The recent development of easily applicable fecal surrogate markers of intestinal inflammation, such as fecal calprotectin (FC) or lactoferrin, has provided a new means for objectively assessing disease activity in patients with chronic inflammatory bowel disease (IBD) (1–7). The level of these neutrophil-derived markers in feces reflects the influx of inflammatory cells into the intestinal mucosa (8). There are limited data on the day-to-day variations in fecal surrogate marker levels in inflammation. Moum et al (9) demonstrated that there is considerable intraindividual variability in the FC levels, but mainly in the higher values and not in the values classifying the patient as either positive or negative. Because the use of fecal surrogate markers is emerging in clinical practice (10), it is also important to be aware of the possible limitations of the FC measurements and the possible influence of diagnostic procedures on the FC results and their interpretation.
METHODS
In a prospective study for evaluating the effect of bowel cleansing for ileocolonoscopy on FC levels, we collected repeated stool samples from 10 pediatric patients undergoing bowel cleansing. The patients provided a baseline sample 0 to 2 days before the procedure to compare the levels during cleansing. The ward's first sample was obtained when the patient started an oral intake of polyethylenglycol, Moviprep, and repeatedly thereafter for every stool sample. We registered the time of the stool sample. A follow-up fecal sample was obtained 2 days after the procedure. The ethics committee of the Helsinki University Hospital approved the fecal sampling.
We stored all of the samples in −20°C and analyzed them within 2 months using the PhiCal ELISA Test (Calpro AS, Oslo, Norway; NovaTec Immunodiagnostica, GmBH, Dietzenbach, Germany) with a BEP 2000 Advance ELISA analyzer (Siemens Healthcare Diagnostics, Munich, Germany). FC levels <100 μg/g were considered normal and values >1000 μg/g were considered to be high (3). The detection limit of the assay was 5 μg/g. All of the samples from an individual patient were run on the same microtitration plate to avoid the possible effect of interassay variation on the results. Statistics were performed using the StatView program (Abacus Concepts, Berkeley, CA).
RESULTS
There were in total 81 fecal samples (a median of 8 samples per patient, range 3–13) from 10 patients (median age 13.1 years, range 8.9–16.2; boys n = 4). For 7 patients, the indication for endoscopy was IBD (ulcerative colitis in 4), whereas for the other 3, the indications were mild elevation in FC or abdominal pain (Table 1). Of the patients with IBD, 5 of 7 were undergoing primary investigations and had no medication, 1 was having a flare when on 5-aminosalicylic acid (5-ASA) and 6-mercaptopurine (6-MP) and 1 was followed up for mild disease and was taking no medication. The background data of the patients are shown in Table 1.
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TABLE 1: Background data of the 10 pediatric patients undergoing bowel cleansing for colonoscopy
Baseline samples (Table 1) were available at 2 to 48 hours (median of 24) in 9, and 72 hours in 1 before the introduction of the cleansing procedure. Six children provided a follow-up sample within 2 days after endoscopy, and 1 child 5 days later.
The FC level varied considerably during the bowel cleansing. A significant decrease in the FC concentration was found between the baseline and lowest values during cleansing (n = 10, P = 0.005, Wilcoxon paired test). The lowest concentration occurred 1 to 15 hours after the baseline sample was collected.
The variation during baseline, the lowest FC values, and the recovery phase (Table 1) from the bowel-cleansing procedure were all statistically significant (n = 7, P = 0.0021, Friedman analysis of variance with repeated measurements). Notably, the FC level remained within normal limits (<100 μg/g; (3)) throughout cleansing in 2 of the 3 patients with a non-IBD disease and increased only mildly in 1 with a borderline value at baseline (from 93 to 189 μg/g; no. 8 in Table 1); however, the FC value for patients with IBD was occasionally normal, although the baseline sample and the first sample taken on the ward were high (>1000 μg/g). Of the 6 patients with a raised FC value at baseline, 5 had at least 1 FC value below the limit <100 μg/g of a raised value and in 1 the lowest value was on this limit (104 μg/g; patient no. 5 in Table 1). The median time to the lowest value was 3 hours (range 1–15). An example of such a patient is shown in Fig. 1. In the follow-up sample, the FC level was comparable to the baseline but 2 patients showed a 9- and 46-fold increase by day 2.
FIGURE 1: Fecal calprotectin levels measured in samples collected during bowel cleansing in stool specimens from 1 child to show individual variation. The cutoff in this study for a normal stool sample was <100 μg/g.
DISCUSSION
Surrogate markers of the presence of intestinal inflammation, such as FC, offer a promising means for revealing endoscopic disease activity in pediatric IBD (1–7). Their use in clinical practice is only now emerging. Therefore, it is important to be familiar with the possible limitations of the measurements. Here, we demonstrate that the FC levels measured during bowel cleansing vary considerably and may even range from high values to normal levels. Thus, for patients with high FC levels, fecal samples taken during the cleansing procedure may be in line with the baseline of the patient or they may misleadingly suggest a low level of inflammation. This variation in FC levels was individual and not predictable. Therefore, it is important in clinical practice (or for research purposes) to advance stool sampling beyond just bowel cleansing to get FC results that better reflect the ongoing level of intestinal inflammation. Otherwise, decision making related to FC levels, for example, estimating the effect of a medication, may be based on misunderstanding the FC levels. In clinical practice an FC sample taken before endoscopy is often an indication for scope and a sample exactly by the same time of the procedure is rarely needed; however, as FC values relate to therapeutic response (3,11) it is important that such evaluation is not based on samples obtained during bowel cleansing.
Furthermore, the cleansing procedure did not alter the baseline FC level when it was assessed 2 to 5 days later. Therefore, it seems unlikely that the procedure itself would induce colonic inflammation in children; however the possibility that an existing inflammation may worsen cannot be completely excluded because the follow-up samples showed a marked elevation in the FC values of 2 of the patients within 2 days. More important, patients with low FC values at baseline did not have elevated values in any of the samples, which would erroneously suggest active inflammation.
FC values are stable in feces and sampling as such is not a critical issue. There was some daily variation in the individual levels of FC, but it mostly occurred in high levels that did not result in misclassifying the patient as either positive or negative (9). As a limitation, daily variation of FC in pediatric patients with IBD on routine care and with no bowel preparation was not assessed, but it is unlikely to be different from the adult observations.
The large variation observed during the cleansing procedure was most likely the result of individual variation related to fluid intake and high fluid content in the sample, but some watery samples had a high FC content as well. Thus, it is important to perform stool sampling either before changing the diet for bowel cleansing or, if needed, at least 2 days after a colonoscopy to obtain a proper view of the patient's FC level.
Acknowledgment
Ms Anne Nikkonen is thanked for excellent assistance in organizing the sample collection and patient data.
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