ORIGINAL ARTICLE
High Within-day Variability of Fecal Calprotectin Levels in Patients with Active Ulcerative Colitis: What Is the Best Timing for Stool Sampling? Margalida Calafat, MD,*,† Eduard Cabré, MD, PhD,* Míriam Mañosa, MD, PhD,* Triana Lobatón, MD, PhD,* Laura Marín, BSc,* and Eugeni Domènech, MD, PhD*
Background: Fecal calprotectin (FC) is considered the best noninvasive way to assess disease activity in ulcerative colitis (UC). However, it is not known which is the more suitable moment for stool sampling in patients with increased stool frequency. The aims of this study were to assess the intraindividual variation of FC within day and to evaluate if the first bowel movement in the morning is the more suitable sample for FC measurement in patients with acute flares of UC. Patients and Methods: Patients admitted because of active UC were invited to collect samples from several bowel movements (including the first in the morning) during the same day providing their ordinal chronology. FC was measured by means of a quantitative rapid point-of-care test based on lateral flow assay immunochromatography.
Results: Eighteen patients were included for a total of 56 stool samples. Most patients had extensive UC and severe disease activity. Within-day FC values varied widely, and the median coefficient of variation was 40% (5%–114%) with a median range of variation of FC values of 3887 mg/kg (69– 9946). The sample from the first stool in the morning obtained the highest individual FC within-day value in 33.3% of cases and the lowest in 38.9%. Conclusions: FC values widely vary between motions in patients with active UC. Stool sample collection from the first bowel movement in the morning does not ensure the highest or lowest within-day FC value. In patients with overt active UC, a single FC determination should not be used as the basis for therapeutic strategies. (Inflamm Bowel Dis 2015;21:1072–1076) Key Words: fecal calprotectin, ulcerative colitis, stool sampling
F
ecal calprotectin (FC), a leukocyte-derived protein, has emerged as one of the most sensitive noninvasive surrogate markers of inflammatory activity in ulcerative colitis (UC). FC is a useful tool for patients in clinical remission because it has proved to be accurate in predicting UC relapse1 and to have a good correlation with endoscopic2 and histological remission,3 allowing for the identification of patients at risk for clinical relapse and the avoidance of endoscopic examination in certain clinical situations. However, its role in active disease is not well established. In mild active disease, FC may differentiate symptoms due to UC or
Received for publication November 13, 2014; Accepted January 19, 2015. From the *Hospital Universitari Germans Trias i Pujol, Ciberehd, Badalona, Spain; and †Universitat Autònoma de Barcelona, Barcelona, Spain. E. Domènech received a research grant (Beca d’intensificació 2013) from the Catalonian Society of Gastroenterology (Societat Catalana de Digestologia) that partly supported this study. AbbVie provided FC-QPOCT kits and devices. The authors have no conflicts of interest to disclose. Guarantor of article: E. Domènech is acting as the submission’s guarantor. Reprints: Eugeni Domènech, MD, PhD, Hospital Universitari Germans Trias i Pujol, IBD Unit, Gastroenterology Department, Carretera del Canyet s/n, 08916 Badalona, Spain (e-mail: [email protected]). Copyright © 2015 Crohn’s & Colitis Foundation of America, Inc. DOI 10.1097/MIB.0000000000000349 Published online 19 March 2015.
1072
| www.ibdjournal.org
2015
to coexisting functional intestinal disorders. In severe active UC, some authors observed a relationship between baseline FC and the risk of colectomy,4 and cutoff FC values were recently agreed on by a panel of experts to define active and severely active UC to aid physicians with treatment modification decisions.5 Moreover, it has been proposed that FC could be useful to monitor response to biological therapies.6–8 However, considerable overlaps in FC values have been observed among patients with different degrees of severity in disease activity,6,9 and many studies found no correlation between baseline FC and response to therapy.7,10,11 Finally, although a homogeneous distribution of FC in human feces has been shown,12 a significant day-to-day13,14 and within-day15 variation in FC has been observed in patients with inflammatory bowel disease (IBD), mainly in mild-to-moderate active disease. This raised a growing interest in stool sampling issues because they might be relevant to FC-based decision-making strategies, and there is no general advice on which stool sample is most suitable for FC measurement. The aims of this study were therefore to assess the intraindividual variations in within-day FC values in patients with acute flares of UC and to evaluate whether the first bowel movement in the morning was the most suitable sample for FC measurement. Inflamm Bowel Dis Volume 21, Number 5, May 2015
Inflamm Bowel Dis Volume 21, Number 5, May 2015
PATIENTS AND METHODS Patients and Data Collection Between June 2013 and July 2014, all patients admitted to our unit because of an acute UC flare were invited to participate in the study after informed consent, regardless of their ongoing treatment, disease extent, and severity. Patients were given 4 numbered stool collector pots and were instructed to take a stool sample from the first bowel movement and from 1 to 3 additional bowel movements within a 24-hour period. Stool sampling was not allowed during bowel cleansing or within 48 hours after colonoscopic examination to avoid the impact of bowel cleansing on FC levels.16 Intestinal bacterial superinfections were ruled out in all cases by means of stool culture and the determination of Clostridium difficile toxins. The study was approved by the Institutional Review Board of the Hospital Universitari Germans Trias i Pujol. In addition to demographic (gender, age) and biological variables (C-reactive protein, hemoglobin, and albumin), many clinical variables were also recorded (date of diagnosis, date of hospital admission, UC extent, and treatment received) including all the parameters of the Simple Clinical Colitis Activity Index17 as follows: bowel frequency, bowel frequency during nighttime, urgency of defecation, rectal bleeding, general well-being, and extraintestinal manifestations. Disease severity was evaluated according to the Montreal’s classification,18 and the endoscopic Mayo subscore was used to assess endoscopic disease severity.19 Regarding stool sampling, the date of collection, number of samples, and the chronologic order of samples for each patient were registered.
FC Measurement
All samples were stored at 2208C, thawed, and analyzed by a quantitative point-of-care test (FC-QPOCT) (Quantum Blue; Bühlmann Laboratories AG, Schönenbuch, Switzerland). This test uses lateral flow assay technology, including an easy-to-use reader system allowing for a quantitative read out. There are 2 FCQPOCT kits: a low-range kit and a high-range kit, which provide quantitative results within minutes, ranging from 30 to 300 mg/kg or 100 to 1800 mg/kg FC, respectively. This FC-QPOCT testing demonstrated a good correlation with the conventional enzymelinked immunosorbent assay test for FC determination20,21 even for Crohn’s disease and UC.2,22 Because all patients had an active disease at the time of sample collection, the high-range FCQPOCT kit was initially used for FC measurement in all samples. All samples scoring .1800 mg/kg were performed again after sample dilution to obtain their exact value. Additionally, when wide variations in FC values of different samples from the same patient were observed, FC measurements were repeated at least twice more.
Statistical Analyses Data are expressed as median and range or absolute and relative frequencies. To assess the correlation of FC values of
Intraindividual Variability of FC in Active UC
different stool samples within a day, the coefficient of variation (CV) was calculated. The Spearman’s rho, Student’s t test, and chisquare test were used as needed to assess the correlation between CV and maximal/minimal FC values in the first sample in the morning and the presence of nocturnal bowel motions, rectal bleeding, urgency/incontinence, C-reactive protein levels, hemoglobin concentration, or disease severity. All statistical analyses were carried out in SPSS12.0 for Windows (SPSS Inc. Chicago, IL).
RESULTS Baseline characteristics of the 18 included patients are summarized in Table 1. Most patients had severe disease activity (72%) at the time of stool sample collection, and all but 2 had extensive UC. Endoscopic assessment was available in 15 cases (83%), with two-thirds of them (66%) showing severe mucosal lesions (Mayo subscore 3). In agreement with this, only 5 patients had normal C-reactive protein levels and 2 had normal hemoglobin concentration. The median number of bowel movements for all patients was 7.5 (2–12), 83% had rectal bleeding, and 72% had nocturnal bowel movements. Fifteen patients (83%) were receiving rectal therapy during admission, and this might account for the very low rate of urgency or incontinence (11%) among the included patients. A total of 56 stool samples were collected (8 patients with 4 samples, 8 patients with 3 samples, and 2 patients with 2 samples) after a median of 4 days since hospital admission (range, 1–12). The median highest FC value was 6927 mg/kg (288–15,870), whereas the median lowest value was 1659 mg/kg (101– 13,870). Within-day FC values varied widely, and the median CV was 40% (5%–114%) with a median range of variation of FC values of 3887 mg/kg (69–9946). Figure 1 shows the individual median, the highest and the lowest values of FC. CV was similar irrespective of mean FC levels (Fig. 2). C-reactive protein
TABLE 1. Characteristics of Patients Male/female Mild/moderate/severe Disease extent (UC) Proctitis/left-sided/extensive Ongoing therapy (at the time of stool samples collection) Aminosalicylates Systemic corticosteroids Thiopurines Anti-TNF agents Rectal corticosteroids or aminosalicylates C-reactive protein, mg/L Hemoglobin, g/dL Albumin, g/L
10/8 1/4/13 0/2/16
14 12 8 3 15 16.5 (1–171) 10.5 (7–14) 34.5 (27–42)
Values expressed as absolute numbers or median (range).
www.ibdjournal.org |
2015
1073
Inflamm Bowel Dis Volume 21, Number 5, May 2015
Calafat et al
FIGURE 1. Median, highest, and lowest intraindividual values of FC.
levels, hemoglobin concentration, median FC values, endoscopic severity score, the total number of bowel movements, or the presence of rectal bleeding, urgency/incontinence, severe activity, or nocturnal bowel movements were not associated with the CV. The sample from the first stool in the morning obtained the highest FC within-day value in 33.3% of patients and the lowest in 38.9%. For the remaining samples, maximal within-day FC values were obtained with the second sample in 44.4%, in the third sample in 18.7% (in 3 out of 16 patients with at least 3
samples), and in the fourth sample in 12.5% (in 1 out of 8 patients with 4 samples). Figure 3 shows the intraindividual variation of FC values regarding the chronology of sample collection. Having the highest or lowest individual FC values at the first stool in the morning was not associated with C-reactive protein levels, hemoglobin concentration, FC values, the total number of bowel movements, or the presence of rectal bleeding, urgency/incontinence, severe activity, or nocturnal bowel movements.
DISCUSSION
FIGURE 2. Correlation between individual mean FC levels and the corresponding SD.
1074
| www.ibdjournal.org
2015
FC has emerged as the best noninvasive surrogate marker of disease activity in UC. There is an increasing use of FC in quiescent disease, mainly for predicting disease relapse and mucosal healing. Recently, some authors posed its usefulness for predicting and/or monitoring response to therapy, suggesting that FC might be relevant for decision making in active disease. However, several factors have been reported as influencing FC, such as colonic cleansing,16 age,23,24 diet, or exercise.24 In addition, it has been hypothesized that in patients with active IBD, the amount of mucus or blood (which can vary widely between motions) could influence FC levels. From this perspective, decisionmaking strategies based on single measurements of FC might be wrong if high intraindividual variability was demonstrated. Little is known about the intraindividual variability of FC in IBD. Moum et al,13 in a study of patients with mild-to-moderate active Crohn’s disease, found significant differences in FC in 63 pairs of stool samples collected in 2 consecutive days. Conversely, a low variability was observed in 3 daily consecutive fecal samples from 93 Crohn’s disease patients in clinical remission.14 Dobrzanski et al,25 in a study published only in abstract form, reported a higher FC diurnal variation between samples collected from all motions during 3 consecutive days in
Inflamm Bowel Dis Volume 21, Number 5, May 2015
Intraindividual Variability of FC in Active UC
FIGURE 3. Intraindividual variation of calprotectin values regarding the chronology of stool sample collection.
outpatients with active UC as compared with inactive UC and active or inactive Crohn’s disease. These data confirm that variability may be only relevant in active disease and particularly in UC in which mucus and rectal bleeding are more often present in stools. Lasson et al15 recently reported their results in 18 patients with mild-to-moderate active UC in whom 2 samples from 2 different parts of the feces were collected at every bowel movement for 2 days. The median CV of FC during the day was 52%, being more pronounced in patients with high FC levels. In agreement with this study, our data clearly show a great within-day variability of FC in patients with active UC, with a median CV of 40%, close to that previously reported; however, we did not find any correlation between CV and median individual FC. The second important finding of our study is that the timing of stool sampling does not seem to be relevant because the highest or lowest FC values may also vary during the day. It could be argued that we did not assess some macroscopic characteristics of stools, such as mucus, blood, or consistency that may influence FC levels. Regarding this issue, Lasson et al were not able to find a correlation between stool consistency, content of blood, or time between bowel movements and CV. Moreover, they found a strong correlation in terms of FC concentrations between random samples of the same bowel movement, although mucus and blood may not be evenly distributed in feces.15 The same authors concluded that patients with no bowel movements during the night should preferably collect stool samples at the first defecation in the morning because they found a significant positive correlation between the level of FC and the time between bowel movements. However, in a similar small series, we did not find any relationship between median FC of the first stool in the morning and rectal
bleeding or nocturnal bowel movements. The differences between Lasson’s study and ours must be mainly due to the characteristics of the population. Although in the former, only outpatients with mild-to-moderate active UC were included, we included inpatients with moderate-to-severe active disease. In addition, most of our patients were on topical therapy, whereas this was an exclusion criterion in the former study. Technical aspects may also explain controversial results between studies, as recently shown in a study comparing different calprotectin assays for the assessment of IBD.26 In this sense, the dilution for FC values .1800 mg/kg that was performed in our study might potentially contribute to the variability of within-day FC values; however, this simple method had been previously performed for enzyme-linked immunosorbent assay measurement of FC.27 The relevance of the great within-day variation of FC will depend on the clinical setting and how we use FC values for decision making. In mild disease, Lasson et al15 found that up to 33% of patients had at least 1 FC measurement below the threshold of disease activity (250 mg/kg), whereas in severe disease, we only observed 15% of patients with at least 1 FC measurement below 1000 mg/kg, a threshold that has been proposed to define severe activity.5 However, it has also been reported that baseline FC higher than 1992 mg/kg may identify those patients with UC who will not respond to intravenous corticosteroids.4 In this sense, 8 of our 18 patients (44%) had values below and over this threshold. Some authors posed the drop of FC between baseline and some weeks of therapy (DFC) as a predictive tool of response to biological agents8; in this scenario, the baseline value may widely vary leading to decreased prediction accuracy of DFC. From this point of view, it seems more reasonable to use semiquantitative FC measurement at least for UC acute severe flares (a situation in www.ibdjournal.org |
2015
1075
Inflamm Bowel Dis Volume 21, Number 5, May 2015
Calafat et al
which FC levels are dramatically elevated and variability is high), because it should be cheaper and more rapid. In summary, FC may vary greatly within day in patients with moderate-to-severe active UC. There is no apparent benefit in sampling the first stool in the morning for FC measurement. Decision-making strategies based on single quantitative FC determinations in severe active UC are not advisable.
ACKNOWLEDGMENTS The authors want to thank the staff of Palex Medical SA (Spain) for their technical support in fecal calprotectin measurements. Author contributions: E. Domènech designed the study, recruited patients, performed statistical analyses, and drafted the article. M. Calafat recruited patients, stored samples, performed fecal calprotectin measurements, collected data, performed statistical analyses, and drafted the article. E. Cabré performed statistical analyses and reviewed the article. M. Mañosa, T. Lobatón, and L. Marín recruited patients, collected data, and reviewed the article.
REFERENCES
1. Mao R, Xiao YL, Gao X, et al. Fecal calprotectin in predicting relapse of inflammatory bowel diseases: a meta-analysis of prospective studies. Inflamm Bowel Dis. 2012;18:1894–1899. 2. Lobatón T, Rodríguez-Moranta F, Lopez A, et al. A new rapid quantitative test for fecal calprotectin predicts endoscopic activity in ulcerative colitis. Inflamm Bowel Dis. 2013;19:1034–1042. 3. Guardiola J, Lobatón T, Rodríguez-Alonso L, et al. Fecal level of calprotectin Identifies Histologic inflammation in patients with ulcerative colitis in clinical and endoscopic remission. Clin Gastroenterol Hepatol. 2014; 12:1865–1870. 4. Ho GT, Lee HM, Brydon G, et al. Fecal calprotectin predicts the clinical course of acute severe ulcerative colitis. Am J Gastroenterol. 2009;104: 673–678. 19262524. 5. Rogler G, Aldeguer X, Kruis W, et al. Concept for a rapid point-of-care calprotectin diagnostic test for diagnosis and disease activity monitoring in patients with inflammatory bowel disease: expert clinical opinion. J Crohns Colitis. 2013;7:670–677. 6. Sipponen T, Savilahti E, Kolho KL, et al. Crohn’s disease activity assessed by fecal calprotectin and lactoferrin: correlation with Crohn’s disease activity index and endoscopic findings. Inflamm Bowel Dis. 2008;14: 40–46. 7. Molander P, af Björkesten CG, Mustonen H, et al. Fecal calprotectin concentration predicts outcome in inflammatory bowel disease after induction therapy with TNF alpha blocking agents. Inflamm Bowel Dis. 2012;18:2011–2017. 8. De Vos M, Dewit O, D’Haens G, et al. Fast and sharp decrease in calprotectin predicts remission by infliximab in anti-TNF naïve patients with ulcerative colitis. J Crohns Colitis. 2012;6:557–562.
1076
| www.ibdjournal.org
2015
9. Schoepfer AM, Beglinger C, Straumann A, et al. Ulcerative colitis: correlation of the Rachmilewitz endoscopic activity index with fecal calprotectin, clinical activity, C-reactive protein, and blood leukocytes. Inflamm Bowel Dis. 2009;15:1851–1858. 10. Wagner M, Peterson CG, Ridefelt P, et al. Fecal markers of inflammation used as surrogate markers for treatment outcome in relapsing inflammatory bowel disease. World J Gastroenterol. 2008;14:5584–5589. 11. Sipponen T, Björkesten CG, Färkkilä M, et al. Faecal calprotectin and lactoferrin are reliable surrogate markers of endoscopic response during Crohn’s disease treatment. Scand J Gastroenterol. 2010;45:325–331. 12. Roseth AG, Fagerhol MK, Aadland E, et al. Assessment of the neutrophil dominating protein calprotectin in feces. A methodologic study. Scand J Gastroenterol. 1992;27:793–798. 13. Moum B, Jahnsen J, Bernklev T. Fecal calprotectin variability in Crohn’s disease. Inflamm Bowel Dis. 2010;16:1091–1092. 14. Naismith GD, Smith LA, Barry SJ, et al. A prospective single-centre evaluation of the intra-individual variability of faecal calprotectin in quiescent Crohn’s disease. Aliment Pharmacol Ther. 2013;37:613–621. 15. Lasson A, Stotzer PO, Ohman L, et al. The intra-individual variability of faecal calprotectin: a prospective study in patients with active ulcerative colitis. J Crohns Colitis. 2015;9:26–32. 16. Kolho KL, Alfthan H, Hämäläinen E. Effect of bowel cleansing for colonoscopy on fecal calprotectin levels in pediatric patients. J Pediatr Gastroenterol Nutr. 2012;55:751–753. 17. Walmsley RS, Ayres RC, Pounder RE, et al. A simple clinical colitis activity index. Gut. 1998;43:29–32. 18. Silverberg MS, Satsangi J, Ahmad T, et al. Toward an integrated clinical, molecular and serological classification of inflammatory bowel disease: report of a Working Party of the 2005 Montreal World Congress of Gastroenterology. Can J Gastroenterol. 2005;19(suppl A):5A–36A. 19. Schroeder KW, Tremaine WJ, Ilstrup DM. Coated oral 5-aminosalicylic acid therapy for mildly to moderately active ulcerative colitis. A randomized study. N Engl J Med. 1987;317:1625–1629. 20. Coorevits L, Baert FJ, Vanpoucke HJ. Faecal calprotectin: comparative study of the Quantum Blue rapid test and an established ELISA method. Clin Chem Lab Med. 2013;51:825–831. 21. Wassell J, Wallage M, Brewer E. Evaluation of the Quantum Blue rapid test for faecal calprotectin. Ann Clin Biochem. 2012;49(pt 1):55–58. 22. Lobatón T, López-García A, Rodríguez-Moranta F, et al. A new rapid test for fecal calprotectin predicts endoscopic remission and postoperative recurrence in Crohn’s disease. J Crohns Colitis. 2013;7: e641–651. 23. Joshi S, Lewis SJ, Creanor S, et al. Age-related faecal calprotectin, lactoferrin and tumour M2-PK concentrations in healthy volunteers. Ann Clin Biochem. 2010;47(pt 3):259–263. 24. Poullis A, Foster R, Shetty A, et al. Bowel inflammation as measured by fecal calprotectin: a link between lifestyle factors and colorectal cancer risk. Cancer Epidemiol Biomarkers Prev. 2004;13:279–284. 25. Dobrzanski C, Pedersen N, Voxen Hansen V, et al. Is there a diurnal variation in faecal calprotectin in inflammatory bowel disease patients? United European Gastroenterol J. 2013;1(suppl 1):A209. 26. Labaere D, Smismans A, Van Olmen A, et al. Comparison of six different calprotectin assays for the assessment of inflammatory bowel disease. United European Gastroenterol J. 2014;2:30–37. 27. Burri E, Manz M, Rothen C, et al. Monoclonal antibody testing for fecal calprotectin is superior to polyclonal testing of fecal calprotectin and lactoferrin to identify organic intestinal disease inpatients with abdominal discomfort. Clin Chim Acta. 2013;416:41–47.
High Within-day Variability of Fecal Calprotectin Levels in Patients with Active Ulcerative Colitis: What Is the Best Timing for Stool Sampling? Margalida Calafat, MD,*,† Eduard Cabré, MD, PhD,* Míriam Mañosa, MD, PhD,* Triana Lobatón, MD, PhD,* Laura Marín, BSc,* and Eugeni Domènech, MD, PhD*
Background: Fecal calprotectin (FC) is considered the best noninvasive way to assess disease activity in ulcerative colitis (UC). However, it is not known which is the more suitable moment for stool sampling in patients with increased stool frequency. The aims of this study were to assess the intraindividual variation of FC within day and to evaluate if the first bowel movement in the morning is the more suitable sample for FC measurement in patients with acute flares of UC. Patients and Methods: Patients admitted because of active UC were invited to collect samples from several bowel movements (including the first in the morning) during the same day providing their ordinal chronology. FC was measured by means of a quantitative rapid point-of-care test based on lateral flow assay immunochromatography.
Results: Eighteen patients were included for a total of 56 stool samples. Most patients had extensive UC and severe disease activity. Within-day FC values varied widely, and the median coefficient of variation was 40% (5%–114%) with a median range of variation of FC values of 3887 mg/kg (69– 9946). The sample from the first stool in the morning obtained the highest individual FC within-day value in 33.3% of cases and the lowest in 38.9%. Conclusions: FC values widely vary between motions in patients with active UC. Stool sample collection from the first bowel movement in the morning does not ensure the highest or lowest within-day FC value. In patients with overt active UC, a single FC determination should not be used as the basis for therapeutic strategies. (Inflamm Bowel Dis 2015;21:1072–1076) Key Words: fecal calprotectin, ulcerative colitis, stool sampling
F
ecal calprotectin (FC), a leukocyte-derived protein, has emerged as one of the most sensitive noninvasive surrogate markers of inflammatory activity in ulcerative colitis (UC). FC is a useful tool for patients in clinical remission because it has proved to be accurate in predicting UC relapse1 and to have a good correlation with endoscopic2 and histological remission,3 allowing for the identification of patients at risk for clinical relapse and the avoidance of endoscopic examination in certain clinical situations. However, its role in active disease is not well established. In mild active disease, FC may differentiate symptoms due to UC or
Received for publication November 13, 2014; Accepted January 19, 2015. From the *Hospital Universitari Germans Trias i Pujol, Ciberehd, Badalona, Spain; and †Universitat Autònoma de Barcelona, Barcelona, Spain. E. Domènech received a research grant (Beca d’intensificació 2013) from the Catalonian Society of Gastroenterology (Societat Catalana de Digestologia) that partly supported this study. AbbVie provided FC-QPOCT kits and devices. The authors have no conflicts of interest to disclose. Guarantor of article: E. Domènech is acting as the submission’s guarantor. Reprints: Eugeni Domènech, MD, PhD, Hospital Universitari Germans Trias i Pujol, IBD Unit, Gastroenterology Department, Carretera del Canyet s/n, 08916 Badalona, Spain (e-mail: [email protected]). Copyright © 2015 Crohn’s & Colitis Foundation of America, Inc. DOI 10.1097/MIB.0000000000000349 Published online 19 March 2015.
1072
| www.ibdjournal.org
2015
to coexisting functional intestinal disorders. In severe active UC, some authors observed a relationship between baseline FC and the risk of colectomy,4 and cutoff FC values were recently agreed on by a panel of experts to define active and severely active UC to aid physicians with treatment modification decisions.5 Moreover, it has been proposed that FC could be useful to monitor response to biological therapies.6–8 However, considerable overlaps in FC values have been observed among patients with different degrees of severity in disease activity,6,9 and many studies found no correlation between baseline FC and response to therapy.7,10,11 Finally, although a homogeneous distribution of FC in human feces has been shown,12 a significant day-to-day13,14 and within-day15 variation in FC has been observed in patients with inflammatory bowel disease (IBD), mainly in mild-to-moderate active disease. This raised a growing interest in stool sampling issues because they might be relevant to FC-based decision-making strategies, and there is no general advice on which stool sample is most suitable for FC measurement. The aims of this study were therefore to assess the intraindividual variations in within-day FC values in patients with acute flares of UC and to evaluate whether the first bowel movement in the morning was the most suitable sample for FC measurement. Inflamm Bowel Dis Volume 21, Number 5, May 2015
Inflamm Bowel Dis Volume 21, Number 5, May 2015
PATIENTS AND METHODS Patients and Data Collection Between June 2013 and July 2014, all patients admitted to our unit because of an acute UC flare were invited to participate in the study after informed consent, regardless of their ongoing treatment, disease extent, and severity. Patients were given 4 numbered stool collector pots and were instructed to take a stool sample from the first bowel movement and from 1 to 3 additional bowel movements within a 24-hour period. Stool sampling was not allowed during bowel cleansing or within 48 hours after colonoscopic examination to avoid the impact of bowel cleansing on FC levels.16 Intestinal bacterial superinfections were ruled out in all cases by means of stool culture and the determination of Clostridium difficile toxins. The study was approved by the Institutional Review Board of the Hospital Universitari Germans Trias i Pujol. In addition to demographic (gender, age) and biological variables (C-reactive protein, hemoglobin, and albumin), many clinical variables were also recorded (date of diagnosis, date of hospital admission, UC extent, and treatment received) including all the parameters of the Simple Clinical Colitis Activity Index17 as follows: bowel frequency, bowel frequency during nighttime, urgency of defecation, rectal bleeding, general well-being, and extraintestinal manifestations. Disease severity was evaluated according to the Montreal’s classification,18 and the endoscopic Mayo subscore was used to assess endoscopic disease severity.19 Regarding stool sampling, the date of collection, number of samples, and the chronologic order of samples for each patient were registered.
FC Measurement
All samples were stored at 2208C, thawed, and analyzed by a quantitative point-of-care test (FC-QPOCT) (Quantum Blue; Bühlmann Laboratories AG, Schönenbuch, Switzerland). This test uses lateral flow assay technology, including an easy-to-use reader system allowing for a quantitative read out. There are 2 FCQPOCT kits: a low-range kit and a high-range kit, which provide quantitative results within minutes, ranging from 30 to 300 mg/kg or 100 to 1800 mg/kg FC, respectively. This FC-QPOCT testing demonstrated a good correlation with the conventional enzymelinked immunosorbent assay test for FC determination20,21 even for Crohn’s disease and UC.2,22 Because all patients had an active disease at the time of sample collection, the high-range FCQPOCT kit was initially used for FC measurement in all samples. All samples scoring .1800 mg/kg were performed again after sample dilution to obtain their exact value. Additionally, when wide variations in FC values of different samples from the same patient were observed, FC measurements were repeated at least twice more.
Statistical Analyses Data are expressed as median and range or absolute and relative frequencies. To assess the correlation of FC values of
Intraindividual Variability of FC in Active UC
different stool samples within a day, the coefficient of variation (CV) was calculated. The Spearman’s rho, Student’s t test, and chisquare test were used as needed to assess the correlation between CV and maximal/minimal FC values in the first sample in the morning and the presence of nocturnal bowel motions, rectal bleeding, urgency/incontinence, C-reactive protein levels, hemoglobin concentration, or disease severity. All statistical analyses were carried out in SPSS12.0 for Windows (SPSS Inc. Chicago, IL).
RESULTS Baseline characteristics of the 18 included patients are summarized in Table 1. Most patients had severe disease activity (72%) at the time of stool sample collection, and all but 2 had extensive UC. Endoscopic assessment was available in 15 cases (83%), with two-thirds of them (66%) showing severe mucosal lesions (Mayo subscore 3). In agreement with this, only 5 patients had normal C-reactive protein levels and 2 had normal hemoglobin concentration. The median number of bowel movements for all patients was 7.5 (2–12), 83% had rectal bleeding, and 72% had nocturnal bowel movements. Fifteen patients (83%) were receiving rectal therapy during admission, and this might account for the very low rate of urgency or incontinence (11%) among the included patients. A total of 56 stool samples were collected (8 patients with 4 samples, 8 patients with 3 samples, and 2 patients with 2 samples) after a median of 4 days since hospital admission (range, 1–12). The median highest FC value was 6927 mg/kg (288–15,870), whereas the median lowest value was 1659 mg/kg (101– 13,870). Within-day FC values varied widely, and the median CV was 40% (5%–114%) with a median range of variation of FC values of 3887 mg/kg (69–9946). Figure 1 shows the individual median, the highest and the lowest values of FC. CV was similar irrespective of mean FC levels (Fig. 2). C-reactive protein
TABLE 1. Characteristics of Patients Male/female Mild/moderate/severe Disease extent (UC) Proctitis/left-sided/extensive Ongoing therapy (at the time of stool samples collection) Aminosalicylates Systemic corticosteroids Thiopurines Anti-TNF agents Rectal corticosteroids or aminosalicylates C-reactive protein, mg/L Hemoglobin, g/dL Albumin, g/L
10/8 1/4/13 0/2/16
14 12 8 3 15 16.5 (1–171) 10.5 (7–14) 34.5 (27–42)
Values expressed as absolute numbers or median (range).
www.ibdjournal.org |
2015
1073
Inflamm Bowel Dis Volume 21, Number 5, May 2015
Calafat et al
FIGURE 1. Median, highest, and lowest intraindividual values of FC.
levels, hemoglobin concentration, median FC values, endoscopic severity score, the total number of bowel movements, or the presence of rectal bleeding, urgency/incontinence, severe activity, or nocturnal bowel movements were not associated with the CV. The sample from the first stool in the morning obtained the highest FC within-day value in 33.3% of patients and the lowest in 38.9%. For the remaining samples, maximal within-day FC values were obtained with the second sample in 44.4%, in the third sample in 18.7% (in 3 out of 16 patients with at least 3
samples), and in the fourth sample in 12.5% (in 1 out of 8 patients with 4 samples). Figure 3 shows the intraindividual variation of FC values regarding the chronology of sample collection. Having the highest or lowest individual FC values at the first stool in the morning was not associated with C-reactive protein levels, hemoglobin concentration, FC values, the total number of bowel movements, or the presence of rectal bleeding, urgency/incontinence, severe activity, or nocturnal bowel movements.
DISCUSSION
FIGURE 2. Correlation between individual mean FC levels and the corresponding SD.
1074
| www.ibdjournal.org
2015
FC has emerged as the best noninvasive surrogate marker of disease activity in UC. There is an increasing use of FC in quiescent disease, mainly for predicting disease relapse and mucosal healing. Recently, some authors posed its usefulness for predicting and/or monitoring response to therapy, suggesting that FC might be relevant for decision making in active disease. However, several factors have been reported as influencing FC, such as colonic cleansing,16 age,23,24 diet, or exercise.24 In addition, it has been hypothesized that in patients with active IBD, the amount of mucus or blood (which can vary widely between motions) could influence FC levels. From this perspective, decisionmaking strategies based on single measurements of FC might be wrong if high intraindividual variability was demonstrated. Little is known about the intraindividual variability of FC in IBD. Moum et al,13 in a study of patients with mild-to-moderate active Crohn’s disease, found significant differences in FC in 63 pairs of stool samples collected in 2 consecutive days. Conversely, a low variability was observed in 3 daily consecutive fecal samples from 93 Crohn’s disease patients in clinical remission.14 Dobrzanski et al,25 in a study published only in abstract form, reported a higher FC diurnal variation between samples collected from all motions during 3 consecutive days in
Inflamm Bowel Dis Volume 21, Number 5, May 2015
Intraindividual Variability of FC in Active UC
FIGURE 3. Intraindividual variation of calprotectin values regarding the chronology of stool sample collection.
outpatients with active UC as compared with inactive UC and active or inactive Crohn’s disease. These data confirm that variability may be only relevant in active disease and particularly in UC in which mucus and rectal bleeding are more often present in stools. Lasson et al15 recently reported their results in 18 patients with mild-to-moderate active UC in whom 2 samples from 2 different parts of the feces were collected at every bowel movement for 2 days. The median CV of FC during the day was 52%, being more pronounced in patients with high FC levels. In agreement with this study, our data clearly show a great within-day variability of FC in patients with active UC, with a median CV of 40%, close to that previously reported; however, we did not find any correlation between CV and median individual FC. The second important finding of our study is that the timing of stool sampling does not seem to be relevant because the highest or lowest FC values may also vary during the day. It could be argued that we did not assess some macroscopic characteristics of stools, such as mucus, blood, or consistency that may influence FC levels. Regarding this issue, Lasson et al were not able to find a correlation between stool consistency, content of blood, or time between bowel movements and CV. Moreover, they found a strong correlation in terms of FC concentrations between random samples of the same bowel movement, although mucus and blood may not be evenly distributed in feces.15 The same authors concluded that patients with no bowel movements during the night should preferably collect stool samples at the first defecation in the morning because they found a significant positive correlation between the level of FC and the time between bowel movements. However, in a similar small series, we did not find any relationship between median FC of the first stool in the morning and rectal
bleeding or nocturnal bowel movements. The differences between Lasson’s study and ours must be mainly due to the characteristics of the population. Although in the former, only outpatients with mild-to-moderate active UC were included, we included inpatients with moderate-to-severe active disease. In addition, most of our patients were on topical therapy, whereas this was an exclusion criterion in the former study. Technical aspects may also explain controversial results between studies, as recently shown in a study comparing different calprotectin assays for the assessment of IBD.26 In this sense, the dilution for FC values .1800 mg/kg that was performed in our study might potentially contribute to the variability of within-day FC values; however, this simple method had been previously performed for enzyme-linked immunosorbent assay measurement of FC.27 The relevance of the great within-day variation of FC will depend on the clinical setting and how we use FC values for decision making. In mild disease, Lasson et al15 found that up to 33% of patients had at least 1 FC measurement below the threshold of disease activity (250 mg/kg), whereas in severe disease, we only observed 15% of patients with at least 1 FC measurement below 1000 mg/kg, a threshold that has been proposed to define severe activity.5 However, it has also been reported that baseline FC higher than 1992 mg/kg may identify those patients with UC who will not respond to intravenous corticosteroids.4 In this sense, 8 of our 18 patients (44%) had values below and over this threshold. Some authors posed the drop of FC between baseline and some weeks of therapy (DFC) as a predictive tool of response to biological agents8; in this scenario, the baseline value may widely vary leading to decreased prediction accuracy of DFC. From this point of view, it seems more reasonable to use semiquantitative FC measurement at least for UC acute severe flares (a situation in www.ibdjournal.org |
2015
1075
Inflamm Bowel Dis Volume 21, Number 5, May 2015
Calafat et al
which FC levels are dramatically elevated and variability is high), because it should be cheaper and more rapid. In summary, FC may vary greatly within day in patients with moderate-to-severe active UC. There is no apparent benefit in sampling the first stool in the morning for FC measurement. Decision-making strategies based on single quantitative FC determinations in severe active UC are not advisable.
ACKNOWLEDGMENTS The authors want to thank the staff of Palex Medical SA (Spain) for their technical support in fecal calprotectin measurements. Author contributions: E. Domènech designed the study, recruited patients, performed statistical analyses, and drafted the article. M. Calafat recruited patients, stored samples, performed fecal calprotectin measurements, collected data, performed statistical analyses, and drafted the article. E. Cabré performed statistical analyses and reviewed the article. M. Mañosa, T. Lobatón, and L. Marín recruited patients, collected data, and reviewed the article.
REFERENCES
1. Mao R, Xiao YL, Gao X, et al. Fecal calprotectin in predicting relapse of inflammatory bowel diseases: a meta-analysis of prospective studies. Inflamm Bowel Dis. 2012;18:1894–1899. 2. Lobatón T, Rodríguez-Moranta F, Lopez A, et al. A new rapid quantitative test for fecal calprotectin predicts endoscopic activity in ulcerative colitis. Inflamm Bowel Dis. 2013;19:1034–1042. 3. Guardiola J, Lobatón T, Rodríguez-Alonso L, et al. Fecal level of calprotectin Identifies Histologic inflammation in patients with ulcerative colitis in clinical and endoscopic remission. Clin Gastroenterol Hepatol. 2014; 12:1865–1870. 4. Ho GT, Lee HM, Brydon G, et al. Fecal calprotectin predicts the clinical course of acute severe ulcerative colitis. Am J Gastroenterol. 2009;104: 673–678. 19262524. 5. Rogler G, Aldeguer X, Kruis W, et al. Concept for a rapid point-of-care calprotectin diagnostic test for diagnosis and disease activity monitoring in patients with inflammatory bowel disease: expert clinical opinion. J Crohns Colitis. 2013;7:670–677. 6. Sipponen T, Savilahti E, Kolho KL, et al. Crohn’s disease activity assessed by fecal calprotectin and lactoferrin: correlation with Crohn’s disease activity index and endoscopic findings. Inflamm Bowel Dis. 2008;14: 40–46. 7. Molander P, af Björkesten CG, Mustonen H, et al. Fecal calprotectin concentration predicts outcome in inflammatory bowel disease after induction therapy with TNF alpha blocking agents. Inflamm Bowel Dis. 2012;18:2011–2017. 8. De Vos M, Dewit O, D’Haens G, et al. Fast and sharp decrease in calprotectin predicts remission by infliximab in anti-TNF naïve patients with ulcerative colitis. J Crohns Colitis. 2012;6:557–562.
1076
| www.ibdjournal.org
2015
9. Schoepfer AM, Beglinger C, Straumann A, et al. Ulcerative colitis: correlation of the Rachmilewitz endoscopic activity index with fecal calprotectin, clinical activity, C-reactive protein, and blood leukocytes. Inflamm Bowel Dis. 2009;15:1851–1858. 10. Wagner M, Peterson CG, Ridefelt P, et al. Fecal markers of inflammation used as surrogate markers for treatment outcome in relapsing inflammatory bowel disease. World J Gastroenterol. 2008;14:5584–5589. 11. Sipponen T, Björkesten CG, Färkkilä M, et al. Faecal calprotectin and lactoferrin are reliable surrogate markers of endoscopic response during Crohn’s disease treatment. Scand J Gastroenterol. 2010;45:325–331. 12. Roseth AG, Fagerhol MK, Aadland E, et al. Assessment of the neutrophil dominating protein calprotectin in feces. A methodologic study. Scand J Gastroenterol. 1992;27:793–798. 13. Moum B, Jahnsen J, Bernklev T. Fecal calprotectin variability in Crohn’s disease. Inflamm Bowel Dis. 2010;16:1091–1092. 14. Naismith GD, Smith LA, Barry SJ, et al. A prospective single-centre evaluation of the intra-individual variability of faecal calprotectin in quiescent Crohn’s disease. Aliment Pharmacol Ther. 2013;37:613–621. 15. Lasson A, Stotzer PO, Ohman L, et al. The intra-individual variability of faecal calprotectin: a prospective study in patients with active ulcerative colitis. J Crohns Colitis. 2015;9:26–32. 16. Kolho KL, Alfthan H, Hämäläinen E. Effect of bowel cleansing for colonoscopy on fecal calprotectin levels in pediatric patients. J Pediatr Gastroenterol Nutr. 2012;55:751–753. 17. Walmsley RS, Ayres RC, Pounder RE, et al. A simple clinical colitis activity index. Gut. 1998;43:29–32. 18. Silverberg MS, Satsangi J, Ahmad T, et al. Toward an integrated clinical, molecular and serological classification of inflammatory bowel disease: report of a Working Party of the 2005 Montreal World Congress of Gastroenterology. Can J Gastroenterol. 2005;19(suppl A):5A–36A. 19. Schroeder KW, Tremaine WJ, Ilstrup DM. Coated oral 5-aminosalicylic acid therapy for mildly to moderately active ulcerative colitis. A randomized study. N Engl J Med. 1987;317:1625–1629. 20. Coorevits L, Baert FJ, Vanpoucke HJ. Faecal calprotectin: comparative study of the Quantum Blue rapid test and an established ELISA method. Clin Chem Lab Med. 2013;51:825–831. 21. Wassell J, Wallage M, Brewer E. Evaluation of the Quantum Blue rapid test for faecal calprotectin. Ann Clin Biochem. 2012;49(pt 1):55–58. 22. Lobatón T, López-García A, Rodríguez-Moranta F, et al. A new rapid test for fecal calprotectin predicts endoscopic remission and postoperative recurrence in Crohn’s disease. J Crohns Colitis. 2013;7: e641–651. 23. Joshi S, Lewis SJ, Creanor S, et al. Age-related faecal calprotectin, lactoferrin and tumour M2-PK concentrations in healthy volunteers. Ann Clin Biochem. 2010;47(pt 3):259–263. 24. Poullis A, Foster R, Shetty A, et al. Bowel inflammation as measured by fecal calprotectin: a link between lifestyle factors and colorectal cancer risk. Cancer Epidemiol Biomarkers Prev. 2004;13:279–284. 25. Dobrzanski C, Pedersen N, Voxen Hansen V, et al. Is there a diurnal variation in faecal calprotectin in inflammatory bowel disease patients? United European Gastroenterol J. 2013;1(suppl 1):A209. 26. Labaere D, Smismans A, Van Olmen A, et al. Comparison of six different calprotectin assays for the assessment of inflammatory bowel disease. United European Gastroenterol J. 2014;2:30–37. 27. Burri E, Manz M, Rothen C, et al. Monoclonal antibody testing for fecal calprotectin is superior to polyclonal testing of fecal calprotectin and lactoferrin to identify organic intestinal disease inpatients with abdominal discomfort. Clin Chim Acta. 2013;416:41–47.
