Fecal Calprotectin and Eosinophil-derived Neurotoxin in Healthy Children Between 0 and 12 Years : Journal of Pediatric Gastroenterology and Nutrition

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Original Articles: Gastroenterology

Fecal Calprotectin and Eosinophil-derived Neurotoxin in Healthy Children Between 0 and 12 Years

Roca, María; Rodriguez Varela, Ana; Donat, Ester∗,‡; Cano, Francisco; Hervas, David§; Armisen, Ana; Vaya, Maria J.; Sjölander, Anders||; Ribes-Koninckx, Carmen∗,‡

Author Information
Journal of Pediatric Gastroenterology and Nutrition: October 2017 - Volume 65 - Issue 4 - p 394-398
doi: 10.1097/MPG.0000000000001542

Abstract

What Is Known

  • Young healthy children have higher fecal calprotectin levels than healthy adults.
  • The use of fecal calprotectin levels as a biomarker is limited in young children and reference values are not steadily fixed.
  • Reference values for fecal eosinophil–derived neurotoxin levels in children have not been established; its use as a biomarker is understudied.

What Is New

  • Nomogram with reference values of fecal calprotectin levels by age and suggestion of 3 different age groups for evaluation of fecal calprotectin levels in clinical practice.
  • Reference values for fecal eosinophil–derived neurotoxin levels in a pediatric population.
  • Fecal calprotectin and fecal eosinophil–derived neurotoxin levels in children 0 to 4 years of age suggest that the main utility of both biomarkers is intraindividual variation monitoring.

A calcium-binding heterocomplex protein with 2 heavy and 1 light chain, calprotectin, was described by Fagerhol in 1980 (1,2). This protein belongs to the S100 (S100 A8/A9) family and is present in tissues and fluids and is especially abundant in neutrophils and monocytes (3). Calprotectin has numerous biological functions such as antimicrobial and antifungal activity (4) and different properties as a regulatory protein in inflammatory reactions (5).

In the intestinal tract, inflammatory diseases with different etiology cause increased mucosal permeability that induces migration of granulocytes and monocytes into the intestinal lumen (6). The activation and death of these cells releases a great amount of calprotectin, which is excreted in the feces (7). High fecal calprotectin (fCP) levels are described in children and adults, in pathological conditions, both inflammatory and neoplastic: Crohn disease, ulcerative colitis, cystic fibrosis, rheumatoid arthritis, bacterial infections, and gastric cancer (6,8). The stability of calprotectin in feces stored for 7 days at room temperature (9) and relatively simple fCP assays makes it a useful noninvasive tool in pediatric care. In young healthy children whose degree of maturity of the intestinal immune system is, however, age-dependent, cut-off levels are not well defined, especially in children younger than 4 years of age. Also, the use of different methodologies in the studies carried out so far makes it difficult to compare published results.

Eosinophils contain cytotoxic granules composed of a number of antimicrobial proteins including eosinophil-derived neurotoxin, a protein with ribonuclease activity, involved in the host defense against invading parasites; in addition, it has been shown to display cytotoxic properties and antiviral activity (10,11). High fecal eosinophil–derived neurotoxin (fEDN) concentrations have been described in active ulcerative colitis, Crohn disease (10,12–14), and non–IgE-mediated cow's milk protein allergy in toddlers (15). EDN is stable in feces for at least 7 days at room temperature (16), facilitating its use as a noninvasive clinical biomarker.

In young children, the use of calprotectin as a biomarker is limited because reference values have not been widely accepted up to now. Moreover, reference values for fEDN levels in children have not been established. The aim of the present study was to investigate fCP and fEDN concentrations in young healthy children between 0 and 12 years of age to establish reference values.

MATERIALS AND METHODS

Patients and Samples

Stool samples were obtained from healthy children ages 0 to 12 years. From January 2015 to February 2016 we recruited healthy children at the Primary Health Care Center of Betera during regular visits scheduled according to the National Health System protocols. In addition, in the same period, children pertaining to families of the hospital staff or patient's relatives were recruited at the Pediatric department of La Fe Hospital. For all children, information on clinical features provided by parents or by the pediatrician, including age, weight and length, sex, vaccines, and physical examination findings were recorded in a specific CRF before the stool sample collection. In addition, gestational age, birth weight, mode of delivery (vaginal or cesarean section), and type of feeding (exclusively breast-feeding or formula-feeding) were recorded for infants up to 6 months. All recruited children met the following inclusion criteria: age 0 to 12 years, no illnesses or vaccines in the prior month to enrollment, no hospital admissions 3 months before enrollment, gestational age >37 weeks, birth weight appropriate, and with no known underlying chronic inflammatory disease. The exclusion criteria were the following: any intake of steroidal or nonsteroidal anti-inflammatory drugs, gastric acidity inhibitors, antibiotics or any other drug during the 2 weeks before recruitment, or a history of signs or symptoms of infection or gastrointestinal disease (diarrhea, vomiting, hematochezia, fever).

Methods

Parents of each child were provided with a specific plastic screw–capped container and were instructed to collect a small amount of feces. The containers with the stool samples were kept in the fridge at home; they were brought to the laboratory no later than 7 days after collection and were then stored at −20°C until analysis. The protein extraction procedure of the samples, as a preliminary step of the analysis, was performed using the Fecal Sample Preparation Kit (Roche Diagnostics, Rotkreuz, Switzerland) according to the manufacturer's instructions.

fCP concentrations were analyzed in the stool samples using the EliA Calprotectin 2 assay (Phadia, Uppsala, Sweden) and concentrations were expressed as milligrams per kilogram of feces. fEDN concentrations in the stool samples were analyzed by using a novel immunofluorescence research assay based on the ImmunoCAP technology and for use in automated Phadia 250 instruments (Phadia). Briefly, a pair of complementary monoclonal antibodies (mAbs) was used where the capture mAb was covalently coupled to the ImmunoCAP solid phase and the detection mAb was labeled with the enzyme β-galactosidase. Purified EDN (Athens Research and Technologies, Athens, GA) was used to develop a calibrator curve consisting of 5 calibrator points with a measuring range of 2 to 200 μg/L. A new software method for EDN, based on the method for ImmunoCAP ECP, was developed for the Phadia 250 instrument for running the assays including calibrator curve fitting and conversion of assay responses to EDN concentrations. The sample and enzyme conjugate incubation times were 30 and 24 minutes, respectively. The incubations were performed at 37°C and were terminated by extensive washing steps using ImmunoCAP Washing Solution. After washing, Development Solution and finally Stop Solution were added (generic standard ImmunoCAP reagents). The generated fluorescence was proportional to the formed EDN-antibody complexes. The fluorescence was measured by the Phadia 250 instrument at the end of the assay run and EDN concentrations were given in μg/L. The fEDN concentrations were subsequently transformed and expressed as milligrams per kilogram of feces.

The assay was verified for precision, dilution linearity, and recovery of spiked EDN. The intra- and interassay coefficients of variation were <6.5%. Samples with fEDN concentrations within the assay measuring range could be diluted at least 20 times with ratios between obtained and expected EDN concentrations ranging from 91% to 109%. The recovery of EDN added to stool samples with known EDN concentrations was >75%.

Ethical Considerations

The study was approved by the Ethical Committee of Universitary and Politecnic La Fe Hospital. Written informed consent was obtained from the parents of all the children who participated in this study before their enrollment.

Statistical Methods

We determined the reference values for fCP and fEDN levels according to the 95th percentile for each age. To estimate this 95th percentile a quantile regression model including a restricted cubic spline for the relation between fCP/fEDN concentration and age was adjusted. With this method, we estimated both the median (50th percentile) and the 95th percentile. To assess the association of sex, type of delivery, and breast-feeding with fCP concentration a linear regression model was adjusted including these variables and age as covariates and fCP concentration as dependent variable. In the case of fEDN concentrations, a quantile regression model for the median was used to avoid bias due to the presence of values under the limit of detection. P values <0.05 were considered statistically significant. All statistical analyses were performed using R (version 3.3.1).

RESULTS

Study Population

The study population included 174 children (87 girls and 87 boys) ages 0 to 12 years. Initially 183 children were recruited in the study, but 9 were excluded due to different reasons: 2 because of incomplete data, 1 because of a lactose-free diet, 2 because of acute infectious diseases, and 4 because of medical treatment (antibiotics, corticosteroids, omeprazole and polyethylene glycol). In total, 174 healthy children fulfilled the inclusion and exclusion criteria and were considered for the final analysis. One fecal sample was obtained from each of them. The median age of all participants was 25 months, First and third quartiles being 5 and 54 months, respectively.

We found no evidence of association between sex and fCP (P = 0.62) or fEDN (P = 0.14) concentrations. Ninety-fifth percentile values ranged from 1519 mg/kg at 0 months to 54.4 mg/kg at 144 months for fCP and from 9.9 mg/kg at 0 months to 0.2 mg/kg at 144 months for fEDN. There was, however, a statistically significant association between age and fCP concentrations and age and fEDN concentrations, older children displaying lower fCP and fEDN concentrations (P < 0.001). We also found a statistically significant association between fEDN and fCP concentrations (rho = 0.52, P < 0.001).

Fecal Calprotectin and Fecal Eosinophil–derived Neurotoxin Concentrations in Infants Ages 0 to 6 Months

Infants from 0 to 6 months (n = 51) had a median age of 1 month (first and third quartile: 0.25 and 4 months, respectively). All of them were born at term; additional characteristics are shown in Table 1. Concentrations of fCP and fEDN ranged from 17.3 to 2681.7 and 0.25 to 9.62 mg/kg, respectively (Fig. 1).

T1
TABLE 1:
Characteristics of children from 0 to 6 months
F1
FIGURE 1:
A, Scatter plot depicting the relation between age in months and fCP levels. Regression lines have been added for the 95th percentile and for the 50th percentile. B, Scatter plot depicting the relation between age in months and fEDN levels. Regression lines have been added for the 95th percentile and for the 50th percentile. fCP = fecal calprotectin; fEDN = fecal eosinophil–derived neurotoxin.

For infants up to 6 months, fCP and fEDN concentrations did not differ significantly between the 2 different feeding groups (breast-fed or infant's formula) (P = 0.66 and P = 0.77, respectively). Likewise, we found no evidence of association between the type of birth (vaginal or cesarean) and fCP or fEDN concentrations (P = 0.95 and P = 0.39, respectively) for this group of children.

Individual fCP and fEDN concentrations are shown in Figure 1.

The Relationship Between Age and Fecal Calprotectin and Fecal Eosinophil–derived Neurotoxin Concentrations

The quantile regression models obtained for the 95th and 50th percentiles of fCP and fEDN concentrations are shown in Supplemental Digital Content, Table S1, https://links.lww.com/MPG/A904. The relation between age and fCP and fEDN concentrations was clearly nonlinear, with a sharp decrease during the first 48 months which progressively stabilized around 20 mg/kg; for fEDN concentrations the trend was similar, but the fEDN concentrations continued to decrease after 48 months. This association between age and both biomarker concentrations is shown in Figure 1.

A nomogram was developed based on the results of the regression analyses (Fig. 2). The nomogram comprises 5 rows, the first is the age in months of the healthy children, the second and third rows are the 95th and 50th percentiles of the fCP concentrations, and the fourth and fifth rows are the 95th and 50th percentiles of the fEDN concentrations.

F2
FIGURE 2:
Nomogram of healthy children (0–12 years) for 95th and 50th percentile of fCP and fEDN levels. Nomogram with estimated reference values based on the 95th and 50th percentile of fCP and fEDN values by age in months for healthy children. fCP = fecal calprotectin; fEDN = fecal eosinophil–derived neurotoxin.

We suggest 3 age groups for both markers: from 0 to 12 months, from 1 to 4 years and from 4 to 12 years. In Table 2 we show reference values for fCP and fEDN corresponding to the lower value of 95th percentile.

T2
TABLE 2:
Fecal calprotectin and fecal eosinophil–derived neurotoxin reference values in healthy children, according to 3 different age groups

DISCUSSION

We have evaluated the concentrations of fCP and fEDN in healthy children between 0 and 12 years old. We show that fCP concentrations in these children are higher than the reference values reported for healthy adults (17). As no reference values for fEDN have been published up to now for any age group, we cannot compare our results with results obtained in other populations. In our study population, fCP and fEDN values varied sharply between different ages. For both parameters a significant and negative correlation with age was obtained, the highest values being found in infants younger than 1 year of age (18). We found no evidence of association between sex and fCP level, as shown in other studies (17).

We considered it more appropriate to provide reference values based on the continuous relation between fCP or fEDN levels and age, to avoid the error linked to continuous variable categorization (19). Therefore, we estimated different equations for the age-specific average (50th percentile) and upper bound (95th percentile) reference values. To simplify the application of these equations, we developed a nomogram. As far as we know, this approach has not been used before for fCP and fEDN levels in healthy children. To be able to compare our data with other studies, and also for applicability in clinical practice we, however, eventually recommend reference values (cut off values) for specific age groups, being aware that this approach introduces a bias in each age group. Also, the selection and definition of the most appropriate age groups will always be arbitrary.

In the neonatal period, the small bowel is more permeable than in adults (20). This fact can be explained by the immaturity of the digestive tract and of the immune system. Although full-term infants are born with sufficiently developed absorptive and digestive function, the gastrointestinal tract undergoes significant postnatal development in the first year (21). So aspects such as the intestinal length are continuous until 3 to 4 years of age. The acquisition of intestinal microbiota occurs during the first year of life, reaching an adult microbiota profile at the end of first year of life. In addition, the sIgA system, the immunological barrier's first line of defense, which binds to antigenic substances, does not completely mature until 4 years of age. Accordingly, it is generally considered that the mucosal barrier is not fully mature until the age of 4 (20).

Therefore, we suggest to divide the children into 3 different age groups (Table 2) and to assign a reference value for each group. This reference value corresponds to the lower value of the 95th percentile for each group, and thus avoiding false-negative results. The variability of fCP and fEDN concentrations in children up to 4 years of age justifies the need to establish 2 different reference values in this age range: 0 to 1 years and from 1 to 4 years. It also suggests that the main utility of both markers in this age range may be intraindividual variation monitoring, allowing assessment of disease progression or response to therapeutic intervention. From the age of 4 and up to 12 years, a unique reference value can be used and the lower variability of fCP and fEDN concentrations in this age group also allows for a better discrimination between true positive and true negative results.

In children younger than 4 years, the use of calprotectin as a marker is limited because widely accepted reference limits have not been established. In a recent study, Oord and Hornung (22) collected 75 samples from healthy children between 0 and 4 years, and these were classified into different groups according to age. As a result, 3 cutoff levels were established based on the 97.5% percentiles: 538 mg/kg for the group 1 to 6 months, 214 mg/kg for the group 6 months to 3 years, and 75 mg/kg for the group 3 to 4 years. In our study, for the group 0 to 1 year we suggest a higher reference value: 910 mg/kg. This can be explained because Oord and Hornung did not include samples from neonates between 0 and 4 weeks of age, whose fCP levels are elevated. Moreover, they used a different fCP analysis method.

Fagerberg et al (17) suggested that the cutoff level for adults as recommended by the manufacturer's instruction (<50 μg/g) could also be used for children ages 4 years and older regardless of sex. In 117 healthy children, ages 4 to 17 years, he, however, reported that fCP concentrations were unevenly distributed, the median values (50th percentile) for the different age groups being respectively 28.2 μg/g for 4 to 6 years, 13.5 μg/g for 7 to 10 years, 9.9 μg/g for 11 to 14 years, and 14.6 μg/g for 15 to 17 years. For the whole group, the median concentration was 13.6 μg/g, which is comparable to the median concentration obtained in our study population ages 4 to 12 years, that is, 17.3μg/g (equivalent to 17.3 mg/kg). We observed median fCP values ranging from 26 (4-year-old children) to 13.9 μg/g (12-year-old children), and thus being below the cutoff level for adults. The 95th percentile value for the 4 to 12 years age range in our study was 54 μg/g. The 95th percentile values provided by Fargerber et al are not comparable to our results as they include data from 3 nonhealthy children and follow-up samples. Overall at recruitment, 11% of the children had fCP concentrations >50 μg/g.

Controversial results have been reported on the relation between type of feeding and the concentration of fCP in infants (23,24). In our study, similar fCP and fEDN concentrations were found in breast-fed babies ages up to 6 months as compared to formula-fed infants, showing that fCP and fEDN levels were not influenced by the feeding pattern in this population.

In younger infants, the wide variation in fCP and fEDN concentrations may be partially explained by the fact that the stool samples were collected from the babies’ diapers. Actually, Olafsdottir et al (25) showed that this method of collection increases calprotectin concentrations up to a maximum of 30% because water is absorbed by the diaper. Although parents were instructed to collect stool samples immediately after defecation occurred, this may not be so in all cases. The wide range of interindividual results found in the first year of life, however, imply that individual factors are accountable for this large variation in fCP concentration. Another potential limitation of our study is that we have to trust that parents complied with the instructions for samples storage and also rely on the information they provided to the study team. In addition, we did not analyze follow-up samples, which could also be considered a limitation of our study. Children with any health condition, whenever detected after recruitment, were, however, excluded from the analysis.

Children in the first months of life show high fCP concentrations, reflecting an increase in granulocytes in the gut lumen. An increased permeability of mucosal barriers could be associated with this migration of granulocytes into the lumen, because there is no accumulation of leukocytes in the healthy children's mucosa at first months of life (24,26). This fact could be related to immaturity of adaptive immunity in infancy (25). Increased permeability of the mucosa in the gastrointestinal tract caused by different types of pathological changes could result in increased migration of granulocytes and monocytes in the gut lumen (6). Moreover, fCP levels may be high in the first months of life because intestinal epithelial barrier function is immature and infants have not developed the ability to regulate the microbiota in the gut (24). As for fCP levels, children also showed high fEDN levels in the first year of life, suggesting eosinophil activation and degranulation in their intestine. As in our study, fEDN concentrations were negatively correlated with age in 25 toddlers studied by Kalach et al (15).

By using an improved analytical method, we have confirmed that young healthy children have higher fCP concentrations than healthy adults. According to our results we provide a nomogram and we suggest 3 different age groups for evaluation of fCP concentrations for their applicability to clinical practice. Moreover, we report, for the first time, reference values for fEDN in a pediatric population, and thus allowing analysis of biomarkers for both neutrophil and eosinophil intestinal inflammation in children. Further studies are required to evaluate the value of this in clinical practice.

Acknowledgments

The authors would like to thank all the participants and their families, and the staff collaborating in samples collection at La Fe Hospital: S. Carrasco, M. San Felix, F. Araujo, and I. Gómez.

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Keywords:

calprotectin; children; eosinophil-derived neurotoxin; feces; reference values

Supplemental Digital Content

Copyright © 2017 by European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition