ethics Committees at the Karolinska Institutet, Stockholm, and Uppsala University, Uppsala, Sweden. The children were included in the study after verbal informed consent in the presence of their parents.
The fecal calprotectin concentrations were unevenly distributed, with a median fecal calprotectin concentration of 13.6 μg/g (95% confidence interval, 9.9–19.5 μg/g) in the 117 healthy children. In the four age groups, the median values were 28.2 μg/g (4–6 years), 13.5 μg/g (7–10 years), 9.9 μg/g (11–14 years), and 14.6 μg/g (15–17 years;Fig. 1). There was no significant correlation between age and fecal calprotectin concentration (r = 0.17;Fig. 2). The median value for boys (13.4 μg/g) and girls (15.5 μg/g) did not differ significantly. Further, 104 children (89%) had a fecal calprotectin concentration <50 μg/g. Accordingly, 13 children (in ages 4.0–17.8 years) had a fecal calprotectin concentration >50 μg /g and they all delivered a follow-up fecal sample from 4 to 14 months later (Fig. 3). All but three children had a concentration of <50 μg/g in the follow-up sample. The three children with elevated fecal calprotectin level in the follow-up sample were: A 17.8-year-old asymptomatic boy with a fecal calprotectin concentration of 60.7 μg/g in his first fecal sample. Ten months later, he reported bloody diarrhea. His calprotectin concentration at that time increased to 240 μg/g. An investigation revealed distal ulcerative proctitis. The second child was a 10.5-year-old girl with a fecal calprotectin concentration of 259 μg/g in the first fecal sample and 112 μg/g in the second sample. The second sample was delivered while she had an upper respiratory tract infection and therefore she was asked to deliver one more follow-up sample. The fecal calprotectin concentration at this follow-up was 14.1 μg/g. The third child was a 15.7-year-old girl who had a concentration of 124 μg/g in the first fecal sample and 77.5 μg/g in the second sample. She had previously been diagnosed lactose intolerant and was asymptomatic on diet.
When we calculated the ninety-fifth percentile on the first samples from the 117 children we found it to be 105.1 μg/g. The corresponding value was 43.4 μg/g if the fecal sample from the boy in whom distal ulcerative proctitis had developed was excluded and the results from the follow-up samples (n = 12 children) were used instead of the first samples.
The median fecal calprotectin concentration also was analyzed in the samples from the 22 excluded children. In the children with upper respiratory tract infection or tonsillitis (n = 12), the median concentration was 16.8 μg/g (range, 3.7–106.6 μg/g). No significant difference was observed between this group and the healthy group regarding the median fecal calprotectin concentrations. In the remaining 10 excluded children (five with abdominal pain or diarrhea, four with menstrual or nasal bleeding, and one with ongoing medication with trimethoprim-sulfamethoxazole), the median fecal calprotectin concentration was 21.0 μg/g (range, 3.3–190.0 μg/g). This value did not differ from the median fecal calprotectin concentration in the 117 healthy children who participated in the study.
In our study, the median fecal calprotectin concentration was 13.6 μg/g (95% confidence interval, 9.9–19.5 μg/g) in 117 healthy children aged 4 to 17 years. There were no differences in fecal calprotectin concentrations attributable to age or sex. Although there is a need for simple and noninvasive tests in pediatric care, there are only few studies on the use of fecal calprotectin as a diagnostic test in children. The improved fecal calprotectin assay has, as far as we know, been used in only one recently published paper in infants and children (20).
The original ELISA method was first described in 1992 by Røseth et al. (9), and the results were provided in “per litre of fecal homogenate.” They found that fecal calprotectin is stable up to 7 days in room temperature as the calcium–calprotectin complex is resistant to heat and proteolytic enzymes (2). Furthermore, they concluded that measurement of fecal calprotectin in a spot sample reflects the average daily excretion of calprotectin. Bunn et al. (21), who used the original method for fecal calprotectin, showed that children with inflammatory bowel disease had fecal calprotectin concentrations that closely correlated to macroscopic and histologic inflammation as well as to technetium 99-labeled white cell scanning score. The reference group in their study comprised 31 healthy children (median age, 6.8 years; range, 1.5–15.3 years) and their median fecal calprotectin concentration was 2.1 mg/L (range, 0.5–6.3 mg/L), which was below the cutoff (<10 mg/L) established for adults in the original method (17).
Use of dissociating agents in the extraction solution in conjunction with a higher dilution of the sample has resulted in the improved fecal ELISA calprotectin method (19). The extraction yield in samples with a calprotectin value <10 mg/L in the original method was increased one- to sixfold, whereas samples with high fecal calprotectin values showed a higher increase. This means that the separation between normal and pathologic values is better with the improved method. The size of the sample is substantially reduced from 5 g to 50 to 100 mg, and the results are expressed as micrograms of fecal calprotectin per gram wet feces. When the improved ELISA assay for fecal calprotectin was studied in 59 healthy adults, the median fecal calprotectin concentration was 26 μg/g (range, 4–262 μg/g) and the cutoff was suggested to be <50 μg/g for adults, but the ninety-fifth percentile was not mentioned (19).
From the beginning, the commercial test was called PhiCal ELISA (Nycomed Pharma AS, Oslo, Norway) but now exists as Calprest and is manufactured by Eurospital SpA, Trieste, Italy. From Nycomed, the following threshold values were supplied for adults: negative test when <50 μg/g, weakly positive test when between 50 and 100 μg/g, and strongly positive when >100 μg/g (13). These threshold values seem to be applicable also in children aged from 4 to 17 years. Although 13 (11%) of the presumed healthy children in our study had a fecal calprotectin concentration higher than the reference value recommended for adults, only three of these children still had an elevated concentration at follow-up. For ethical reasons, we chose not to investigate the healthy children further. Children with apparent gastrointestinal symptoms such as abdominal pain or diarrhea during the last month of the study were excluded.
In this healthy population, latent disease could theoretically be present in some of the children and hence could influence the fecal calprotectin concentrations. This was found in one of the presumed healthy children who had a slightly increased fecal calprotectin concentration at the time of the first sampling, with an increase at follow-up in association with the development of ulcerative proctitis. The variation of calprotectin excretion in feces may also be explained by normal biologic variability with day-to-day variation that previously has been described in adults (16,22). Husebye et al. (22) noted that two populations emerged in adults with normal findings at colonoscopy: one group with remarkably low and stable fecal calprotectin values within the recommended cutoff of 50 μg/g and one group with labile values also beyond this limit.
In our study, children with menstrual or nasal bleeding were excluded to avoid measuring calprotectin from neutrophils in blood that could have contaminated the fecal sample. It has been estimated that bleeding volume of at least 100 mL daily may cause an elevated fecal calprotectin concentration (14). Children taking nonsteroidal anti-inflammatory drugs also were excluded because this medication has been reported to induce enteropathy and elevated fecal calprotectin excretion (23). Interestingly, our results showed that the median fecal calprotectin concentration in the healthy group of children was not different from the median in children with upper respiratory tract infection or tonsillitis. This observation may be explained by the fact that these children probably had nonpurulent infections caused by virus.
Healthy children aged between 4 and 17 years apparently exhibit a similar pattern of fecal calprotectin excretion as in adults. In infants, however, the fecal calprotectin concentrations seem to be age dependent. Surprisingly high values have been found, especially in healthy infants 0 to 3 months of age, with a median fecal calprotectin concentration of 265 μg/g (20,24). The explanation for this observation could be a migration of neutrophils through the mucosal membrane during the development of oral tolerance and regulation of the microbial flora (9).
Our conclusion is that the suggested cutoff level for the improved assay for fecal calprotectin in adults (i.e., <50 μg/g) can be used for children aged from 4 to 17 years independent of sex. A fecal calprotectin concentration >50 μg/g warrants follow-up. We suggest that studies on the feasibility of this test for investigation of gastrointestinal disorders in children could be based on the reference values found in this study.
The authors thank Inger-Lise Johansson and Åsa Andersson, nurses in school health services, Västerås and Petra Wahlén, Centre for Clinical Research, Västerås, for assistance.
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