Diagnostics and Prognostics in IBD Dig Dis 2013;31:336–344 DOI: 10.1159/000354689
Diagnostics and Prognostics of Inflammatory Bowel Disease with Fecal Neutrophil-Derived Biomarkers Calprotectin and Lactoferrin Taina Sipponen Department of Medicine, Clinic of Gastroenterology, Helsinki University Central Hospital, Helsinki, Finland
Abstract Crohn’s disease (CD), ulcerative colitis (UC), and colitis unclassified, collectively defined as inflammatory bowel disease (IBD), are the consequence of chronic inflammatory reactions in the gastrointestinal tissue. Endoscopy with biopsies is the mainstay in the diagnosis of this inflammation and is also important in the assessment of disease activity and monitoring of treatment. Furthermore, mucosal healing is increasingly becoming a therapeutic target for treatment of IBD and the golden standard of assessing it is endoscopy. However, due to the costs, invasiveness, and to limited endoscopic capacity, the need is strong for reliable surrogate markers of intestinal inflammation. Bowel contents, being in close contact with intestinal mucosa, can take up molecules that are measurable from stool samples and thus can serve as markers of inflammation. The fecal neutrophil-derived biomarkers, especially calprotectin and lactoferrin, have several features of an ideal test for detecting intestinal inflammation: they are noninvasive, simple, and low in cost. The utility of these biomarkers in distinguishing IBD from nonin-
© 2013 S. Karger AG, Basel 0257–2753/13/0314–0336$38.00/0 E-Mail [email protected] www.karger.com/ddi
flammatory conditions such as irritable bowel syndrome is well documented. They correlate closely with endoscopic activity both in CD and UC. They allow serial monitoring of disease activity and of treatment success, and can even serve in predicting clinical relapse in unsymptomatic patients or sustained remission after induction with TNF-α-blocking agents. In this review an overview will be given to the role of fecal neutrophil-derived biomarkers calprotectin and lactoferrin in diagnostics and prognostics of IBD. © 2013 S. Karger AG, Basel
Introduction
Inflammatory bowel diseases (IBD), the main types being ulcerative colitis (UC) and Crohn’s disease (CD), are lifelong, chronic intestinal inflammatory conditions marked by recurrent episodes of inflammation in the gastrointestinal tract. Conventional laboratory tests such as C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) are widely used in diagnosis and monitoring of IBD, but these tests are insufficiently sensitive to detect intestinal inflammation. Endoscopy with biopsies remains the gold standard for detecting and quantifying intestinal inflammation [1, 2]. It allows assessment of the Taina Sipponen, MD, PhD Helsinki University Central Hospital Clinic of Gastroenterology PO Box 340, FI–00029 Helsinki (Finland) E-Mail taina.sipponen @ hus.fi
Downloaded by: UCSF Library & CKM 169.230.243.252 - 9/29/2014 10:39:37 AM
Key Words Calprotectin · Lactoferrin · Disease activity · Ulcerative colitis · Crohn’s disease
Calprotectin
Overview of Calprotectin Calprotectin was first isolated from human granulocytes by Fagerhol et al. [5]. It is a 36-kDa calcium- and zinc-binding protein complex consisting of one light and two heavy polypeptide chains [6]. Calprotectin is one of the phagocytic S100 proteins that are endogenous molecules released by activated or damaged cells under conditions of cell stress [7]. Calprotectin is a major component of human neutrophil granulocytes, constituting up to 60% of their cytosol protein [5], but it is also expressed in activated macrophages and monocytes [8, 9]. Upon neutrophil activation or upon endothelial adhesion of monocytes, calprotectin is released and is besides feces, detectable in serum and other body fluids, for example synovial fluid, saliva, and urine [10–12].
Fecal Biomarkers in IBD
Fecal Calprotectin FC is already present in meconium even at low gestational ages, and it is – apparently as a normal phenomenon – higher (even >10-fold) in infants in the first year of life than in children over 1 year [13, 14]. From approximately that age up to 17-year-old healthy children, calprotectin concentrations seem to be similar to that of healthy adults [15, 16]. In adults, a day-to-day variation in calprotectin excretion in serial samples has been demonstrated both in IBD patients and in controls [17, 18]. However, a recent study in a large cohort of clinically quiescent CD patients showed a low day-to-day variation in calprotectin concentrations obtained on 3 consecutive days [19]. Roseth et al. [20] developed a method for extraction of calprotectin from feces and quantification by an enzymelinked immunoassay (ELISA) and showed that FC concentrations in UC and CD patients were significantly higher than those in healthy volunteers or in non-IBD controls [20]. In the presence of Ca2+, calprotectin is very resistant to degradation and at room temperature stable in stool for up to 7 days [20], making transport of samples to the laboratory by ordinary mail possible. An improved immunoassay for FC was later published [21]. According to the manufacturer, the normal level of FC is usually suggested to be 250 μg/g had a sensitivity of 71% and specificity of 100% in detecting endoscopically active UC [51]. Diagnosing Active CD with Fecal Calprotectin In CD, symptoms do not always correlate with inflammatory activity in the bowel. Continuing inflammation in symptomless patients may indicate a stage of active ongoing inflammation which can progress to clinical relapse or complications. Thus, a reliable surrogate marker for continuing inflammation is in CD probably even more important than in UC. A Finnish study compared FC levels to clinical and endoscopic activity in CD [56]. All patients with clinically active disease (CDAI >150) had an elevated calprotectin, indicating that their symptoms were a result of intestinal inflammation and not a sign of Sipponen
Downloaded by: UCSF Library & CKM 169.230.243.252 - 9/29/2014 10:39:37 AM
Fecal Calprotectin in Diagnostics of IBD FC has proved to be a useful tool in discriminating between IBD and irritable bowel syndrome (IBS) in several studies. Tibble et al. [29] measured FC in 602 consecutive patients with abdominal symptoms. At an FC cutoff level of 10 mg/l (50 μg/g converted to a newer assay), sensitivity was 89% and specificity was 79% for organic bowel disease. In other studies, sensitivity in adults has ranged from 63 to 100% and specificity from 80 to 100% in differentiating IBD from functional disorders [4, 15, 30–35]. Accordingly, the positive predictive value has been between 70 and 100% and the negative predictive value between 51 and 91% [15, 30–35]. Comparable results have been detected in pediatric studies [15, 32, 36, 37]. A metaanalysis based on prospective studies has evaluated the diagnostic precision of FC for IBD [22]. A total of 663 patients (both adults and children) had CD, 361 UC, and 183 patients were labeled as having IBD. For diagnosis of IBD, a sensitivity of 89% and a specificity of 81% were calculated from the data of eight studies [4, 15, 30, 36, 38–41]. Another meta-analysis of 13 studies found that initial screening of suspected IBD by FC would result in a 67% reduction in the number of adults requiring endoscopy [42]. The screening performance of FC and fecal hemoglobin has been compared in chronic diarrhea patients undergoing colonoscopy [43]. Increased calprotectin levels were significantly associated with colorectal inflammation, whereas fecal hemoglobin levels were not. At an FC cutoff level of 100 μg/g, the sensitivity for any colorectal inflammation was 83% and the specificity 83%. In a more recent study of chronic nonbloody diarrhea patients, FC correlated closely with findings of endoscopic and histological inflammation [44]. Performance of FC against other fecal biomarkers has been studied and FC seems to perform equal to FL [33] or slightly better than FL or PMN-E [35, 45] in detecting inflammation in patients with lower gastrointestinal symptoms. In one study a granulocyte-specific protein, S100A12, however, showed even better sensitivity and specificity in distinguishing active IBD from IBS than calprotectin [46], but this finding needs confirmation in other studies. For the diagnosis of IBD, FC appears in several studies to be superior to serological markers such as CRP, ESR, ANCA, or ASCA [4, 16, 34, 45, 47–49] and combining of serological markers ASCA/pANCA with FC or FL seems to only marginally increase diagnostic accuracy compared to either fecal test alone [34].
Table 2. Fecal calprotectin in monitoring treatment response of IBD Reference (first author), study design
Patients, n disease
Therapy
Pretreatment median calprotectin, μg/g (range)
Posttreatment median calprotectin, μg/g (range)
Roseth, 2004 [55] retrospective
18 IBD
data not available (NA)
3,000 (850 – 14,800)
18 (1 – 50)
Sipponen, 2008 [59] prospective
15 CD
infliximab or adalimumab induction
1,173 (88 – 15,326)
130 (13 – 1,419)
Wagner, 2008 [88] prospective
38 IBD
various medications
5,430a (151 – 14,170)b
1,720a (38 – 3,390)b
Diagnostics and Prognostics of Inflammatory Bowel Disease with Fecal Neutrophil-Derived Biomarkers Calprotectin and Lactoferrin Taina Sipponen Department of Medicine, Clinic of Gastroenterology, Helsinki University Central Hospital, Helsinki, Finland
Abstract Crohn’s disease (CD), ulcerative colitis (UC), and colitis unclassified, collectively defined as inflammatory bowel disease (IBD), are the consequence of chronic inflammatory reactions in the gastrointestinal tissue. Endoscopy with biopsies is the mainstay in the diagnosis of this inflammation and is also important in the assessment of disease activity and monitoring of treatment. Furthermore, mucosal healing is increasingly becoming a therapeutic target for treatment of IBD and the golden standard of assessing it is endoscopy. However, due to the costs, invasiveness, and to limited endoscopic capacity, the need is strong for reliable surrogate markers of intestinal inflammation. Bowel contents, being in close contact with intestinal mucosa, can take up molecules that are measurable from stool samples and thus can serve as markers of inflammation. The fecal neutrophil-derived biomarkers, especially calprotectin and lactoferrin, have several features of an ideal test for detecting intestinal inflammation: they are noninvasive, simple, and low in cost. The utility of these biomarkers in distinguishing IBD from nonin-
© 2013 S. Karger AG, Basel 0257–2753/13/0314–0336$38.00/0 E-Mail [email protected] www.karger.com/ddi
flammatory conditions such as irritable bowel syndrome is well documented. They correlate closely with endoscopic activity both in CD and UC. They allow serial monitoring of disease activity and of treatment success, and can even serve in predicting clinical relapse in unsymptomatic patients or sustained remission after induction with TNF-α-blocking agents. In this review an overview will be given to the role of fecal neutrophil-derived biomarkers calprotectin and lactoferrin in diagnostics and prognostics of IBD. © 2013 S. Karger AG, Basel
Introduction
Inflammatory bowel diseases (IBD), the main types being ulcerative colitis (UC) and Crohn’s disease (CD), are lifelong, chronic intestinal inflammatory conditions marked by recurrent episodes of inflammation in the gastrointestinal tract. Conventional laboratory tests such as C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) are widely used in diagnosis and monitoring of IBD, but these tests are insufficiently sensitive to detect intestinal inflammation. Endoscopy with biopsies remains the gold standard for detecting and quantifying intestinal inflammation [1, 2]. It allows assessment of the Taina Sipponen, MD, PhD Helsinki University Central Hospital Clinic of Gastroenterology PO Box 340, FI–00029 Helsinki (Finland) E-Mail taina.sipponen @ hus.fi
Downloaded by: UCSF Library & CKM 169.230.243.252 - 9/29/2014 10:39:37 AM
Key Words Calprotectin · Lactoferrin · Disease activity · Ulcerative colitis · Crohn’s disease
Calprotectin
Overview of Calprotectin Calprotectin was first isolated from human granulocytes by Fagerhol et al. [5]. It is a 36-kDa calcium- and zinc-binding protein complex consisting of one light and two heavy polypeptide chains [6]. Calprotectin is one of the phagocytic S100 proteins that are endogenous molecules released by activated or damaged cells under conditions of cell stress [7]. Calprotectin is a major component of human neutrophil granulocytes, constituting up to 60% of their cytosol protein [5], but it is also expressed in activated macrophages and monocytes [8, 9]. Upon neutrophil activation or upon endothelial adhesion of monocytes, calprotectin is released and is besides feces, detectable in serum and other body fluids, for example synovial fluid, saliva, and urine [10–12].
Fecal Biomarkers in IBD
Fecal Calprotectin FC is already present in meconium even at low gestational ages, and it is – apparently as a normal phenomenon – higher (even >10-fold) in infants in the first year of life than in children over 1 year [13, 14]. From approximately that age up to 17-year-old healthy children, calprotectin concentrations seem to be similar to that of healthy adults [15, 16]. In adults, a day-to-day variation in calprotectin excretion in serial samples has been demonstrated both in IBD patients and in controls [17, 18]. However, a recent study in a large cohort of clinically quiescent CD patients showed a low day-to-day variation in calprotectin concentrations obtained on 3 consecutive days [19]. Roseth et al. [20] developed a method for extraction of calprotectin from feces and quantification by an enzymelinked immunoassay (ELISA) and showed that FC concentrations in UC and CD patients were significantly higher than those in healthy volunteers or in non-IBD controls [20]. In the presence of Ca2+, calprotectin is very resistant to degradation and at room temperature stable in stool for up to 7 days [20], making transport of samples to the laboratory by ordinary mail possible. An improved immunoassay for FC was later published [21]. According to the manufacturer, the normal level of FC is usually suggested to be 250 μg/g had a sensitivity of 71% and specificity of 100% in detecting endoscopically active UC [51]. Diagnosing Active CD with Fecal Calprotectin In CD, symptoms do not always correlate with inflammatory activity in the bowel. Continuing inflammation in symptomless patients may indicate a stage of active ongoing inflammation which can progress to clinical relapse or complications. Thus, a reliable surrogate marker for continuing inflammation is in CD probably even more important than in UC. A Finnish study compared FC levels to clinical and endoscopic activity in CD [56]. All patients with clinically active disease (CDAI >150) had an elevated calprotectin, indicating that their symptoms were a result of intestinal inflammation and not a sign of Sipponen
Downloaded by: UCSF Library & CKM 169.230.243.252 - 9/29/2014 10:39:37 AM
Fecal Calprotectin in Diagnostics of IBD FC has proved to be a useful tool in discriminating between IBD and irritable bowel syndrome (IBS) in several studies. Tibble et al. [29] measured FC in 602 consecutive patients with abdominal symptoms. At an FC cutoff level of 10 mg/l (50 μg/g converted to a newer assay), sensitivity was 89% and specificity was 79% for organic bowel disease. In other studies, sensitivity in adults has ranged from 63 to 100% and specificity from 80 to 100% in differentiating IBD from functional disorders [4, 15, 30–35]. Accordingly, the positive predictive value has been between 70 and 100% and the negative predictive value between 51 and 91% [15, 30–35]. Comparable results have been detected in pediatric studies [15, 32, 36, 37]. A metaanalysis based on prospective studies has evaluated the diagnostic precision of FC for IBD [22]. A total of 663 patients (both adults and children) had CD, 361 UC, and 183 patients were labeled as having IBD. For diagnosis of IBD, a sensitivity of 89% and a specificity of 81% were calculated from the data of eight studies [4, 15, 30, 36, 38–41]. Another meta-analysis of 13 studies found that initial screening of suspected IBD by FC would result in a 67% reduction in the number of adults requiring endoscopy [42]. The screening performance of FC and fecal hemoglobin has been compared in chronic diarrhea patients undergoing colonoscopy [43]. Increased calprotectin levels were significantly associated with colorectal inflammation, whereas fecal hemoglobin levels were not. At an FC cutoff level of 100 μg/g, the sensitivity for any colorectal inflammation was 83% and the specificity 83%. In a more recent study of chronic nonbloody diarrhea patients, FC correlated closely with findings of endoscopic and histological inflammation [44]. Performance of FC against other fecal biomarkers has been studied and FC seems to perform equal to FL [33] or slightly better than FL or PMN-E [35, 45] in detecting inflammation in patients with lower gastrointestinal symptoms. In one study a granulocyte-specific protein, S100A12, however, showed even better sensitivity and specificity in distinguishing active IBD from IBS than calprotectin [46], but this finding needs confirmation in other studies. For the diagnosis of IBD, FC appears in several studies to be superior to serological markers such as CRP, ESR, ANCA, or ASCA [4, 16, 34, 45, 47–49] and combining of serological markers ASCA/pANCA with FC or FL seems to only marginally increase diagnostic accuracy compared to either fecal test alone [34].
Table 2. Fecal calprotectin in monitoring treatment response of IBD Reference (first author), study design
Patients, n disease
Therapy
Pretreatment median calprotectin, μg/g (range)
Posttreatment median calprotectin, μg/g (range)
Roseth, 2004 [55] retrospective
18 IBD
data not available (NA)
3,000 (850 – 14,800)
18 (1 – 50)
Sipponen, 2008 [59] prospective
15 CD
infliximab or adalimumab induction
1,173 (88 – 15,326)
130 (13 – 1,419)
Wagner, 2008 [88] prospective
38 IBD
various medications
5,430a (151 – 14,170)b
1,720a (38 – 3,390)b
