Inflammatory bowel disease (IBD) diagnosis is currently based on clinical criteria together with biochemical and instrumental investigations. A prompt diagnosis is advisable in order to offer restitution to well-being and good quality of life to the patient. The recognition and diagnosis of IBD and its differential diagnosis from other acute and chronic bowel diseases is crucial for offering proper treatment and good prognosis. IBD therapy should induce a rapid symptom control, normalize biochemical indexes and resolve endoscopic lesions. With these targets in mind, we can hope to clear the disease or at least to slow the progression, to reduce the need for steroids and to avoid complications and surgeries in the patients.

If IBD is suspected, laboratory tests can guide further investigation and contribute to the differential diagnosis. Blood count, electrolytes, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) and stool cultures are routine exams which can be easily performed on peripheral blood samples and are useful to detect acute ongoing inflammation. However, they are not specific to detect intestinal inflammation. Serum and fecal inflammation markers that reflect the intestinal damage might be useful both in terms of diagnosing the disease and for the management and prognosis.

Anemia is frequent in IBD. It may stem from multiple sources, the most obvious being acute, chronic, or occult bleeding, but the role of chronic inflammation also needs to be considered [3,4]^[1][2]. The biochemical assessment of iron metabolism allows discrimination of iron-deficiency anemia which is characterized by a low serum iron and ferritin with high serum levels of transferrin and total iron binding capacity, from anemia induced by chronic inflammation which has a normal/high ferritin, low transferrin, and total iron binding capacity. Thromboembolic disease has been described in IBD, and especially in ulcerative colitis, possibly secondary to colonic inflammation. During active disease phases [5]^[3], not only inflammatory parameters (CRP, ESR, platelet count) but also coagulation parameters (thrombin–antithrombin complexes, fibrinogen, FgDP, and FbDP) increase, the coagulation and fibrinolytic cascades are activated in inflamed ulcerative colitis, with the hemostatic arm favoring coagulation. This hypercoagulable state may persist also when the acute phase subsides. CRP is inexpensive and easy to perform. CRP is synthesized by hepatocytes following the release of IL-6, TNF alfa and IL 1 beta. The molecular structure consists of five subunits that make up a pentameric appearance. Its production is quite rapid and its short half-life (18 h) makes the probe useful to rapidly detect inflammatory changes and to follow up the process [8]^[4]. However, there is considerable heterogeneity in CRP generation based on the genetics of an individual patient [9]^[5]. CRP is a poor parameter of inflammation, especially in ulcerative colitis; about 50% of ulcerative colitis patients have normal CRP during a disease flare [10]^[6].

2.2. Immunological and Other Antibody Markers

p-ANCAs (perinuclear anti-neutrophil cytoplasmic antibodies) have been described in ulcerative colitis patients for thirty years, though the exact epitope remains unknown [12,13]^[7][8]. Examined by indirect immunofluorescence, the neutrophil labeling pattern of these antibodies shows a perinuclear staining distinguishable from the one produced by ANCA in Wegener granulomatosis, which exhibits a diffuse fluorescent neutrophil cytoplasm labeling. P-ANCAs have a sensitivity of 52% and a specificity of 91% in differentiating ulcerative colitis from Crohn’s disease [14]^[9]. High similarity in anti-OmpC and anti-Pseudomonas fluorescence-associated peptide I2 antibodies has been demonstrated in discordant monozygotic twins. Twin pairs, but not discordant dizygotic twin pairs, suggest that both Anti-OmpC and anti-I2 stand for a genetically determined loss of tolerance [26]^[10]. The role of anti-glycan antibodies in disease pathogenesis has also been investigated in unaffected first-degree relatives. Both qualitative and quantitative analysis revealed that unaffected first-degree relatives have increased antibody response to microbial antigens. This impaired immunological response towards enteric microorganisms may result from genetic predisposition [29]^[11]. New insights into the pathogenesis of IBD involve an inappropriate and persistent inflammatory response to commensal gut microbiota in genetically susceptible individuals. Indeed, studies show that the intestinal microbiota in IBD patients is distinct from that found in healthy subjects and bacteria play an important role in the onset and perpetuation of the inflammatory process. Microbial biomarkers hold promise in assessing disease activity, treatment effectiveness and in personalizing treatment strategies.  In Crohn’s disease, a loss of beneficial bacteria, such as Faecalibacterium prausnitzii and an increase in Escherichia coli, have been observed. Particular strains of E. coli, such as enteroadherent E.coli, may be associated with disease in a subset of Crohn’s disease patients with ileal involvement [32]^[12]. The composition of the fecal microbiota has been less characterized in ulcerative colitis patients, but a lower abundance of Roseburia hominis (p < 0.0001) and Faecalibacterium prausnitzii (p < 0.0001) was found in ulcerative colitis patients compared to controls by real-time PCR analysis [31]^[13]. Longitudinal studies involving a large cohort of European IBD patients confirmed greater dysbiosis and lower microbial diversity in Crohn’s than in ulcerative colitis patients [33]^[14].

2.3. Cytokines

In the inflamed mucosa, immune cell recruitment produces cytokines and this leads to a stimulation and amplification of the inflammatory cascade and some of them are now the preferred target of biological drugs. They may contribute to our understanding of the inflammatory events and features of IBD, but the increased expression of proinflammatory cytokines in the intestinal mucosa is not always accompanied by increased concentrations of cytokines in the circulation. The levels of IL-33, a member of the IL-1 family, and its ligand-related protein, ST2, were increased in the mucosa as well as in the serum of ulcerative colitis patients, and a good correlation was found with disease severity [35]^[15]. Oncostatin M, an IL6 family cytokine quickly released during degranulation, has been implicated in the pathogenesis of IBD and as an emerging marker for non-responsiveness to anti TNF alfa therapy. Serum Oncostatin M looks promising because increased levels have been found in first-degree relatives of IBD families, in newly diagnosed patients and in patients with recurrent disease after surgery [38]^[16]. IL-10 is regarded as one of the anti-inflammatory cytokines associated with ulcerative colitis. This was first demonstrated in IL-10 knock-out mice, which develop spontaneous enterocolitis [39]^[17]. The IL-10 cytokine maintains the intestinal homeostasis by inhibiting the immune response during inflammation. The measurement of circulating cytokines has received much attention in the past decades; however, the inconsistency of the results does not recommend their use outside research. This might be due to the local effect of some cytokines, to different conditions of sample storage or to the different type of assay.

2.4. MicroRNA (miRNA)

MiRNAs are a group of small noncoding RNAs, ~18–22 nucleotides, which act as regulators for post-transcriptional gene expression. The miRNAs circulate in the human peripheral blood but can also be found in urine, saliva, and feces [41]^[18]. MiRNAs affect the intestinal barrier and inflammatory reactions, so most recent research in the IBD field has measured circulating miRNAs in body fluids such as blood or feces and in homogenized tissue biopsies using microarray profiling techniques, quantitative reverse transcription PCR, and next-generation sequencing [43]^[19]. MiR-21, which increases the paracellular permeability of the intestinal epithelium and the level of TNF alfa, is possibly the most intriguing, with associations between miR-21 and IBD replicated in several studies [46,47]^[20][21].

2.5. Other Markers

Extraintestinal manifestations, as well as osteo-articular, cutaneous, ocular, hepatic, pancreatic, nephrological, endocrinological, hematological, pulmonary, and thromboembolic ones are well recognized as being associated with IBD. Arthropathy represents the most frequent extraintestinal manifestation of IBD, reported in 10–35% of patients [50]^[22]. Biochemical markers are not useful for the diagnosis when the two different inflammatory events coexist. Serum human cartilage glycoprotein 39 (YKL-40) was investigated in IBD patients with articular symptoms. Hepatobiliary diseases in IBD may range from abnormal liver function tests to primary sclerosing cholangitis. Altered liver function tests have been reported in 11% of a large cohort of Swedish patients, especially related to intestinal inflammation, and was usually reversible after the disease was brought under control [52]^[23]. The occurrence of liver steatosis and increased transaminases has also been demonstrated in a multicenter study involving IBD patients and controls [53]^[24]. Primary sclerosing cholangitis has a prevalence in ulcerative colitis ranging from 2.5 to 7.5%. 82% of PSC patients are p-ANCA positive [54]^[25]. Most patients are asymptomatic at the time of diagnosis, and routine tests show increased alkaline phosphatase and gamma–glutamyl transferase. Intestinal permeability reflects the integrity of the intestinal mucosal barrier, which enables the passage of luminal substances by unmediated diffusion [55]^[26]. Intestinal permeability can be assessed non-invasively in vivo by measuring the urinary excretion of orally administered sugars such as lactulose/mannitol, glucose and sucralose or radioactive probes such as 51Cr-EDTA. An increased lactulose/mannitol ratio, together with increased CRP levels, were independent predictors of a final diagnosis of small bowel disease in 261 consecutive patients referred to a tertiary referral center with chronic diarrhea [56]^[27]. In Crohn’s disease abnormal permeability is detected in 95% of patients with small bowel Crohn’s disease, while in Crohn’s colitis, the sensitivity is lower [57]^[28].

3. Fecal Markers

3.1. Calprotectin and Lactoferrin

The presence of intestinal inflammation increases mucosal permeability, resulting in more leukocytes passing through the mucosa and penetrating into the intestinal lumen. Leukocytes can be retrieved in stools and detected under the microscope, but since their degranulation is quick, only fresh stools can be analyzed [59,60]^[29][30]. Some leukocyte proteins (such as lactoferrin and calprotectin) are durable, however, and can be used as surrogate markers of leukocyte activity. Calprotectin is a calcium and zinc-binding protein of the S-100 protein family, which comprises 60% of the cytosolic protein in human neutrophils, and lactoferrin is a component of the granules of neutrophilic granulocytes [61]^[31]. Lactoferrin, like other neutrophil proteins such as elastase, myeloperoxidase and lysozyme, increases in concentration during the active phases of the disease by comparison with the periods of remission. Lactoferrin is stable, and its extracellular release is the most efficient [62]^[32]. Calprotectin levels can help differentiate between inflammatory and non-inflammatory bowel conditions such as diverticulosis and irritable bowel syndrome. In a prospective study of 870 consecutive patients referred for colonoscopy, elevated calprotectin levels (>50 mg/dL) were detected in 85% of patients with colorectal cancer, and 81% of those with inflammatory conditions, but also in 37% of patients with normal or trivial endoscopic findings. In patients referred for chronic diarrhea, sensitivity and negative predictive value were 100% in detecting either inflammation or cancer [63]^[33]. In gastrointestinal infections, especially of bacterial origin, fecal calprotectin concentrations are strongly elevated and correlate with disease severity [64,65]^[34][35]. Viral infections, including the coronavirus disease induced by SARS-CoV2, show abnormal calprotectin levels, although less elevated than in IBD and bacterial infections [66]^[36]. Since optimal calprotectin cut-offs are not established, clinicians may be challenged in the interpretation of intermediate concentrations of 150–250 µg/g (declared as a grey zone by STRIDE-II recommendations) [67]^[37]. On the contrary, values higher than 250 should prompt further evaluation such as endoscopy. Calprotectin is now considered a biomarker for inflammation in the gastrointestinal mucosa with implications for clinical decisions, but its biology in the gut needs further studies. In the healthy mucosa, calprotectin has a broad spectrum of immunomodulatory properties which may drive the generation of reactive oxygen species during gut injury. Therefore, not only increased fecal calprotectin concentrations are a landmark of neutrophilic inflammation, but also gut inflammation induces epithelial calprotectin expression and secretion.

3.2. HMGB1

The nuclear protein High-Mobility Group Box 1 (HMGB1) is actively secreted from immune cells in the extracellular matrix, where it behaves as a proinflammatory cytokine. HMGB1 was measured in stools of 40 IBD pediatric patients and 13 controls. HMGB1 protein levels were significantly increased (p < 0.001) in the stools of patients, but were undetectable in the controls; fecal HMGB1correlated well with fecal calprotectin levels (r: 0.77 in CD, r: 0.70 in UC; p < 0.01) [76]^[38].

3.3. S100B

S100 proteins have been demonstrated to exert a protective role in the gastrointestinal tract. S100B is typically expressed by enteroglial cells and can be detected in feces. Its role as a non-invasive indicator of gastrointestinal inflammation has been tested prospectively in 48 IBD patients. Unlike calprotectin, S100B was significantly decreased in IBD patients compared to non IBD-patients. At the onset of disease, the lowest levels were found, suggesting that S100B in feces could have a potential diagnostic value for IBD [78]^[39].

3.4. MiRNAs

MiRNAs can also be found in feces [79]^[40]; significant miRNA expression changes were observed in IBD patients for all studied miRNAs with the highest expression of miR-155 and miR-223 in testing and validation cohorts. miR-21, miR-155, and miR-223 display significant levels and could potentially be considered biomarkers for IBD [48]^[41].

3.5. Novel Markers

The search of new stool protein/peptide biomarkers for diagnosing IBD has been performed with novel proteomic methods: MALDI-TOF/MS (m/z 1000–4000) analysis for peptides and LTQ-Orbitrap XL MS analysis for proteins have shown interesting differences between IBD patients and controls [80]^[42]. The MALDI-TOF/MS spectra showed numerous features in IBD patients, unlike controls.

4. Conclusions

Traditional biochemical tests remain helpful in guiding strategies for the diagnosis of IBD. Fecal calprotectin determination is useful to rule out the presence of intestinal inflammation and to avoid unnecessary invasive procedures. New potential indices are promising, but at the moment, the accuracy for diagnosing ulcerative colitis or Crohn’s disease is suboptimal and not ready for use in clinical practice. The influence of disease location, symptom duration, type of therapy, the use of concomitant drugs and the baseline inflammation all contribute to the variability of the results. The uniqueness of each patient will probably be targeted in further studies by a panel of markers rather than one marker alone.