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Original Articles: Gastroenterology

Fecal Calprotectin: A Quantitative Marker of Colonic Inflammation in Children With Inflammatory Bowel Disease

Fagerberg, Ulrika Lorentzon*,‡; Lööf, Lars; Lindholm, Johan§; Hansson, Lars-Olof; Finkel, Yigael*

Author Information
Journal of Pediatric Gastroenterology and Nutrition: October 2007 - Volume 45 - Issue 4 - p 414-420
doi: 10.1097/MPG.0b013e31810e75a9
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Abstract

INTRODUCTION

In a decades-long trend, Western children increasingly have been diagnosed with inflammatory bowel disease (IBD) (1,2). Because of the chronic nature of IBD, with episodes of relapse and remission, affected individuals require long-term and usually lifelong monitoring of disease activity. Endoscopy is considered the gold standard for diagnosis of IBD, including differentiation between ulcerative colitis (UC) and Crohn disease (CD), and is also a tool for estimation of disease activity and efficacy of therapy. However, endoscopy is unsuitable for frequent use because it is an invasive and expensive procedure that requires careful bowel preparation and most often general anesthesia when used on children.

Individualized medication and careful monitoring are necessary to achieve optimal growth and well-being of children with IBD. Methods other than endoscopy, such as leukocyte scintigraphy and assessing 111In-labeled white cells in feces, have been used for the measurement of disease activity in IBD, but these techniques are costly and invasive as well. Moreover, they are inappropriate for regular use in children because of exposure to radiation. Clinical indices have been developed for longitudinal estimation of disease activity and for evaluation of anti-inflammatory therapies in IBD. Nevertheless, clinical disease activity and indices do not necessarily reflect the degree or extent of mucosal inflammation (3,4). In addition, compilation of clinical disease indices is time-consuming, making them less applicable to daily clinical practice. Simple, inexpensive, and objective tools for assessment of mucosal inflammation are therefore desirable. Previous studies have indicated that fecal calprotectin may be a promising marker.

Calprotectin is a calcium- and zinc-binding protein and originates from neutrophils, monocytes, and macrophages. This protein can be measured in feces, as well as in various body fluids, by enzyme-linked immunosorbent assay methods (ELISA) (5,6). The reference values differ between the fecal calprotectin ELISAs. For the ELISA described by Tøn et al (6), reference values have been established at <50 μg/g in adults (6) and in children from 4 years old and older (7). Previous studies have shown that fecal calprotectin is a useful and sensitive marker to detect gastrointestinal inflammation and to differentiate inflammatory diseases from functional gastrointestinal disorders (8–10). Thus, fecal calprotectin is now becoming an established diagnostic tool in children with gastrointestinal symptoms. Fecal calprotectin also has been shown to correlate well to the endoscopic and microscopic disease activity in IBD. This assumption has been based primarily on 1 study in adults (11) and 1 study in children (12) performed with the ELISA method described by Roseth et al (5).

The aims of this study were to confirm the validity of fecal calprotectin (measured with the method described by Tøn et al (6)) as a marker for quantitative assessments of colonic inflammatory activity in children with IBD, and to evaluate fecal calprotectin concentrations at complete, microscopic remission of inflammation.

PATIENTS AND METHODS

The study was performed at the Department of Pediatric Gastroenterology and Nutrition, Astrid Lindgren Children's Hospital, Karolinska University Hospital, Stockholm, and the Department of Pediatrics, Central Hospital, Västerås, Sweden. The children mainly were recruited from the outpatient clinic, and 58 children with suspected or previously confirmed IBD were asked to deliver a fecal sample before they underwent a planned colonoscopy. Of these children, 39 fulfilled the criteria to be included in the IBD study group: They had a complete colonoscopy, the time interval between delivery of a fecal sample and start of bowel cleansing was ≤2.5 weeks, and IBD had been diagnosed previously or at the study colonoscopy. Seven children with IBD had to be excluded because they exceeded the time interval between fecal sampling and colonoscopy. Twelve children with suspected IBD had a noninflamed colonic mucosa and no other sign of IBD at the time of investigation. These children comprised a control group.

In the IBD study group the median age was 13.8 years (range, 6.7–18.8 years) and the median time between delivery of fecal sample and performance of bowel cleansing was 2 days (range, 0–17 days). Differentiation between CD (n = 27), UC (n = 10), and indeterminate colitis (IC) (n = 2) was made according to accepted clinical, endoscopic, microscopic, and radiological criteria for diagnosis. The colonoscopies were performed at disease onset in 12 cases (CD n = 9, UC n = 2, IC n = 1) and during follow-up in 27 cases (CD n = 18, UC n = 8, IC n = 1). In the CD group, the median time since onset of symptoms was 17 months (range, 2–89 months), in the UC group 20 months (range, 1–120 months), and in the IC group 10 months (range, 4–15 months). The IBD patients used the following medications: sulfasalazine or 5-aminosalicylic acid (CD n = 17, UC n = 8, IC n = 1), azathioprine (CD n = 11, UC n = 1, IC n = 1), steroids (CD n = 6, IC n = 1), antibiotics (CD n = 3), and enteral nutrition (CD n = 3). Some of the patients had >1 ongoing treatment at the time of inclusion, whereas others were still untreated. Clinical assessment of IBD activity was based on the patient's history, clinical examination, and routine laboratory tests by the clinicians. Based on these data, the IBD patients were grouped into 2 categories: symptomatic (n = 23) and asymptomatic (n = 16). To be included in the asymptomatic group, the patient had to meet the following criteria: no reported symptoms, no abdominal mass, no glucocorticoid therapy, and normal laboratory blood tests with hemoglobin >115 g/L, erythrocyte sedimentation rate ≤12 mm/hour, C-reactive protein <8 mg/L, orosomucoid <1.15 g/L, and albumin ≥37 g/L. Bacterial gastrointestinal infections were excluded by negative fecal cultures. In 2 cases the fecal samples for culture were missing, but both of these children had negative serological tests for Yersinia and Salmonella and the histopathology did not arouse any suspicion of bacterial infection.

In the control group the median age was 12.8 years (range, 6.6–17.3 years) and the diagnoses were functional bowel disorder (n = 5), lactose intolerance (n = 2), cow's milk–protein intolerance (n = 1), or gastrointestinal symptoms of a temporary nature (n = 4). Among the controls, an interval of up to 1 month was accepted between delivery of fecal sample and start of bowel cleansing because they were not expected to have a fluctuating degree of inflammation over time. The median time interval was calculated to be 3 days (range, 1–31 days). During the study the clinicians, the endoscopists, the histopathologist, and the nursing staff were blinded to the fecal calprotectin results.

Endoscopies

The endoscopies were performed under general anesthesia by experienced pediatric endoscopists. The macroscopic appearance of the colonic mucosa was evaluated according to a previously used model (12,13) (Table 1), but an increased number of colonic segments were scored (ie, cecum, ascending colon, right flexure, transverse colon, left flexure, descending colon, sigmoid, and rectum). Intubation of terminal ileum failed in a few instances. Consequently, the scores from the terminal ileum were not included in the model. The macroscopic severity score was equivalent to the highest regional score in any colonic segment (possible range, 0–3). The macroscopic extent score was defined as the number of colonic segments with a regional score ≥1 (possible range, 0–8). Finally, a combined macroscopic extent and severity score was calculated from the sum of the 8 regional scores (possible range, 0–24).

T1-5
TABLE 1:
Macroscopic scoring system*

Microscopic Assessment

An experienced gastrointestinal histopathologist, who was blinded to clinical data and to the patients' fecal calprotectin concentration, performed the microscopic assessments of colorectal inflammation in all biopsy specimens. According to a previously used model (12,13), the severity of changes in the crypts, enterocytes, and cellularity of the lamina propria (mononuclear cells and neutrophils) were graded in each biopsy specimen and the possible sum of grades varied from 0 to 12 (Table 2). We modified the model by taking tissue samples from an increased number of colonic segments for microscopic assessment (ie, cecum, ascending colon, right flexure, transverse colon, left flexure, descending colon, sigmoid, and rectum). The mean sum of grades for all specimens taken in a segment was then converted into a regional microscopic score to define the severity of microscopic inflammation in each segment (Table 3). In exceptional cases, when no tissue sample was available from a segment (ie, in 13/312 of the examined segments), the score was replaced by a median value of the calculated scores for the individual. Finally, the individual's regional microscopic scores were transformed into a microscopic extent, a microscopic severity, and a microscopic combined extent and severity score in the same way as previously described for the macroscopic scores in the endoscopies. In addition, we tested our own model for calculation of microscopic inflammation. In our model all of the available colonic biopsy specimens taken from the patient were graded according to Table 2 with assessments of changes in the crypts, enterocytes, and cellularity of the lamina propria (mononuclear cells and neutrophils). These grades were then summarized and divided directly by the number of biopsy specimens taken. In this way, we avoided the conversion step to a regional score and a possible source of error.

T2-5
TABLE 2:
Microscopic grading system in each colonic biopsy specimen*
T3-5
TABLE 3:
Conversion of microscopic grade into microscopic regional score*

Fecal Calprotectin

The stool samples were prepared and analyzed for calprotectin according to the manufacturer's instructions (Calprest, Eurospital, Trieste, Italy). Stool was collected in screw-capped plastic containers and sent by mail on the same day or next day 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/volume ratio of 1:50. The samples were mixed for 30 seconds by means of a vortex and homogenized for 25 minutes. One milliliter of the homogenate was transferred to a tube and centrifuged for 20 minutes at 10,000g. Finally, the supernatant was collected and frozen at −20°C. The supernatants were thawed and calprotectin was analyzed with the quantitative calprotectin ELISA. Calprotectin was expressed as micrograms per gram of feces. The biochemist performing the assay was unaware of all clinical details.

Statistical Analysis

Statistical analyses were performed using the Statistical Package for Social Sciences version 11.0 for Windows (SPSS, Chicago, IL). Nonparametric methods were used because of skewed distribution of the data. The descriptive statistics were calculated as median values with ranges given as the minimum and maximum values or with 95% confidence interval (CI). Spearman rank-order correlation test was applied for correlations between variables. The Mann-Whitney U test was used for comparison of 2 groups. P < .05 was considered statistically significant. The reliability of the fecal calprotectin method to detect microscopic colonic inflammation in IBD patients was expressed in terms of sensitivity, specificity, positive predictive values, negative predictive values, and observed agreement. For fecal calprotectin, receiver operating characteristic curve, as well as area under the curve, with 95% CI was calculated. An area under the curve of 0.5 means that the test is not better than chance alone in discriminating between inflammation and mucosal healing, whereas a test with an area under the curve of 1.0 indicates perfect discriminative capability.

Ethical Considerations

The study was granted ethical approval by 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 from the children and their parents.

RESULTS

The median fecal calprotectin concentration was 264 μg/g (95% CI, 101–382 μg/g) in the IBD group compared with 16.5 μg/g (95% CI, 6.9–28.2 μg/g) in the control group (P < .001). A significant difference in fecal calprotectin concentrations also was found within the IBD group when the IBD cases were grouped into 2 categories; in patients with IBD symptoms, the median concentration was 392 μg/g (95% CI, 278–440 μg/g) and in asymptomatic IBD patients the corresponding value was 32.9 μg/g (95% CI, 9.4–237 μg/g) (P < .001).

The fecal calprotectin concentrations correlated significantly to the scores for both macroscopic as well as microscopic inflammatory activity in the IBD group. The correlations and median values of the scores are presented in Table 4. The strongest correlation was found between fecal calprotectin and the microscopic combined extent and severity score (Spearman ρ = 0.75; 95% CI, 0.57–0.86; P < .001) (Fig. 1). The correlation between the macroscopic and the microscopic extent and severity scores also was significant (Spearman ρ = 0.60, P < .0003).

T4-5
TABLE 4:
Correlations between fecal calprotectin concentrations and the different scores*
F1-5
FIG. 1:
Correlation between fecal calprotectin concentrations and the microscopic combined extent and severity scores in children with IBD from Crohn disease (
TABLE 1
), ulcerative colitis (
TABLE 1
), and indeterminate colitis (
TABLE 1
).

Our own model for calculation of microscopic inflammation was tested as well, and resulted in a similar correlation to fecal calprotectin concentrations (Spearman ρ = 0.79; 95% CI, 0.63–0.88; P < .001). Furthermore, we compared the inflammatory scores between the symptomatic and asymptomatic patients with IBD. The macroscopic scores differed significantly between these groups, with P < .007 for extent score, P < .001 for severity score, and P = .003 for the macroscopic combined extent and severity score. The 3 corresponding microscopic scores also differed significantly between the symptomatic and asymptomatic patients with IBD (P < .001).

In the control group the median microscopic combined extent and severity score was 0 (range, 0–2). Thus, a score ≤2 was considered to be normal and a reasonable cutoff to define a noninflamed colonic mucosa or a complete, microscopic mucosal healing. This cutoff level was applied in the group of asymptomatic IBD patients, in which we found 9/16 of the children (UC n = 5, CD n = 4) appeared to have a complete microscopic healing of the colonic mucosa. Their median fecal calprotectin concentration was 9.9 μg/g (95% CI, 5.9–41.9 μg/g) with fecal calprotectin concentrations <50 μg/g in 8 of the children and a borderline concentration (ie, 50–100 μg/g) at 84.7 μg/g in 1 child. Among the remaining asymptomatic IBD patients (7/16), subclinical colonic inflammation was present in 6 children with CD and 1 child with UC. For these children, the median value of the microscopic score for combined extent and severity was 7 (range, 6–11) and the median fecal calprotectin concentration was 237 μg/g (95% CI, 11.9–368 μg/g). Accordingly, the fecal calprotectin concentrations differed significantly (P = .004) between the patients with noninflamed and inflamed mucosa, even though all were asymptomatic.

The 4 variables (severity of changes in the crypts, enterocytes, mononuclear cells, and neutrophils) included in the template for grading of microscopic inflammation (Table 2) also were analyzed separately and related to the fecal calprotectin excretion. The correlations were found to be significant in the crypts (Spearman ρ = 0.70; 95% CI, 0.50–0.83; P < .001), enterocytes (Spearman ρ = 0.57; 95% CI, 0.31–0.75; P < .001), mononuclear cells (Spearman ρ = 0.77; 95% CI, 0.61–0.87; P < .001), and neutrophils (Spearman ρ = 0.81; 95% CI, 0.66–0.89; P < .001).

Using <50 μg/g as an upper reference limit for fecal calprotectin and ≤2 as a cutoff for the combined microscopic extent and severity score, we calculated the accuracy with which the fecal calprotectin method detected microscopic colonic inflammation in children with IBD. We found that the sensitivity was 93% (95% CI, 0.76–0.99), the specificity was 73% (95% CI, 0.39–0.94), the positive predictive value was 90% (95% CI, 0.73–0.98), and the negative predictive value was 80% (95% CI, 0.44–0.97). The observed agreement between the microscopic scorings and the fecal calprotectin concentrations was 87% (95% CI, 0.73–0.96). The receiver operating characteristic curve showed that the area under the curve was 0.87 (95% CI, 0.72–1.0). The maximal sum of sensitivity and specificity for fecal calprotectin was achieved at 85.7 μg/g, which indicated that this concentration was the optimal cutoff for discrimination between inflamed and noninflamed colonic mucosa in children with IBD (Fig. 2). With this somewhat higher cutoff, the sensitivity remained unchanged at 93%, whereas there were improvements in the specificity of 82% (95% CI, 0.48–0.98), the positive predictive value of 93% (95% CI, 0.76–0.99), the negative predictive value of 82% (95% CI, 0.48–0.98), and the observed agreement of 90% (95% CI, 0.76–0.97). Active inflammation in the terminal ileum could be suspected in 10 symptomatic patients with CD. Two of them had elevated fecal calprotectin concentrations (278 and 491 μg/g, respectively), although their microscopic extent and severity score was ≤2 at colonoscopy. One of these patients had inflammation and stenosis in the terminal ileum and jejunum, requiring a surgical procedure. The other patient had shown colitis and an increased uptake in the terminal ileum region at leukocyte scintigraphy 1 year before, but at the follow-up colonoscopy the ileocecal valve could not be intubated. The remaining 8 symptomatic patients with CD had increased fecal calprotectin concentrations and colitis, but also signs of ileitis from biopsy specimens (n = 5, with score 1 in terminal ileum) and small bowel enema (n = 3). None of the other 17 patients with CD had inflammation in their biopsy specimens from terminal ileum.

F2-5
FIG. 2:
Receiver operating characteristics curve for fecal calprotectin (solid line) demonstrates the accuracy with which the test discriminates between healed and inflamed colonic mucosa in patients with IBD. The optimal cutoff level turned out to be <86 μg/g. The area under the curve was 0.87 and demonstrates that the probability of the test classifying correctly is 87%. The dotted diagonal line shows the likelihood of correct classification merely by chance.

DISCUSSION

Fecal calprotectin is considered to be a useful tool for the detection of gastrointestinal inflammation, as demonstrated by several studies (8–10). The present study confirms previous reports that fecal calprotectin also can be used as a quantitative marker of colonic inflammation in children with IBD (12). The magnitude of calprotectin excretion in feces was related to both the extent and the severity of macroscopic and microscopic inflammation in children with IBD. A majority of the children appeared to have mild to moderate inflammation, which was demonstrated by the median values of the combined macroscopic and microscopic extent and severity scores. Furthermore, we found normalized fecal calprotectin concentrations in a small group of patients with IBD who had achieved endoscopic remission with microscopic mucosal healing. The method that we used in the present study (6) has an increased extraction of calprotectin and a better separation between high and low calprotectin values when compared with the in-house method used in several of the previous studies (5,8,11,12). It is a user-friendly method because fecal calprotectin is stable at room temperature for at least 4 days and the patients can send their samples by regular mail. The model we used for scoring of inflammation has been described previously (12,13), but may have some weaknesses. For example, biopsy specimens may not be representative of the segment if the inflammation is patchy. Additionally, endoscopic scoring of inflammation is by necessity limited to those parts of the gastrointestinal tract that can be reached. In the hope of obtaining a better estimate of the presence of colonic inflammation, we performed histological analysis on an increased number of biopsy specimens and we also evaluated our own model for calculation of the microscopic inflammation.

The correlations between fecal calprotectin and the inflammatory scores were very much in agreement with the results from 2 similar studies performed with the fecal calprotectin ELISA method described by Roseth et al (5). The first study included 62 adult patients with UC and the fecal calprotectin concentrations correlated to the microscopic inflammation (ρ = 0.7; P < .001) and to the macroscopic inflammation (ρ = 0.57; P < .001) (11). In the second study 13 children with IBD (UC n = 9, CD n = 2, IC n = 2) were included. Fecal calprotectin correlated also in this study to the microscopic inflammation (ρ = 0.74; P < .01) and to the macroscopic inflammation (ρ = 0.65; P < .05) when defined as combined extent and severity scores (12).

Roseth et al (14) found the correlation between fecal calprotectin and the 3-day excretion of 111In-labeled granulocytes to be significant (ρ = 0.8; P < .0001) in adults with IBD. Furthermore, fecal calprotectin excretion was correlated to the scorings of disease activity from 99Tc–labeled white cell scanning in 14 children (CD n = 10, UC n = 3, allergic colitis n = 1) (12). Several of these patients were known to have inflammation in the small bowel. The results were analogous (ρ = 0.80; P = .001) and these studies further support the assumption that fecal calprotectin is a valid marker of gastrointestinal inflammation in IBD.

Calprotectin is mainly derived from neutrophils, but it is also known to exist in monocytes and in macrophages (15,16). In accordance, we found the fecal calprotectin concentrations to correlate significantly with the cellularity (mononuclear cells and neutrophils) in the lamina propria and the severity of changes in the crypts and the enterocytes. It is well known that microscopic inflammation can be present in the mucosa without obvious macroscopic signs (17). Accordingly, we also found a stronger correlation between fecal calprotectin concentrations and the scores for microscopic inflammation than with the scores for macroscopic inflammation. This was also the case in 2 previous studies (11,12).

The results from our study confirm that fecal calprotectin can be used as a quantitative marker of microscopic gastrointestinal inflammation and even for detection of subclinical colonic inflammation in children with IBD. This was demonstrated by slightly elevated fecal calprotectin concentrations in the group of asymptomatic children with mild microscopic inflammation. The significance of subclinical inflammation is thus far not understood, but it may be associated with relapse. The value of fecal calprotectin as a predictor of relapse has been studied in adults (18), but no studies of pediatric populations have been published. Tibble et al (18) found a 13-fold increased risk of clinical relapse within 12 months in patients with IBD who were in clinical remission and showed elevated fecal calprotectin levels (corresponding to >250 μg/g in our method).

Even though the number of children with microscopic remission was limited in the present study, the results indicate that fecal calprotectin may be used as a marker of mucosal healing. Our results are supported by a report on adult IBD patients in clinical remission who were investigated with colonoscopy when their fecal calprotectin concentration was <50 μg/g (19). Mucosal healing was achieved macroscopically in 44/45 and microscopically in 38/45 among these patients. The remaining 7 patients had low-grade infiltration in the lamina propria. In the present study elevated fecal calprotectin concentrations were discovered in 2 symptomatic patients with CD, although they had noninflamed colonic mucosa, but in both of these cases the fecal calprotectin could be suspected to originate from inflammation in the small intestine. Accordingly, the calculated specificity and negative predictive value of fecal calprotectin, to distinguish mucosal healing from microscopic inflammation in colon, may be even better in patients with isolated CD colitis or UC.

Until now, therapeutic trials of anti-inflammatory and immunosuppressive drugs have most frequently used disease-activity indices to define disease activity and remission. The indices also have been used when diagnostic tests and other fecal markers have been evaluated. Disease-activity indices are usually based on blood tests, patients' report of symptoms, and physical signs. It is well known that routine blood tests have low sensitivity and can be normal despite the presence of mucosal inflammation (10). Nonetheless, the child must suffer repeated venepunctures and discomfort to enable the counting of disease-activity indices. Symptoms can be neglected or underreported, especially if the child or adolescent is afraid of therapies or investigations associated with discomfort. In the pediatric version of the Crohn Disease Activity Index, growth has been added to clinical signs, but neither height nor signs of perirectal disease actually change rapidly enough to make them useful for assessing clinical outcome during therapy (20). Taken together, these weaknesses account for a poor correlation between traditional disease-activity indices and the actual microscopic mucosal inflammation (3,4).

So far, there has been no suitable fecal marker of inflammatory activity and mucosal healing for routine use. Measurement of fecal calprotectin seems to be a promising method for IBD monitoring and for follow-up in therapeutic trials. In daily practice fecal calprotectin has potential to guide the clinician in decisions about treatment and colonoscopic investigations, and also may be helpful to discover when symptoms originate from something other than inflammation. If fecal calprotectin concentrations are elevated in spite of a normal gastroscopy and colonoscopy, then this may indicate inflammation in the small intestine. However, fecal calprotectin is an unspecific marker of inflammation, and consequently the concentrations also may be elevated for other reasons, such as bacterial infections in the gastrointestinal or the upper respiratory tract, medication with nonsteroidal anti-inflammatory drugs, or liver cirrhosis (9,21).

In conclusion, fecal calprotectin can be used as a quantitative marker of colonic inflammation in pediatric IBD. This noninvasive method seems to be useful for the detection of subclinical mucosal inflammation as well as mucosal healing, and should facilitate objective assessment and monitoring of IBD activity in children.

Acknowledgments

The authors thank Rumjana Djilali Merzoug (Department of Clinical Chemistry), Kajsa Nyquist, and the staff at the Endoscopy Unit (Department of Gastroenterology, Astrid Lindgren Children's Hospital) at Karolinska Hospital, Stockholm. We also are grateful to Kent Nilsson and John Öhrvik (Centre for Clinical Research) and the staff at the Gastroenterology Unit (Department of Pediatrics) at the Central Hospital, Västerås, Sweden. Prof. Magne Fagerhol, of Oslo, Norway, kindly supplied the reagents for fecal calprotectin.

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Keywords:

Colitis; Colonoscopy; Feces; Inflammatory bowel diseases; Leukocyte L1 antigen complex

© 2007 Lippincott Williams & Wilkins, Inc.