Received: 18 January 2021 DOI: 10.1002/ueg2.12069 REVIEW ARTICLE - Accepted: 26 February 2021 International consensus on methodological issues in standardization of fecal calprotectin measurement in inflammatory bowel diseases Ferdinando D'Amico1,2 | David T. Rubin3 | Paulo Gustavo Kotze4 | Fernando Magro5 | Britta Siegmund6 | Taku Kobayashi7 | Pablo A. Olivera8 | Peter Bossuyt9 | Lieven Pouillon9 | Edouard Louis10 | Eugeni Domènech11,12,13 | Subrata Ghosh14 | Silvio Danese1,15 | Laurent Peyrin‐Biroulet2 1 Department of Biomedical Sciences, Humanitas University, Milan, Italy Abstract 2 Department of Gastroenterology and Inserm Background: Fecal calprotectin (FC) is a non‐invasive marker of gut inflammation U1256, Nutrition – Genetics and Exposure to which is frequently used to guide therapeutic decisions in patients with inflamma- Environmental Risks, University Hospital of Nancy, University of Lorraine, Vandoeuvre‐lès‐ tory bowel diseases (IBD). Each step of FC measurement can influence the results, Nancy, France leading to misinterpretations and potentially impacting the management of IBD 3 Section of Gastroenterology, Hepatology and Nutrition, University of Chicago Department patients. To date, there is high heterogeneity between FC measurements and no of Medicine, Chicago, Illinois, USA current method is universally accepted as a standard. 4 IBD Outpatient Clinics, Catholic University or Aims: Our aim was to provide clear position statementsabout the pre‐analytical and Paraná (PUCPR), Curitiba, Brazil 5 the analytical phases of FC measurement to homogenize FC levels and to minimize Department of Gastroenterology, Centro Hospitalar São João, Porto, Portugal variability and risk of misinterpretation through aninternational consensus. 6 Medizinische Klinik m. S. Gastroenterologie, Materials & Methods: Fourteen physicians with expertise in the field of IBD and FC Infektiologie und Rheumatologie, Charité ‐ from 11 countries attended a virtual international consensus meeting on July 17th, Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt‐ 2020. A systematic literature was conducted and the literature evidence was shared Universität zu Berlin, and Berlin Institute of and discussedamong the participants. Statements were formulated, discussed, and Health, Berlin, Germany 7 voted. Statements were considered approved if all participants agreed. Center for Advanced IBD Research and Treatment, Kitasato University Kitasato Results: Nine statements were formulated and approved. Based on the available Institute Hospital, Tokyo, Japan evidence, quantitative tests should be preferred for measuring FC. Furthermore, FC 8 Gastroenterology Section, Department of Internal Medicine, Centro de Educación measurement, if possible, should always be performed with the same method and Médica e Investigaciones Clínicas (CEMIC), factors influencing FC levels should be taken into account when interpreting the Buenos Aires, Argentina results. 9 Imelda GI Clinical Research Center, Imelda General Hospital, Bonheiden, Belgium Discussion: FC has an increasingly important role in the management of patients 10 Department of Gastroenterology, CHU Liège with IBD. However, large multicenter studies should be conducted to define the University Hospital, Liège, Belgium reproducibility and to confirm the diagnostic accuracy of the available FC tests. 11 Gastroenterology Department, Hospital - This is an open access article under the terms of the Creative Commons Attribution‐NonCommercial‐NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made. © 2021 The Authors. United European Gastroenterology Journal published by Wiley Periodicals LLC. on behalf of United European Gastroenterology. United European Gastroenterol J. 2021;9:451–460. wileyonlinelibrary.com/journal/ueg2 451 452 - UNITED EUROPEAN GASTROENTEROLOGY JOURNAL Universitari Germans Trias i Pujol, Badalona, Catalonia, Spain Conclusion: FC concentrations guide clinicians' treatment decisions. Our state- 12 Departament de Medicina, Universitat ments have a relevant impact in daily practice and could be applied in clinical trials Autònoma de Barcelona, Barcelona, Spain to standardize FC measurement. 13 Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas KEYWORDS (CIBEREHD), Madrid, Spain fecal calprotectin, inflammatory bowel disease, measurement, standardization 14 NIHR Biomedical Research Centre, University of Birmingham and University Hospitals NHS Foundation Trust, Birmingham, Key Summary UK Summarise the established knowledge on this subject. 15 IBD Center, Humanitas Research Hospital, ‐ FC is a surrogate non‐invasive marker of gut inflammation. IRCCS, Milan, Italy ‐ FC is closely correlated with endoscopic and histological activity of disease. Correspondence ‐ High variability exists between FC measurements. Laurent Peyrin‐Biroulet, Inserm Nutrition – ‐ There is no globally accepted cut‐off of FC. Genetics and Exposure to Environmental Risks and Department of Gastroenterology, Nancy University Hospital, University of What are the significant findings of this study? Lorraine, 1 Allée du Morvan, Vandoeuvre‐lès‐ ‐ Stool consistency can influence FC extraction. Nancy 54511, France. ‐ Quantitative tests are recommended for FC measurement. Email: peyrinbiroulet@gmail.com ‐ Serial FC measurement in an individual patient should be performed with the same FC Funding information test. Amgen ‐ Interpretation of FC measurement results should include the evaluation of factors that may influence the test. INTRODUCTION date, no standardization regarding FC measurement is available. Thus, following a comprehensive literature review,8 we aimed to Crohn's disease (CD) and ulcerative colitis (UC) are chronic provide clear position statements about the pre‐analytical and the inflammatory bowel diseases (IBD) with a remitting and relapsing analytical phases of FC measurement to homogenize FC levels and course requiring periodic follow‐up.1,2 Endoscopy is the gold stan- to minimize variability and risk of misinterpretation through an in- dard for IBD monitoring as it directly visualizes the mucosa to ternational consensus. 3 assess the presence of inflammation. However, endoscopy is expensive, time‐consuming, can cause discomfort to patients and requires bowel preparation.4–6 To overcome these limitations, the METHODS use of fecal calprotectin (FC) has been proposed.7 FC is considered a surrogate non‐invasive marker of the infiltration of neutrophils in A virtual consensus meeting was organized on July 17th, 2020 to the intestinal mucosa and an increase in FC levels is associated with define the optimal method for measuring FC in order to reduce intestinal inflammation.8 FC plays an increasingly crucial role in the the heterogeneity and homogenize the measurements. Fourteen management of IBD patients. It is closely correlated with endo- physicians with expertise in the field of IBD and FC (FDA, DTR, scopic and histological activity of disease and allows to distinguish PGK, FM, BS, TK, PO, PB, LP, EL, ED, SG, SD, and LPB) from IBD from irritable bowel syndrome (IBS), assesses disease activity eleven countries worldwide (Argentina, Belgium, Brazil, England, and response to therapy, and predicts disease recurrence.7,9,10 France, Germany, Italy, Japan, Portugal, Spain, United States) Despite its proven clinical utility, high variability exists between FC attended the meeting. A systematic search of the scientific litera- measurements, preventing the definition of a globally accepted cut‐ ture was conducted in the PubMed/MEDLINE, EMBASE [Excerpta 11,12 off for the interpretation of FC results. FC measurement con- Medica Database], and Cochrane databases up to January 2020 to sists of two main phases, a pre‐analytical and an analytical phase.8 identify all studies reporting data on FC measurement. Results The pre‐analytical phase involves timing of stool sampling, collec- from the literature search were recently published in extenso.8 8,13 tion, storage, and FC extraction. The test to be used and the The literature evidence was shared and discussed among the threshold for result interpretation must be decided during the participants. Subsequently, statements were formulated and dis- analytical phase.8,13 Each step can influence FC results and cussed. Statements were considered approved if all participants contribute to the heterogeneity of FC measurement.8 It is important agreed. If a statement was not unanimously accepted, it was re‐ to emphasize that an error in this process can cause impaired discussed and amended until all participants approved it. All ex- assessment of disease activity, impacting the medical decision‐ perts were involved in drafting the manuscript and approved its making process and leading to an over‐ or under‐treatment. To final version. D'AMICO ET AL. - 453 TABLE 1 Statements for pre‐analytical and analytical phases of fecal calprotectin measurement Statement 1 Feces should be collected in a dedicated clean container without additives to avoid any accidental contamination. Statement 2 The most appropriate timing for stool sampling is unclear. Statement 3 The analysis of a single stool sample is usually sufficient for FC measurement. Statement 4 Stool consistency can influence FC extraction. Statement 5 Stool storage at room temperature is preferably limited to 3 days, although up to one week is still acceptable. If the sample cannot be processed within 7 days, feces should be stored in a freezer at −20 C° for an optimal conservation. Statement 6 Quantitative tests are recommended for FC measurement. The ELISA tests and automated ELISA tests have an accurate analytical performance and should be preferred. Point of care tests and home tests represent valid alternatives to ELISA tests. Statement 7 There is insufficient evidence to support the use of one specific FC test over another. Statement 8 The tests for FC measurement are not interchangeable because there is a high variability between the different methods. If possible, serial FC measurement in an individual patient should be performed with the same FC test. Statement 9 Interpretation of FC measurement results should include the evaluation of factors that may influence the test. Abbreviations: ELISA, enzyme‐linked immunosorbent assay; FC, fecal calprotectin. RESULTS values.17 All patients were asked to collect up to four stool samples within 24 h. In most cases, the highest FC value was obtained with the All statements on the pre‐analytical and the analytical phases of FC second stool sample of the day (44%) and a high variability between the measurements are shown in Table 1. different samples was found (median coefficient of variation = 40%).17 Similarly, Kristensen et al. examined the FC values of three stool samples (morning, evening, and next morning) from 50 IBD patients, Pre‐analytical phase reporting a high coefficient of variation (39.4%, 95% CI 31.1%– 47.7%).18 Two other studies enrolling patients with active disease Statement 1: Feces should be collected in a dedicated clean container evaluated the variability of FC concentrations during the day, report- without additives to avoid any accidental contamination. ing variations of 52% and 61% between measurements.11,16 It was The most common method of fecal sampling is with a dedicated noted that the longer the time between bowel movements, the higher container that is equipped with a spoon on the lid. This allows pa- the FC values, suggesting that the first sample in the morning could be tients to handle the stool in a hygienically safe and convenient way. preferred as it guaranteed the longest interval in patients without night The spoon usually collects less than 5 g of feces, while the containers evacuations.16 A prospective observational cohort study including 45 13 can generally contain up to 60 g of feces. Of note, the amount patients with IBD (of which 21 with clinically active disease) showed needed for the analysis is definitely lower, about 100 mg.12,14,15 A that variability in FC concentrations ranged from 64% to 77% based on crucial aspect of this step is to avoid sample contamination with time of collection (morning, afternoon, evening).19 Interestingly, if liquids (e.g., water or urine) in order to prevent sample dilution, but to samples were stratified according to time of collection and FC reassure patients that if they are passing liquid stool, it should still be threshold, a relatively small variability rate (7%) was found analyzing collected. Lasson et al. found that stool consistency was significantly the first feces in the morning with a cut‐off of 250 µg/g.19 The available correlated with FC levels (median r = 0.68, range: −0.68 to 0.87; evidence is not sufficient to recommend the correct timing of FC p = 0.01), noting that the more liquid the stools, the higher the FC measurement and further studies on large populations are needed to concentrations.16 It is important to underline that the presence of address this topic. It is important to emphasize that within‐day vari- liquid stools is not a contraindication to the FC assay as it is the most ability is less relevant when considering a cut‐off which is significantly frequent situation in patients with active disease. However, stool higher than the values generally used in clinical practice to define consistency should be taken into account when interpreting the re- disease activity (>300 µg/g). For now, patients' convenience may drive sults. To date, it is not known whether the impact of liquid stools on the collection timing. Given this variability, patients should be advised FC values is due to technical issues related to greater difficulty in to collect stool samples approximately at the same time in successive collecting liquid stool, to issues with sampling heterogeneity, or to FC measurements. the increased disease activity in these patients. Statement 3: The analysis of a single stool sample is usually Statement 2: The most appropriate timing for stool sampling is sufficient for FC measurement. unclear. The need to repeat the FC assay is heavily debated. A single‐ Several studies investigated the most appropriate timing for stool center prospective study recruiting 98 CD patients in clinical sampling. Calafat and colleagues enrolled 18 patients with acute UC remission assessed the FC variability on three consecutive days.15 flares to assess the intra‐individual variations in within‐day FC Consistency between FC levels was high as evidenced by the intra‐ 454 - UNITED EUROPEAN GASTROENTEROLOGY JOURNAL class correlation (ICC) value (0.84, 95% CI: 0.79–0.89). On the other sample cannot be processed within 7 days, feces should be stored in a hand, a relevant day‐to‐day variability was described in UC patients freezer at −20 C° for an optimal conservation. with a median coefficient of variation of 40.8% between two Stool storage is an essential step in FC measurement as the delay consecutive days.16 An observational case‐control study by Cremer between stool sampling and analysis can lead to sample deterioration and colleagues enrolling patients with active and inactive disease and to invalidate FC results. Røseth et al. investigated the tempera- confirmed the intra‐individual variability of FC concentrations be- ture impact on stool storage in 111 patients (33 healthy subjects, 40 tween two different bowel movements within 1–6 days period, hospital controls, 21 CD, and 17 UC).24 No significant difference in reporting a median variation coefficient of 36%.12 At the same time, FC concentrations was found between fresh samples and those the FC amount in different punches of the same stool sample was stored at room temperature (+20°C) for 7 days, suggesting that FC also analyzed, finding a low median intra‐stool variability (17%). was resistant to intestinal proteolysis for up to one week. On the Another study including 63 patients with active CD found a high other hand, a study including patients with active UC showed that FC variability among the samples collected on two consecutive days levels remained stable at room temperature for up to 3 days, while (kappa 0.355; p < 0.0002).20 Interestingly, the variability between after 7 days a mean reduction of 28% was reported (p < 0.01; 95% CI measurements was greater with increasing FC levels. Furthermore, 0.10–0.47).16 Padoan et al. compared the stability of the samples only in 6 cases (9%) a contrasting value (remission vs. disease activity, stored at room temperature with those stored at 4°C. After one using a FC threshold of 200 mg/L) between the first and second week, a greater sample deterioration was detected in samples at measurement was found. These data are of crucial importance as room temperature (p = 0.004), suggesting that temperature could they indicate that variability rarely affects therapeutic decisions. alter FC concentrations.25 Stool freezing at minus 20°C has been Consequently, despite the intra‐individual variability between FC proposed to limit the progressive sample deterioration. Oyaert et al. measurements, repetition of FC assessment on two consecutive days assessed the effect of freezing on six extraction assays, revealing that is not to be recommended.20 Instead, it may be prudent to repeat the in most cases (4/6, 66.6%) no reduction in FC occurred after freezing procedure within a few weeks in case of borderline values that could at minus 20°C.26 Finally, Pelkmans et al. compared FC results in fresh influence the therapeutic decision, or in the suspicion of measure- fecal extracts and in aliquots frozen at minus 20°C of the same ment inaccuracy and factors invalidating the result. sample, confirming stability and comparability of the obtained Statement 4: Stool consistency can influence FC extraction. values.27 Based on these data, it would be preferable to process and The extraction phase allows to separate the calprotectin from analyze the stool samples as soon as possible. The physical charac- 8 the feces. This procedure requires that feces are first weighed and teristics of calprotectin allow it to be stable at room temperature for then positioned inside a buffer to obtain protein extraction. Weighing 3 days in optimal conditions and up to one week in acceptable con- can be manually performed, but currently it is preferable to use de- ditions with a minimal reduction in FC concentrations. In case of vices that are specific to each measuring kit in order to collect a further delay between collection and analysis, the unprocessed stool 8 predefined amount of feces and to reduce sample manipulation. sample should be frozen to avoid its deterioration or sampling should There is high variability among commercially available extractors. be repeated. Whitehead et al. found different FC values with three different ex- tractors (Roche extraction device, Schebo device, Immunodiagnostik device) compared to manual extraction method and the lowest FC Analytical phase values were detected by analyzing liquid feces, suggesting how stool consistency could influence test results.21 Similarly, Juricic et al. Statement 6: Quantitative tests are recommended for FC measure- compared two extractors (EliA™ Calprotectin EliA SEK device and ment. The enzyme‐linked immunosorbent assay (ELISA) tests and fCAL®Turbo Calex®Cap “N”), detecting significant differences in FC automated ELISA tests have an accurate analytical performance and values based on stool consistency (27.0, 77.0, and 277.0 mg/kg vs. should be preferred. Point of care (POC) tests and home tests 103.5, 249.0, and 203.5 mg/kg in hard, normal, and liquid feces represent valid alternatives to ELISA tests. respectively, p < 0.001).22 Liquid stools have been associated with Growing evidence shows that different FC values can be asso- both increase and decrease in FC levels and should therefore be ciated with different outcomes in IBD patients, including endoscopic avoided to prevent measurement errors. To overcome this technical activity or remission and histological remission of disease.8,9,28,29 issue, feces should be diluted in the extraction buffer and eventually Thus, it is of extreme relevance for clinicians to quantify FC con- centrifuged allowing the detection of sedimented particles. In addi- centrations. For this reason, quantitative tests are recommended tion, the use of a mix of four extracts from the same fecal sample over qualitative tests as the latter only express a positive or negative showed to be well correlated with the standard extraction of a single result according to the range and cut‐off provided by the manufac- punch (r2 = 0.8517), indicating how this technique could homogenize turer without indicating the FC value. The ELISA test is the most the sample to be analyzed and reduce the intra‐individual frequently used quantitative test for FC measurement and has been variability.23 used for other purposes for almost 30 years.24,30 ELISA tests have an Statement 5: Stool storage at room temperature is preferably accurate analytical performance but are time‐consuming as they are limited to 3 days, although up to 1 week is still acceptable. If the commonly performed as a batch‐like procedure every 1 or D'AMICO ET AL. - 455 2 weeks.8,31,32 Automated ELISA tests are currently available allow- were also found in predicting moderate‐severe endoscopic disease ing to analyze the samples at any time, even individually. Importantly, activity in both CD and UC patients. As regards to mild disease activity, the diagnostic accuracy of these tests is equivalent to the traditional acceptable performance values were identified in patients with UC, ELISA method.33–40 Recently, point of care rapid tests and home tests while low sensitivity values were reported in CD patients. Overall, all have been developed. POC tests use specific lateral flow immuno- assays were significantly correlated (p < 0.05) and no method proved to 41–50 assays. Patients provide a stool sample which is collected, and an be superior to the others. Further comparative studies on large IBD extract of the sample is prepared for analysis by a healthcare pro- populations are needed to assess whether a specific ELISA test, POC fessional. Subsequently, the extracted sample is inserted into a test, or home test has better diagnostic accuracy compared to the reader and the quantitative or semi‐quantitative results are dis- others and should be preferred. played on a connected computer within 30 min. POC tests are highly Statement 8: The tests for FC measurement are not inter- correlated with traditional ELISA tests41–50 and their direct and in- changeable because there is a high variability between the different direct costs are lower than traditional ELISA tests.30 The home tests methods. If possible, serial FC measurement in an individual patient allow to measure FC directly at home. It is imperative that patients should be performed with the same FC test. have a smartphone compatible with the measurement test and that A head‐to‐head trial compared the performance of two ELISA they download an application to perform data analysis.51 In addition tests (CalproTM and Calprest®) in assessing disease activity in 116 to collecting the feces, patients carry out the extraction process and patients with UC.65 Both tests accurately predicted endoscopic disease after placing the feces in the specific reading cassette, they proceed activity defined as Mayo endoscopic score (MES) of 2–3 (AUC = 0.79 with the sample analysis through the camera of their smartphone. for CalproTM and AUC = 0.80 for Calprest®). However, different FC Finally, the result is forwarded to the patient's healthcare team to cut‐offs were identified (148 mg/kg for Calprest® and 208 mg/kg for remotely monitor FC levels. The home tests provide semi‐ CalproTM) and a difference of up to 30% in FC levels was found be- quantitative results and are sufficiently correlated with both stan- tween the tools when FC values between 200 and 1000 mg/kg were dard ELISA and POC tests.52–58 Interestingly, the correlation is high analyzed. Similarly, Oyaert and colleagues investigated the accuracy of when FC values ≤500 μg/g are taken into consideration (agreement 6 automated ELISA tests in distinguishing between functional and interval of 560, 455, and 458 μg/g with IBDoc®, QuantonCal®, and organic bowel disorders.66 A total of 105 patients with IBD, other TM CalproSmart respectively), while the agreement is low when FC gastroenterological diseases, or rheumatological conditions were concentrations are >500 μg/g (agreement interval of 1160, 2392, enrolled, and all stool samples were extracted with the same device to TM and 1310 μg/g with IBDoc®, QuantonCal®, and CalproSmart reduce the variability risk. All tests showed a sensitivity of 100% and a respectively).57,59 This aspect suggests that home tests can be used in specificity ranging from 58% to 78% at the manufacturer threshold of daily clinical practice as threshold values ≤500 μg/g generally guide 50 μg/g. The areas under the curves (AUC) were very high (from 0.974 the therapeutic decisions.60,61 It should be emphasized that the use to 0.998) for all tests and no statistically significant difference was of home tests requires high patient compliance and adequate training found. On the other hand, total imprecision values ranged from 1.5% to to avoid reduced test adherence or measurement errors.55,62 23.3% and the difference between the tests increased with increasing Statement 7: There is insufficient evidence to support the use of FC concentrations. For this reason, it is legitimate to assume that the one specific FC test over another. tests currently available to measure FC are not interchangeable. For Multiple tests are available for FC monitoring and several studies patient follow‐up, preferentially, the same assay should be used. It have compared their operating characteristics in order to identify the would be desirable to have a reference stool sample for FC at the optimal method to be used in clinical practice. A study by Mirsepasi‐ initiation of a new treatment which can be compared with the endo- Lauridsen et al. compared the accuracy of 3 ELISA tests (EK‐CAL®, scopic evaluation at baseline, ensuring the reliability of the biochemical CALPROTM, and HK325®) in diagnosing IBD.63 CALPROTM ELISA test finding. This could allow for the evaluation of FC trend and could be achieved higher rates of specificity compared to the other tests but the used for patient monitoring, predicting any recurrence of disease and study was neither designed nor powered to demonstrate the superi- facilitating interpretation of the results. ority of one method over the others. A head‐to‐head comparative trial Statement 9: Interpretation of FC measurement results should evaluated the characteristics of 3 FC home tests.59 No difference in include the evaluation of factors that may influence the test. terms of agreement with ELISA tests was found, but fewer reading FC is a marker of gut inflammation, but it is not specific for IBD as errors (defined as an image of the test cassette not leading to quanti- some gastrointestinal diseases, medication, and lifestyle can alter its tative result) were found with the IBDoc® compared with Cal- concentrations.8 Increased FC values have been found in patients with TM proSmart and QuantonCal® (1.9% vs. 5.8% and 4.8%, p < 0.05 for IBS, colon polyps, colonic diverticular disease, colon cancer, gastroin- both comparisons). Moreover, a prospective cohort study investigated testinal bleeding, gastrointestinal infections, microscopic colitis, the performance of 6 tests (3 POC tests, 2 ELISA tests, and one auto- proctitis after radiation therapy, pouchitis, rheumatologic diseases, mated ELISA test) for the diagnosis and follow‐up of 62 patients with and liver cirrhosis (Table 2).67–77 In addition, some medications such as 64 suspected or confirmed diagnosis of IBD. The tests showed a sensi- non‐steroidal anti‐inflammatory drugs (NSAIDs) and proton pump in- tivity of 75%–83% and a specificity of 68%–95% for the diagnosis of hibitors (PPI) have been associated with increased FC levels (up to IBD considering a FC cut‐off of 50 μg/g. High sensitivity and specificity 520 µg/g in NSAIDs users and 150 µg/g in PPI users).78–82 Prolonged 456 - UNITED EUROPEAN GASTROENTEROLOGY JOURNAL T A B L E 2 Factors associated with increased fecal calprotectin pregnant women could be associated with a beneficial effect on disease concentration activity.93 Finally, interpretation of FC values should take into Range of FC increase consideration all factors that may influence the FC results. In daily Gastrointestinal diseases (µg/g) clinical practice, physicians should evaluate the alignment between FC Colorectal neoplasia 70 57–133 concentrations and endoscopic and histologic data. The correlation between these findings could support the reliability of the fecal test. Colon polyps68 1–117.7 On the other hand, if no correlation is found or if there are factors Colonic diverticular disease69 <15–60 influencing FC levels, FC measurement should be repeated after a few Bacterial and viral gastrointestinal infections72 0–994 weeks. 71 Gastrointestinal bleeding <20–429 73 Liver cirrhosis 21–357 RESEARCH GAPS 67 Irritable bowel syndrome 16–294 Microscopic colitis74 130–480 Although FC has an increasingly important role in the management of 75 patients with IBD, some research questions need to be further Proctitis after radiation therapy 50–270 addressed. The standardization of FC measurement could allow to Pouchitis76 55–110 reduce the heterogeneity between the studies and to obtain more Medication reliable data. First, it is necessary to define whether the timing of Non‐steroidal anti‐inflammatory drugs 82 5–520 stool collection can affect the measurement results, clarifying 79 whether the repetition of the FC measurement is useful or not Proton pump inhibitors 50–150 (Figure). Second, the best strategy for stool storage at room tem- Lifestyle perature should be investigated and a comparative study evaluating Obesity90 5–185 FC concentrations in stool samples analyzed within 3 or 7 days Physical inactivity87 25–60 should be conducted to address this question. Third, it is necessary to Other define how to deal with liquid stools as this is the most frequent situation in IBD patients, defining the best approach for stool Age < 9 years85 18–213 collection and homogenization. Fourthly, large multicenter studies Age > 65 years85 14–118 should be conducted to define the reproducibility and to confirm the Bowel preparation for colonoscopy84 51–17,379 diagnostic accuracy of the available FC tests. Fifthly, to date, there is Rheumatologic diseases 77 14–513 no generally accepted FC cut‐off to distinguish activity or disease remission. The homogenization of the FC assay could allow clinicians Perianal disease91 207–1705 to identify and validate a threshold that predicts disease activity, Stoma88 <150–1130 guiding therapeutic decisions and representing a new treat‐to‐target Abbreviation: FC, fecal calprotectin. for IBD patients. use of NSAIDs can lead to NSAID‐induced enteropathy which is CONCLUSION characterized by intestinal erosions and ulcers.82 For this reason, NSAIDs should be discontinued at least two weeks before FC mea- FC is increasingly recognized as a treatment target for UC and CD 82 surement. The rationale for the increase in FC after PPI treatment is and is commonly used in daily practice and clinical trials for the unknown, however it may be related to the inhibition of gastric acid management of IBD patients. Several pre‐analytical and analytical production, resulting in intestinal bacterial overgrowth.78,83 Similarly, variables can influence the FC measurement. We summarize PPIs should be suspended 4 weeks prior to FC measurement, allowing available data and propose recommendations concerning FC for adequate drug wash out and preventing any measurement bias.80 measurement in order to standardize the method. The main Furthermore, patients' age, including younger (<9 years) and older limitation of our statements is the absence of a validated method- patients (>65 years), body mass index (obesity), physical inactivity, ology to reach consensus such as the Delphi method. To the best of perianal disease, presence of an ostomy, and bowel preparation have our knowledge, this is the first international consensus addressing 84–91 been reported as factors associated with increased FC values. FC this topic. A uniformly accepted protocol for FC measurement could is strongly correlated with disease activity in pregnant women with allow to reduce inter‐assay variability and increase measurement IBD and is frequently used for monitoring these patients.92 However, a reliability. Furthermore, large studies using a standardized method trend of progressive reduction in FC levels was found during pregnancy are needed to identify a FC cut‐off, predicting disease activity and of IBD women suggesting that the immune changes occurring in guiding therapeutic decisions. D'AMICO ET AL. - 457 FC: fecal calprotectin FIGURE 1 Research gaps that could be filled with standardization of fecal calprotectin measurement A CK N O WL ED GM E N T S Medical, EA Pharma, Kyorin Pharmaceutical, Mochida Pharmaceutical, We thank Materia Prima for organizing the international virtual Nippon Kayaku, Otsuka Holdings, Sekisui Medical, Thermo Fisher meeting and supporting the paper. This study was funded by Amgen Scientific, Zeria Pharmaceutical. Paulo Gustavo Kotze reports personal via Materia Prima. fees from Abbvie, Janssen, Pfizer and Takeda; research grants from Pfizer and Takeda. Pablo A. Olivera received consulting fees from C ON F L I C T O F I N T E R ES T Abbvie, Takeda, and Janssen and lecture fees from Takeda and Ferdinando D’Amico declares no conflict of interest. David T. Rubin has Janssen. Peter Bossuyt received has received financial support for received grant support from Takeda; and has served as a consultant for research from AbbVie, Mundipharma, Pfizer, Janssen, Amgen, and Abbvie, Abgenomics, Allergan Inc., Biomica, Boehringer Ingelheim Ltd., Mylan; lecture fees from AbbVie, Takeda, Pfizer, and Janssen; advisory Bristol‐Myers Squibb, Celgene Corp/Syneos, Check‐cap, Dizal board fees from Abbvie, Takeda, Hospira, Janssen, Celltrion, BMS, Pharmaceuticals, GalenPharma/Atlantica, Genentech/Roche, Gilead Roche, Arena, MSD, Mundipharma, Roche, Pfizer, Sandoz, and Pentax. Sciences, GlaxoSmithKline Services, Ichnos Sciences S.A., InDex Lieven Pouillon received advisory board fees from Janssen and Takeda; Pharmaceuticals, Janssen Pharmaceuticals, Lilly, Narrow River Mgmt, presentation fees from Abbvie and Ferring; and personal fees from Pfizer, Prometheus Laboratories, Reistone, Shire, Takeda, and Techlab Abbvie, Ferring, Norgine, and Takeda. Edouard Louis reports grants Inc. Fernando Magro has served as a speaker and received honoraria from European Commission for BIOCYCLE Research Programme; from Merck Sharp & Dohme, Abbvie, Vifor, Falk, Laboratorios Vitoria, grants and personal fees from Abbvie, Takeda, Pfizer and Janssen and Ferring, Hospira, and Biogen. Britta Siegmund has served as Consultant personal fees from MSD, Ferring, Falk, and Celgene. Eugeni Domènech for Abbvie, Boehringer, Celgene, Falk, Janssen, Lilly, Pfizer, has served as a speaker and has received research and educational Prometheus, Takeda and received speaker's fees from Abbvie, CED funding and advisory fees from MSD, AbbVie, Takeda, Kern Pharma, Service GmbH, Falk, Ferring, Janssen, Novartis, Takeda (BS served as Pfizer, Janssen, Celgene, Adacyte Therapeutics, Otsuka Pharmaceuti- representative of theCharité). Subrata Ghosh declares consulting fees cals, Ferring, Shire Pharmaceuticals, Tillots, Thermofisher, Grifols, and from Pfizer, Janssen, AbbVie, Takeda, Bristol‐Myers Squibb, Receptos, Gebro. Silvio Danese has served as a speaker, consultant, and advisory Celgene, Gilead, Eli Lilly and Boehringer Ingelheim and speaker fees board member for Schering‐Plough, AbbVie, Actelion, Alphawasser- from AbbVie, Janssen, Takeda, Ferring, Shield, and Falk Pharma; man, AstraZeneca, Cellerix, Cosmo Pharmaceuticals, Ferring, outside of the submitted work. Taku Kobayashi has served as a Genentech, Grunenthal, Johnson and Johnson, Millenium Takeda, speaker, a consultant or an advisory board member for AbbVie, MSD, Nikkiso Europe GmbH, Novo Nordisk, Nycomed, Pfizer, Phar- Ajinomoto Pharma, Asahi Kasei Medical, Astellas, Alfresa Pharma, macosmos, UCB Pharma and Vifor. Laurent Peyrin‐Biroulet has served Celltrion, Covidien, EA Pharma, Eisai, Eli Lilly, Ferring Pharmaceuticals, as a speaker, consultant and advisory board member for Merck, Abb- Gilead Sciences, Janssen, JIMRO, Kyorin Pharmaceutical, Mitsubishi vie, Janssen, Genentech, Mitsubishi, Ferring, Norgine, Tillots, Vifor, Tanabe Pharma, Mochida Pharmaceutical, Nippon Kayaku, Pfizer, Hospira/Pfizer, Celltrion, Takeda, Biogaran, Boerhinger‐Ingelheim, Takeda Pharmaceutical, Thermo Scientific, Zeria Pharmaceutical, and Lilly, HAC‐ Pharma, Index Pharmaceuticals, Amgen, Sandoz, For‐ward received research funding from AbbVie, Alfresa Pharma, Asahi Kasei Pharma GmbH, Celgene, Biogen, Lycera, Samsung Bioepis, Theravance. 458 - UNITED EUROPEAN GASTROENTEROLOGY JOURNAL A U TH O R’ S C O N T R I B U T IO N 6. Shafer LA, Walker JR, Waldman C, Yang C, Michaud V, Bernstein Ferdinando D'Amico, wrote the first draft and created table and figure, CN, et al. Factors associated with anxiety about colonoscopy: the preparation, the procedure, and the anticipated findings. Dig Dis Sci. discussed the statements and contributed to the final manuscript. Da- 2018;63:610–8. vid T Rubin, critically reviewed the content of the paper, Paulo Gustavo 7. Mumolo MG, Bertani L, Ceccarelli L, Laino G, Fluri GD, Albano E, et al. Kotze, critically reviewed the content of the paper, discussed the From bench to bedside: fecal calprotectin in inflammatory bowel statements and contributed to the final manuscript. Fernando Magro, diseases clinical setting. World J Gastroenterol. 2018;24:3681–94. 8. D’Amico F, Nancey S, Danese S, Peyrin‐Biroulet L. A practical guide critically reviewed the content of the paper, discussed the statements for faecal calprotectin measurement: myths and realities. J Crohns and contributed to the final manuscript. Britta Siegmund, critically Colitis. 2021;15(1):152–61. reviewed the content of the paper, discussed the statements and 9. D’Amico F, Bonovas S, Danese S, Peyrin‐Biroulet L. Review article: contributed to the final manuscript. Taku Kobayashi, critically reviewed faecal calprotectin and histologic remission in ulcerative colitis. Aliment Pharmacol Ther. 2020;51:689–98. the content of the paper, discussed the statements and contributed to 10. Guardiola J, Lobatón T, Rodríguez‐Alonso L, Ruiz‐Cerulla A, Arajol C, the final manuscript. Pablo A. Olivera, critically reviewed the content of Loayza C, et al. Fecal level of calprotectin identifies histologic the paper, discussed the statements and contributed to the final inflammation in patients with ulcerative colitis in clinical and endo- manuscript. Peter Bossuyt, critically reviewed the content of the paper, scopic remission. Clin Gastroenterol Hepatol. 2014;12:1865–70. discussed the statements and contributed to the final manuscript. 11. Toyonaga T, Kobayashi T, Nakano M, Saito E, Umeda S, Okabayashi S. 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