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Journal of Pediatric Gastroenterology & Nutrition:
doi: 10.1097/MPG.0b013e3180308d8e
Original Articles: Gastroenterology

Fecal Lactoferrin Is a Sensitive and Specific Marker of Disease Activity in Children and Young Adults With Inflammatory Bowel Disease

Walker, Thomas R*,†,‡; Land, Michelle L; Kartashov, Alex; Saslowsky, Tracee M; Lyerly, David M§; Boone, James H§; Rufo, Paul A*,†,‡

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Author Information

*Center for Inflammatory Bowel Disease

Combined Program in Pediatric Gastroenterology and Nutrition

Clinical Research Center, Children's Hospital

Harvard Medical School, Boston, MA

§TECHLAB Incorporated, Blacksburg, VA

Received 8 March, 2006

Accepted 1 December, 2006

Address correspondence and reprint requests to Paul A. Rufo (e-mail: paul.rufo@childrens.harvard.edu).

Supported by a restricted grant from TECHLAB, Inc (MLL), Clinical Investigator Awards K23 DK02729 (P.A.R.), and T32 DK07477 (T.R.W), and a Career Development Award from the Crohn's and Colitis Foundation (T.R.W.). Dr. Rufo was a Pfizer Fellow in the Clinical Investigator Training Program of the Harvard-Massachusetts Institute of Technology Division of Health Sciences and Technology and the Beth Israel Deaconess Medical Center.

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Abstract

Background and Aims: Fecal lactoferrin (FLA) is a neutrophil-derived surrogate marker of intestinal inflammation that is elevated in patients with inflammatory bowel disease. However, the correlation between FLA levels and serological markers of disease activity has not been previously reported, to our knowledge. In the present study we evaluated the ability of FLA levels to reflect disease activity in pediatric patients with inflammatory bowel disease. We further assessed the relationship between FLA levels and customary laboratory and clinical measures of inflammation.

Patients and Methods: Fecal specimens were collected from 148 consecutive pediatric patients (79 with Crohn disease, 62 with ulcerative colitis, and 7 with irritable bowel syndrome) and 22 healthy control individuals. Lactoferrin was measured by enzyme-linked immunosorbent assay (IBD-SCAN, TECHLAB, Inc). Disease activity was assessed at the time of sample provision by laboratory measures (including erythrocyte sedimentation rate [ESR] and albumin) and previously validated disease activity indices (Pediatric Crohn Disease Activity Index, Kozarek, Harvey Bradshaw Activity Index).

Results: Lactoferrin levels were significantly higher in patients with ulcerative colitis (1880 ± 565 μg/mL) (mean ± SE) or Crohn disease (1701 ± 382 μg/mL) than in healthy control individuals under 21 years of age (1.17 ± 0.47 μg/mL, P < 0.001). Lactoferrin levels correlated significantly with ESR, hematocrit, albumin, and platelet count (P < 0.001). Receiver operating characteristic curve analysis revealed that FLA levels were comparable to ESR in detecting patients with clinically active disease (P < 0.001). Patients who experienced a clinical flare within 2 months of specimen collection displayed higher lactoferrin levels (845 ± 452 μg/mL) than did those who remained in clinical remission (190 ± 90 μg/mL, P = 0.003).

Conclusions: Data presented here demonstrate that FLA is a sensitive and specific biochemical marker of inflammation for use in the diagnosis and interval assessment of pediatric patients with IBD, and its level correlates well with both clinical disease activity indices and ESR. Elevated levels of FLA may also identify patients at greater risk for the development of subsequent clinical flares.

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INTRODUCTION

Inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn disease (CD), represent a spectrum of diseases characterized by an idiopathic and chronic inflammation affecting the gastrointestinal (GI) tract. Pediatric and adult patients with IBD may present with a variety of clinical symptoms (including abdominal pain and diarrhea) that can be nonspecific. The pathogenesis of IBD remains unknown, and it is generally thought to be caused by a combination of genetic, environmental, and host immune factors. Recent studies suggest that the incidence of these disorders in children may be as high as 7/100,000 per year (1).

Tissue samples from patients with active IBD demonstrate a chronic inflammatory process characterized by an accumulation of activated polymorphonuclear neutrophils (PMNs) in the lamina propria and intestinal lumen. These activated cells are capable of mounting a potent host innate immune response through the release of proteins with proinflammatory properties. Among these is lactoferrin, an 80-kDa member of the transferrin family (2–8). Lactoferrin has been identified in the secretions overlying most mucosal surfaces that interact directly with external pathogens including saliva, tears, vaginal secretions, and mammalian breast milk (2,3,9). Although fecal lactoferrin (FLA) levels were initially studied as a cost-effective way of discriminating patients with acute and self-limited (primarily viral) infectious diarrhea from those with bacterial diarrheal disease, recent studies have demonstrated that elevated FLA is also a sensitive and specific marker of intestinal inflammation in patients with chronic intestinal disease (10–17).

Clinicians rely on several biochemical studies to help either support a diagnosis of IBD or gauge a patient's disease activity during a particular office visit. These studies include complete blood counts and measurement of acute-phase reactants such as erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) (18–20). Though clinically useful, these biochemical tests lack specificity. In addition, measurement of these serum markers requires phlebotomy, a procedure that is invasive and often technically difficult and anxiety provoking in pediatric patients. With that in mind, investigators have worked to develop alternative cost-effective and reliable surrogate markers of intestinal inflammation that may limit the need for more invasive testing. Ultimately, these markers could result in a more expedient diagnosis of IBD or identification of disease flare.

The goal of this study was to evaluate the degree to which quantitative FLA levels reflect disease activity in pediatric patients with known or suspected IBD. Previous studies have demonstrated that FLA levels differ significantly in patients with inflammatory and noninflammatory diarrheal disease (10). More recent studies have further demonstrated that quantitative FLA levels may be useful in assessing disease activity in adults and children with IBD and pouchitis (10,13,16,21). Our data validated this finding in a large pediatric population. We further extended these findings to demonstrate, for the first time as far as we are aware, the relationship between FLA levels and existing biochemical markers of inflammation. We also determined the correlation between FLA levels and disease severity, as defined by existing and previously validated disease activity indices.

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PATIENTS AND METHODS

Consecutive pediatric and young adult patients (≤21 years of age) with known or suspected IBD or irritable bowel syndrome (IBS) were recruited from the inpatient and ambulatory services at Children's Hospital Boston from March 1998 to September 2003. Accrual of study participants was delayed by research resources, and there was no systematic exclusion to bias the selection. Patients characterized as having clinically active or inactive disease were asked to participate. Classification of IBD was based on endoscopic and histological criteria (22). Ulcerative colitis was defined as endoscopically and histologically confluent mucosal inflammation beginning in the rectum and limited to the colon, and an absence of granulomata in histopathologic sections. Crohn disease was defined either as endoscopically or histologically discontinuous colonic disease, or radiological, endoscopic, or histological evidence of extracolonic or granulomatous mucosal disease. Fecal specimens were collected from 148 patients (79 with CD and 62 with UC). Stool samples collected from an age-matched control population of 22 healthy individuals were included in our analysis. Seven patients with IBS also submitted samples for assay of FLA. Patients with UC and a colectomy, those with concurrent or recent (within 2 months) culture-positive or toxin-positive bacterial colitis, and those with hepatitis A, B, or C or HIV were not eligible for participation.

This study protocol was approved by the Committee on Clinical Investigation at Children's Hospital. Written informed consent was obtained from all parents or legal guardians. Patient assent was also obtained when appropriate. Disease activity was determined prospectively by study personnel at the time of enrollment, using relevant disease activity indices and available laboratory data that were extracted from the medical record.

Stool samples were collected from inpatients in the GI service at Children's Hospital Boston. Ambulatory patients who were unable to provide a fecal specimen at the time of enrollment were permitted to collect samples at home and return them (refrigerated) using a commercial overnight delivery service in accordance with IPAA regulations. For study participants mailing fecal samples by overnight courier, concurrent disease activity was assessed by written questions (included in the sample mailing kit and/or collected by telephone).

Radiological data extracted from upper GI contrast studies were scored (presence or absence of disease) within anatomic regions of the esophagus, stomach, and small intestine. Histological data were similarly coded for each section of the GI tract when available. Radiological and endoscopic data were not typically obtained coincident with study enrollment and did not necessarily reflect the location, degree, or extent of mucosal inflammation at the time of FLA sample collection. In patients with multiple previous studies, data were coded from the radiological or endoscopic study occurring most closely to the time when the fecal sample was collected.

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Data Collection

Pediatric Crohn Disease Activity Index (PCDAI) and Harvey Bradshaw Activity Index (HBAI) scores were calculated for patients with CD. Not all patients had these data available for subsequent analysis. The PCDAI categorizes patients into 1 of 3 categories: inactive (<10), mildly active (10–30), and moderate to severe (>30) (23). A modified HBAI, used in a previous publication from our group, characterizes patients as having inactive (<4) or active (≥4) disease (10,24). Kozarek scoring for UC discriminates patients as having inactive (0–3), mild (4–6), or moderate to severe (≥7) disease (25). We also used the Physician's Global Assessment (PGA) in our analyses. The PGA is based on the integration of all available clinical and laboratory information and therefore has the potential to be the most valid assessment of a patient's clinical status. The PGA has been used successfully in many previous clinical trials (26–30). At the same time we recognized that these assessments carry with them a degree of interprovider variability. “Active disease” was defined as clinical deterioration significant enough to warrant a change in medical management, such as increasing the dose of an existing medication or adding a new medication to the patient's regimen. “Inactive disease” was assigned when no change in medical therapy was instituted during the most recent physician encounter. Patients who were being actively weaned from steroid therapy (“steroid weaning”) at the time of specimen collection were considered to have active disease in our analysis. Unlike the PCDAI, HBAI, or Kozarek scores, which were obtained prospectively, the PGA was assigned retrospectively, based on the treating physician's assessment at the time of sample collection and at 2 months after enrollment. In all cases the designation of clinical disease activity was determined by investigators blinded to FLA measurements. Physician's Global Assessments of this type have been used successfully by investigators in several previously reported studies (31–38).

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Lactoferrin Assay

Collected fecal specimens were labeled, stored at –80°C, and batched for subsequent analysis. Fecal lactoferrin was quantified by use of a commercially available polyclonal-based enzyme-linked immunosorbent assay (IBD-SCAN; TECHLAB, Inc) and reported as microgram per milliliter of feces, as previously described (10). Mean lactoferrin levels are expressed as mean ± standard error (SE). Previous studies have demonstrated that the microgram per milliliter wet weight correlated with clinical data and showed no advantage to expressing the results based on dry weight and/or milligrams of fecal protein (data on file at TECHLAB). All lactoferrin measurements were performed by technicians who were blinded to the study participants' diagnoses and disease activity. A cutoff point of ≥7.25 μg/mL for indicating elevated FLA has been previously established and validated for use in patients with IBD (10,21). Fecal lactoferrin is stable at room temperature for as long as 5 days (39).

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Statistical Analysis

Data were collected in an SPSS database (SPSS, Chicago), and subsequent analysis was performed by use of SAS (SAS Institute, Cary, NC). Fecal lactoferrin levels were not normally distributed in our study population. Accordingly, we used the Mann-Whitney test in statistical assessments. We present raw FLA measurements when reporting means, distribution, and sensitivity and specificity values. Log transformation permitted the use of parametric testing in studies evaluating the correlation between FLA levels and disease activity indices and biochemical parameters. Fecal lactoferrin was compared across diagnoses (IBD, CD, and UC) and level of disease activity. The relationship between FLA levels and other biochemical markers of inflammation was assessed by Spearman rank correlation. Receiver operating characteristic (ROC) curves were generated to determine the sensitivity and specificity of FLA in discriminating patients with IBD from healthy control individuals and the ability of FLA to discriminate the presence or absence of active disease in patients with IBD. Area under the curve (AUC) with 95% confidence interval was calculated for each ROC curve, and AUCs were compared between different markers of inflammation by use of the statistical package Medcalc. Mean values are reported together with standard deviations, unless otherwise specified. P < 0.05 were considered significant.

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RESULTS

FLA Levels Discriminated Pediatric Patients With IBD From Healthy Control Participants

A total of 170 (101 male, 69 female) individuals with a mean age of 13.4 years (range 2–21 years) provided single fecal specimens for use in this study (Table 1). Of the 170 study participants, 107 had IBD (56 CD, 51 UC), and blood work was completed within 1 week of specimen collection. These data were used in analyses correlating FLA levels with existing biochemical parameters of intestinal inflammation. Levels of FLA were significantly greater (Fig. 1) in patients with UC (1880 ± 565 μg/mL) (mean ± SE) or CD (1701 ± 382 μg/mL) than in healthy control individuals. The upper limit of normal for healthy control individuals was 7.25 μg/mL, corresponding to a log FLA 0.086. Mean FLA levels measured in healthy control individuals (1.17 ± 0.47 μg/mL) fell below the 7.25-μg/mL cutoff and were statistically indistinguishable from those measured in the 7 patients with IBS (2.08 ± 0.90 μg/mL).

Table 1
Table 1
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Fig. 1
Fig. 1
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Similarly, we did not detect a significant difference in FLA levels between patients with UC and those with CD (P = 0.603). The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) in distinguishing individuals with IBD from those without IBD (IBS and healthy control individuals) were 83.5%, 96.6%, 99%, and 55%, respectively. All 7 patients with IBS, 21 of 22 healthy control individuals (1 individual had FLA = 8.09), and 23 of 141 patients with IBD had normal (<7.25 μg/mL feces) FLA levels. Only 2 of 23 patients with IBD and normal FLA levels were scored as active according to either the HBAI (HBAI ≥4) or the PGA.

Receiver operating characteristic curves comparison demonstrated that FLA levels displayed greater sensitivity and specificity (AUC 0.932 ± 0.026, P < 0.001) for identifying patients with IBD than did ESR (AUC 0.750 ± 0.085, P = 0.04) (Fig. 2). Including all patients without IBD (IBS and healthy control individuals), the AUC was 0.952 ± 0.015, P < 0.001. Despite the small number of patients with IBS, we next evaluated FLA measurements as a screening tool for discriminating 87 patients with active GI symptoms (HBAI score >2) as having IBD or IBS. In this symptomatic population we found that the sensitivity of elevated FLA was 97% (91%–99%, 95% CI) and the specificity was 100% (57%–100%). The corresponding AUC of the ROC in this subset of patients was 0.983 ± 0.013 (P < 0.001, data not shown).

Fig. 2
Fig. 2
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FLA Levels Correlated With Disease Activity in Patients With IBD

We next examined the correlation between FLA and clinical disease severity in subsets of patients with IBD, CD, or UC (Table 2). Disease activity was assessed according to several previously validated disease activity indices. PGA and HBAI and scores were available for 130 and 135 of 141 patients with IBD, respectively. PGA was assigned retrospectively (and blinded to FLA levels) based on the assessment of a patient's treating gastroenterologist and characterized into 1 of 3 categories: active, inactive, and steroid weaning. By use of the PGA 84 patients were classified as having active disease and 55 as having inactive disease. Fecal lactoferrin levels were higher in patients with active disease (2782 ± 509 μg/mL) than in those with inactive disease (250 ± 94 μg/mL, P < 0.001) when stratified by PGA. Patients with active IBD (defined as an HBAI score ≥ 4) had significantly higher FLA (2803 ± 571 μg/mL) than did those with inactive disease (695 ± 276 μg/mL, P < 0.001). Fecal lactoferrin levels correlated well with continuous HBAI scores (Spearman correlation coefficient of 0.575, P < 0.001).

Table 2
Table 2
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We next examined FLA levels in patients with CD stratified by PCDAI. We first compared levels in those with inactive disease (PCDAI <10) and those with mildly active disease (PCDAI 10–30). There was no difference in FLA levels in these 2 populations. Therefore, for further analysis, these values were combined, and they were termed inactive.

When we stratified patients with CD by PGA, we found that FLA levels measured in patients with active disease were significantly higher (2472 ± 574 μg/mL) than in patients with inactive disease (398 ± 172 μg/mL, P < 0.001). Similarly, we found that patients with moderate to severe CD (PCDAI >30) displayed significantly greater FLA levels (6177 ± 1793 μg/mL) than did those with inactive disease (658 ± 260 μg/mL, P = 0.004). FLA levels were also higher in patients with active CD (2465 ± 691 μg/mL) than in those with inactive disease (1055 ± 429 μg/mL) when assessed by HBAI (P = 0.002). In an additional analysis we compared FLA levels with continuous PCDAI scores and found a highly significant Spearman correlation (0.598) between disease activity as assessed by PCDAI and FLA measurements (P < 0.001).

FLA levels were equally sensitive predictors of disease activity in patients with UC. Physician's Global Assessment, modified HBAI, and Kozarek scores were available for 61, 60, and 39 of a total of 62 patients with UC, respectively (Table 2). FLA levels differed substantially in patients with active UC (3215 ± 926 μg/mL) in comparison with levels observed in patients with inactive UC (84 ± 33 μg/mL), as assessed by PGA (P < 0.001). Similarly, FLA levels were significantly higher (3121 ± 904 μg/mL) in patients with active UC than in patients with inactive disease (79 ± 34 μg/mL, P < 0.001) when categorized by HBAI. Spearman analysis revealed FLA levels to be highly correlated (0.701) with HBAI scoring (P < 0.001). Finally, FLA levels (93 ± 58 μg/mL) distinguished patients with clinically inactive disease (Kozarek score 0–3) from those with moderately active disease (Kozarek score 4–6) (2940 ± 1728 μg/mL, P = 0.006) and those with severe disease (Kozarek score >6) (3035 ± 979 μg/mL, P < 0.001). FLA levels in patients with moderately active (Kozarek score 4–6) and severely active (>6) disease did not differ significantly and were grouped together in comparison with patients with inactive disease (<4).

As previously noted, the PGA used in this study categorized patients receiving steroid therapy as having active disease irrespective of the presence or absence of clinical symptoms. We therefore completed a subgroup analysis that compared FLA levels in 15 patients with active disease and completing steroid tapers, 69 patients with active disease but not receiving steroid therapy, and 55 individuals with clinically inactive disease at the time of study enrollment. Interestingly, we found that patients with IBD and who were being weaned from steroid therapy displayed log-transformed FLA levels (2.22 ± 0.15 μg/mL) that were significantly higher than those measured in individuals with clinically inactive disease (1.32 ± 0.15 μg/mL, P = 0.003) but less than those measured in patients with clinically active disease but not receiving steroids (3.04 ± 0.091, P < 0.001). To compare data from patients with UC and CD, we examined log-transformed FLA measurements in each category described above. As shown in Figure 3, and as expected, FLA levels differed significantly in patients with active disease, those with inactive disease, and those who were being weaned from steroids. However, they did not discriminate patients with UC from those with CD (Fig. 3).

Fig. 3
Fig. 3
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Existing data suggest that FLA levels should rise with increasing levels of intestinal inflammation (10,40). In the present study we attempted to determine whether FLA levels identified active disease. Our data revealed that an FLA level >100 displayed a sensitivity of 87%, a specificity of 68%, a PPV of 83%, and an NPV of 77% for identifying pediatric patients with IBD and active disease (assessed by PGA) in our study population. Parallel calculations completed for an elevated ESR (>20) revealed a sensitivity of 83%, a specificity of 61%, a PPV of 89%, and an NPV of 65%. These data suggest that comparable information concerning disease activity was derived from either FLA levels or ESR.

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FLA May Predict Recurrence in Patients With IBD Who Are in Clinical Remission

In our study population 141 patients carried a diagnosis of IBD and 55 were in remission (assessed by PGA) at the time of enrollment. We retrospectively examined the clinical course of these patients (blinded to FLA) during the subsequent 2-month period. Of the patients who were in remission at the time of enrollment, 10% (5/55) went on to experience a clinical flare in their disease. Subgroup analysis revealed that FLA levels in the 5 patients (4 CD, 1 UC) in whom active disease subsequently developed (845 ± 452 μg/mL) were significantly higher than those observed in the 50 patients who remained in clinical remission (190 ± 90 μg/mL, P < 0.01). Concomitant ESR values that were available in 4 of these 5 patients at the time of sample collection were 14, 19, 23, and 79 (normal <20 mm/h). Although our sample size was small, these data suggest that elevated FLA levels may identify patients at risk for disease recurrence, or at least those who may merit closer follow-up. Larger prospective studies are necessary to further address this intriguing hypothesis.

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FLA Levels Correlated With Biochemical Markers of Inflammation

Clinicians typically define disease activity in their pediatric and adult patients with IBD using a combination of clinical and laboratory dimensions incorporating the use of ESR, hematocrit (Hct), serum albumin (Alb), and platelet count (Plt). We wanted to determine how well a patient's quantitative FLA levels compared with commonly acquired laboratory indices. To that end, we used a Spearman linear regression to measure the correlation between FLA levels and biochemical studies that were completed within 1 week of fecal sample collection (Table 3). Analyzing all of the study participants, we found significant correlations between FLA levels and ESR, Hct, Alb, and Plt. Correlations between FLA levels and ESR, Alb, Hct, and Plt were significant (P < 0.05) in patients with active or inactive disease as defined by PGA. These correlations were present when similar subgroup analyses were performed in patients with either UC or CD. Receiver operating characteristic curves comparing the relative abilities of FLA and ESR to discern disease activity in patients with IBD revealed that both markers are sensitive and specific indices of disease activity (defined by PGA, Fig. 4). Fecal lactoferrin levels displayed comparable sensitivity and specificity profiles in distinguishing patients with active IBD from those with inactive IBD (AUC 0.9 ± 0.4 when compared with serum ESR, AUC 0.8 ± 0.05). However, the difference between these surrogate markers of intestinal inflammation reached statistical significance only in patients with UC (P < 0.05).

Table 3
Table 3
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Fig. 4
Fig. 4
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DISCUSSION

The data collected in this large pediatric population demonstrate that FLA levels correlate with disease activity (assessed prospectively) determined by previously validated disease activity indices. Our data also suggest that FLA levels may be elevated in patients who are clinically well but subsequently experience a flare in disease activity. The data are consistent with findings reported in previous adult series (10,13,16,21).

The acute and chronic active inflammation observed in patients with IBD is characterized by an influx of activated neutrophils into and across the mucosal lining, resulting in the formation of crypt abscesses in the intestinal lumen. Clinicians have used the identification of fecal leukocytes as a way to differentiate patients with inflammatory (ie, invasive bacterial infection, IBD) from those with noninflammatory (ie, viral, toxigenic) diarrheal illness. Fecal leukocyte analysis, however, is limited by its lack of sensitivity, its dependence on a fresh stool sample, and the skill of the laboratory technician (41–44). By contrast, measuring FLA, a product of activated neutrophils (43), is a simple and reliable method of detecting and quantifying inflammatory cells in the stool of patients whose conditions are being evaluated for IBD (10,13,16,21).

Several factors support the inclusion of FLA levels in the diagnosis and interval assessment of IBS. First, because lactoferrin can be measured in feces, it is likely to be a more direct measure of intestinal inflammation than are serum markers such as ESR (as demonstrated in the present study) that reflect systemic inflammation. Second, because FLA measurements are collected noninvasively, they are especially attractive for use in pediatric patients, in whom phlebotomy is both anxiety provoking and technically difficult. Third, the relative ease of sample collection and the prospect of monitoring FLA in samples collected and then sent through the mail (similarly to samples for ova and parasites examination) or collected at a patient's bedside, makes FLA measurements especially well suited for serial assay in the same patient over time. Finally, quantitative FLA levels are easily and reliably measured in the stool by use of a commercially available diagnostic test that has been cleared by the US Food and Drug Administration (10,17,42).

It should be noted that there are limitations to the use of FLA measurements and other stool-based assays that clinicians must keep in mind when applying surrogate markers in the diagnosis and treatment of their patients with IBD. It may be more difficult for parents to collect samples from less cooperative pediatric patients. In addition, elevated FLA levels indicate intestinal inflammation that may be caused by infections and by clinical flares in IBD activity. Therefore, clinicians need to consider obtaining stool cultures from patients presenting with rectal bleeding or symptoms of colitis.

Most fecal markers studied to date, including α-1-antitrypsin, elastase, lysozyme, esterase, and myeloperoxidase, have not been fully validated for clinical use, and it remains unclear how closely their levels reflect the presence or extent of intestinal inflammation (14,45). By contrast, fecal levels of calprotectin, another neutrophil-derived protein, seem to reflect active intestinal inflammation in patients with IBD (10,45,46). Both FLA and calprotectin show similar results for patient groups with intestinal inflammation. However, within the normal population, a significant number of people with baseline lactoferrin levels have elevated fecal calprotectin levels (47). Fecal calprotectin levels are significantly higher in patient populations with IBD than in patients with IBS, and they seem to be correlated with clinical and endoscopic disease activity in patients with UC and, to a lesser degree, with CD (40,46,48–53). However, the clinical application of fecal calprotectin as a screening tool in pediatric and adult populations has been hindered by an apparent lack of consensus with respect to normative values and units of measurement in published reports (49,54,55). Large-scale comparative trials in pediatric patients are needed to evaluate the relative utility of these biomarkers in clinical practice.

We further evaluated the potential utility of FLA measurements in predicting patients who are at risk for disease flares by comparing the short-term (2-month) clinical outcome in patients with inactive disease at the time of specimen collection. We found that FLA levels were significantly increased in patients who then experienced flares within 2 months of sample collection in comparison with patients who remained in remission. Although our sample size was relatively small, the results were significantly different, and they raise the possibility that elevations in FLA may presage clinical flares. This question is being addressed in a multicenter study evaluating the utility of serial lactoferrin measurements in pediatric and adult patients with active and inactive IBD. Our data further demonstrate that quantitative FLA measurements are highly correlated with commonly used biochemical metrics of inflammation, including ESR, hematocrit, Alb, and Plt count.

An inherent weakness of our study, and perhaps all studies evaluating the use of surrogate markers in the assessment of patients with IBD, is the lack of an accepted gold standard of disease activity against which to measure their validity. Although endoscopic and histological assessments are generally accepted as definitive benchmarks, their application is limited by their cost, invasive nature, and potential interrater reliability. As such, we were unable to correlate FLA levels with disease location and severity in this study. Similarly, we were not able to correlate FLA measurements, clinical activity, and disease location in subgroups of patients with primarily small or large bowel IBD. In our study population 5 of 62 (8%) patients with UC had disease limited to the left colon, and no patients had isolated proctitis, which was consistent with previous reports of disease distribution in pediatric patients (1). Patients with CD limited to the large or small intestine were similarly rare (4/79 and 13/79, respectively). Future studies using simultaneous FLA and endoscopic assessment will be necessary to fully define the degree to which FLA is influenced by the extent and localization of endoscopic inflammation.

The development of valid, noninvasive, and readily obtainable surrogate markers of intestinal disease activity will contribute significantly to patient care. In addition, the application of surrogate markers, such as FLA, could play an important role in pharmaceutical trials by reducing both the cost and the length of time necessary to determine the effects of novel anti-inflammatory therapies and by limiting the need for more invasive diagnostic studies. Further studies are needed to define how to optimize the inclusion of FLA measurements into the prospective management of IBD in children and adults.

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Acknowledgments

The authors thank the Children's Hospital Clinical Research Program and General Clinical Research Center (MO1 RR-02172) for assistance with this study, Dr. Richard J. Grand for his time and effort in revising this manuscript, and the Children's Hospital Center for Inflammatory Bowel Disease, the Wolpow Family Fund, and the community of patients and providers for their support in completing this clinical study.

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Keywords:

Fecal lactoferrin; Ulcerative colitis; Crohn disease; Inflammatory bowel disease; Irritable bowel syndrome

© 2007 Lippincott Williams & Wilkins, Inc.

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