What Is Known
- Fecal calprotectin is a well-integrated objective marker of inflammation that is useful in monitoring inflammatory bowel disease.
- Fecal calprotectin analyses are limited by day-to-day variation and by the consistency of the feces.
- Many adolescents find the stool-collecting uncomfortable leading to reduced compliance and hence suboptimal monitoring.
- In a few studies on adults, serum calprotectin has been shown to correlate with clinical disease activity.
What Is New
- The level of serum calprotectin correlates with the severity of inflammation assessed by endoscopy in adolescents with ulcerative colitis.
- This study supports serum calprotectin as a potential biomarker for disease monitoring of adolescent patients.
Patients diagnosed with inflammatory bowel disease (IBD) during childhood often present extensive disease and a severe disease course that requires intensive treatment (1–5). Accordingly, tight monitoring of the disease activity and high treatment adherence are needed. The golden standard to assess disease activity is a colonoscopy including biopsies, which provides a macroscopic and microscopic description of the mucosa. However, endoscopic procedures are unpleasant for patients, time consuming, expensive, and require general anesthesia in children and adolescents. Therefore, noninvasive surrogate biomarkers are needed as indicators of the present mucosal inflammation. Calprotectin is a member of the protein S100 family and is mainly found in the cytosol of neutrophils. Levels of fecal calprotectin (FC) have been shown to correlate with endoscopically assessed inflammation in patients being evaluated for possible IBD (6–12) and can be measured noninvasively in stools and analyzed at low costs. Furthermore, FC has been shown to be a useful predictor for clinical relapse in adolescents with IBD (13). However, FC exhibits significant within-day and day-to-day variation (14,15). Furthermore, certain features of collecting and managing the fecal sample before arrival at the laboratory can influence FC levels and may, therefore, limit its use in clinical practice. In the outpatient clinic, stool samples are usually collected by the patient and sent by mail to the laboratory. The analysis of FC is dependent on which part of the stool the patient has chosen for collection (ie, with or without blood), and furthermore, the analysis of the concentration of FC depends on the consistency of the feces (16). In recent studies, FC has been shown to be stable for up to 3 days at room temperature. When the samples were stored for up to 7 days, a decrease in the concentration was registered (14). Hence, delays by mail or/and extreme weather temperatures can reduce the precision of the test result.
In the outpatient clinic, clinicians often experience a reluctance from adolescence to collect stool sample despite the use of special collecting kits and reminders from the hospital. Ultimately, this can lead to adolescent patients not adhering to sample collection and hence suboptimal monitoring of the disease. Therefore, we see the need of a substitute biomarker for ease and optimizing the monitoring of adolescents with IBD. Calprotectin in serum (SC) is elevated in various diseases characterized by inflammation such as arthritis (17) and cystic fibrosis (18). However, in contrast to FC, only few studies have addressed the use of SC in IBD (19–23) and to our knowledge, none have investigated SC in adolescents with IBD. The aim of our study was to assess the role of SC as a biomarker in adolescents with IBD in relation to standard disease parameters.
The study is a mixed method study investigating SC in adolescents with IBD. Using cross sectional data, we correlated SC with endoscopically assessed mucosal inflammation. Using both cross sectional and longitudinal data, we investigated the relationship between FC, symptom scores, and standard inflammatory blood markers.
Data were collected from two studies:
Study A: Cross Sectional Data
From July, 2017 to April, 2018, data were collected from UC patients (<18 years old) included in a prospective study investigating the inflammatory profile in pediatric onset UC. Prior (maximum 24 hours) to an endoscopic assessment of the intestinal mucosa, symptom scores were recorded, and fecal and blood sample were collected. FC and standard biochemical analyses were measured in feces and blood, and an additional blood sample was allowed to coagulate, centrifuged, and stored in a biobank (−80o C) and later analyzed for SC in May 2018. The physician performed the endoscopy and reported the results of the procedure in a uniform standardized fashion described below. The study was performed at the Department of Pediatrics, Hvidovre University Hospital, Copenhagen. The study was approved by the National Ethical Committee jr. nr.: H-16049534.
Study B: Longitudinal Data
Data from IBD patients included in the study Web-based Monitoring of Adolescents with Inflammatory Bowel Disease(24) were collected. In short, this study evaluated a patient-based eHealth telemonitoring system (young.constant-care.com) among adolescents with IBD (10–17 years old) during a 2½-year period August, 2013 to to April, 2016. Participants were treated with Infliximab (IFX, anti-Tumor Necrosis Factor alpha agent), and followed at the Pediatric Department, Hvidovre University Hospital, Denmark. Before IFX infusions, a blood sample was taken for the analysis of standard biochemical biomarkers, and additional serum was stored in a biobank (−80 oC) and analyzed for SC in May 2018. As part of the study, patients registered symptom scores on the study website and collected fecal samples every week starting 4 weeks after the infusion and until the next infusion. The infusion was initiated either because of increased disease activity (based on the web-based monitoring) or because of reaching the threshold of 12 weeks treatment intervals. For this study, the last symptom score and FC level before an infusion were used in combination with standard biochemical biomarkers and SC. Thereby, we were able to collect repetitive blood and fecal samples, as well as symptom scores within a range of a few days related to the day of treatment. The study was approved by the National Ethical Committee Jr. nr.: H-22013061.
From both studies, the following variables were collected from patient files: diagnosis, sex, age, treatment, disease extension, age at diagnosis, ongoing infections, and intercurrent diagnosis.
Calprotectin was measured directly in diluted samples with a SC Enzyme-Linked Immuno Sorbent Assay (ELISA) kit (Calpro, Lysaker, Norway) following the instructions of the manufacturer. Briefly, aliquots of the diluted samples were applied to microtiter plates coated with antibodies to calprotectin. After incubation, bound calprotectin was quantified by incubation with alkaline phosphatase-conjugated antibodies to calprotectin followed by development with para-nitrophenylphosphate. SC was analyzed in the Department of Autoimmunology and Biomarkers, State Serum Institute, Denmark.
FC was analyzed at the time of the sample collection in both study A and study B. Samples from study A was collected at the hospital and stored at 5 °C before being transferred to the laboratory the following day. Samples in study B were collected by the patient/parents and mailed (in average 1–2 days delay) to the laboratory. All samples were analyzed at the time of arrival at the laboratory. Fecal samples from study A were analyzed with Bühlmann FC ELISA kit (BÜHLMANN fCAL ELISA, Switzerland), Hvidovre University Hospital, following the instructions of the manufacturer (range 0–1800 mg/kg). Fecal samples from study B were measured by Calpro sandwich ELISA kit (Calpro Ltd., Lysaker, Norway), State Serum Institute, Copenhagen, Denmark (range 0–2500 mg/kg).
Standard Biomarkers in Blood
Standard biochemical analyses included C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), hemoglobin (HgB), albumin (ALB), leucocytes (LEU), and neutrophils (NEU).
Endoscopic Assessment of the Intestinal Mucosa
The grade of intestinal inflammation was assessed by the physician performing the endoscopy (study A) and registered in an Ulcerative Colitis Endoscopic Index of Severity (UCEIS) (25). Each segment of the colon was assessed according to predefined segments and the final score represents the most affected area of the colon (range 0–8).
Clinical symptoms were scored using the Pediatric Ulcerative Colitis Activity Index (PUCAI) (26) either by the study investigator (study A) or the patient (study B). CD patients in study B registered symptoms in the abbreviated Pediatric Crohn's Disease Activity Index (aPCDAI) (27). PUCAI range 0 to 85 points and disease activity was categorized as none (<10), mild (10–34), moderate (35–64), and severe (>64) disease activity. aPCDAI range 0 to 60 points and was categorized as none (<10), mild (10–15), moderate (16–25), and severe (>25) disease activity. Demographics, diagnosis (IBD and non-IBD inflammatory conditions), and possible presence of ongoing infections (bacteria, virus or parasites) were collected from participants’ medical records.
Descriptive characteristics were presented as median and range for continuous variables and frequencies and percentages for categorical variables. According to the nonparametric distribution of data, scatter plots and Spearman correlation coefficients were used to investigate the correlation between SC and disease parameters (UCEIS, symptom score, FC, and CRP) in data from study A.
Data from study B were collected at consecutive treatments with a maximum of 14 days between the SC and FC, symptoms, and CRP. Linear Mixed Effect Model (MEM) was used to analyze the association between SC and disease parameters (FC, symptom score, CRP) in the repetitive data. The MEM model included a random intercept for subjects with a correlation between measures specified by a first order autocorrelation structure.
The role of FC in relation to UCEIS and symptom scores was illuminated by Spearman correlations between FC and UCEIS and PUCAI in data from study A and by MEM to analyze the association between FC and symptom score on data from study B. P-values < 0.05 were considered as significant. Statistical analyses were performed using SAS Enterprise Guide 9.4 and the statistical computing program R 3.2.2. R Core Team 2015.
Patient demographics and clinical characteristics are presented in Table 1. In total, SC was analyzed in 95 samples (19 from study A and 76 from study B). None of the patients previously had a colectomy. Two participants in study A were diagnosed with non-IBD inflammatory conditions (chronic recurrent multifocal osteomyelitis and uveitis inclusive juvenile idiopathic arthritis); however, none of the two participants presented activity in the intercurrent disease at the time of the endoscopy. None of the participants in study B were diagnosed with other non-IBD inflammatory conditions. One patient in study B had an ongoing Staphylococcus skin infection treated with antibiotics at the time of two consecutive IFX infusions and presented the following biomarker values: SC 160 and 290 ng/L; FC 0 and 30 mg/kg, and CRP 0.3 and 0.3 mg/L.
Relationship Between Serum Calprotectin and Standard Disease Parameters
Study A:The Spearman correlation coefficient between SC and UCEIS was positive (r = 0.56, P = 0.01). Similarly, the correlations between SC and PUCAI and between SC and CRP were positive (r = 0.64, P = 0.003 and r = 0.97, P < 0.0001, respectively). We found no significant correlation between SC and FC (Fig. 1 ). Presentation of scatterplot and Spearman correlation coefficient with a maximum of SC = 3750 ng/ml are presented in supplemental material in supplemental Figure 1 (Supplemental Digital Content, http://links.lww.com/MPG/B550).
Study B: UC patients: The associations between SC and FC and between SC and CRP were significant (P = 0.004, estimate 0.32, CI 0.12–0.52) and (P = 0.0001, estimate 0.002, CI 0.001–0.003). MEM analysis between SC and PUCAI showed an insignificant positive estimate (P = 0.76, estimate 0.001, CI −0.003 to 0.004).
CD patients: The association between SC and CRP was positive and significant (P = 0.02, estimate 0.002, CI 0.0003–0.003). MEM analysis between SC and FC and between SC and aPCDAI showed insignificant positive estimates (Fig. 1 ). The correlation coefficients and MEM estimates between SC and the standard disease parameters are presented in Table 2.
Relationship Between Fecal Calprotectin and Ulcerative Colitis Endoscopic Index of Severity and Symptom Scores
Data from study A analyzed with the Spearman correlation coefficient between FC and UCEIS and between FC and PUCAI were insignificant (r = 0.45, P = 0.06) and (r = 0.23, P = 0.35). Data from study B analyzed with MEM analysis showed no association between FC and PUCAI (P = 0.53, estimate 0.002, CI −0.005 to 0.009) whereas MEM analysis between FC and abbrPCDAI was significant, however, with a negative estimate (P = 0.003, estimate −0.009; CI = −0.01 to −0.003). Supplemented material in Figure 2 (Supplemental Digital Content, http://links.lww.com/MPG/B550).
Relationship Between C-reactive protein and Ulcerative Colitis Endoscopic Index of Severity
Analyzed on data from study A, the Spearman correlation coefficient between CRP and UCEIS was insignificant (r = 0.15, P = 0.55) and (r = 0.23, P = 0.35).
In this pilot study, we investigated the role of SC in adolescents with IBD. The mixed method design consisted of a correlation and association between SC and standard disease parameters in cross sectional and longitudinal data.
We found a positive correlation between SC and UCEIS as well as between SC and PUCAI. Estimates in the MEM analyses of the repetitive measures of SC and PUCAI showed also positive, although insignificant, correlation. Overall, this demonstrated that in UC patients, a more severe inflammatory state in the intestine and severe clinical symptoms (ie, active disease) was reflected by higher SC levels. Similar findings have been reported in previous studies (19–22). Meuwis et al (20) demonstrated a positive correlation between SC and endoscopic assessment as well as between SC and symptoms in adult CD patients participating in the STORI trial. Furthermore, CD patients with active disease had significantly higher SC levels compared with patients with inactive disease. Three previous studies (19,20,22) have found a significant difference in the level of SC when comparing IBD patients to controls (both adults and children). Only one study by Fukunaga et al (23) found conflicting results as they could only demonstrate a mild correlation in UC patients but none in CD patients. Overall, taking our as well as previous studies into consideration, SC can be a promising biomarker in the monitoring of UC.
Like Meuwis et al (20) and Leach et al (19), we found a positive correlation between SC and CRP. As both CRP and SC reflect inflammation and both can be categorized as acute phase reactants (28), this correlation was expected. However, we noticed that CRP correlated with neither to UCEIS nor to PUCAI leading to the conclusion that SC could supplement the monitoring of IBD.
Interestingly, our results indicate that SC may be a better surrogate biomarker of the intestinal inflammation than FC. Contrary to this, Kalla et al (22) found FC to be superior to SC as a screening tool in discriminating IBD from non-IBD patients. Furthermore, several studies have cemented FC as a stable surrogate biomarker of intestinal inflammation (6–12). FC has the advantage of detecting calprotectin located in the gastrointestinal tract whereas SC to a greater extent may represent inflammation from other sites as well. Consequently, SC probably does not pose the ability to be used as a diagnostic screening tool in IBD like FC. To determine, however, whether SC should complement or could replace FC in the monitoring of IBD after diagnosis, larger studies are needed. A normal range in healthy, non-IBD subjects should additionally be defined as well as within-day and day-to-day variation.
Adolescent patients are seen regularly in the outpatient clinic, where they undergo a clinical examination and blood sampling. Therefore, adding SC to the blood panel instead of measuring calprotectin in feces could increase the adherence to this biomarker. Addressing the delay from collecting the feces sample until the FC result becomes available, FC home tests have been introduced in adult IBD (29). However, the major issue of not delivering fecal samples seems to be related to the actual handling of the feces, why the home-test solution appears less feasible in the adolescent patient population. We believe that SC potentially can improve the monitoring of adolescent patients who fail to deliver fecal samples for FC.
Strengths and Limitations
The main limitation of the current study is the small number of samples, which can lead to lack of power. The small number increases the risk of type II errors, and furthermore, a few extreme samples or a lack of high and low observations can easily affect the results. Another limitation is that the analysis of FC in study A was performed by ELISA kit from Bühlmann whereas the SC was analyzed by an ELISA kit from CALPRO A/S. The FC results in study A had a maximum of 1800 mg/kg (n = 3) when even higher levels may have been present. The strength of the present study is that we have investigated SC in relation to the golden standard of inflammation (endoscopic assessment of the intestine) and have presented longitudinal data, which accounts for the dynamics of IBD.
We did not find a significant correlation between FC and UCEIS, which may raise some concerns about the quality of our data, as a relationship between FC and the mucosal intestinal inflammation is presented unambiguously in the literature. The lack of significance can either be caused by a lack of power (type II error) with too few samples not representing the whole spectrum of SC and FC values or caused by the circumstance that FC does not correlate that well with UCEIS in adolescents with UC. Larger studies are needed to evaluate this.
We found a correlation between SC and the endoscopically assessed inflammation of the intestine. Hence, SC can potentially improve the disease monitoring of adolescent patients, who are noncompliant to fecal sampling. However, to determine the use of SC in the monitoring of IBD, larger studies including endoscopic and histopathologic evaluation of the mucosa status and healing are needed.
1. Van Limbergen J, Russell RK, Drummond HE, et al. Definition of phenotypic characteristics of childhood-onset inflammatory bowel disease. Gastroenterology
2. Jakobsen C, Bartek J, Wewer V, et al. Differences in phenotype and disease course in adult and paediatric inflammatory bowel disease-a population-based study. Aliment Pharmacol Ther
3. Malmborg P, Grahnquist L, Ideström M, et al. Presentation and progression of childhood-onset inflammatory bowel disease in northern Stockholm County. Inflamm Bowel Dis
4. Gower-Rousseau C, Dauchet L, Vernier-Massouille G, et al. The natural history of pediatric ulcerative colitis: a population-based cohort study. Am J Gastroenterol
5. Vernier-Massouille G, Balde M, Salleron J, et al. Natural history of pediatric Crohn's disease: a population-based cohort study. Gastroenterology
6. D’Haens G, Ferrante M, Vermeire S, et al. Fecal calprotectin
is a surrogate marker for endoscopic lesions in inflammatory bowel disease. Inflamm Bowel Dis
7. Mosli MH, Zou G, Garg SK, et al. C-reactive protein, fecal calprotectin
, and stool lactoferrin for detection of endoscopic activity in symptomatic inflammatory bowel disease patients: a systematic review and meta-analysis. Am J Gastroenterol
8. Bunn SK, Bisset WM, Main MJ, et al. Fecal calprotectin
: validation as a noninvasive measure of bowel inflammation in childhood inflammatory bowel disease. J Pediatr Gastroenterol Nutr
9. Carlsen K, Riis LB, Elsberg H, et al. The sensitivity of fecal calprotectin
in predicting deep remission in ulcerative colitis. Scand J Gastroenterol
10. Theede K, Holck S, Ibsen P, et al. Level of fecal calprotectin
correlates with endoscopic and histologic inflammation and identifies patients with mucosal healing in ulcerative colitis. Clin Gastroenterol Hepatol
11. Patel A, Panchal H, Dubinsky MC. Fecal calprotectin
levels predict histological healing in ulcerative colitis. Inflamm Bowel Dis
12. Zittan E, Kelly OB, Kirsch R, et al. Low fecal calprotectin
correlates with histological remission and mucosal healing in ulcerative colitis and colonic Crohnʼs disease. Inflamm Bowel Dis
13. Van Rheenen PF. Role of fecal calprotectin
testing to predict relapse in teenagers with inflammatory bowel disease who report full disease control. Inflamm Bowel Dis
14. Lasson A, Stotzer PO, Öhman L, et al. The intra-individual variability of faecal calprotectin: a prospective study in patients with active ulcerative colitis. J Crohn's Colitis
15. Calafat M, Cabré E, Mañosa M, et al. High within-day variability of fecal calprotectin
levels in patients with active ulcerative colitis. Inflamm Bowel Dis
16. Nishimuta M1, Inoue N, Kodama N, et al. Moisture and mineral content of human feces-high fecal moisture is associated with increased sodium and decreased potassium content. J Nutr Sci Vitaminol (Tokyo)
17. Nordal HH, Fagerhol MK, Halse AK, et al. Calprotectin (S100A8/A9) should preferably be measured in EDTA-plasma; results from a longitudinal study of patients with rheumatoid arthritis. Scand J Clin Lab Invest
18. Gray RD, Imrie M, Boyd AC, et al. Sputum and serum calprotectin are useful biomarkers during CF exacerbation. J Cyst Fibros
19. Leach ST, Yang Z, Messina I, et al. Serum and mucosal S100 proteins, calprotectin (S100A8/S100A9) and S100A12, are elevated at diagnosis in children with inflammatory bowel disease. Scand J Gastroenterol
20. Meuwis MA, Vernier-Massouille G, Grimaud JC, et al. GETAID (Groupe d’Étude Thérapeutique Des Affections Inflammatoires Digestives). Serum calprotectin as a biomarker for Crohn's disease. J Crohns Colitis
21. Boschetti G, Garnero P, Moussata D, et al. Accuracies of serum and fecal S100 proteins (calprotectin and calgranulin C) to predict the response to TNF antagonists in patients with Crohn's disease. Inflamm Bowel Dis
22. Kalla R, Kennedy NA, Ventham NT, et al. Serum calprotectin: a novel diagnostic and prognostic marker in inflammatory bowel diseases. Am J Gastroenterol
23. Fukunaga S, Kuwaki K, Mitsuyama K, et al. Detection of calprotectin in inflammatory bowel disease: fecal and serum levels and immunohistochemical localization. Int J Mol Med
24. Carlsen K, Houen G, Jakobsen C, et al. Individualized infliximab treatment guided by patient-managed ehealth in children and adolescents with inflammatory bowel disease. Inflamm Bowel Dis
25. Travis SPL, Schnell D, Krzeski P, et al. Reliability and initial validation of the ulcerative colitis endoscopic index of severity. Gastroenterology
26. Turner D, Otley AR, Mack D, et al. Development, validation, and evaluation of a pediatric ulcerative colitis activity index: a prospective multicenter study. Gastroenterology
27. Turner D, Griffiths AM, Walters TD, et al. Mathematical weighting of the pediatric Crohn's disease activity index (PCDAI) and comparison with its other short versions. Inflamm Bowel Dis
28. Meuwis M, Vernier-massouille G, Grimaud JC, et al. ScienceDirect serum calprotectin as a biomarker for Crohn's disease. J Crohn's Colitis
29. Vinding KK, Elsberg H, Thorkilgaard T, et al. Fecal calprotectin
measured by patients at home using smartphones--a new clinical tool in monitoring patients with inflammatory bowel disease. Inflamm Bowel Dis