INTRODUCTION

Calprotectin is a 36-kDa calcium- and zinc-binding protein belonging to the S100 protein family. This noncovalent heterodimeric complex consists of a light chain (S100A8) and 2 heavy chains (S100A9) composed of 93 and 113 amino acids, respectively. Each monomer is characterized by its ability to bind to 2 Ca2+ ions with different affinities and to other divalent cations, such as Zn2+, Mn2+, Fe2+, and Ni2+ (1–3). Calprotectin is present in the cytoplasm of neutrophils, where it represents approximately 5% of the total protein and 30%–60% of the proteins of the cytosol. It is also expressed by monocytes, dendritic cells, activated macrophages, keratinocytes, and certain mucosal epithelial cells. At the intracellular level, calprotectin exerts regulatory functions that are crucial for immune defense through an interaction with zinc-dependent metalloproteinases and the activation of proinflammatory cytokine synthesis, as well as through interactions with cell-signaling pathways associated with innate immunity, leading to the modulation of tissue adhesion by the rearrangement of microtubules, thus promoting leukocyte recruitment, the transport of arachidonic acid toward the sites of inflammation, and the generation of reactive oxygen species (3). After release into the extracellular environment, calprotectin can be found in plasma, urine, saliva, intestinal secretions, and stool, where it develops direct antimicrobial properties through the chelation and sequestration of Zn2+ and Mn2+ ions, depriving microorganisms of these nutrients and blocking cell growth (4). It also contributes to the inflammation process through neutrophil chemotaxis. The luminal concentration of calprotectin is thus directly related to the migration of granulocytes to the mucosa. Measuring fecal calprotectin (FC) levels as a biomarker of mucosal inflammation was first suggested at the end of the 1990s by Røseth et al (5), who showed a close correlation between fecal excretion of leukocytes labeled with 111indium, the gold-standard method for identification of mucosal inflammation, and FC concentrations. Since the early 2000s, many studies have confirmed these results in patients with abdominal symptoms to help differentiate those with inflammatory bowel disease (IBD) from those with irritable bowel syndrome (IBS), and prioritize colonoscopy. FC is also used to assess the inflammatory activity of the disease and the response to treatment, as well as the risk of relapse, whether during treatment monitoring or postoperatively. Of note, FC levels do not increase during nonintestinal inflammation. This review will discuss the analytical aspects of test performance that have the potential to affect the results and use of this biomarker in the clinical setting.

MEASUREMENT OF FC

When bound to calcium, calprotectin undergoes conformational changes that allow it to resist the proteolytic intestinal or fecal environment. Although early studies reported a stability of native stool samples of over 7 days at room temperature, it now seems to be preferable to limit storage to 2–3 days. Storage at 4 °C is preferred; however, conflicting results have been reported when storage is prolonged to 7 days, depending on the assay (6,7). Finally, the storage of extracted stools at 4 °C seems to be more appropriate if the measurement is performed within 1 week (6). Samples can be stored at −20 °C for longer periods, up to 1 year, and no alterations in the concentration have been reported after freezing and thawing (8).

Sampling is frequently performed at the patient's home. In such circumstances, patients must be informed not only about the limited time of storage but also about the utmost importance of the quality of the sample collection, which must not be contaminated by urine or toilet water. Patients need to be informed that a liquid stool sample is not a contraindication to the measurement of FC, especially as this is a frequent situation for patients with IBD flare-ups. Several studies have reported intraindividual variability in FC concentrations depending on the period of stool collection (morning, noon, or evening), probably related to the duration of transit. It is therefore preferable to collect the sample from the first bowel movement of the day (9). Such intraindividual variation rarely modifies the therapeutic decision. Thus, it is not necessary or recommended to perform the measurement several days in a row (9). A sample of a few grams is sufficient for the assay. Regardless of the method used for measurement, the first step requires preparation of a fecal extract. The gold-standard method consists of weighing a known quantity of stool and diluting it in the appropriate volume of extraction buffer. As this protocol is long and tedious, most manufacturers offer extraction devices. However, incorrect results can be obtained when using such devices with liquid, mucous-containing, or, on the contrary, very dry stool samples because there is a risk of undersampling for samples not retained in the grooves.

Various commercial assays can be used, all based on immunoenzymatic methods, but without standardization. Enzyme-linked immuno assay has been used for many years but is time-consuming and involves the performance of serial assays. It has been replaced by assays adapted to various machines (fluorescence, chemiluminescence, immunoturbidimetry, etc), allowing results to be rendered on an ad hoc basis. All assays use polyclonal or monoclonal antibodies that target various epitopes of the heterodimer, thus explaining the variability in the stability and results. However, the diagnostic performance of the various methods is similar (10) and there is no reason to favor one method over another. Owing to the absence of standardization, assays are not interchangeable. Thus, patients must be monitored using the same method to limit interassay variation, which could lead to misinterpretation. For example, a decrease in FC concentration obtained when switching to a different technique does not always indicate a response to treatment, and vice versa. Moreover, special vigilance is required when changing methods. Rapid immunochromatographic tests, initially semiquantitative and more recently quantitative, have been developed but should not be prioritized, especially when FC concentrations are high (9).

Almost all techniques recommend 50 μg/g stool sample as the cutoff for the normal concentration for adults and children older than 4 years, without a distinction of sex (8). Moderate increases in FC levels, up to 100 μg/g, have been described for subjects older than 65 years (11). The concentration of FC is also physiologically higher for neonates, infants, and young children (12), with high intraindividual and interindividual variability, probably related to the high permeability of the intestinal tract and the immunoinflammatory responses induced by implantation of the intestinal microbiota and the first contact with food allergens (13). FC concentrations should therefore be interpreted with caution in the pediatric population and each laboratory should define its own reference values. As calprotectin is also found in serum during an inflammatory process, the question arose of the influence of digestive bleeding on FC levels. Tibble et al (14) showed that it would take bleeding of approximately 300 mL/24 hours to reach a concentration of 150 μg/g. This type of interference is therefore not significant. Finally, there are contradictory data on the role of certain drugs, such as nonsteroidal anti-inflammatory drugs and proton-pump inhibitors, which could induce an increase in FC levels. Thus, it is probably preferable, when possible, to stop taking these drugs at least 2 weeks before performing the assay (9).

The billing cost for FC varies among country and is influenced by the level of coverage by social security organizations. For example, it usually costs 75–150$ in North America with a reimbursement of 16.63$ in the United States, 43.5€ (public laboratory)–70€ (private laboratory) in France, 15.9€ (public laboratory)–30.2€ (private laboratory) in Germany, and 54.9 Swiss franc, totally reimbursed in Switzerland.

INDICATIONS AND BIOCLINICAL INTERPRETATION OF THE FC ASSAY

Given its various biological functions, it is not surprising that FC is now recognized as a sensitive marker for identifying and monitoring digestive inflammation. However, FC will not help in identifying the etiology because its levels increase in various clinical situations associated with mucosal inflammation (e.g., gastrointestinal infections, colonic polyps and diverticulosis, colon cancer, gastrointestinal bleeding, microscopic colitis, proctitis after radiotherapy, pouchitis, rheumatological diseases, etc.) (9). A specific attention should be given in patients with surgical situations such as intestinal anastomosis or ileostomy, especially in those with diarrhea as FC may vary significantly over time. The bioclinical interpretation of the concentration should thus always consider the clinical history of the patient, their symptomatology, and factors that could affect the results.

Differential diagnosis between IBD and IBS

Chronic diarrhea is one of the most frequent reasons for consultation in gastroenterology. It is therefore important to discriminate symptomatic patients with IBS from those with a distinct pathology, in particular IBD, when the possibility of an organic disease is unclear. The main forms of IBD are ulcerative colitis (UC) and Crohn's disease (CD). Both are chronic inflammatory disorders characterized by relapsing-remitting clinical behavior and grouped based on the location of the inflammation: in CD, the inflammation can be in any part of the intestine, whereas in UC, the inflammation is limited to the colon. From the first studies using first-generation assays, Tibble et al (15) showed that high concentrations of FC accurately identify an organic intestinal disease in symptomatic patients, regardless of its origin (IBD, neoplasia, infectious enteritis, etc.), with a positive predictive value of 76%, a negative predictive value of 89%, and an odds ratio of 27.8 (95% confidence interval [CI]: 17.6–43.7). These results are far superior to those of serum markers of inflammation. Several meta-analyses and systematic reviews have now confirmed these first results and indicate an overall sensitivity of 80%–98% and a specificity of 68%–96% with an FC threshold ranging from 30 to 100 μg/g in adults, mostly 50 μg/g (16,17). In most cases, a cutoff of 50 μg/g is retained, with a global sensitivity of 0.81 (95% CI: 0.75–0.86) and a high specificity of 0.87 (95% CI: 0.78–0.92). Thus, it seems to be the most powerful cut-off value in clinical practice because patients with higher FC concentrations are 6 times more likely to have IBD (positive likelihood ratio, 6.0; 95% CI: 3.0–9.5) (18). No effect of lesion location has been observed (19). FC measurement may thus be considered a useful screening tool in the initial assessment of symptomatic patients for identifying those who are more likely to need an endoscopic assessment to identify IBD (20). Such an approach would significantly reduce the number of negative endoscopies and therefore the cost of the management of these patients (16). However, this approach could also delay the diagnosis for approximately 5% of patients with IBD who have FC concentrations below 50 μg/g. Although there is a consensus for the normal threshold, the concentration that allows confirmation of mucosal inflammation is still debated. FC levels may be moderately elevated in certain patients with IBS and such results should be considered as false positives. Conversely, certain patients with an active organic disease (IBD or other) have FC concentrations between 50 and 150 μg/g (or even 250 μg/g, depending on the method). For these patients, the risk of flare-ups is low, and it seems legitimate to ask for a reevaluation of FC within 1 month before directing the patient to an endoscopy examination. An algorithm to help with the interpretation of the FC assay and guide the clinical decision has been proposed by Jukic et al (3). A version adapted for the laboratory is proposed in Figure 1. Of course, the upper value of the gray zone has to be adapted according to the method of measurement.

Figure 1.:

Algorithm for the interpretation of the FC concentration for patients with a history and/or symptoms suggestive of intestinal inflammation. *The threshold has to be adapted according to the method used for FC measurement and may be extended to 250 μg/g. FC, fecal calprotectin; IBD, inflammatory bowel disease; IBS, irritable bowel syndrome; NSAID, nonsteroidal anti-inflammatory drug.

Similar performance of the FC assay has been reported for children but with lower specificity (16). A meta-analysis of 8 studies performed on 715 children suspected of having IBD reported a sensitivity of 98% (95%–100%) and a specificity of 68% (50%–86%), probably related to the frequency of enteropathy of infectious origin (21). This population of infants, and even more so of neonates, represents a target for the development of noninvasive biomarkers. This is particularly important in preterm infants for the diagnosis of necrotizing enterocolitis. However, a recent study did not allow us to confirm threshold values to identify intestinal distress in this population (22).

ASSESSMENT OF DISEASE ACTIVITY AND RESPONSE TO TREATMENT IN IBD

IBDs are chronic diseases characterized by repeated cycles of disease activation, strongly affecting the quality of life. An evaluation of intestinal inflammation is therefore crucial for appropriate clinical management, not only at the time of diagnosis but also throughout the course of the disease, especially in CD, in which the symptomatology imperfectly reflects mucosal inflammation. Endoscopy with histology is the reference method for evaluating mucosal inflammation during IBD, and this is of utmost importance because mucosal healing is now considered to be the optimal therapeutic target to improve the natural history of the disease (23,24). However, colonoscopy is an expensive and invasive procedure that requires a high level of training, which limits its use in the context of regular monitoring of the disease. Today, FC measurement can be used as an objective and reliable marker not only of IBD flare-ups but also of mucosal healing (25). Numerous studies have confirmed the study of Røseth et al, which suggested that FC concentrations significantly correlate not only with disease activity but also with endoscopic and histological scores better than other biological parameters, such as CRP levels or platelet or leukocyte counts (3,26,27). Indeed, Schoepfer et al (26) showed a close correlation between FC concentrations and the endoscopic score for 122 patients with CD. It was thus possible to discriminate between inactive disease (healed mucosa) and mild inflammatory disease (104 ± 138 vs 231 ± 244 μg/g, P < 0.001), between mild and moderate disease (231 ± 244 vs 395 ± 256 μg/g, P = 0.008), and, finally, between moderate and severe disease activity (395 ± 256 vs 718 ± 320 μg/g, P < 0.001). Similar results have been reported for UC using the Modified Baron score to assess endoscopic severity of the disease. In patients with endoscopic stages 0 and 1, considered to correspond to remission generally associated with mucosal healing, FC remained within normal values, i.e., ≤16 (10–30) μg/g and 35 (25–48) μg/g, respectively. By contrast, patients with active disease have increased levels of FC concentration with values of 102 (44–159) μg/g at endoscopic stage 2, 235 (176–319) μg/g at endoscopic stage 3, and 611 (406–868) μg/g at endoscopic stage 4 (27). The decision threshold to define clinical remission with mucosal healing depends on the study, but FC concentrations beyond the cutoff considered to be normal (up to 150 or even 250 μg/g) could be used with a sensitivity of more than 80% (28). The recent update of the STRIDE recommendations (Selecting Therapeutic Targets in Inflammatory Bowel Disease, STRIDE II) published by the International Organization for the Study of Inflammatory Bowel Diseases confirmed these data and proposed <150 μg/g as a threshold value associated with remission (24). Once again, the diversity of methods and the lack of standardization lead to the proposal of a gray zone for analysis that can extend up to 250 μg/g. This cutoff has been confirmed in a study of 87 patients with Crohn's disease as FC concentrations of greater than 250 μg/g were associated with the presence of large ulcers and FC concentrations of 250 μg/g or less predicted endoscopic remission (29). A meta-analysis of 19 studies reported a sensitivity of 0.88 (95% CI: 0.84–0.90), with a specificity of 0.73 (95% CI: 0.66–0.79), for FC for the identification of endoscopic relapses (30). Certain patients develop functional intestinal disorders and the entanglement of these 2 conditions is estimated to be ∼30% in the case of IBD, which can interfere with its therapeutic monitoring. The European Crohn's and Colitis Organization has therefore recommended the measurement of FC in the monitoring of patients with IBD to better discriminate between symptomatic patients experiencing flare-ups from those more likely to present with functional intestinal disorders (31). Moreover, the multicenter, phase 3, randomized CALM study that compared the efficacy of a tight control algorithm of disease activity with stringent criteria including measurement of FC (cutoff: 250 μg/g) to current clinical management clearly showed that the treatment algorithm led to superior outcomes compared with the algorithm based on clinical management alone in patient with early Crohn's disease because it allows rapid therapeutic adaptation (32). An algorithm to help interpret calprotectin assays and guide clinical decision-making has been proposed by Jukic et al (3) and a version adapted for the laboratory is proposed in Figure 2. Currently, a significant decrease in the FC concentration toward acceptable levels associated with the regression of symptoms is considered to be an objective to be achieved in the monitoring of these diseases, regardless of the therapeutic approach used (24). However, patients' symptoms always need to be considered because various gastrointestinal diseases and some medication-related and even lifestyle factors can alter FC concentrations.

Figure 2.:

Algorithm for the interpretation of the FC concentration for patients with an established diagnosis of IBD. FC, fecal calprotectin; IBD, inflammatory bowel disease; IBS, irritable bowel syndrome.

Prediction of relapse

The natural history of IBD is characterized by periods of remission and clinical flare-ups. Having a high-performance biomarker to predict relapses would help to quickly adapt treatments while avoiding overtreatment of people at low risk of recurrence. An initial study showed the relative risk of relapse within a year to be multiplied by 14 (P < 0.0001) for patients with UC when the FC concentrations was over 150 μg/g stool (33). Other studies have proposed higher thresholds and Kallel et al (34) reported a threshold of 340 μg/g for a risk of relapse multiplied by 18. Finally, a meta-analysis of 6 studies including 318 patients with UC and 354 with CD reported a combined sensitivity of 78% (95% CI: 72%–83%) and a specificity of 73% (95% CI: 68%–77%) using thresholds varying between 120 and 340 μg/g (35). In addition to this result, the monitoring of FC concentrations should contribute to identifying the risk of relapse. A prospective study performed on patients with UC in remission on infliximab treatment and for whom FC was evaluated monthly reported a normal fecal concentration (<50 μg/g) throughout the duration of the study for patients without clinical flare-ups (36). Similar results were reported for various types of treatment. Therefore, less frequent monitoring (half-yearly or even yearly) may be proposed for asymptomatic patients with FC concentrations regularly below 50 μg/g. On the contrary, patients who relapsed had high FC concentrations as soon as 2–3 months before the clinical flare-up. Modeling of the receiver operating curve suggested a threshold of 300 μg/g, with a sensitivity of 58.3% and a specificity of 93.3% for the risk of relapse. Interestingly, if 2 consecutive measurements performed 1 month apart differed by more than 300 μg/g, the sensitivity of the biomarker to identify the risk of relapse increased to 61.5% and its specificity to 100% (36). Finally, Theede et al (37) showed that the measurement of FC is sufficient to determine the risk of relapse during UC 6–12 months in advance and to assess mucosal healing.

Despite therapeutic developments, the natural history of IBD is still frequently associated with the need for surgical management. Although colectomy is largely curative for patients with UC, except for those who developed pouchitis, surgery is not curative for CD and relapses are frequent. Colonoscopy is the reference diagnostic method to identify postoperative recurrence, with the limitations already described and limited data regarding the optimal time for monitoring. Numerous studies have now evaluated the performance of FC as a diagnostic tool to detect postsurgical relapses in patients with CD, whether in the adult or pediatric population (38,39). A meta-analysis of 9 studies suggested a threshold >150 μg/g to have the best overall accuracy for predicting postoperative endoscopic recurrence, with a sensitivity of approximately 70% (40). However, the concentration of FC may vary significantly over time after ileocecal resection, especially in patients with diarrhea. In those patients, a single measurement of FC may thus be of limited clinical utility to predict endoscopic recurrence (41). FC measurement is thus now part of the management of recurrences in CD after intestinal resection to identify patients for whom endoscopy and the escalation of treatment would be appropriate.

HELP IN THE DECISION TO STOP TREATMENT

Biotherapies associated with immunomodulators are widely used because they are effective in inducing and maintaining remission. The STORI study assessed the risk of relapse among 115 patients with CD in stable remission under combined treatment with anti-TNFα and an immunomodulator (thiopurines or methotrexate), who stopped infliximab. After a median of 28 months of follow-up, 52 patients experienced a relapse. Multivariate analysis of the results identified a number of risk factors for relapse, among them is a FC concentration at inclusion >300 μg/g (42). The FC assay, combined with the clinical monitoring of patients, should therefore contribute to the identification of patients at low risk of relapse, for whom treatment could be interrupted.

CONCLUSION

Fecal biomarkers are now widely used to support the diagnosis and management of digestive pathologies, particularly IBD. FC is the most highly conclusive and documented of such biomarkers. Studies performed over the last decade have improved our understanding of its molecular functions, revealing a broad spectrum of antibacterial and immunoregulatory properties. The measurement of FC is easily accessible. The first studies were performed by enzyme-linked immuno assay using a manual extraction technique. Various methods are now available and extraction devices have been developed to save analytical time. However, these devices are not suitable for all types of samples. Finally, although the analytical performance of the various assays is good, they can provide significantly different results and there is an urgent need for international standardization. It is therefore essential that each laboratory controls all aspects of the assay (preanalytical and analytical) and defines the thresholds and interpretations appropriate to their own clinical environment. This noninvasive marker was first validated for differential diagnosis between IBD and IBS-type functional pathologies for patients with suggestive symptoms to identify those requiring endoscopy. However, FC is not a marker of IBD but rather of mucosal inflammation, which can be observed not only during various clinical situations, such as IBD, but also in gastrointestinal infections or malignant tumors. Interpretation should thus always consider the clinical environment. This biomarker has now been validated for the management of IBD to evaluate the response to treatment through the evaluation of mucosal inflammation. This allows the detection, or even anticipation, of relapses, as well as healing, and thus assists the clinician in decisions concerning the escalation or reduction of medication. However, there is still a lack of consensus concerning the optimal thresholds for interpretation. This leads to a gray zone for intermediate concentrations of FC ranging from 50 to 250 μg/g, which are difficult to interpret and thus require close monitoring. By contrast, concentrations below <50 μg/g exclude IBD with a negative predictive value >95%, whereas those >250 μg/g indicate the need for a close monitoring and endoscopy, especially in patients with persistent symptoms to confirm a flare-up. Although this biomarker does not replace clinical evaluation and endoscopic examination, it efficiently contributes to the short-term and long-term management of IBD.

CONFLICTS OF INTEREST

Guarantor of the article: Nathalie Kapel, PharmD, PhD.

Specific author contributions: N.K. and L.B.-T. wrote the first paper draft and all authors further contributed to the writing and approval of the manuscript.

Financial support: None to report.

Potential competing interests: None to report.

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