Journal of Pediatric Gastroenterology & Nutrition:
Rapid Test for Fecal Calprotectin Levels in Children With Crohn Disease
Kolho, KL*; Turner, D.†; Veereman-Wauters, G.‡; Sladek, M.§; de Ridder, L.||; Shaoul, R.¶; Paerregaard, A.#; Dias, J. Amil**; Koletzko, S.††; Nuti, F.‡‡; Bujanover, Y.§§; Staiano, A.||||; Bochenek, K.¶¶; Finnby, L.##; Levine, A.***; Veres, G.†††
*Children's Hospital, Helsinki University Central Hospital, University of Helsinki, Helsinki, Finland
†Pediatric Gastroenterology Unit, Shaare Zedek Medical Center, Hebrew University of Jerusalem, Israel
‡Pediatric Gastroenterology Unit, University Hospital UZ Brussels, Belgium
§Polish-American Children's Hospital, Jagiellonian University School of Medicine, Krakow, Poland
||Pediatric Gastroenterology Unit, Erasmus MC-Sophia Children's Hospital, Rotterdam, The Netherlands
¶Meyer Children's Hospital, Rambam Medical Center, Haifa, Israel
#Department of Pediatrics, Hvidovre Hospital, Hvidovre, Denmark
**Department of Pediatrics, Hospital, S. Joao, Porto, Portugal
††Dr von Haunersches Kinderspital, Ludwig Maximilians University Munich, Munich, Germany
‡‡Department Pediatrics, La Sapienza Hospital, Rome
§§Edmon & Lili Safra Children's Hospital, Sheba Medical Center, Tel Aviv University, Tel Aviv, Israel
||||Department of Pediatrics, University of Naples “Federico II,” Naples, Italy
¶¶Department of Pediatric Gastroenterology and Nutrition, Medical University of Warsaw, Warsaw, Poland
##Unger-Vetlesens Institute, Lovisenberg Diakonale Hospital, Oslo, Norway
***Pediatric Gastroenterology Unit, Wolfson Medical Center, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel
†††1st Department of Pediatrics, Semmelweis University, Budapest, Hungary.
Address correspondence and reprint requests to Kaija-Leena Kolho, MD, PhD, Children's Hospital, University of Helsinki, Box 281, FIN-00029 HUS, Helsinki, Finland (e-mail: firstname.lastname@example.org).
Received 16 October, 2011
Accepted 23 February, 2012
The present study received financial support from European Crohn's and Colitis Organization (A.L.), Finnish Pediatric Research Foundation Helsinki (K.L.K.), and University Central Hospital Research Fund (K.L.K.).
The authors report no conflicts of interest.
ABSTRACT: Assessment of fecal calprotectin, a surrogate marker of mucosal inflammation, is a promising means to monitor therapeutic response in pediatric inflammatory bowel disease, especially if the result is readily available. We tested the performance of a novel calprotectin rapid test, Quantum Blue, versus the conventional enzyme-linked immunosorbent assay in 134 stool samples from 56 pediatric patients with Crohn disease. The intraclass correlation coefficient analysis reflected good agreement (intraclass correlation coefficient 0.97 [95% confidence interval 0.95–0.98]) but agreement was better in lower values, where dilutions were not required. Using a cutoff of 100 μg/g for normal values, the percentage agreement between the 2 tests was 87%. The optimal cutoff values to guide clinical decisions in the therapy of inflammatory bowel disease have yet to be determined.
The recent development of easily applicable fecal surrogate markers of intestinal inflammation, such as fecal calprotectin (FC) or lactoferrin, has provided new means for objective assessment of disease activity also in pediatric patients with chronic inflammatory bowel disease (IBD) (1–4). The fecal levels of these neutrophil-derived markers reflect the influx of inflammatory cells into the intestinal mucosa and relate to the mucosal lesions found during endoscopy and the grade of histological inflammation (3–5). Active inflammation in the lower gastrointestinal tract is unlikely if their levels are low (4,6). FC is the most accurate tool to detect the presence of active mucosal inflammation in the colon when compared with serum inflammatory markers and with clinical scores (4,7,8), but is also applicable in small bowel Crohn disease (CD) (9).
In adults, low FC concentration is associated with mucosal healing in patients with CD responding to therapy (10,11). There are few studies on the use of fecal markers in the assessment of therapeutic response in children (12–15), but these reports show that FC is a promising, noninvasive means to objectively classify individual responses. To be applicable in clinical practice, however, a test should be simple and the results rapidly available for the clinician. Thus, a rapid test for FC may be valuable in clinical decision making. We prospectively studied the performance of a novel rapid test in pediatric patients with CD.
All of the patients were participants in the ongoing European Growth, Relapse and Outcomes With Therapy (GROWTH) CD study. This prospective multicenter study enrolls children with newly diagnosed CD, and follows them for 2 years at 3- to 6-month intervals, assessing risk factors for a complicated disease course.
In this substudy, frozen stool samples were transported via express delivery to Norway and tested with a conventional enzyme-linked immunosorbent assay (ELISA) test and the results were compared with those obtained with a novel rapid test, Quantum Blue (QB) (Bühlmann Laboratories, Basel, Switzerland). Clinical disease activity was scored with the Pediatric Crohn Disease Activity Index (PCDAI (16)).
Extraction of Stool Samples
Calprotectin was extracted from feces according to instructions of the manufacturer. Fecal sample (80 mg) was filled into the cap of the ROCHE extraction device (Hoffman-La Roche, Basel, Switzerland) and 4 mL of extraction buffer was added (1:50 dilution). After vortexing for 2 minutes and allowing the samples to sediment for 10 minutes, 1 mL of the extract was transferred to 2-mL NUNC tubes. The extracts were stored at −20°C until assayed.
All of the samples were run in duplicate according to the instructions of the manufacturer. Samples with a higher FC concentration than the highest control were repeated in a 2-fold dilution. After 30-minute incubation, the plates were washed before application of 100 μL of enzyme conjugate and incubated for 30 minutes. The plates were washed again, and 100 μL of substrate was added. The plates were read at 450 nm after 15 minutes with a Tecan ELISA analyzer and Magellan software (both Männendorf, Switzerland).
Rapid test uses the same antibodies as the ELISA, recognizing a structural epitope of a complete calprotectin molecule. Catching antibodies are conjugated with gold particles and after binding to soluble calprotectin, migrate down a nitrocellulose membrane until immobilized by a secondary antibody. The color intensity of a red line (from gold particles) is determined after 12 minutes by the QB reader and converted to calprotectin concentration using a built-in standard curve. The dynamic range of this test was 30 to 300 μg/g.
For the QB, the same fecal extracts were used as for the ELISA. Depending on the results from the ELISA, extracts >300 μg/g were diluted 20 + 900 μL (1:46) and/or 20 + 1800 μL (1:91).
If the results from the ELISA and the QB dichotomized the value differently with a cutoff for a normal or a raised value (>150 μg/g for the study purpose), the samples were reanalyzed.
The local ethical committees approved the protocol. All of the participants/their guardians signed an informed consent form.
Data are presented as mean ± standard deviation or median (interquartile range [IQR]) as appropriate for the normality distribution. Values obtained using the 2 assays were compared using paired analysis, as appropriate. Spearman r was used for correlations. Agreement between the assays was determined using 3 methods. First, the intraclass correlation coefficient (ICC) was calculated using 2-way mixed model of analysis. Unweighted κ was used to reflect the agreement of normal versus abnormal tests results, and finally, the calprotectin values of the 2 assays were compared using the 95% limits of agreement (17). The limits of agreement show how much one method of measurement differs from another by calculating the mean difference ± 1.96 standard deviation versus mean individual value; if the difference is not clinically important, the 2 methods are said to agree (18). Limits of agreement can differentiate fixed from proportional bias (ie, change in the magnitude of the difference between the values across the range). The result is a visual depiction of the variance, and the location of the variance along the scale score. All of the analyses were performed using SPSS version 15 (SPSS Inc, Chicago, IL) with level of statistical significance of 0.05.
There were 132 samples obtained from 56 children with CD (median age of 13 years, range 1–18; boys n = 28) included in the present study. Fecal samples were derived from a total of 10 European pediatric gastroenterology units (3–29 samples per center). Thirty of the fecal samples were obtained at the time of diagnosis, whereas the others, 8 to 72 weeks following introduction of therapy. There were 1 to 6 samples from each patient (median 2). Ten samples were reanalyzed as ELISA and QB dichotomized them differently as a normal or raised value. Subgroup analyses using only the first sample per subject yielded similar results in all of the analyses, indicating low risk for repeated measures bias. Furthermore, there was no difference in the test performance between baseline and follow-up samples: the ICC value related to disease onset was 0.96 (95% confidence interval [CI] 0.93–0.97) and to samples obtained later 0.97 (0.96–0.98). Thus, all of the analyses were performed on the entire sample set.
Median FC value using the ELISA assay was significantly higher using the QB (317 μg/g [IQR 81–830; range 0–1862] vs 172 μg/g [IQR 50–840; range <30–1656, respectively; P = 0.001]. In 28 samples (21%), the FC value was undetectable in QB, but in 15 of these, the ELISA values were measurable and ranged from 27 to 163 μg/g (median of 36 μg/g). Thirteen samples (9.8%) had undetectable values with ELISA.
The results of the ELISA and the QB test were in excellent correlation with each other (Spearman r = 0.94, P < 0.001) (Fig. 1). It is clear from the scatterplot that agreement was better in values <300 μg/g, when dilutions were not required. The ICC analysis reflected excellent agreement (ICC 0.97 [95% CI 0.95–0.98]). Good agreement was reflected from the limit of agreement figure (mean difference of 52 μg/g, which is not significantly different from zero), but the limits increased with increasing values, suggesting proportional bias (Fig. 2).
The correlation of calprotectin values obtained using both assays with PCDAI was extremely low (n = 85 samples with available PCDAI [median 5, range 0–45]; r = −0.053 for the ELISA; P = 0.632 and r = 0.002; P = 0.988 for the QB).
Using a cutoff of 100 μg/g for normal values (13–15), the percentage agreement between the 2 tests was 87% with a moderate κ of 0.72 (95% CI 0.60–0.84). The corresponding values were identical with a cutoff value of 150 μg/g.
Surrogate markers of the presence of intestinal inflammation, such as FC, are promising means for reflecting endoscopic disease activity in pediatric IBD (2–4,9). Their use in clinical practice is already emerging and will likely increase if the test is easy to perform and the results are readily available. We have demonstrated that a rapid test has comparable performance to a conventional ELISA for detecting abnormal values of FC. Thus, rapid testing with same-day results may become feasible in clinical practice in children as shown in adult IBD (19). The rapid test applied here, however, needed several dilutions of the samples for higher FC, decreasing the agreement to ELISA and the time to test result. With higher FC levels, the agreement between ELISA and QB was not as good as with lower values most likely as a result of several dilutions, suggesting that it may be easier to use for screening purposes (is FC elevated or normal) than for monitoring patients with high FC levels. Although high values were somewhat divergent, results of both tests suggested active inflammation and would therefore comparably guide the clinical decision making. The future goal, however, would be to have a rapid test with no need for serial dilutions and easy enough to even be done by the patient at home.
Rapid test was comparable to ELISA also during therapy, but there are few studies on FC related to therapeutic response in pediatric IBD (13,15). Active inflammation in the lower gastrointestinal tract is unlikely if FC levels are within the normal range. When in clinical remission, however, the FC level may be elevated in up to two-thirds of the pediatric patients with IBD (14), and there are no guidelines yet for adjustment of therapy according to FC levels. FC and PCDAI did not correlate in our study. This is in line with earlier data showing that clinical activity indices have less correlation to the presence of mucosal inflammation than fecal neutrophil biomarkers (20).
In conclusion, fecal surrogate markers provide noninvasive means to study the presence of intestinal inflammation, thus possibly improving the assessment of the disease activity and therapeutic response. To apply such a test in clinical practice, reliable testing is needed with no major delay in getting the results. We show here that the rapid calprotectin test has good performance in children with CD and is feasible for monitoring therapeutic responsiveness; however, the cutoffs to guide clinical decisions on therapeutic response need to be determined.
Ms Orit Shevah is thanked for assistance in organizing the patient data. Calprotectin ELISA and QB rapid test kits were kindly supplied by Bühlmann Laboratories, Basel, Switzerland.
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