Journal of Pediatric Gastroenterology & Nutrition:
Fecal Calprotectin: Validation as a Noninvasive Measure of Bowel Inflammation in Childhood Inflammatory Bowel Disease
Bunn, Susan K.*; Bisset, W. Michael†; Main, Margaret J. C.*; Gray, Elizabeth S.‡; Olson, Shona§; Golden, Barbara E.*
*Department of Child Health, University of Aberdeen, Foresterhill, †Royal Aberdeen Children's Hospital, Cornhill Road; ‡Department of Pathology, University of Aberdeen, Foresterhill; and §Department of Radiology, Aberdeen Royal Infirmary, Foresterhill, Aberdeen, United Kingdom
Received April 10, 2000; accepted January 25, 2001.
Financial support was provided by the Sanofi Winthrop Foundation, Surrey, UK.
Address correspondence and reprint requests to Dr. Barbara Golden, Senior Lecturer, Department of Child Health, University of Aberdeen, Foresterhill, Aberdeen, AB25 2ZD, U.K. (e-mail: b.e.golden@abdn. ac.uk).
This article accompanies an editorial. Please see Bjarnason I, Sherwood R. Fecal calprotectin: a significant step in the noninvasive assessment of intestinal inflammation. J Pediatr Gastroenterol Nutr 2001;33:11–3.
Background: Calprotectin is an abundant neutrophil protein, which is extremely stable in feces. This study aimed to validate fecal calprotectin as a marker of bowel inflammation against invasive measures in children with inflammatory bowel disease (IBD), including colitis and small bowel Crohn disease.
Methods: Fecal calprotectin was measured using a simple enzyme-linked immunosorbent assay in 36 spot stool samples from 22 children before colonoscopy and from 14 children before technetium-99 ( 99 Tc) scanning. Using standard scoring systems, the severity of inflammation was assessed macroscopically and histologically at six standard sites in those who underwent colonoscopy and also at six standard sites in those who underwent 99 Tc scanning. The subscores from each site were summated to give combined severity and extent scores for macroscopic and for histologic inflammation in the group undergoing colonoscopy and total inflammation in the group undergoing 99 Tc scanning.
Results: In the 22 children who underwent colonoscopy, median fecal calprotectin was 4.9 mg/L (0.1–272.5 mg/L) (range). Disease groups included six normal cases, nine ulcerative colitis cases, two isolated Crohn colitis cases, two indeterminate colitis cases, and three allergic colitis cases. Fecal calprotectin correlated closely with colonic macroscopic inflammation (r = 0.75, P < 0.001) and histologic inflammation (r = 0.85, P < 0.001). Of the 14 children undergoing 99 Tc scanning, 10 had Crohn disease, 3 had ulcerative colitis, and 1 had allergic colitis. Median fecal calprotectin was 9.1 mg/L (0.3–141.7 mg/L), and this correlated closely with the 99 Tc scanning score (r = 0.80, P = 0.001).
Conclusion: Fecal calprotectin correlates closely with the best invasive measures of colonic and small bowel inflammation in childhood inflammatory bowel disease. As a sensitive objective measure of bowel inflammation that is risk-free and noninvasive, fecal calprotectin lends itself particularly to the monitoring of and assessment of therapeutic interventions in children with inflammatory bowel disease.
The medical treatment of children with inflammatory bowel disease (IBD) is hampered by the lack of a reliable, objective, noninvasive index of bowel inflammation. Accurate assessment of bowel inflammation is of utmost importance for optimizing treatment on an individual patient basis and for evaluating therapeutic trials.
Clinical assessment of disease activity and hematologic inflammatory indices has been shown to correlate poorly with colonoscopy and histology in adults (1) and children (2). Clinical disease activity scoring systems were introduced to make this assessment more objective and reproducible. However, in adult studies, they exhibit a high degree of interobserver variation (3,4) and correlate poorly with bowel inflammation assessed using invasive methods (4).
Colonoscopic examination with histology is regarded as the most accurate objective measure of colonic inflammation, against which all new potential tests should be assessed. An accurate, objective measure of bowel inflammation for Crohn disease is more controversial. Barium small bowel series show anatomic lesions but are not a measure of inflammation. It has been suggested that fecal excretion of indium-11 ( 11 In)–labeled granulocytes should be used for Crohn disease (5), but the accepted best measure in routine pediatric practice is technetium-99–labeled ( 99 Tc) white cell scanning. However, these investigations are invasive, have inherent risks, and cannot be repeated frequently in children.
Calprotectin belongs to a group of Ca 2+ binding proteins in the S100 family. It consists of two heavy chain and one light polypeptide chain, with a total molecular mass of 36.5 kd (6). It is found in abundance in neutrophil granulocytes, in which it accounts for 5% of total protein and 60% of the protein in the cytosol fraction. Lower concentrations are found in monocytes and reactive macrophages (7). Calprotectin is present in stool, and its fecal concentration is stable for up to 7 days at room temperature (8). Therefore, fecal samples for calprotectin analysis can be collected at home and mailed to the laboratory. A simple enzyme-linked immunosorbent assay method for quantifying fecal calprotectin has been developed, which can be performed on small (5-g) spot stool samples (8). The assay is available as a commercial kit (Eurospital S.p.A., Trieste, Italy) for which very small quantities (100 mg) of stool can be used.
Studies in adults indicate that the concentration of fecal calprotectin is higher than in control values in IBD, in colorectal carcinoma, and even after short-term treatment with nonsteroidal antiinflammatory drugs (9,10). A strong, positive correlation between fecal calprotectin concentration and fecal excretion of 111 In-labeled neutrophils has recently been shown (11,12). This supports the hypothesis that the increase in fecal calprotectin seen in IBD is primarily a result of increased migration of neutrophils into the gut lumen through inflamed mucosa. A recent study by the group who initially described and validated the fecal assay (11), has also shown a strong correlation between endoscopic–histologic grading of inflammation during colonoscopy and fecal calprotectin concentration in 62 adults with ulcerative colitis (UC). These authors (11) conclude that fecal calprotectin concentration reflects inflammation at the tissue level. More recently, we showed a close correlation between fecal calprotectin and disease activity assessed using a modified Lloyd–Still score in children with IBD (13).
The current study aimed to validate fecal calprotectin as a marker of bowel inflammation in children with IBD by comparison with the best markers of inflammation, by colonoscopy and histology in children with colitis only and 99 Tc-labeled white cell scanning in all subgroups of children with IBD, including small bowel Crohn disease.
MATERIALS AND METHODS
Ethical approval was obtained from the Grampian Health Board and University of Aberdeen Joint Ethical Committee for this research. Full verbal and written explanation of the study was given to parents and children, and written consent was obtained.
Subjects and Fecal Collection
Forty-two children were recruited in the pediatric gastroenterology outpatient department at the Royal Aberdeen Children's Hospital when it was decided that colonoscopy or 99 Tc-labeled white cell scanning was clinically indicated. The children were asked to collect a spot sample of feces (at least 10 g) at home, place it in a specimen container, and send it to the Department of Child Health laboratory in a prepaid envelope, together with a health questionnaire completed on the same day. Children and parents found the collection of fecal samples to be straightforward and acceptable, and no child or family either refused to take part or failed to return the fecal sample.
Twenty-eight children underwent colonoscopy of at least the cecum. They collected the fecal sample just before starting bowel-clearance medication. Of these children, data are presented for 22. The data obtained from the other six children, who underwent colonoscopy but were subsequently shown to have small bowel Crohn disease, were excluded. This was because colonoscopy does not assess small bowel disease and is, therefore, not a measure for such disease. Fecal calprotectin in small bowel Crohn disease was assessed by comparison with 99 Tc-labeled white cell scan findings because this test assesses small bowel disease. Fourteen children underwent 99 Tc-labeled white cell scanning, of which eight had small bowel Crohn disease.
Patients undergoing colonoscopy examination
Consecutive children underwent colonoscopy of least the cecum, all during general anesthetic. Demographics of the study population are shown in Table 1. All diagnoses of Crohn colitis and UC had been or were subsequently confirmed by histologic examination of intestinal biopsy specimens. The six normal children were confirmed as having no underlying disease process after colonoscopy, histology, and other test results were available. All colonoscopies were performed by one experienced pediatric gastroenterologist (WMB) who was blind to the fecal calprotectin results.
Using a scoring system described by Saverymuttu et al. (14) (Table 2), inflammation was scored macroscopically immediately after colonoscopic examination. The six standard sites evaluated were the cecum, the ascending colon, the transverse colon, the descending colon, the sigmoid colon, and the rectum. One mucosal biopsy specimen was taken from each of the six sites, from the area that seemed to be macroscopically the most inflamed. Each biopsy specimen was assessed by an experienced pediatric histopathologist (ESG), who was blind to the fecal calprotectin results, for severity of the changes in the enterocyte and crypts and the cellularity of the lamina propria, using a standardized scoring system, also described by Saverymuttu et al. (14) (Table 3).
Patients undergoing 99 Tc-labeled white cell scanning examinations
Demographics of the 14 consecutive children who underwent 99 Tc-labeled white cell scanning are shown in Table 1. All diagnoses of Crohn disease and UC had been or were subsequently confirmed by histologic examination of intestinal biopsy specimens. Eight of nine children with Crohn disease were known to have had small bowel inflammation previously, and five had active small bowel disease seen on scanning.
The 99m Tc-labeled white cell scanning was performed in the Nuclear Medicine Department of Aberdeen Royal Infirmary. White cell labeling was performed using standard techniques. 400 millibecquerels (MBq) 99m Tc hexamethyl-proplyleneamineamide–labeled white cells were injected into each patient. Anterior images were acquired at 1 hour, and anterior and posterior images at 4 hours after injection.
The grading and scoring of the inflammation was performed by three experienced observers, individually and then together on a consensus basis. The observers were blind to clinical information and to the calprotectin results. The inflammation was scored at six standard sites using a scoring system described by Saverymuttu et al. (14), shown in Table 4. The standard sites were “rectum and sigmoid,” “descending colon,” “transverse colon,” “ascending colon and cecum,” “terminal ileum,” and “other small bowel.” The individual scores from two observers showed close correlation, but the third observer consistently “over scored” in comparison to the others, especially with respect to the extent of inflammation. The three observers therefore scored the images together to give a consensus view. At the Aberdeen Royal Infirmary, nuclear medicine images are routinely reported dually, and a consensus view is considered to be a valid approach to assess disease extent and distribution by scintigraphy.
Calculation of extent, severity, and “combined extent and severity” scores
To score for inflammation extent (area involved), severity, and total inflammation separately, three separate scores were generated, each from the macroscopic regional scores, the histologic regional scores, and the 99m Tc-labeled white cell scan regional scores. The number of regions for which a score of 1 or greater was made generated an “extent score.” Therefore, for macroscopic, histologic and 99m Tc-labeled white cell scanning inflammation (all of which were scored from 0–3 in six regions) the extent score could range from 0 to 6. Taking the highest regional score generated a “severity score.” Therefore, for macroscopic, histologic, and 99m Tc-labeled white cell scanning inflammation, the severity score could range from 0 to 3. A “combined extent and severity score” was generated by the summation of the regional scores. Therefore, for macroscopic, histologic, and 99m Tc-labeled white cell scanning inflammation, the combined extent and severity score for each could range from 0 to 18.
Preparation of Samples and Laboratory Methods
All samples were stored at < −20°C from receipt at the laboratory. Fecal concentrations of calprotectin were determined by an in-house enzyme-linked immunosorbent assay, as previously described (8). Purified calprotectin standard, an immunoglobulin G (IgG) fraction of rabbit anticalprotectin and alkaline phosphatase–conjugated anticalprotectin were `supplied by Professor M. K. Fagerhol, Oslo, Norway.
Briefly, the stool samples were thawed, 5 g of each were mixed with 10 ml fecal extraction buffer (Tris-buffered isotonic saline with 10 mmol/L calcium chloride and 0.25 mmol/L thimerosal at pH 8.0), homogenized using a Stomacher 80 (Seward Medical, London, UK) for 60 seconds at normal speed, and centrifuged at 45,000 g for 60 minutes. The supernatants were gently aspirated and stored at −20°C. For the analysis of calprotectin, microtiter plate wells were first coated with an immunoglobulin G fraction of rabbit anticalprotectin diluted 1:2000 in phosphate-buffered saline with 0.25 mmol/L thimerosal, pH 7.4. These could be stored at 4°C for up to 1 month. Batches of supernatants were thawed and duplicate “samples” prepared by diluting each supernatant 1:50, 1:250, and 1:1250 in assay buffer (Tris-buffered isotonic saline with 0.5 mmol/L magnesium chloride, 2.5 mmol/L potassium chloride, 0.25 mmol/L thimerosal, 0.05% Tween 20, and 10 g/L bovine serum albumin at pH 8.0). Standards (3.75–60 mg/L) were prepared by diluting the purified calprotectin in assay buffer. Before use, the plates were washed in buffer (assay buffer minus albumin). Fifty microliters of sample, standard, or blank were added to each well; then, 50 μl alkaline phosphatase–conjugated anticalprotectin was added. The plate was then shaken at room temperature for 30 minutes. After washing and the addition of substrate (p-nitrophenylphosphate) and additional incubation, optical densities were recorded at 410 nm. A software program was used to calculate calprotectin concentrations of the samples from the curve relating optical density to standard concentration. Coefficients of variation were less than 2% within and less than 15% between assays. The method used in this study differs from that described previously (8) by the use of a stomacher rather than a homogenizer before centrifugation of the sample. Use of the stomacher was validated using samples from 30 children (with IBD and controls). No significant difference in fecal calprotectin concentration was found using the two methods of homogenization.
Statistical Analysis and Graphs
Data are shown as the median and (range). Statistical analysis was performed using SPSS statistical package software (SPSS Inc., Chicago, IL). Statistical comparisons were performed using the Mann-Whitney U test. Simple regression analysis was used to calculate correlation coefficients for parametric data and Spearman rank correlation coefficient was used for nonparametric data. A probability value of 0.05 or less was considered statistically significant.
Fecal Calprotectin and Colonoscopy and Histology Scores
We previously demonstrated the normal pediatric range of fecal calprotectin to be 2.1 mg/L (0.5–6.3 mg/L) (13). For the colonoscopy study group (n = 22), fecal calprotectin was 4.9 mg/L (0.9–272.5 mg/L). In the two children with isolated Crohn colitis, fecal calprotectin concentration was 13.3 mg/L (0.9–25.7 mg/L). In the nine children with UC it was 18.3 mg/L (3.7–272.5 mg/L). In the two children with indeterminate colitis it was 1.5 mg/L (0.7–2.3 mg/L). In the three children with allergic colitis it was 0.8 mg/L (0.6–6.0 mg/L) and in the six normal children it was 0.6 mg/L (0.1–11.7 mg/L).
For the total study group undergoing colonoscopy, fecal calprotectin concentration correlated closely with macroscopic and histologic inflammation (Table 5, Fig. 1). The correlations between fecal calprotectin and extent score, severity score, and combined severity and extent score were similar but closer for histologic scores (r = 0.79 to 0.85, P < 0.001) than for macroscopic scores (r = 0.70 to 0.75, P < 0.01) (Table 5).
For the 13 children with IBD in the colonoscopy study group (UC, indeterminate colitis, or isolated Crohn colitis), fecal calprotectin was closely and significantly correlated to colonoscopy and histologic inflammation scores (Table 5, Fig. 2 and 3). Again, fecal calprotectin concentration correlated more closely with histologic scores (r = 0.70 to 0.82, P < 0.01) than with macroscopic scores (r = 0.56 to 0.65, P < 0.05), and the correlations between fecal calprotectin and extent score, severity score, and combined severity and extent score were similar (Table 5). The effect of drug treatment was investigated by separating those children with IBD and those in whom colonoscopy was used as a diagnostic procedure and who were given no treatment and those in whom the diagnosis of IBD was already made and who were receiving treatment (Fig. 2). In those children in whom colonoscopy was used as a diagnostic procedure, all but one had high inflammation scores and high fecal calprotectin concentrations so that correlation could not be performed. However, in those children receiving treatment, there was a range of inflammation scores and fecal calprotectin concentrations. The children receiving treatment showed a correlation between fecal calprotectin and histologic severity score, extent score, and combined extent and severity score of 0.74, 0.81, and 0.83, respectively, which reached significance for the severity score (P = 0.05) and the combined extent and severity score (P = 0.04) (Fig. 2).
The sensitivity and specificity of fecal calprotectin, using the 95th percentile of the pediatric normal range (6.3 mg/L) as a cut-off to identify those with and without inflammation, were assessed against the histologic inflammation scores. Histologic inflammation was used, rather than macroscopic inflammation because it is recognized as the most sensitive marker of colonic inflammation (2,4,11). To apply these tests, results of the inflammation scores had to be dichotomized into “negative” and “positive.” For this purpose, the cut-off used was the highest score observed in the normal children undergoing colonoscopy. Those above this cut-off were considered to be positive and those below considered to be negative for inflammation. For histology severity score, the upper limit of normal was 1 and for histology combined extent and severity score the upper limit of normal was 6. Defining the upper limit of normal for histology extent score proved impossible because one normal child had very mild (score 1) histologic inflammation in each of the six colonic regions, giving her an extent score of 6 (the maximum possible). Although there was mild histologic inflammation present throughout the colon, the histopathologist thought the “inflammation” could be considered within the boundaries of normal for this child. The sensitivity and specificity of fecal calprotectin therefore were only assessed with respect to severity and combined extent and severity colonic inflammation scores.
The sensitivity and specificity of fecal calprotectin in identifying those children with IBD with and without severity of colonic histologic inflammation over the upper limit of normal (score of 1) were 90% and 100%, respectively. The sensitivity and specificity of fecal calprotectin in identifying those children with IBD with and without combined extent and severity of colonic histologic inflammation over the upper limit of normal (score of 6) were 100% and 80%, respectively.
Fecal Calprotectin and 99 Tc-Labeled White Cell Scan Scores
For the entire group undergoing 99 Tc-labeled white cell scans (n = 14), the median fecal calprotectin concentration was 9.1 mg/L (range, 0.3–141.7 mg/L). The median severity score was 2.0 (range, 0–3), the median extent score was 2.5 (range, 0–4), and the median combined severity and extent score was 2.5 (range, 0–9).
Fecal calprotectin showed a strong positive correlation with all of the 99 Tc-labeled white cell scan scores. The correlations for severity score (r = 0.77, P = 0.001), extent score (r = 0.73, P < 0.01), and combined severity and extent of inflammation score (r = 0.80, P = 0.001) (Fig. 4) were similar.
The sensitivity and specificity of fecal calprotectin, using the 95th percentile of the pediatric normal range (6.3 mg/L)) as a cut-off to identify those with and without inflammation, were assessed against the 99 Tc-labeled white cell scan scores (Fig. 4). A 99 Tc-labeled white cell scan score of 0 was considered to be negative and more than 0 was positive. The sensitivity and specificity of fecal calprotectin in identifying those children with IBD with and without inflammation seen on a 99 Tc-labeled white cell scan were 90% and 100%, respectively. The sensitivity and specificity values were identical for extent, severity, and combined extent and severity of inflammation seen on white cell scans.
We previously demonstrated that fecal calprotectin concentration is significantly higher in children with IBD (11.8 mg/L [0.6–272.5 mg/L]) than in controls (2.1 mg/L [0.5–6.3 mg/L]) and that there is no difference between the values in UC (11.5 mg/L [0.6–272.5 mg/L]) and Crohn disease (14.0 mg/L [0.7–59.7 mg/L]). Additionally, we demonstrated that fecal calprotectin concentration correlated with clinical disease activity as assessed by a modified Lloyd-Still score (r = −0.61, P < 0.001) (13). We have now demonstrated in childhood IBD, for colonic and small bowel inflammation both, that fecal calprotectin concentration correlates closely with the best invasive measures of bowel inflammation—colonoscopy with histology for colitis and 99 Tc-labeled white cell scanning for “total bowel inflammation,” including small bowel inflammation. Fecal calprotectin concentration correlates equally with the severity of inflammation (maximum inflammation score found), extent of inflammation (number of regions scoring > 1), and combined severity and extent (summation of each individual regional inflammation score) identified by the invasive tests in colonic and small bowel disease. This suggests that calprotectin is a marker influenced by length of bowel affected and the severity of inflammation. We have also shown that fecal calprotectin concentrations of more than 6.3 mg/L (95th percentile of the pediatric normal range) have a sensitivity of 100% and a specificity of 80% in identifying in which children with IBD significant inflammation will and will be detected by colonic histology and a sensitivity of 90% and specificity of 100% in detecting in which children with IBD inflammation will and will not be detected by 99 Tc-labeled white cell scanning. These results indicate that fecal calprotectin concentration can identify which children with IBD have active bowel inflammation and in those who do, the concentration of fecal calprotectin reflects the extent or severity of that inflammation.
Fecal calprotectin cannot replace invasive tests, which will always be necessary to obtain tissue samples, to investigate complications of IBD, and to identify disease distribution. However, if the invasive tests are performed merely to assess severity of inflammation or response to treatment, fecal calprotectin measurement provides an excellent alternative. As a simple, noninvasive, objective, child-friendly test, fecal calprotectin measurement has great potential for the serial monitoring of children with IBD, and in the assessment of interventions, especially in the context of therapeutic trials.
It is well recognized that histology of colonic biopsy specimens is the most sensitive marker of colitis (2,4,11) and that macroscopic examination of the colon underestimates both the extent and the degree of inflammation compared with histology (2). It is therefore interesting that fecal calprotectin concentration correlated more closely to histologic than to macroscopic colonic inflammation (Table 5). This suggests that fecal calprotectin concentration may show that inflammation that is not detectable macroscopically during colonoscopy.
The role of calprotectin in health and in IBD is not known. In health, fecal calprotectin concentration is at least 10-fold higher in feces than in plasma (6,8); this finding is compatible with data suggesting that most neutrophils terminate their circulating life by migrating though the gut wall (15). In vitro, calprotectin has bacteriostatic and fungistatic properties, with minimum inhibitory concentrations comparable with those of antibiotics (16). Recent studies suggest that calprotectin inhibits microbial growth by competing for zinc (17,18). Calprotectin also has an antiproliferative action in various tumor cell lines, possibly because of its inhibition of casein kinase II (19). Therefore, it has been suggested that calprotectin may have a role in the gastrointestinal tract in the control of gut flora (8) and, specifically in the setting of mucosal inflammation in IBD, in the prevention of bacterial translocation and the control of epithelial dysplastic to neoplastic progression (20). In adult studies, the very close correlation between spot fecal calprotectin and fecal excretion of 111 In-labeled granulocytes in Crohn disease and UC (11,12) supports the hypothesis that fecal calprotectin concentration reflects the migration of neutrophils through inflamed gastrointestinal mucosa into the gastrointestinal tract in these conditions. This hypothesis also explains why fecal calprotectin is an equally good marker of small bowel and colonic inflammation and an equally good marker in Crohn disease and UC. Although the immunologic pathogeneses of UC and Crohn disease are different, they have many “downstream” inflammatory processes in common. Macrophages and neutrophils are increased in the intestinal mucosa in Crohn disease and UC, and they are likely to be the effectors of mucosal injury in both conditions. However, it is likely that, although fecal calprotectin measurement is a sensitive test of intestinal inflammation in IBD, it is not specific because any cause of increased intestinal neutrophils will result in increased fecal calprotectin concentration. There is no information on the consequences of gastroenteritis or of upper respiratory tract infections on fecal calprotectin in the literature. However, pilot studies at our center indicate that fecal calprotectin concentration is increased in bacterial and viral gastroenteritis but unchanged during viral upper respiratory tract infections (unpublished, 2000).
Measurement of bowel inflammation is important in the assessment of children with IBD. Because these disorders are relapsing conditions, repeated assessments are often necessary. Many methods of assessing bowel inflammation have been proposed, including laboratory indices and clinical scores, but they are limited by subjectivity, low sensitivity, and nonspecificity (1,2,4). Invasive tests provide an objective assessment of bowel inflammation, but patient safety and comfort severely limit their frequency. It has been suggested that an inflammatory mediator released directly into the gut lumen from the inflammatory process might be an ideal test of bowel inflammation in IBD (21). Several groups have attempted to develop fecal markers as noninvasive tests of bowel inflammation. Neutrophil elastase (22–24), leukocyte esterase (25), interleukin 1β(26), interleukin 1 receptor antagonist (20), tumor necrosis factor α(27,28), PAF-acether (29), lactoferrin (30), and eosinophil cationic protein (31,32) have all been shown to be excreted in increased amounts in the feces of patients with IBD, in greater amounts in active than in inactive disease. However, their instability in feces precludes their routine clinic use. α1-Antitrypsin is relatively stable in feces, and its fecal concentration is increased in IBD. However, high concentrations result from the increased permeability of the inflamed gut to plasma proteins and, therefore, as a marker of gastrointestinal inflammation, it is too indirect. It shows a variable relation to clinical disease activity (reviewed in Kjeldsen and Schaffalitzky (33) and in Beck (34) and does not correlate with intestinal inflammation as measured by fecal excretion of 111 In-labeled granulocytes (35). Calprotectin is stable in stools, directly associated with the inflammatory process, and easy to measure and has the potential to fulfil all criteria for an “ideal” test: simple, inexpensive, safe, noninvasive, convenient, acceptable to patients and staff, objective, reliable, and amenable to serial measurements to permit the assessment of therapeutic interventions.
That the concentration of a neutrophil and macrophage protein in a spot fecal sample correlated so closely with objectively assessed bowel inflammation using invasive methods in children with IBD is surprising. However, in the initial methodological study of fecal calprotectin, Roseth et al. (8) demonstrated that fecal calprotectin was distributed remarkably uniformly throughout any one stool, with the correlation coefficient between a spot sample value from a stool, and the value from the same homogenized stool being 0.90 to 0.95. In a recent publication by the same group, Roseth et al. (12) demonstrated that the correlation between fecal calprotectin from a spot fecal sample and the 3-day excretion of 111 In (r = 0.80, P < 0.0001) was almost as good as that between the daily excretion of calprotectin and 3-day fecal excretion of 111 In (r = 0.87, P <0.0001) in UC and Crohn disease.
Our results are in agreement with the work of other groups who have quantified neutrophil proteins in feces. The concentrations of fecal neutrophil elastase (22–24) and lactoferrin (30) are higher in IBD than in controls and higher in active than in inactive disease, though they have not been validated against invasive measures of bowel inflammation. In contrast to these measures, it would seem that the source of calprotectin may include activated macrophages and neutrophils. The relative contribution of these two sources is unknown, though that of neutrophils presumably predominates because of the much greater number of neutrophils.
Fecal calprotectin correlates closely with the best objective, invasive measures of bowel inflammation in children with colonic and small bowel IBD. This study validates the measurement of fecal calprotectin as a sensitive and specific measure of inflammation in children with IBD and an accurate, objective method of quantifying the inflammation present. There will always be a requirement for invasive tests to assess distribution of inflammation, histologic features, and complications, but fecal calprotectin provides a noninvasive, risk-free means of diagnosing and monitoring inflammation. As such, fecal calprotectin measurement comes close to the requirements of the ideal test. It lends itself particularly to monitoring of bowel inflammation on an outpatient basis and in the assessment of response to therapeutic interventions. Further studies are continuing to assess fecal calprotectin measurement as a screening test for pediatric IBD in the outpatient setting.
The authors thank Professor Magne Fagerhol (Ullevaal University Hospital, Oslo) and Dr. Arne Roseth (Aker University Hospital, Oslo) for providing reagents and advice; Lorna Rankin (University of Stirling, Stirling) for technical assistance; and the Department of Public Health at Aberdeen University for statistical advice.
1. Holmquist L, Ahren C, Fallstrom SP. Clinical disease activity and inflammatory activity in the rectum in relation to mucosal inflammation assessed by colonoscopy. A study of children and adolescents with chronic inflammatory bowel disease. Acta Paediatr Scand 1990; 79 (5): 527–34.
2. Beattie RM, Nicholls SW, Domizio P, et al. Endoscopic assessment of the colonic response to corticosteroids in children with ulcerative colitis. J Pediatr Gastroenterol Nutr 1996; 22 (4): 373–9.
3. de Dombal FT, Softley A. IOIBD report no 1: Observer variation in calculating indices of severity and activity in Crohn's disease. International Organisation for the Study of Inflammatory Bowel Disease. Gut 1987; 28 (4): 474–81.
4. Gomes P, du B, Smith CL, et al. Relationship between disease activity indices and colonoscopic findings in patients with colonic inflammatory bowel disease. Gut 1986; 27 (1): 92–5.
5. Becker W, Fischbach W, Weppler M, et al. Radiolabelled granulocytes in inflammatory bowel disease: diagnostic possibilities and clinical indications (review). Nucl Med Commun 1988; 9 (10): 693–701.
6. Dale I, Fagerhol MK, Naesgaard I. Purification and partial characterization of a highly immunogenic human leukocyte protein, the L1 antigen. Eur J Biochem 1983; 134 (1): 1–6.
7. Dale I, Brandtzaeg P, Fagerhol MK, et al. Distribution of a new myelomonocytic antigen (L1) in human peripheral blood leukocytes. Immunofluorescence and immunoperoxidase staining features in comparison with lysozyme and lactoferrin. Am J Clin Pathol 1985; 84 (1): 24–34.
8. Roseth AG, Fagerhol MK, Aadland E, et al. Assessment of the neutrophil dominating protein calprotectin in feces. A methodologic study. Scand J Gastroenterol 1992; 27 (9): 793–8.
9. Roseth AG, Kristinsson J, Fagerhol MK, et al. Faecal calprotectin: a novel test for the diagnosis of colorectal cancer? Scand J Gastroenterol 1993; 28 (12): 1073–6.
10. Meling TR, Aabakken L, Roseth A, et al. Faecal calprotectin shedding after short-term treatment with non-steroidal anti-inflammatory drugs. Scand J Gastroenterol 1996; 31 (4): 339–44.
11. Roseth AG, Aadland E, Jahnsen J, et al. Assessment of disease activity in ulcerative colitis by faecal calprotectin, a novel granulocyte marker protein. Digestion 1997; 58 (2): 176–80.
12. Roseth AG, Schmidt PN, Fagerhol MK. Correlation between faecal excretion of indium-111-labelled granulocytes and calprotectin, a granulocyte marker protein, in patients with inflammatory bowel disease. Scand J Gastroenterol 1999; 34 (1): 50–4.
13. Bunn S, Bisset W, Golden B. Faecal Calprotectin—an objective measure of bowel inflammation in childhood inflammatory bowel disease. J Pediatr Gastroenterol Nutr 2001; 32: 171–7.
14. Saverymuttu SH, Camilleri M, Rees H, et al. Indium 111-granulocyte scanning in the assessment of disease extent and disease activity in inflammatory bowel disease: I. A comparison with colonoscopy, histology, and fecal indium 111-granulocyte excretion. Gastroenterology 1986; 90 (5): 1121–8.
15. Fliedner SH, Cronkite EP, Robertson JS. Granulopoiesis, senescence and random loss of neutrophilic granulocytes in human beings. Blood
16. Steinbakk M, Naess-Andresen CF, Lingaas E, et al. Antimicrobial actions of calcium binding leucocyte L1 protein, calprotectin. Lancet 1990; 336 (8718): 763–5.
17. Clohessy PA, Golden BE. Calprotectin-mediated zinc chelation as a biostatic mechanism in host defence. Scand J Immunol 1995; 42 (5): 551–6.
18. Sohnle PG, Hahn BL, Santhanagopalan V. Inhibition of Candida albicans growth by calprotectin in the absence of direct contact with the organisms. J Infect Dis 1996; 174 (6): 1369–72.
19. Murao S, Collart FR, Huberman E. A protein containing the cystic fibrosis antigen is an inhibitor of protein kinases. J Biolog Chem 1989; 264 (14): 8356–60.
20. Rugtveit J, Brandtzaeg P, Halstensen TS, et al. Increased macrophage subset in inflammatory bowel disease: apparent recruitment from peripheral blood monocytes. Gut 1994; 35 (5): 669–74.
21. Cellier C, Sahmoud T, Froguel E, et al. Correlations between clinical activity, endoscopic severity, and biological parameters in colonic or ileocolonic Crohn's disease. A prospective multicentre study of 121 cases. The Groupe d'Etudes Therapeutiques des Affections Inflammatoires Digestives. Gut 1994; 35 (2): 231–5.
22. Andus T, Gross V, Caesar I, et al. PMN-elastase in assessment of patients with inflammatory bowel disease. Dig Dis Sci 1993; 38 (9): 1638–44.
23. Adeyemi EO, Neumann S, Chadwick VS, et al. Circulating human leucocyte elastase in patients with inflammatory bowel disease. Gut 1985; 26 (12): 1306–11.
24. Bohe M, Genell S, Ohlsson K. Protease inhibitors in plasma and faecal extracts from patients with active inflammatory bowel disease. Scand J Gastroenterol 1986; 21 (5): 598–604.
25. Brouwer J. Semiquantitative determination of fecal leukocyte esterase by a dip-and-read assay (letter). Clin Chem 1993; 39 (12): 2531–2.
26. Saiki T, Mitsuyama K, Toyonaga A, et al. Detection of pro-and anti-inflammatory cytokines in stools of patients with inflammatory bowel disease. Scand J Gastroenterol 1998; 33 (6): 616–22.
27. Braegger CP, Nicholls S, Murch SH, et al. Tumour necrosis factor alpha in stool as a marker of intestinal inflammation [see comments]. Lancet 1992; 339 (8785): 89–91.
28. Nicholls S, Stephens S, Braegger CP, et al. Cytokines in stools of children with inflammatory bowel disease or infective diarrhoea. J Clin Pathol 1993; 46 (8): 757–60.
29. Denizot Y, Chaussade S, Nathan N, et al. PAF-acether and acetylhydrolase in stool of patients with Crohn's disease. Dig Dis Sci 1992; 37 (3): 432–7.
30. Uchida K, Matsuse R, Tomita S, et al. Immunochemical detection of human lactoferrin in feces as a new marker for inflammatory gastrointestinal disorders and colon cancer. Clin Biochem 1994; 27 (4): 259–64.
31. Berstad A, Borkje B, Riedel B, Elsayed S. Increased fecal eosinophil cationic protein in inflammatory bowel disease. Hepato-Gastroenterol 1993; 40 (3): 276–8.
32. Bischoff SC, Grabowsky J, Manns MP. Quantification of inflammatory mediators in stool samples of patients with inflammatory bowel disorders and controls. Dig Dis Sci 1997; 42 (2): 394–403.
33. Kjeldsen J, Schaffalitzky D. Assessment of disease severity and activity in inflammatory bowel disease (review). Scand J Gastroenterol 1993; 28 (1): 1–9.
34. Beck IT. Laboratory assessment of inflammatory bowel disease (review). Dig Dis Sci 1987; 32 (suppl 12): 26S–41S.
35. Fischbach W, Becker W, Mossner J, et al. Faecal alpha-1-antitrypsin and excretion of 111indium granulocytes in assessment of disease activity in chronic inflammatory bowel diseases. Gut 1987; 28 (4): 386–93.
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