Expert recommendations regarding the use of fecal calprotectin in daily clinical practice: statements of a taskforce process from the Hellenic Group for the Study of Inflammatory Bowel Disease (EOMIFNE)

Giorgos Bamiasa, Smaragdi Fessatoub, Christina Kapizionic, Konstantina Kitsoud, Georgios Kokkotisa, Afroditi Kourtib, Panagiotis Markopoulose, Eleni Orfanoudakif, Spyros Siakavellasg, Maria Tzouvalah

Sotiria” Hospital, National and Kapodistrian University of Athens; Attikon University General Hospital, National and Kapodistrian University of Athens; Athens Medical Center; National and Kapodistrian University of Athens; “Metaxa” Memoriam Hospital; General Hospital of Chania, Crete; “Hippocration” General Hospital of Athens, National and Kapodistrian University of Athens; “Agios Panteleimon” General Hospital, Nikaia, Piraeus, Greece

aGI Unit, 3rd Department of Internal Medicine, Sotiria Hospital, National and Kapodistrian University of Athens (Giorgos Bamias, Georgios Kokkotis); bDepartment of Pediatric Gastroenterology, Hepatology and Nutrition, 3rd Department of Paediatrics, Attikon University General Hospital, National and Kapodistrian University of Athens (Smaragdi Fessatou, Afroditi Kourti); cDepartment of Gastroenterology, Athens Medical Center (Christina Kapizioni); dNational and Kapodistrian University of Athens (Konstantina Kitsou); eDepartment of Gastroenterology, “Metaxa” Memoriam Hospital (Panagiotis Markopoulos); fDepartment of Gastroenterology, General Hospital of Chania, Crete (Eleni Orfanoudaki); gLiver-GI Unit, 2nd Academic Department of Internal Medicine, “Hippocration” General Hospital of Athens, National and Kapodistrian University of Athens (Spyros Siakavellas); hDepartment of Gastroenterology, “Agios Panteleimon” General Hospital, Nikaia, Piraeus, (Maria Tzouvala), Greece

Correspondence to: Panagiotis Markopoulos, Department of Gastroenterology, “Metaxa” Memoriam Hospital, Athens, Greece, e-mail: panosmarkmd@yahoo.gr
Received 6 November 2025; accepted 6 January 2026; published online 13 February 2026
DOI: 10.20524/aog.2026.1040
© 2026 Hellenic Society of Gastroenterology

Abstract

Fecal calprotectin (FC) has become an indispensable tool in everyday clinical practice for the diagnosis and management of inflammatory bowel disease (IBD). Nonetheless, specific and clear recommendations for its use are scarce. On behalf of the Hellenic Group for the Study of IBD (EOMIFNE), a group of experts have formulated 18 statements with the aim to provide evidence for the best use of FC and offer practical guidance in common clinical scenarios. Statements address issues for the proper application of FC measurement in the diagnostic workup of patients with gastrointestinal symptoms, and as a screening tool for the management of established IBD, including pediatric patients. Thus, the present manuscript aims to propose a standardized approach for the optimal use of FC in patient care, also taking into account non-medical concerns.

Keywords Fecal calprotectin, inflammatory bowel disease, EOMIFNE, guidelines

Ann Gastroenterol 2026; 39 (2): 161-173

Introduction

Calprotectin constitutes the major cytosolic protein of neutrophils, and is released from those cells in response to pathogen-associated molecular patterns or danger-associated molecular patterns. Its concentration is an indicator of tissue infiltration by neutrophils, which signifies the presence of active inflammation. Accordingly, fecal calprotectin (FC) is secreted by infiltrating neutrophils that are present in the intestinal tissue during inflammatory processes, including inflammatory bowel disease (IBD); thus, measurement of FC has emerged in recent years as an accurate and valuable noninvasive biomarker for gastrointestinal inflammation [1,2]. Those characteristics have led to the inclusion of FC in numerous national and international guidelines, and the proposal for its use: a) as a screening tool for the diagnosis of IBD; and b) as a monitoring instrument in patients with established IBD [3-5].

Despite the ample existing literature that supports the applicability and clinical relevance of measurement of FC in patients with IBD, the test is not currently reimbursed in Greece. This may in part be due to the fact that the optimal use of FC in everyday practice has not yet been clearly defined, including its applicability in specific clinical scenarios. To address these unmet needs, the Hellenic Group for the Study of IBD (EOMIFNE) organized a taskforce with the goal of formulating expert recommendations that aim: a) to clarify “gray areas” and dictate clinical decisions about the optimal use of FC for the management of Greek patients; b) to serve as a practical guide for gastroenterologists, but also other specialists (primary care physicians, rheumatologists, internists); and c) facilitate regulatory agencies in decision-making regarding the implementation of FC measurements in the reimbursement plan of the National Health system in Greece.

Methodology

A taskforce was organized consisting of two senior IBD experts as chairpersons (GB and MT), and several active EOMIFNE members who were assigned specific predefined subtopics of major interest related to the use of FC. The core members of this taskforce considered that strict application of the GRADE methodology was impractical, and proposed the use of a hybrid approach, which consisted of a systematic literature review in the context of a modified GRADE system, expert discussion, and voting to form consensus recommendations. Details regarding the way the level of evidence and strength of recommendations were derived are summarized in Table 1.

Table 1 Details on how levels of evidence and strength of recommendations were derived using a hybrid approach, incorporating both the modified GRADE system and voting outcomes

thumblarge

For each topic of interest, a thorough literature search was conducted for relevant publications in the PubMed, EMBASE and MEDLINE databases. The selection of published papers for further analysis was based on specific inclusion and exclusion criteria. In particular, only articles written in the English language were included. In addition, priority was given to papers with higher levels of evidence, such as randomized controlled trials (RCTs) and meta-analyses (MA). Finally, more recent publications were also given higher priority.

This evidence-based process resulted in the development of a draft containing several conditional recommendations for the use of FC. This first draft of statements was circulated for additional comments among all EOMIFNE members. A final online meeting was then scheduled, with the participation of an expert panel of 30 adult and pediatric gastroenterologists. During this meeting, additional refinement of the statements took place, followed by voting for each statement. Final approval was granted only to those statements that achieved an 80% rate of agreement among the expert panelists.

Results

Technical aspects of FC measurement

We recommend the use of fresh stool samples for FC measurements. If this is not feasible, samples can be stored at 4oC for up to 1 week until final analysis. (Strong recommendation, Consensus 100%)

Studies consistently report significant FC degradation in samples stored at room temperature (25oC), which can begin as early as within the first 24 h [6,7]. Consequent decreases in FC concentration correlate to the duration of storage prior to analysis [6-10] and can reach up to 50% after 1 week [7]. To mitigate this reduction, samples should be stored at 4°C until analysis, as data support a clear superiority of refrigeration [6,7,10], with reported preservation of stable calprotectin values for up to 1 week post-collection [6]. Calprotectin extraction using buffers has been shown to enhance the maintenance of stable values after 1 week when stored at 4°C [5,6], but there are equivocal results for their storage at room temperature. However, the longevity of FC regarding its degradation in the commercially available extraction buffers is highly dependent on the assay manufacturer’s specifications. Hence, all procedures should be conducted in accordance with the respective instructions [7]. Deep freezing (<-20°C) of samples is superior to refrigeration [9], and it is preferred for sample preservation longer than 1 week, since stable calprotectin values can be maintained for 6-12 weeks.

We recommend that a single measurement of FC at a specific time point is adequate for clinical decision-making, as day-to-day, intra-day, and intra-assay variability do not significantly impact the result. (Strong recommendation, Consensus 96%)

Significant intra-day variability in FC values has been observed, although not uniformly. In several studies, diurnal fluctuation of FC was reported to be significant [8,11-13] and was attributed to various factors. Lasson et al reported statistically significant elevations in FC levels during evening bowel movements and in looser stools, and a trend for higher values in bloody stools [8], although the correlation between elevated FC values and evening bowel movements was not confirmed in other studies [13,14]. It is noteworthy that this intra-day variation is more profound in cohorts of patients with active IBD [12,13], even though it does not result in clinically meaningful variations. In contrast to diurnal variation, intra-assay and day-to-day variability appear to be negligible. In particular, when FC levels from the same bowel movement were measured at 2 distinct time points, to calculate intra-assay variability, the resulting values demonstrated a strong intraclass correlation of 0.79 [10] and 0.91 [15]. Day-to-day variability was reported in only 1 cohort of 63 patients, with low clinical relevance (9% for the 200 μg/g and 5% for the 50 μg/g threshold [16]. In other studies, day-to-day variability was either absent [15,17] or not statistically significant [14]. It should be noted that variation may be particularly pronounced at higher FC values, yet instances of deviations concerning patient positivity are infrequent. Therefore, it is reasonable to conclude that a single measurement of FC is adequate in clinical practice.

We recommend that quantitative FC testing, when available, should be preferred over qualitative assays. Enzyme-linked immunosorbent assays (ELISA) and automated ELISA platforms in certified laboratories offer the most accurate measurements. Point-of-care (POC) and at-home testing devices are considered reliable alternatives. (Strong recommendation, Consensus 97%)

Currently, various immunobiochemistry methods that target proteinic epitopes in the stools are available for the measurement of FC concentration. Pretreatment of stool samples is required before measuring FC, to allow for the extraction of the protein in the appropriate buffer [18]. The standard method is the weighing method, which includes weighing of a certain amount of stool to achieve the required concentration in a buffer; however, this is time-consuming and impractical for a busy laboratory to adopt [18]. To address these issues, multiple specialized, commercially available extraction/measurement kits have been developed [18]. In a relevant study, the fluorescence-immunoassay EliA Stool Extraction Kit (EliA SEK, Thermo Fisher, Uppsala, Sweden) and the Calex® Cap (Calex, Bühlmann Laboratories AG, Schönenbuch, Switzerland) extraction device, which uses particle-enhanced turbidimetric immunoassay (PETIA) with BühlmannfCAL® Turbo assay (Bühlmann Laboratories AG, Schönenbuch, Switzerland), were compared against the weighing extraction method with favorable results [19]. To minimize the effect of liquid stool consistency in the evaluation of FC concentration, an additional step of dilution of a standard volume of liquid stool in the respective buffer volume, and centrifugation to exclude the sediment of the analysis can be considered [20,21]. It is also recommended to perform 4 extractions from 4 different locations of the stool specimen to ensure a representative FC concentration estimation on further analysis, as extraction from a single stool location leads to high heterogeneity for the same sample [19].

FC can be estimated using both qualitative (reporting a positive/negative result), and quantitative (reporting the exact concentration of FC in stools) methods [19]. Quantitative measurements are recommended in patients with IBD, given the high clinical relevance of accurate calculations of the precise concentration of FC [22]. The ELISA method, is considered the gold standard for FC measurement [23]. Multiple automated ELISA-based platforms have been developed (fluoro-enzyme immunoassays, chemiluminescence immunoassays, and particle-enhanced turbidimetric immunoassays), that need less laboratory manipulation time and can even be carried out individually for each sample. These assays are reliable, and correlate significantly with the standard ELISA methods, as they provide equivalent results [8,9,13,24-34].

Recently, POC devices have been developed, with strong correlation to ELISA platforms and good accuracy profile, thereby providing reliable results and enabling on-site quantitative or semi-quantitative (within a range of values) FC level estimation [23,35-43] within 30 min [37]. Another useful tool for monitoring patients with IBD are at-home, patient-processed testing devices, which also utilize immunochromatography and produce semi-quantitative results [44]. The result is uploaded to a corresponding application of the specific kit via a smartphone camera and sent to the treating physician. These assays demonstrate significant correlation to ELISA-based assays and POC devices [44-51]. It should be noted that proper patient training regarding the use of these medical devices is crucial for producing reliable results.

We recommend that the same assay should be used for sequential measurements of FC during follow up, given the high variability among different methods. (Strong recommendation, Consensus 100%)

Inter-assay differences in FC measurements values are commonly observed, and are characteristic of nearly all assay comparisons. In a study that compared 4 different assays—EliA calprotectin CN (Thermo Fisher Scientific), EliA calprotectin CN2 (Thermo Fisher Scientific), Liaison DiaSorin calprotectin (DiaSorin, S.p.A.), and Bühlmann fCAL turbo (Bühlmann Laboratories AG)—a high correlation was reported, although significant proportional bias was observed, especially at high FC values [26]. Similar findings for the comparison between FC-specific immunoassays have also been reported by other groups [32-34,40].

Consequently, although variability exists among the available assays, there is no conclusive evidence supporting the superiority of any particular assay. Thus, we do not advocate for the use of any specific assay. On the other hand, given the aforementioned inter-assay variability it is highly recommended that serial measurements of the same patient should preferably be made using the same assay.

FC as a screening tool for IBD (Table 2)

Table 2 Summary of recommended cutoff values and corresponding clinical interpretations by scenario

thumblarge

We recommend against the use of FC testing as a screening test in asymptomatic individuals. (Strong recommendation, strong level of evidence, Consensus 100%)

FC testing is a valuable tool for assessing intestinal inflammation, particularly in differentiating IBD from functional disorders such as irritable bowel syndrome. However, its application is not universal, and certain clinical scenarios warrant caution or alternative approaches [52]. Routine FC testing in asymptomatic individuals is not recommended. Elevated FC levels in the absence of symptoms may not correlate with significant pathology, and can lead to unnecessary investigations. Studies have shown that, while FC is a sensitive marker for inflammation, its specificity decreases in asymptomatic populations, leading to potential overdiagnosis of an inflammatory syndrome [5,53]. In patients with acute diarrhea (<4 weeks), especially when infectious etiologies are suspected, FC testing is not advised as a primary diagnostic tool. Acute infections can increase FC levels, but this does not distinguish between self-limiting infections and chronic inflammatory conditions. The Infectious Diseases Society of America suggests that FC/lactoferrin detection should not be used to establish the cause of acute infectious diarrhea, and there are insufficient data on the value of FC measurement in this context [54].

For patients over 50 years of age, or those presenting with alarm symptoms (e.g., unexplained weight loss, rectal bleeding, anemia), relying solely on FC testing is inadequate. While FC can be elevated in conditions other than inflammation, such as polyposis or neoplasms, its accuracy in detecting gastrointestinal malignancy is low. Endoscopy remains the gold standard for evaluating mucosal pathology and detecting intestinal pathology in high-risk groups, and should not be replaced by FC testing [54].

We recommend the use of FC testing with a cutoff of 50 μg/g to distinguish between IBD and functional disorders in patients with chronic (>4 weeks) abdominal symptoms suggestive of intestinal inflammation and a low risk for gastrointestinal malignancy. (Strong recommendation, strong level of evidence Consensus 100%)

In patients with chronic (>4 weeks) abdominal symptoms and low risk for malignancy, FC testing can help to distinguish between IBD and functional disorders. Incorporating FC testing into clinical practice for evaluating patients with lower gastrointestinal symptoms can reduce the number of unnecessary referrals for endoscopy, thereby decreasing healthcare costs [1,53-55]. FC concentrations in healthy individuals typically range between 10 and 50µg/g of stool, though this range can vary, depending on study cohorts and the assays used. The optimal cutoff value for FC testing, above which patients should be referred for colonoscopy, remains a topic of debate [53,55].

Although, the first meta-analysis demonstrated a cut-off level of 100 μg/g as the one with the highest accuracy to detect patients with IBD [56,57], subsequent evidence has supported a lower threshold, above which patients should be referred for further investigations. More specifically, a subsequent meta-analysis by van Rheenen et al [58] determined that the optimal cutoff point is 50 μg/g, with FC values below this threshold yielding pooled sensitivity and specificity of 0.93 and 0.96, respectively, making it unlikely that these patients have IBD. Additionally, several other studies have stipulated that threshold, including the British Society of Gastroenterology guidelines, as the likelihood of an IBD diagnosis at that range of values is very low [55,59-61].

Moreover, several essays have determined that higher levels of FC (>100 μg/g), are associated with higher diagnostic accuracy, but lower negative predictive values, resulting in higher rates of false negative results [62-64] leading to delayed diagnosis of IBD [58]. An even lower FC cutoff concentration of 40 μg/g has been shown to result in a very low false-negative rate (≤1%), according to a meta-analysis subsequently performed by Menees et al [65]. On the other hand, only 1 recent meta-analysis indicated that the difference in sensitivity and specificity between FC testing cutoffs of 50 μg/g and 100 μg/g was not statistically significant, suggesting that a threshold of 100 μg/g could be adopted as a pre-endoscopy screening tool [66]. In view of these conflicting results, in most cases, a cutoff of 50 μg/g is selected, as it seems to be the most reliable cutoff value.

FC as a monitoring tool in IBD (Table 2)

We recommend measuring FC at least twice annually, as a monitoring tool in patients with IBD in clinical remission. (Strong recommendation, moderate level of evidence, Consensus 100%)

Although no dedicated RCT exists to compare a symptom-alone vs. a biomarker plus symptom-based monitoring strategy in asymptomatic patients with ulcerative colitis (UC), indirect data from observational studies suggest that the latter strategy may offer advantages in the management of these patients. In a meta-analysis of 17 observational studies, including more than 1200 UC patients, performed in the context of the recent American Gastroenterological Association guidelines, it was shown that patients with elevated FC compared to those without had a relative risk of 4.4 (95% confidence interval [CI] 3.48-5.47) to experience disease relapse within a year [3]. The cutoff used by most, but not all, studies was FC>150 μg/g. In another recent meta-analysis of 24 prospective studies that aimed to identify the ideal cutoff for FC to predict relapse in IBD, an FC of 152 μg/g was the threshold shown to perform best. A subgroup analysis of the effect of IBD type (UC or Crohn’s disease [CD]) on that outcome showed similar results for both types [67].

Concerning CD, there is a similar lack of dedicated RCTs comparing symptoms alone versus a biomarker in conjunction with symptom monitoring strategy in asymptomatic patients with CD. The landmark CALM study had such a design, but it was conducted in patients with active disease on study entry [68]. Likewise, the more recently published STARDUST trial followed a similar design pattern, with the addition of early endoscopic assessment; nevertheless, the subjects had active symptoms at study initiation [69]. Thus, only indirect data are available to address the optimal use of FC as a biomarker for the management of patients with asymptomatic CD. As mentioned above, in a recent MA of 24 prospective studies that aimed to identify the ideal cutoff for FC to predict relapse in IBD, an FC of 152 μg/g was the threshold with the best performance, while subgroup analysis did not exhibit a significant difference between UC and CD patients [67]. Furthermore, from a collection of 12 cohort studies with close to 1000 CD patients it was reported that, in those with overt elevation of FC (>200 μg/g), there was an almost 5-fold increased risk for disease relapse [4]. However, there is the caveat that there was a high level of heterogeneity between these studies.

Taking into account the aforementioned evidence, it seems reasonable to incorporate FC in the follow up of asymptomatic patients with IBD, with a proposed frequency of at least twice yearly to correspond with the usual schedule of clinical monitoring.

We recommend endoscopic and/or imaging assessment of disease activity, rather than empiric treatment adjustment, in patients with IBD in clinical remission but persistently elevated FC>150 mg/g. (Strong recommendation, moderate level of evidence Consensus 97%)

There is no RCT addressing the question of whether different cutoffs for any biomarker, including FC, should affect therapeutic decisions with regard to treatment modifications and long-term outcomes. Nonetheless, taking into consideration the correlation between FC and important outcomes such as endoscopic healing, we reviewed studies assessing whether there is an optimal cutoff to define endoscopic activity, which is a recognized surrogate marker for worse long-term outcomes, demanding action from treating physicians.

In a retrospective study including 181 UC patients, FC was significantly correlated with both Mayo endoscopy score (MES) and UC endoscopic index of severity (UCEIS), although the association was stronger for the latter. Receiver operating characteristic (ROC) curve analysis indicated an FC cutoff level of 187.0 mg/kg for predicting complete mucosal healing (MH), defined as an MES value of 0 or a UCEIS value of 0. The sensitivity and specificity of a cutoff value of 187.0 mg/kg for complete MH were 0.857 and 0.891, respectively. FC predicts complete mucosal healing and correlates better with the UCEIS than with the MES in patients with UC [70]. Theede et al identified an FC cutoff of 192 μg/g for predicting MH by either an MES or UCEIS of 0, with similar sensitivity (75% and 79%, respectively) and specificity (88% and 87%, respectively) [71]. In a recent cross-sectional study from the UK, including UC patients, the ideal thresholds for FC to identify MH, defined as an MES of 0 and a UCEIS <2, were 112 and 148 μg/g, with accuracy of 86.9% and 86.8%, respectively [72].

Regarding CD, there is again a lack of RCTs, but we have results from 11 cohort studies showing that, for the endpoint of endoscopically active disease (defined as an SES-CD score ≥3) or endoscopic remission (SES-CD<3), the optimal sensitivity and specificity for a cutoff of 150±50 mg/g were found to be 81% (95%CI 74-87%) and 72% (95%CI 61-81%), respectively [4]. Moreover, from a recent meta-analysis including 25 studies and over 2500 CD patients, albeit with marked heterogeneity in FC cutoffs (the majority being >200 μg/g) and endoscopic endpoints, FC shows a pooled sensitivity of 81% and specificity of 74% for detecting endoscopic active disease, possibly denoting that FC is a valuable diagnostic tool across different phenotypes of CD [73]. Another recent cross-sectional study of 153 CD patients reported an optimal FC threshold of <92.9 μg/g, with a specificity of 89% and a sensitivity of 77% for detecting endoscopic activity, and proposed the combination of FC with intestinal ultrasound as an even better intervention [74]. In an older MA of 25 prospective observational studies assessing the role of FC in identifying disease activity in IBD patients, the authors found that FC had a pooled sensitivity of 85% and specificity of 75% in suggesting endoscopic activity. In a subgroup analysis studying 3 different cutoff levels (50, up to 100 and >100 μg/g) the authors suggested that the best sensitivity (90.6%) was achieved at 50 μg/g—thus, this is the ideal threshold to be used in clinical practice—whereas the best specificity (78.2%) was found at levels >100 μg/g [75]. The interpretation of this finding is that the higher the cutoff, the lower the sensitivity and the higher the specificity. Thus, we decided, based on additional data from the aforementioned studies, to propose a larger threshold in order to diminish false positive results and subsequently decrease unnecessary colonoscopies.

Nevertheless, given that the reliability of the test in a scenario with low pretest probability—as in the case where there is a discrepancy between symptoms and FC measurement—is lower, repeat measurement of FC values as an alternative in due time is a reasonable decision.

We suggest that treatment adjustment in symptomatic patients with IBD should not rely on symptoms alone, but should also take FC measurements into consideration. (Strong recommendation, moderate level of evidence, Consensus 96%)

We suggest further assessment, rather than empiric treatment adjustment, in symptomatic patients with IBD and FC >150 μg/g. (Strong recommendation, moderate level of evidence, Consensus 88%)

No RCT could be retrieved to compare a symptom-alone versus a biomarker/symptom-based monitoring strategy in symptomatic patients with UC. Nonetheless, indirect data from observational studies indicate that the latter strategy could offer advantages in the management of patients with UC. Symptoms have long been shown to be insufficient to reliably detect endoscopic activity. Indeed, in an individual patient data analysis from 6 registrational studies attempting to assess the relationship of Patient Reported Outcomes (PROs) with endoscopic activity, it was shown that there is a substantial percentage of patients (~10-15%) with Rectal Bleeding Subscore (RBS) 2/3 and Stool Frequency Subscore (SFS) 2/3 who had endoscopic remission (MES 0/1) [76]. In a patient-level analysis of 22 prospective observational cohort studies and 1 post hoc analysis of a phase 3 study, Dulai et al attempted to incorporate FC in clinical practice, along with the assessment of PROs. They only assessed predefined FC cutoff levels of ≤50(±10) μg/g or ≥250(±20) μg/g. Indeed, they showed that in the scenario of RBS 2/3 + SFS 2/3, an FC≥250 μg/g greatly diminishes the false positive rate to a percentage of less than 5%, thus potentially avoiding endoscopy [77]. This study shows that incorporating a more objective biomarker in addition to symptoms in the management of patients with UC improves important clinical outcomes.

In CD results are available from the CALM study, as mentioned earlier. This was an open-label RCT recruiting adult patients with moderate to severely active, non-stricturing, non-penetrating CD. Furthermore, these patients were symptomatic (with compounding biochemical and endoscopic results indicative of inflammation) at recruitment, and were also naïve to advanced treatment. After initial treatment with steroids, participants were randomized to a “tight control” group in which treatment escalation was based on biomarkers (including FC>250 mg/g) and/or symptoms, or a “clinical management” group in which treatment escalation was based on symptoms alone. Assessment was carried out per study protocol every 3 months. A total of 244 patients were included, with the “tight control” group showing better results than the “clinical management” group (37% vs. 23%) in achieving all study endpoints by 48 weeks [71]. On the other hand, in the STARDUST trial, timely escalation of ustekinumab treatment in CD patients, based on biomarkers and early endoscopic response, along with clinical symptoms, did not result in significantly better endoscopic outcomes at week 48 than symptom-driven decisions alone [72]. Thus, there seems to be a gap in our knowledge concerning the exact patient phenotype that will benefit the most from additional monitoring.

Currently, there is no consensus regarding the ideal FC cutoff that may accurately indicate the presence of inflammation in symptomatic patients with UC and CD. Indirect data suggest that a threshold of 150 μg/g may indicate the need for endoscopic assessment, and possibly a change in therapy, as it can reliably detect moderate-to-severe inflammation in the presence of symptoms, with an acceptable false positive rate. A higher cutoff may further lower false positive rates, but may inappropriately increase false negative cases. On the other hand, a lower cutoff would increase sensitivity. However, taking into consideration that this is a high pretest probability scenario (symptoms indicative of a flare plus raised FC), the fall in the percentage of false negatives would not be adequate to justify a further decrease in our proposed cutoff. We aimed to propose a cutoff that would provide an acceptable equilibrium between sensitivity, specificity, positive and negative predictive values.

There is also the probability that the combination of FC with novel utilities, such as intestinal ultrasound, will facilitate clinical-decision making for each individual patient.

Use of FC in special populations (Table 2)

We recommend against the use of FC measurement as a substitute for the endoscopic assessment of postoperative recurrence (POR) at 6-12 months after ileocecal resection in patients with CD. (Strong recommendation, low level of evidence, Consensus 100%)

Despite the evolution in IBD treatment, a high proportion of patients with CD (about 50% in the first decade) will still require surgical treatment. After surgery, risk-stratified care management should be implemented. High-risk patients receive treatment to prevent POR, and endoscopic evaluation is recommended 6-12 months postoperatively, aiming to optimize treatment and prevent further disease progression and complications [78].

FC has been tested in the postoperative setting, demonstrating satisfactory accuracy in detecting and/or predicting POR. Several prospective observational studies have shown a positive association between FC concentration and severity of POR (Rutgeerts score), with only rare exceptions [79-82]. Two meta-analyses have evaluated FC’s performance in the detection of POR. Qiu et al, in 2015, included 10 studies with a total of 613 patients, and reported a pooled sensitivity of 82% (95%CI 73-89%), and a negative likelihood ratio of 0.29 (95%CI 0.197-0.44), although specificity was modest 61% (95%CI 51-71%) [83]. The authors did not propose a definitive FC threshold because of the diverse cutoff values in the included studies. A subsequent meta-analysis in 2018 that included 9 studies and 588 patients found an FC value of >150 μg/g to have the best overall accuracy for predicting POR, with a pooled sensitivity of 70% (95%CI 59-81%), specificity 69% (95%CI 61-77%) and an area under the ROC curve of 0.73 [84].

However, the available evidence is not sufficient to recommend FC measurement as a sole reliable alternative to colonoscopic evaluation 6-12 months postoperatively. In particular, the low sensitivity of FC still suggests the need for endoscopic assessment. This may be attributed to certain limitations in existing studies, including substantial heterogeneity regarding the timing of measurements, POR definition (Rutgeerts Score ≥i2 or ≥i2b), type of assay used, and variability of cutoff values. Further, larger, well designed prospective studies are needed to help overcome these limitations. Currently, FC can be considered in the postoperative setting to assess POR along with other imaging modalities [intestinal ultrasound (IUS), magnetic resonance enterography, small bowel capsule endoscopy] in case endoscopy is not feasible or acceptable, or prior to index postoperative endoscopic evaluation, at 3-6 months (as a single measurement, or even better as serial measurements) to guide the timing of endoscopy [5]. Elevated FC values (typically/commonly >100-150 μg/g) suggest POR and an earlier colonoscopy should be considered, whereas lower values may support delayed or standard colonoscopy timing [80, 84]. Besides incorporating FC monitoring into routine postoperative care, serial measurements thereafter can help track inflammatory activity over time and reduce the need for frequent colonoscopies.

We recommend against the use of FC measurement as a substitute for the endoscopic assessment of inflammatory activity in symptomatic patients with UC who have undergone total proctocolectomy with ileal pouch anal anastomosis (IPAA). (Strong recommendation, low level of evidence, Consensus 100%)

In patients with UC who have a history of IPAA there is emerging evidence in favor of the use of FC in diagnosing pouchitis [85-87]. Although studies have shown a positive correlation of FC with the pouchitis disease activity index, as well as its subscores, several limitations—including small sample sizes, and significant variability and heterogeneity in study design and methodology, as well as in the FC assays used—have not allowed for standardization of its use [88,89].

Pouchoscopy remains the gold standard, not only allowing the distinction between inflammatory and functional pouch disorders (which may both be present, with non-specific overlapping symptoms), but also providing the opportunity to exclude causes of secondary inflammation. FC may be used for the early detection of subclinical inflammation in asymptomatic patients, or in case of symptom onset—either complementary to pouchoscopy (offering values for future comparison), or if an empiric treatment approach is preferred in order to diminish unnecessary antibiotic use [88]. In this case a low cutoff value for ruling out pouchitis (such as FC<50 μg/g) may be used [90].

We suggest the use of FC testing in patients with spondyloarthritis and concomitant gastrointestinal (GI) symptoms as a screening tool for latent IBD. (Conditional recommendation, low level of evidence, Consensus 80%)

Spondyloarthritis (SpA) is a family of inflammatory rheumatic diseases that include ankylosing spondylitis (AS), psoriatic arthritis (PsA), reactive arthritis, IBD-related arthritis (enteropathic), enthesitis-related juvenile idiopathic arthritis, and undifferentiated types of spondyloarthritis that share a common etiology and immunopathogenesis with IBD [91].

Compared to other non-SpA rheumatic and musculoskeletal disorders, up to two thirds of patients with differentiated SpA have been found to have subclinical GI inflammation [92]. FC is a biomarker that can detect gut inflammation with high sensitivity and specificity in this scenario. In a meta-analysis by Fauny et al, 21.2-70.7% of SpA patients were found to have elevated FC levels. Higher FC levels correlated with both the presence of macroscopic (11-80% of patients) and microscopic (41.7-100%) inflammation, but not with GI symptoms [92]. Despite this high frequency of endoscopic/microscopic inflammation, only 6-13% of patients with SpA will eventually develop evident IBD during their disease course [92].

FC measurement could help clinicians to decide who would benefit from a more invasive examination (endoscopy, imaging, capsule endoscopy) and, in case of abnormal findings, a tighter control or a more effective treatment targeting both gut and joint inflammation should be implemented. This management could also aid in reducing unnecessary investigations. However, patients with prominent symptoms suggestive of IBD (e.g., persistent/intermittent abdominal pain or diarrhea >4-6 weeks) should undergo an earlier colonoscopy without the need for a prior elevated FC measurement.

As for optimal cutoff levels, no recommendation can be made because there is insufficient evidence. Values of 266 μg/kg and 132 μg/kg at baseline have been correlated with CD diagnosis (sensitivity 100% and 66.7%, specificity 78.7% and 76.9%, respectively) whereas lower values (85 and 100 μg/kg) were associated with the presence of asymptomatic intestinal inflammation [92]. The same applies to the optimal frequency of testing. However, when interpreting FC values in this population, the possible effect of concomitant use of non-steroidal anti-inflammatory drugs (NSAIDs) or biologics [93-95] should be taken into consideration. NSAIDs might cause mild to moderate reversible FC elevations [96], while anti-tumor necrosis factor alpha agents, effective for both SpA and IBD, may lower FC values and conceal possible coexisting IBD [97]. Caution should also be taken in SpA patients under treatment with anti-interleukin (IL)-17, since it has been associated with GI disorders and even new-onset IBD, although this has not been confirmed as statistically significant on meta-analysis [98]. Clinical screening for IBD and FC testing in the presence of symptoms or risk factors should be applied prior to anti-IL-17 initiation [99].

We recommend the use of FC testing to monitor disease activity during pregnancy in patients with IBD. (Strong recommendation, high level of evidence, Consensus 100%)

No difference has been reported in FC concentrations between pregnant and non-pregnant healthy women. In contrast to other serum biomarkers (hemoglobin, erythrocyte sedimentation rate, C-reactive protein [CRP], albumin, iron), whose values are often affected by the physiological adaption that occurs during pregnancy, FC remains stable without significant fluctuation throughout all trimesters [100,101]. Therefore, it is considered an accurate and reliable noninvasive biomarker for the initial evaluation of chronic GI disorders during pregnancy.

Concerning IBD patients, although pregnancy has occasionally been associated with diminished inflammation [102], close monitoring is mandatory, given the potentially unfavorable impact of disease activity on maternal and fetal outcomes. FC has been proven superior to other biomarkers, since it correlates with disease activity as measured by physician global assessment and clinical disease scores in all trimesters [103]. FC values increase up to 3 months before a clinically evident flare, increasing clinician awareness and leading to subsequent timely management (investigation and treatment modifications) so as to prevent worse outcomes during gestation [104]. Along with symptomatic assessment, the measurement of other serum biomarkers and the emerging use of IUS, FC is a valuable tool for close monitoring of women with IBD during preconception and at each trimester [105]. Thus, endoscopy, despite being a relatively low-risk procedure, may be limited to those patients with an absolute indication for further examination.

FC use in pediatric patients with IBD (Table 2)

  • A) For children older than 4 years, we suggest using an FC cutoff level of 100 mg/g to distinguish IBD from functional GI disorders (FGID)

  • B) No recommendation can be made regarding the FC cutoff level in children younger than 4 years old

  • C) We recommend endoscopy in pediatric patients when there is strong suspicion of IBD, regardless of FC levels (strong recommendation, high level of evidence, Consensus 96%)

It is well recognized that FC values vary with age, and are higher in children than in adults. Numerous studies have examined a broad range of age groups and utilized various statistical methods, identifying a distinction between children under 4 years old and those over 4 years old [106,107,108]. Davidson and Lock, in a large study, evaluated FC levels in 8676 healthy children within 2 different age groups, revealing a median FC of 77 mg/g for children aged 1-3.9 years, 62 mg/g for those aged 4-17.9 years, and 61 mg/g for individuals over 18 years. There were no notable differences observed between the age groups of 4-17.9 years and those over 18 years [106]. In 2 more recent studies, Roca et al created valuable nomograms from regression analysis results. In the first study, 174 healthy children aged 0-12 years were categorized into 3 distinct age groups: 0-12 months, 1-4 years and 4-12 years. The cutoff values determined for these 2 groups, based on the lowest 95th percentile for FC in each category, were 910, 286 and 54 mg/g, respectively. Significant variability was observed among individuals below 12 months old [109]. In the second study, which included 212 healthy children aged 4-16 years, it was found that the median concentration of FC for the whole study population, i.e. 18.8 mg/kg, was below the cutoff value of 50 mg/kg proposed for adults, but approximately 20% of healthy children had concentrations above 50 mg/kg [110]. In the biggest study to date, Kolho et al evaluated FC levels in 11,255 healthy children aged 0-18 years. The median level of FC was 51 mg/kg in infants <1 year of age. This was 3-4-fold higher when compared to yearly age groups from 1-10 years (total number of children 5691). Across yearly age groups from 11-18, the median values varied from 11-19 mg/kg (total number of children 5325). Thus, they concluded that FC values in children beyond the first year of life are in general low, and are comparable in children and adolescents [111].

GI symptoms are common in primary care, with FGID being more frequent than organic (OGID). Both types can present with similar symptoms, making diagnosis challenging. Distinguishing between OGID and FGID can be challenging, as their symptoms often lack specificity and tend to overlap. A recent systematic review and meta-analysis by An et al, which included 18 studies, found that for distinguishing patients with OGID (including IBD) from those with FGID (16 studies), the estimated sensitivity of FC testing was 81% and the specificity 81% [66]. For distinguishing IBD from FGID (10 studies), sensitivity was 88% and specificity 72%. Assuming a population prevalence for OGID of 1%, the positive predictive value was 4.2% and the negative predictive value 100%. The difference in sensitivity and specificity between FC testing cutoffs of 50 μg/g and 100 μg/g was not statistically significant. The meta-analysis concluded that FC testing can reduce unnecessary colonoscopies and specialist referrals for children with FGID, and should be incorporated into clinical practice for evaluating patients with lower GI symptoms. In another systematic review and meta-analysis, Holtman et al concluded that FC, along with CRP and albumin, are more reliable indicators of the need for endoscopy in a child compared to symptoms, while among the former measures FC was more reliable than blood tests [112]. Furthermore, Degraeuwe et al, in their meta-analysis of 9 studies and over 800 patients, found that with an FC cutoff level of 50 μg/g there would be 17% false positive and 2% false negative results, concluding that in high-prevalence circumstances, FC can be used as a noninvasive biomarker of pediatric IBD with only a small risk of missing cases, and can help in selecting patients for endoscopic evaluation [113].

We recommend including FC measurement, at least twice annually, for the assessment of pediatric patients with IBD in clinical remission (strong recommendation, high level of evidence, Consensus 100%)

Monitoring FC in IBD patients soon after the initiation of treatment can provide an objective indication of potential clinical progress [52]. Maintaining low levels during treatment is associated with a higher probability of sustaining clinical remission for an additional year [114]. According to Molander et al, FC levels below 100 μg/g in IBD patients after the induction phase with infliximab treatment may serve as a positive prognostic indicator for achieving clinical remission [115]. Taking into account the aforementioned evidence, as well as extrapolation from adult data, it seems reasonable to incorporate FC in the follow up of pediatric patients with IBD in clinical remission, with a proposed interval of at least twice yearly.

We suggest endoscopic evaluation in pediatric patients with IBD in clinical remission with persistent FC measurements of >300 μg/g (strong recommendation, low level of evidence, Consensus 92%)

Further studies on FC levels have demonstrated that, in pediatric cases of IBD, the values for predicting disease flares in conservatively treated patients range between 400 and 800 μg/g [114-116]. Notably, FC levels tend to increase prior to any clinical or endoscopic sign of relapse, as observed in a cohort of adult IBD patients who achieved remission confirmed by endoscopy and were monitored using FC levels. The researchers concluded that FC can serve as a useful tool for identifying patients who need more frequent follow up in clinical settings [115]. Thus, in pediatric IBD patients in clinical remission with persistent FC values ≥300 μg/g, endoscopic assessment seems to be a reasonable option. We arbitrarily selected this cutoff, considering that pediatric studies report a flare prediction at higher concentrations (400-800 μg/g) but also recognizing the risk of missing subclinical inflammation at lower values. Thus, this was considered a reasonable compromise threshold, balancing sensitivity and specificity and aiming to reduce unnecessary procedures.

We recommend against the use of FC in children: 1) presenting with an episode of acute gastroenteritis to distinguish between bacterial and viral pathogens; 2) with a suspicion of intestinal polyps; and 3) as a biomarker for the diagnosis or monitoring of celiac disease (strong recommendation, moderate level of evidence, Consensus 100%)

Sykora et al indicated that FC can help distinguish between bacterial and viral causes of acute gastroenteritis in children under the age of 3 [117]. Their study demonstrated that when FC is used in conjunction with CRP, the diagnostic accuracy for differentiating between bacterial and viral acute gastroenteritis reached as high as 94%. Similarly, Duman et al found that FC levels were significantly elevated in patients with positive results from stool culture, particularly in cases of confirmed bacterial gastroenteritis. In assessing bacterial acute gastroenteritis, they reported that the area under the ROC curve for FC was 0.867, with a sensitivity of 88.9% and specificity of 76.0% at a cutoff of 710 mg/L [118]. Conversely, among children with acute gastroenteritis requiring hospitalization, no significant differences were observed in FC performance between those with a rotavirus and those with Salmonella enteritis infections. The higher levels of FC in rotavirus acute gastroenteritis noted in this study can be partially attributed to the study’s context, as only hospitalized patients, who are likely to exhibit more severe GI inflammation than those treated in primary care, were included. To conclude, although some studies suggest that FC may differentiate bacterial from viral gastroenteritis in children, the results are inconsistent, largely derived from hospitalized cohorts, and not validated in primary care or large-scale prospective trials. Furthermore, the significant overlap of FC levels between bacterial and viral infections, as well as the influence of confounding factors (e.g., age, stool consistency, comorbidities), limit its diagnostic accuracy in everyday pediatric practice.

Until recently, there have only been a few sporadic reports and series documenting cases of children with chronic jejunal polyps and elevated FC levels [119,120]. Nevertheless, an increase in FC levels alone cannot differentiate polyps from IBD without colonoscopy. FC levels are markedly higher in patients diagnosed with celiac disease, particularly among those exhibiting elevated serological markers or typical symptoms. However, no relationship has been identified with histological results. Additionally, conflicting findings have emerged regarding the link between FC and anti-tissue transglutaminase levels. Although the average FC values across the studies fluctuated around 100 μg/g, celiac disease patients showed significantly higher results at the time of diagnosis compared to control subjects. This difference diminished within 4 to 12 months after the initiation of a gluten-free diet [121,122]. Thus, the ambivalence of the above-described results concerning the use of FC in these conditions does not allow for a recommendation in favor of using this biomarker.

Concluding remarks

The EOMIFNE taskforce aimed to generate practical and concise statements that will allow for a standardized approach to the incorporation of FC into everyday patient care. We believe that, with the use of a robust hybrid methodology utilizing existing knowledge, along with expert consensus, while also taking account of the specific characteristics of the Greek healthcare system, we have managed to provide herein a clear guiding framework for the optimal use of FC as a useful diagnostic tool, leading to improved patient outcomes.

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Notes

Conflicts of Interest: Nothing to declare