Introduction

Calprotectin is a major granulocyte-derived alarmin protein that natively occurs as a dimeric and tetrameric Mrp-8/Mrp-14 complex. While serum calprotectin is an emerging biomarker for rheumatoid arthritis and juvenile idiopathic arthritis, fecal calprotectin is already established as the gold standard for diagnostics and monitoring of inflammatory bowel diseases. However, standardization of fecal calprotectin assays differs significantly among providers leading to varying clinical cut-offs. One suspected reason is that the oligomeric state of calprotectin can lead to different quantitative results. However, controlling calprotectin's oligomeric state has been challenging.

Aims & Methods

The aim of this work was to produce pure calprotectin as calibrator material with a controllable oligomeric state. Recombinant Mrp8/Mrp14-fusion protein was expressed solubly in E. coli and the oligomeric state assessed by size-exclusion chromatography and label-free differential scanning fluorimetry. Direct comparison to native calprotectin regarding antibody affinities were measured and its use as calibrator material in various immunoassay formats was tested.

Results

The fusion protein was purified as a calprotectin dimer mimic and could successfully be transformed into a calprotectin tetramer upon calcium addition. Affinities to monoclonal and polyclonal calprotectin antibodies are comparable to native calprotectin. Spiking of different concentrations of recombinant calprotectin showed linear correlations in ELISA and turbidimetric assays. Turbidimetric measurements of 17 human serum samples based on native and recombinant calprotectin calibrators revealed a perfect correlation (slope=1.009; R²=0.999).

Conclusion

The recombinant protein shows immunological properties comparable to native calprotectin and it can be purified in large quantities. Its oligomeric state can be controlled by calcium addition at similar concentration levels as native calprotectin. It therefore presents a promising tool to overcome the prevalent fecal calprotectin standardization problem and prevent future standardization discrepancies for serum calprotectin.

Disclosure

The authors Alexander Ohmann, Dmitrii Guschin, Joana Afonso and Christian-B. Gerhold are employees of BÜHLMANN Laboratories AG.

Introduction

The correlation between fecal calprotectin (FC) levels and small bowel (SB) inflammation in patients with Crohn's Disease (CD) remains a subject of debate.

Aims & Methods

This study aims to investigate the relationship between FC levels and SB inflammatory activity in patients diagnosed with SB CD.
We conducted a retrospective cohort study involving patients with SB CD who underwent small bowel capsule endoscopy (SBCE) and FC testing. Patients with colon inflammation were excluded. Participants were categorized into four groups based on their SB inflammatory status: no inflammation (all three SB tertiles showed Lewis Score (LS) <135); proximal SB inflammation (first and/or second SB tertiles had LS ≥ 135, with no inflammation in the third SB tertile); distal SB inflammation (third SB tertile had LS ≥ 135, with no inflammation in the proximal SB); pan-SB inflammation (both proximal and distal SB with inflammation).

Results

A total of 87 patients were included in the study, with a median age of 35 years, 75.9% being female. The distribution of inflammation among the patients was as follows: no inflammation in 21.8%; proximal inflammation in 4.6%; distal inflammation in 33.3%; pan-SB inflammation in 40.4%.
FC median values exhibited an ascending trend along the SB axis: no inflammation 58 µg/g (33-180); proximal SB inflammation 65 µg/g (30-128); distal SB inflammation 122 µg/g (42-507); pan-SB inflammation 400 µg/g (205-852).
Statistically significant differences in FC levels were observed between pan-SB inflammation vs. isolated proximal inflammation (p = 0.014), and isolated distal inflammation (p = 0.012).
Additionally, FC showed a positive correlation with LS in the second (r = 0.464, p<0.001) and third tertiles (r = 0.435, p<0.001), but no correlation was noted in the first tertile.
FC demonstrated good predictive capacity for identifying inflammation in the distal SB and pan-SB vs. no inflammation and proximal inflammation, with an area under the curve of 0.790 (p < 0.001) and an optimal cutoff value of 228 µg/g.

Conclusion

FC levels correlate positively with the presence of SB lesions in the second and third tertiles of the small bowel. However, it is not a reliable marker for detecting inflammation in the first tertile, highlighting the importance of conducting a SBCE in these patients.

Introduction

The biomarker fecal calprotectin (FC) is an efficacious tool for evaluating the level of disease activity in Crohn's and Ulcerative colitis, as well as for discriminating between inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS). Fecal Alpha-1 Antitrypsin (A1AT) is an excellent marker for intestinal protein loss and for an increased mucosal permeability.

Aims & Methods

The aim of this investigation was to determine the relationship between these two inflammatory fecal markers.
The study was performed retrospectively. We've collected test results for both markers between 2018 and 2022 from 459 patients who performed both tests (67.4% females; mean age 35.1±22.7). Linear correlation evaluation between FC and A1At was performed using the Pearson Correlation Coefficient (PCC).

Results

Out of the 459 patients, 274 were diagnosed with IBD, 61 with IBS and the rest with other, non-GI disorders. Out of these, 37.7% were positive for both markers, 45.3% were negative for both markers, 7.6% were positive for A1AT and not FC and 9.4% were positive for FC and not A1AT. Linear correlation between the two markers using six categories for data scoring, was found to be significant by PCC test (R=0.83, P values < 0.001) and sensitivity and sensitivity, specificity and accuracy were 80.1%, 85.6% and 83.0% respectively. Out of the 274 patients in the IBD group, 39.2% were positive for both markers, 45.8% were negative for both markers, 5.4% were positive for A1AT and not FC and 7.6% were positive for FC and not A1AT. Linear correlation between the two markers was found to be significant by PCC test (R=0.87, P values < 0.0001) and sensitivity, specificity and accuracy were 83.7%, 89.8% and 86.9% respectively.

Conclusion

Our study demonstrates that FC had a significant relationship with A1AT in patients with IBD and IBS. Furthermore, A1AT might be used as a surrogate marker for FC in the diagnosis of IBD, IBS-IBD discrimination and disease severity and treatment follow up. In addition to that, both markers used together may be used as a new clinical score for the assessment of IBD and IBS severity.

Introduction

Infiltration of neutrophils into the intestinal tissue is a crucial component of the inflammatory response in Crohn‘s disease (CD). CPa9-HNE, a calprotectin fragment released by human neutrophil elastase during NETosis, has been shown to strongly correlate with endoscopic disease activity in inflammatory bowel disease (IBD) and serves as an indicator for pharmacodynamic response. Furthermore, emerging data suggests that neutrophil extracellular traps (NETs) significantly contribute to the pathogenesis of fibrosis.

Aims & Methods

For the first time, we investigated the association of CPa9-HNE with histological grading of both inflammation and fibrosis. The cohort included 62 patients with stenotic Crohn‘s disease. Inclusion criterion was the presence of stenotic disease requiring bowel resection, as confirmed by radiographic assessment. Upon surgery, the resected specimens were collected and histologically graded according to transmural fibrosis burden, and inflammation (D'Haens score). Serum was drawn at baseline (before resection). CPa9-HNE was measured, reflecting a human neutrophil elastase-derived fragment of calprotectin. Spearman’s rank correlation was applied.

Results

CPa9-HNE showed an overall positive correlation to the D'Haens score (ρ=0.279, p<0.005). Significant positive correlation was observed between CPa9-HNE and the presence of neutrophils, both in the epithelium and the lamina propria (ρ=0.309, ρ=0.349, p<0.005 both), as well as with epithelial damage (ρ=0.262, p<0.005). We also observed a positive correlation between CPa9-HNE and transmural fibrosis burden, specifically fibrosis in the submucosa (ρ=0.294, p<0.005)

Conclusion

This study showed that CPa9-HNE, a biomarker of neutrophil activity, correlated with the presence of neutrophils in the intestinal tissue, as well as epithelial damage. Interestingly, CPa9-HNE also showed association to submucosal fibrosis, emphasizing the importance of considering neutrophil activity when targeting the fibro-inflammatory axis. These findings highlight the potential use of CPa9-HNE as a complementary tool for histological grading of inflammation and fibrosis burden, and could be used to further explore the role of neutrophil extracellular traps in intestinal fibrosis.

Disclosure

MSA, TKA, MP, MAK, ACBJ and JHM are employees of Nordic Bioscience. MAK, ACBJ and JHM are shareholders of Nordic Bioscience.

Introduction

Fecal calprotectin (FC) is a key non-invasive biomarker used to monitor inflammatory bowel disease (IBD). Despite its clear clinical value, patient adherence to the test remains suboptimal, mainly due to reluctance related to stool collection. The objective of the ACCALMI study was to compare the acceptability of a new stool collection device, similar to the one used for fecal immunochemical tests (FIT), with a standard stool container, and to evaluate its impact on patient compliance.

Aims & Methods

In this prospective, single-center study, 156 patients with an established diagnosis of IBD and who had been prescribed an FC test as part of their routine care, were randomly assigned to either the standard container group or the alternative device group (Procise Stool Collection Device™), which includes a collection tube with a thin spiral-shaped probe facilitating stool collection. Acceptability was assessed using a 4-category ordinal scale (from "poor" to "very good") and a visual analog scale (VAS, from 0 to 10). Analyses were conducted in both per-protocol and intention-to-treat (ITT) populations, with missing data handled using the maximum bias method. Factors associated with improved FC acceptability, and compliance with FC testing, were analyzed.

Results

The alternative collection device significantly improved acceptability as measured by the ordinal scale (odds ratio [OR] 2.69; p < 0.001). In the alternative group, acceptability ratings were significantly higher with only 1.4% versus 7.0% rating it as ‘poor’, 12.5% versus 46.6% as ‘average’, 66.7% versus 40.8% as ‘good’, and 19.4% versus 5.6% as ‘very good’. Measured using the VAS, the average acceptability was 7.9/10 in the group using the alternative device compared to 6.1/10 in the standard group, with a significant difference of 30.4% (95% CI [19.7 - 44.7%], p < 0.001), although it did not reach the threshold of 30% that was set a priori. The ITT analysis led to the same conclusions. Among the patients who had already undergone FC testing and were randomized to the "alternative device" group, 55% indicated a preference for the alternative device, compared to 7.2% for the conventional device, while 37.7% found them similar. Embarrassment and discomfort associated with the standard container were alleviated by the alternative device. A long home-to-hospital distance (> 50 km) and the use of the alternative device were the only factors associated with better acceptability in the multivariate analysis. Compliance was similar between both groups and remarkably high (92%).

Conclusion

The ACCALMI study demonstrated that the alternative stool collection device, similar to the one used in FIT for colorectal cancer screening, is more accepted by IBD patients than the standard container, thus improving the acceptability of FC testing. These findings support the integration of patient-centered innovations in diagnostic and follow-up testing to improve the monitoring of IBD.

Disclosure

Acknowledgments: Collaboration with Biosynex®.

Introduction

Ulcerative colitis (UC) is a chronic inflammatory bowel disease (IBD) presenting in remitting ulcerations of the colonic mucosa and submucosa. Mucosal healing is therefore an important treatment target for optimal disease management. While fecal calprotectin is a common biomarker used to monitor mucosal healing in UC, patient compliance is low due to a preference for serological markers over fecal ones.

Aims & Methods

This study aimed at investigating the association of serum calprotectin [CPa9-HNE], a non-invasive neo-epitope biomarker of neutrophil activity, with both clinical and endoscopic disease activity in UC. The cohort included 49 patients with active UC and 50 and healthy controls (HC). Endoscopic and clinical disease activity was assessed using the Ulcerative Colitis Endoscopic Index of Severity (UCEIS) and the full Mayo score. Serum calprotectin [CPa9-HNE] was measured, reflecting a human neutrophil elastase-derived fragment of calprotectin. Spearman’s rank correlation was applied. Group differences were evaluated using Kruskal-Wallis with Dunn‘s test, bonferroni corrected for MCP. Diagnostic and discriminative capabilities were assessed using receiver operating characteristic (ROC) statistics with are under the curve (AUC).

Results

CPa9-HNE was significantly elevated in patients with UC compared to HC (ng/ml [IQR]: 32.4 [29.0–52.3] vs. 26.0 [22.6–29.0], p<0.0001) and presented acceptable discriminative capabilities (AUC [95% CI]: 0.77 [0.67–0.87], p<0.0001). CPa9-HNE showed a moderate positive correlation to UCEIS and Mayo (ρ=0.42; ρ=0.48, both p<0.01). When patients were stratified according to endoscopic disease activity (UCEIS), CPa9-HNE was significantly elevated in patients with severe disease compared to those with mild disease (ng/ml [IQR]: 55.6 [40.9–66.4] vs. 28.9 [24.6–31.1], p<0.01) and could accurately discriminate between the two groups​. Similarly, when patients were grouped according to the full Mayo score, CPa9-HNE was significantly elevated in patients with mild vs. severe disease (29.1 [24.3–32.4] vs. 62.9 [55.2–67.5], p<0.01) and moderate vs. severe disease (32.0 [29.2–39.2] vs. 62.9 [55.2–67.5], p<0.01), and could accurately discriminate between the groups.

Conclusion

CPa9-HNE was elevated in patients with UC compared with healthy subjects. Furthermore, CPa9-HNE accurately discriminated between patients with mild and severe endoscopic disease based on the UCEIS score, as well as between patients with mild, moderate, and severe clinical disease activity according to the full Mayo score. These findings highlight the potential use of CPa9-HNE as a non-invasive tool to monitor both endoscopic and clinical disease activity in UC, with the potential of guiding treatment decisions and better aligning with patient preferences.

Disclosure

MSA, MPE, MAK, ACBJ, and JHM are employees of NB.