Other Laboratory Markers

Many proinflammatory cytokines, which are stimulated in the acute phase response as TNF-α, interferon-β, and IL-1β, IL-6, and IL-8, may also be elevated in serum of IBD patients. Other potential serum markers comprise adenosine deaminase, soluble ST2 and tryptophan. Currently, none of these molecules is used in routine practice, and their use is exclusively for basic science studies.

Cellular components of blood may also show inflammation. In contrast with infectious diarrhea, the platelet count may be elevated in active CD, whereas the platelet volume is low. Platelets increase in IBD contributes to the high-coagulated status and the high risk of thrombosis. In active IBD, the white blood cell count may be elevated, but it is not specific, it may be influenced by corticotherapy. Red blood cell distribution has showed to be a good predictor of disease activity in CD patients without anemia.

Serum albumin may be low, but it is also affected by nutritional status. These changes may alarm the clinicians about ongoing inflammation.

Fecal Calprotectin

Undoubtedly, the incorporation into routine of fecal calprotectin (FC) as a noninvasive, “gold standard” marker of intestinal inflammation has been a high advance in the management of IBD patients. The molecular pattern proteins S100A8/S100A9, which are damage-associated, collectively called calprotectin and S100A12, are also steady in stool, and are increased in active IBD. Calprotectin is a 36-kDa protein that mixes zinc and calcium and inhibits metalloproteinases. It has antimicrobial effects and induces apoptosis in cell cultures. It is resistant to bacterial degradation in the gut and steady in feces, up to 7 days at room temperature. Its quantification in stool is by enzyme-linked immunosorbent assays. Calprotectin makes up 60% of granulocyte cytosolic protein, and is directly proportional with neutrophil migration toward the intestinal tract, making it a sensitive marker of inflammation. FC can be produced by monocytes and possibly epithelial cells and is remarkably elevated in several conditions, including neoplasia, polyps, other inflammatory colitis (nonsteroidal antiinflammatory enteropathy, microscopic colitis, or allergic colitis), advancing age, celiac disease, and infections. FC has shown to be able to identify patients with symptoms of IBD who should be further investigated for a possible IBD diagnosis and to monitor disease activity or to evaluate the response to therapy; in addition, it can predict a relapse and postoperative recurrence and has a good correlation with endoscopic and histologic activity. However, there are not any well-established cutoff levels because they vary according to the patient’s condition.

Fecal Lactoferrin

Lactoferrin is an iron-binding glycoprotein found in neutrophil granules with antimicrobial properties and is activated in acute inflammation. It is steady for 5 days, resistant to freeze–thaw cycles and degradation; moreover, it is also measured by enzyme-linked immunosorbent assays, easing its use as a laboratory test. Contrasting with calprotectin, lactoferrin is specific to neutrophils. A large number of studies included in a review have assessed the usefulness of fecal lactoferrin by reflecting endoscopic and histologic severity in IBD. However, the results are inconsistent and it is necessary to validate the cutoffs.

Fecal S100A12

S100A12 is similar to calprotectin in its calcium-binding properties and it has high sensitivity and specificity (86% and 96%, respectively). In addition, it is easy to collect and detect, steady for 7 days at room temperature, inexpensive, and has good compliance. This protein activates the nuclear factor-κB pathway and increases cytokine release. Although S1000A12 is also detectable in serum, the fecal essay is more sensitive and specific for IBD. Several studies in pediatric and adult IBD patients have demonstrated its correlation with disease activity, response of treatment, mucosal healing, and disease relapse.

Fecal Myeloperoxidase

Myeloperoxidase, an enzyme that works in the oxygen-dependent killing microorganisms, is released from the primary granules of neutrophils during acute inflammation. The concentration of this enzyme is also proportional to the number of neutrophils within that region. Myeloperoxidase might be used as a surrogate marker for the determination of outcomes of successful treatment for IBD patients, but it has not a good correlation with the severity of the endoscopic inflammation. Later, fecal myeloperoxidase levels have been related to histologic indices of UC patients. Further investigations are necessary to identify the clinical role of fecal myeloperoxidase in IBD.

Rectal Nitric Oxide

In response to acute proinflammatory cytokines, leukocytes and epithelial cells express inducible nitric oxide (NO) synthase, which leads to the production and accumulation of significant quantities of NO. The levels of NO, an endogenously produced gas, have been explored in serum, exhaled directly in the intestinal lumen (NO gas) in IBD patients. According to this, the level of rectal NO has been correlated with the disease activity and the quantity of loose stools in IBD patients and great decrease in response to antiinflammatory treatment. Moreover, low rectal NO levels are predictive of a poor clinical response to steroid treatment.

Other fecal biomarkers are being investigated to be used in IBD patients. Although promise exist, these alternatives have shown less consistent results, lower correlation to disease activity, and overlap among patients with active and inactive disease. These include lysozyme, leukocyte esterase, elastase, TNF-α, IL-1B, IL-4, IL-10, α1-antitrypsin, and α-2-macroglobulin. M2-pyruvate kinase may be the most promising of these developing fecal biomarkers.

Usefulness in the Diagnosis of Inflammatory Bowel Disease

Gastroenterologist help many patients with nonspecific abdominal pain or diarrhea present in organic or functional disorders. The usefulness of the biomarkers in this context is to screen patients who would benefit from invasive and expensive techniques like colonoscopy or radiographs. A metaanalysis of studies of diagnostic accuracy demonstrated that FC can distinguish IBD from irritable bowel syndrome in adults between with a sensitivity of 93% and specificity of 96%. In studies of children and teenagers, the sensitivity was similar to adults (92%); however, the specificity was lower (76%), probably because healthy children up to 9 years old had higher levels of FC. This study showed FC is a useful screening tool for the investigation of suspected IBD and reduces the number of unnecessary endoscopic procedures. A recent systematic review and metaanalysis have revealed CRP and FC of 0.5 mg/dL or less or 40 μg/g or less, respectively, exclude IBD in patients with symptoms with a 99% probability. The cutoff values of these studies range from 24 to 150 μg/g of stool.

The Role of Biomarkers to Evaluate Disease Activity

Currently, it is known endoscopic disease activity is better correlated with biomarkers than with indices of clinical disease activity. A recent review with 28 studies showed the capacity of FC to determine endoscopic disease activity in IBD patients. In CD patients the correlation between the CD Endoscopic Index Severity (CDEIS) and FC is considerably better than its correlation with CD Activity Index. A value of 272 μg/g has demonstrated to be optimal distinguishing endoscopic remission (CDEIS <3). Another study showed levels of 250 μg/g or less predicted endoscopic remission (CDEIS ≤3) with 94.1% sensitivity and 62.2% specificity. A cutoff value of 250 μg/g indicated the presence of large ulcers in CD (sensitivity of 60.4% and specificity of 79.5%). Moreover, FC has demonstrated improved usefulness for activity monitoring that other blood biomarkers and the clinical activity index (CRP, blood leukocytes, and CD Activity Index). It was the only marker that significantly discriminated inactive endoscopic disease from mild activity (104 ± 138 vs 231 ± 244 μg/g; P <.001), mild from moderate activity (231 ± 244 vs 395 ± 256 μg/g; P = .008), and moderate from high activity (395 ± 256 vs 718 ± 320 μg/g; P <.001). However, the correlation between the FC levels and endoscopic activity seems better when the disease has ileocolonic or colonic involvement than when it is a pure ileal location.

It seems FC levels show better correlation with endoscopically and histologically disease activity in UC than in CD. In UC, an FC of greater than 250 μg/g presents a high sensitivity and specificity (71% and 100.0%, respectively) for active mucosal disease activity (Mayo Score >0). FC levels significantly correlates with symptom scores in UC ( r = 0.561; P <.001), but not in CD. Similar to CD, endoscopic disease activity correlates better with FC than other blood biomarkers and the clinical activity index (CRP, platelets, blood leukocytes, hemoglobin, and Lichtiger index). FC is the only marker that can be discriminated among different grades of endoscopic activity (grade 0, 16 [10–30] μg/g; grade 1, 35 [25–48] μg/g; grade 2, 102 [44–159] μg/g; grade 3, 235 [176–319] μg/g; grade 4, 611 [406–868] μg/g; P <.001 for discriminating the different grades). A cutoff of 57 μg/g has got high sensitivity (91%) and specificity (90%) to detect endoscopically active disease (modified Baron Index ≥2). Notably, histologic features of inflammation can be identified reliably based on their fecal level of calprotectin. Patients with active histologic inflammation have got a significantly higher average level of FC than those without active histologic inflammation (278 vs 68 μg/g; P = .002). Moreover, a recent study has demonstrated FC levels have got a good correlation with the degree of disease activity according to magnetic resonance enterography and with surgical pathology damage in ileal CD (Chiorean’s score). A cutoff value of 166.5 μg/g is predictive for a diagnosis of inflammation. No relationship is found for CRP. In the same way, a good association between FC and small bowel inflammation score in capsule endoscopy (Lewis score) has been shown. A cutoff value of 76 μg/g is useful to determine appreciable visual inflammation in small bowel with a sensitivity of 59% and a specify of 41%.

CRP is often increased in active transmural CD more than mild to moderate UC, responses based on extent of disease (less elevated in proctocolitis and left colitis and isolated small bowel CD) and it is a predictor of surgery in subgroups of patients with either UC or CD. CRP elevations (>0.8 mg/dL) are associated with clinical disease activity and endoscopic inflammation in both CD and UC, and with severe active histologic inflammation only in CD patients. Several studies have described a good correlation between CRP and activity endoscopy. In CD patients on anti-TNF therapy, the CDEIS correlates better with CRP than with clinical indices (CD Activity Index and Harvey); however, CRP is not demonstrated to be reliable to identify endoscopic remission.

The Role of Biomarkers to Prediction of Relapse

CF is able to detect subclinical mucosal inflammation and identify patients with risk of relapse. Different studies have demonstrated elevated CF in patients with clinical remission is associated with an increase of risk of relapse. For example, an FC concentration greater than 150 μg/g showed a 2-fold and 14-fold increase in the relapse risk, respectively, in those patients with CD and UC in clinical remission. A Spanish study demonstrated that FC concentration was higher among the patients with relapse than in those that remained in clinical remission (444 μg/g [95% CI, 34–983] vs 112 μg/g [95% CI, 22–996]; P <.01). Finally, an FC of greater than 200 μg/g in CD and greater than 120 μg/g in UC were associated with a 4-fold and 6-fold increase in the probability of disease activity outbreak respectively. Nevertheless, FC seems to be more useful in predicting relapse in UC and CD with colon involvement compared with isolated ileal CD, as mentioned.

Finally, FC has demonstrated to be a good predictor of relapse in IBD patients under maintenance anti-TNFα therapy. In patients treated with infliximab, high levels of FC during maintenance therapy predict relapse within the following 2 months (332 ± 168 vs 110 ± 163 μg/g in relapsing and nonrelapsing disease, respectively). An FC of greater than 160 μg/g is predictive of relapse. In contrast, low FC levels are associated with a good response to treatment and long-term remission. Similarly, patients on adalimumab maintenance therapy with low FC levels have got less probability of relapse than those with high FC levels (45 vs 625 μg/g). Small FC levels exclude relapse within at least 4 months after testing. The cutoff value to predict relapse is 204 μg/g.

Similarly, high CRP levels (>15 mg/L) have been correlated with high severity clinical relapse in CD patients. In quiescent CD, a higher CRP (>10 mg/L), fertilizing disease behavior, and disease confined to the colon are independent predictors of relapse.

The Role of Biomarkers in the Evaluation of the Response to Treatment

Several studies have demonstrated the usefulness of the biomarkers determining the efficacy of treatment accurately and noninvasively. In clinical practice, the normalization of FC levels (<50 μg/g) in IBD patients after medical treatment is a marker that predicts mucosal healing. Greater changes in FC after initiation of new therapy in active disease are correlated with better treatment response. It has suggested for deescalation of any drug or cessation of corticosteroids or mesalamine, a confirmation of biological remission with FC and CRP could be enough.

In UC patients in clinical remission but with an FC of greater than 50 μg/g, the intensification of mesalamine shows a significant decrease of FC level. Kolho and colleagues analyzed the ability of FC to reflect the response to glucocorticoid therapy. In acute UC, the levels of FC are high in patients that require colectomy, but not in corticosteroid and infliximab nonresponders. A decrease of FC concentration in week 2 in UC patients undergoing induction infliximab therapy have been correlated with endoscopic remission in week 10.

The pivotal studies such as CHARM, ACCENT 1, and SONIC have evaluated the association between the response to treatment with anti-TNF antibodies and the decrease in CRP levels. A high stool frequency as well as a high CRP and low serum albumin are related to treatment failure in UC patients. A high CRP level during relapse (>15 mg/L) has shown a better response to infliximab therapy and more severe clinical course in CD patients. Magro and colleagues demonstrate that baseline CRP values are higher in primary non response to infliximab patients when they are compared with those with sustained response (26.2 vs 9.6 mg/L; P = .015). Moreover, in this study, lower CRP levels at week 14 (<3 mg/L) are associated with sustained response (78% vs 57% achieved clinical response; P = .053). According to this, the quick normalization of CRP levels correlates with sustained long-term response to infliximab (CRP <3 mg/L at week 4) and adalimumab (CRP <10 mg/L at week 12). In contrast, elevated CRP (>0.5 mg/dL) may be an indicator of low infliximab levels (<1 μg/mL) and, in consequence, it could predict loss of response and clinical relapse.

The Role of Biomarkers to Predict Postoperative Recurrence

Surgery in CD is frequent, 80% of patients during their lifetime will need surgery and 70% of these patients will require second intestinal resection. Postoperative recurrence is common and clinical symptoms do not reflect recurrent endoscopic inflammation exactly. Treatment should be personalized and based on the risk of recurrence. Rapid detection of postoperative recurrence and subsequent quickly intervention will prevent new surgeries. Today, repetitive ileocolonoscopy is the gold standard to monitor the recurrence but it has many inconveniences. It is desirable to develop a good strategy by using biomarkers to determinate early postoperative recurrence.

FC usually returns to the normal level within 2 months of the surgery, and its increase is associated with recurrence. It has suggested a cutoff for FC of greater than 200 μg/mL is capable of identifying endoscopic recurrence at 1 year after surgery with 63% and 75% of sensitivity and specificity respectively. Another study demonstrated that patients without significant recurrence (Rutgeerts score i0-1) had fewer CF levels than those with postoperative recurrence (Rutgeerts score 2–4; 98 μg/g [30–306] vs 234.5 μg/g [100–612], respectively). The cutoff of value was 203 μg/g. The POCER study (Post-Operative Crohn’s Endoscopic Recurrence) explored the best strategy to prevent recurrence (defined as Rutgeerts ≥2) in postoperative CD patients. Whereas FC levels of greater than 100 μg/g indicated endoscopic recurrence, CRP did not correlate with disease recurrence.

Few studies have evaluated the role of CRP in the prediction of postoperative CD recurrence and the data are not consistent. Although Regueiro and colleagues did not find a correlation between CRP and endoscopic scores, Sorrentino and colleagues showed a significant correlation between the procedures; nonetheless, the sample size in this study was small. A recent study with 86 CD patients found that patients in endoscopic remission had a lower CRP value than patients with recurrence (3 ± 0.7 vs 8.5 ± 1.4 mg/L; P = .0014). However, there are not enough data supporting the use of CRP to determine postoperative recurrence.

FC seems better surrogate marker of endoscopy activity in recurrent CD than other clinical and serologic markers like CRP. The overall accuracy seems greater for FC (defined as >100 μg/g) than for hsCRP (defined as 1 mg/L; 77% vs 53%, respectively). FC values of 100 μg/g or less strongly suggest recurrent disease and no need for further endoscopic procedures.

The Role of Biomarkers to Monitored Ileo–pouch–anal Anastomosis

The role of FC for early detection of pouchitis in patients with UC who have undergone proctocolectomy with ileo–pouch–anal anastomosis is highlighted. FC has been shown to distinguish reliably between inflamed and noninflamed pouches and correlate with the severity of pouchitis. A recently prospective study has demonstrated that FC and lactoferrin levels are elevated 2 months before the diagnosis of pouchitis. Patients with pouchitis after restorative proctocolectomy for UC have higher levels of FC than those without pouchitis. A cutoff value of 56 μg/g for FC has shown a sensitivity of 100% and a specificity of 84% to predict pouchitis, whereas a cutoff value of 50 μg/g for lactoferrin has reached a sensitivity of 90% and a specificity of 86%. These fecal biomarkers can be useful for the early diagnosis of pouchitis and the detection of patients for colonoscopy assessment and therapeutic adjustment.