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.
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Keywords:© 2001 Lippincott Williams & Wilkins, Inc.
Inflammatory bowel disease; Children; Calprotectin; Feces; Disease activity; Technetium-99-labeled white cell scanning; Colonoscopy