# Fecal Lactoferrin: Novel Test to Differentiate between Irritable and Inflamed bowel?



## David in Seattle

Alimentary Pharmacology & Therapeutics. 2010;31(12):1365-1370. © 2010 Blackwell Publishing

Abstract and Introduction
Abstract

Background Distinguishing between inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS) can be challenging.
Aims To investigate the utility of faecal lactoferrin as a marker of inflammation in patients with IBD, IBS and controls.
Methods Disease activity in IBD patients was assessed using the modified Harvey–Bradshaw Activity Index. Stool samples were analysed using an ELISA assay.
Results We recruited 137 patients with IBS, 126 with ulcerative colitis (UC) and 104 with Crohn's disease (CD), and 98 healthy volunteers. The median ± IQ lactoferrin concentration (μg/g faecal weight) was 0 ± 1.4 for IBS patients, 6.6 ± 42 for UC patients, 4 ± 12.7 for CD patients and 0.5 ± 2 for healthy controls. Lactoferrin levels were significantly higher in IBD patients compared with IBS/healthy controls (P < 0.001). The median lactoferrin concentrations were significantly higher in active UC & CD patients compared with inactive patients (P < 0.001 and P = 0.002 respectively). The sensitivity, specificity, positive and negative predictive values of lactoferrin in distinguishing active IBD from IBS/healthy controls were 67% and 96%, 87% and 86.8% respectively.
Conclusions Lactoferrin is useful to differentiate between IBD and IBS, and can be used as an adjunct to blood parameters to determine IBD patients who have ongoing inflammation.
Introduction

Inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS) are common entities in the Western population.[1–3] Both conditions may present with similar clinical features such as diarrhoea and abdominal pain. Patients with IBD oscillate between periods of active and inactive disease and may even present with concomitant functional IBS. The discrimination of IBS from active IBD can be resourcefully challenging for clinicians and may delay effective treatment. Some investigations may also be perceived as uncomfortable or invasive for the patient. Clinical criteria such as ROME II IBS have been devised to aid the diagnosis of IBS.[4, 5] The determination of inflammatory activity is crucial for patients with IBD for the diagnosis, monitoring and step up of therapy. Clinical indices are widely used, but are hampered by the subjective nature of symptom reporting and have been shown to be poorly correlated with mucosal activity.[6] Colonoscopy is the accepted gold standard for investigation of the colon, but is invasive and associated with risks.[7] Whilst there is emerging evidence of activation of the mucosal innate defence system toward a pro-inflammatory response in IBS patients, the absence of endoscopic and histological inflammation remains an accepted approach to the diagnosis of IBS by the bedside.[8]

Lactoferrin (LF) is an iron binding glycoprotein secreted by most mucosal membranes and a major component of secondary granules of polymorphonuclear neutrophils, a component of the inflammatory response.[9, 10] Elevated LF has been used as a marker of active IBD[11–16] and for monitoring patients for response to treatment.[17] Some studies report a high sensitivity of LF for active IBD in comparison with IBS. However, the use of LF for the distinction of inactive IBD and IBS is less clear.[13, 14, 18, 19] In Table 1, the comparative studies of patients with IBD and IBS using LF are tabulated.[11, 13–16, 19]

The aim of this study was to investigate the clinical utility of LF as a marker of GI inflammation in patients with active and inactive IBD compared with patients with diarrhoea predominant IBS and healthy controls.

Methods
Patients

Consecutive patients were recruited from the out-patient clinic. Patients with inflammatory bowel disease were questioned about their general well being, the frequency of bowel habit, the presence/absence of abdominal pain or blood in the stool. Patients with established IBD were given a Harvey–Bradshaw Index (HBI) for Crohn's disease and a (previously validated) modified HBI for Ulcerative colitis (Appendix 1). [15] Patients with HBI of ≥4 were considered to have active disease. All patients who had diarrhoea with the presence of abdominal discomfort and who fulfilled the Rome II criteria for diarrhoea predominant IBS were also recruited.[4, 5] All patients were investigated and treated according to the British Society of Gastroenterology guidelines.[4] Colonoscopy was requested based on clinical need. Healthy controls were recruited after exclusion of disease with a questionnaire. All participants were requested to return a stool sample in a container provided. Ethical approval was obtained from the North Sheffield Ethics Committee.
Stool Analysis

Analysis was performed blind to the clinical details of the patient. Stool samples were frozen at −20 °C immediately on receipt. Quantitative ELISA (IBD SCAN) faecal lactoferrin test was performed on each thawed sample. The stool analysis kits were provided by ScheBo Biotech UK Limited and Techlab, USA (Blacksburg, VA, USA). A cut off level of >7.25 μg/g was deemed positive based on the manufacturer's guide.
Statistical Analysis

The data were analysed using SPSS version 15 (SPSS Inc, Chicago, IL, USA). Nonparametric tests (Mann–Whitney U-test) were used to compare lactoferrin concentrations between groups as the data were not normally distributed. Kendal tau correlations were performed to assess the relationship between lactoferrin concentration and disease activity (HBI). Assistance was also sought from the University of Sheffield statistics department.


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## David in Seattle

Results

Four hundred and sixty-five patients were recruited between November 2006 and October 2008. The mean age in the IBS, UC and Crohn's group was 42 years, 58 years and 56 years respectively. The median LF levels were significantly higher in patients with IBD compared with patients with IBS (P < 0.001) and healthy controls (P < 0.001). In Table 2, the mean and median LF values for each group are tabulated, whilst in Figure 1, the distribution of LF values in all patients is shown.

Figure 1.

Faecal lactoferrin concentrations in all patients.


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## David in Seattle

Among patients with IBD, there was a trend towards higher LF values in patients with UC compared with patients with CD (P  = 0.051). As for stratification based on severity of symptoms/disease activity, the median LF (μg/g) levels were significantly higher in patients with active disease (HBI ≥4) compared with patients with inactive disease for both UC and CD (P < 0.001 and P = 0.002 respectively). Analysis of LF levels in IBD patients based on disease activity is tabulated in Table 3.

Comparisons were also made between patients with inactive IBD (HBI <4) and those with diarrhoea predominant IBS. Patients with inactive IBD had significantly higher LF levels compared with patients with IBS. The median LF (μg/g) ± IQ for patients with inactive UC and that for patients with inactive CD was 3.1 μg/g (8.5) and 1 μg/g (5.8) respectively compared to 0 μg/g (1.4) for patients with IBS (P < 0.001 and P = 0.002 respectively).

The correlation between LF values and the disease activity (Harvey–Bradshaw Index) was fair. The correlation coefficient for patients with UC was 0.4, whereas it was 0.2 for patients with CD and 0.3 for any diagnosis of IBD.

The sensitivity and specificity of LF for active IBD vs. IBS patients were 67% and 96% respectively with positive and negative predictive values of 92% and 80% respectively. Similar calculations for active UC and CD patients are tabulated in Table 4. ROC curves were calculated to illustrate the trade off between the sensitivity and specificity for each group as shown in Figure 2a–c.

Figure 2.

(a) ROC curve for all patients, area under the curve 0.75. (b) ROC curve for patients with active inflammatory bowel disease compared to patients with Irritable bowel syndrome (area under the curve 0.84). (c) ROC curve for patients with active vs. inactive inflammatory bowel disease (area under the curve 0.81).


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## David in Seattle

Discussion

This study, the largest to date on the use of LF (n = 465), has shown that LF has a high sensitivity and specificity for the discrimination of patients with active IBD against patients with IBS and healthy controls. In addition, LF levels were significantly higher in patients with inactive IBD than in patients with IBS, making it a valuable investigative tool in patients where the differentiation is difficult, based on clinical history alone. Whilst the poor correlation between symptom reporting and disease activity in IBD has been demonstrated before,[6] previous LF studies making similar comparisons have shown conflicting results with some studies failing to show a difference between inactive IBD and IBS.[13, 18, 19]

We have also demonstrated a significant difference in LF levels in patients with active IBD compared with patients with inactive IBD. This suggests that LF could be used in conjunction with other parameters (clinical and blood inflammatory markers) to determine the subset of patients who have active disease or who may require a step up of therapy. A paediatric study (n = 5) showed that LF levels is potentially useful as a biomarker for response to anti-tumour necrosis factor (anti-TNF) therapy.[20] It has also been suggested that the course of LF may be an early predictor of a relapse.[11, 20] In a recent study, LF predicted post-operative recurrence in Crohn's disease with greater accuracy than C-reactive protein, platelet count or endoscopic appearance.[17]

In our study, the median LF levels in IBD patients were comparatively lower than in other studies in the published literature.[14, 15] This could be explained by the larger number of patients with inactive disease. The inclusion of patients into this study was based on recruitment from routine out-patient clinics from a single centre as opposed to specially selected patients with severe symptoms.

A perceived limitation of our study is the lack of correlation with endoscopic and histological grading. In our study, colonoscopy was only performed based on clinical need and represented less than 30% of the population group. In addition, the endoscopies which were performed as routine care were performed by a number of endoscopists. Similarly biopsies from these patients were also analysed by a number of histopathologists, which made meaningful comparisons difficult. Previous investigators have demonstrated that LF has a good correlation with endoscopic grading.[14]

LF is an inexpensive and non-invasive test that can provide the clinician with a marker to differentiate between IBD (particularly active disease) and IBS and stratify patients who require endoscopic investigations. In addition, LF can also be used as an adjunct to blood parameters and clinical symptoms to determine IBD patients who have ongoing inflammation.


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## David in Seattle

References

   1. Saito YA, Schoenfeld P, Locke GR 3rd. The epidemiology of irritable bowel syndrome in North America: a systematic review. Am J Gastroenterol 2002; 97: 1910–5.
   2. Drossman DA, Camilleri M, Mayer EA, Whitehead WE. AGA technical review on irritable bowel syndrome. Gastroenterology 2002; 123: 2108–31.
   3. Carter MJ, Lobo AJ, Travis SP. Guidelines for the management of inflammatory bowel disease in adults. Gut 2004; 53(Suppl 5): V1–16.
   4. Spiller R, Aziz Q, Creed F, et al. Guidelines on the irritable bowel syndrome: mechanisms and practical management. Gut 2007; 56: 1770–98.
   5. Manning AP, Thompson WG, Heaton KW, Morris AF. Towards positive diagnosis of the irritable bowel. Br Med J 1978; 2: 653–4.
   6. Crama-Bohbouth G, Pena AS, Biemond I, et al. Are activity indices helpful in assessing active intestinal inflammation in Crohn's disease? Gut 1989; 30: 1236–40.
   7. Bowles CJ, Leicester R, Romaya C, Swarbrick E, Williams CB, Epstein O. A prospective study of colonoscopy practice in the UK today: are we adequately prepared for national colorectal cancer screening tomorrow? Gut 2004; 53: 277–83.
   8. Langhorst J, Junge A, Rueffer A, et al. Elevated human beta-defensin-2 levels indicate an activation of the innate immune system in patients with irritable bowel syndrome. Am J Gastroenterol 2009; 104: 404–10.
   9. Baveye S, Elass E, Mazurier J, Spik G, Legrand D. Lactoferrin: a multifunctional glycoprotein involved in the modulation of the inflammatory process. Clin Chem Lab Med 1999; 37: 281–6.
  10. Levay PF, Viljoen M. Lactoferrin: a general review. Haematologica 1995; 80: 252–67.
  11. Walker TR, Land ML, Kartashov A, et al. Fecal lactoferrin is a sensitive and specific marker of disease activity in children and young adults with inflammatory bowel disease. J Pediatr Gastroenterol Nutr 2007; 44: 414–22.
  12. Sipponen T, Savilahti E, Kolho KL, Nuutinen H, Turunen U, Farkkila M. Crohn's disease activity assessed by fecal calprotectin and lactoferrin: correlation with Crohn's disease activity index and endoscopic findings. Inflamm Bowel Dis 2008; 14: 40–6.
  13. Schoepfer AM, Trummler M, Seeholzer P, Seibold-Schmid B, Seibold F. Discriminating IBD from IBS: comparison of the test performance of fecal markers, blood leukocytes, CRP, and IBD antibodies. Inflamm Bowel Dis 2008; 14: 32–9.
  14. Langhorst J, Elsenbruch S, Koelzer J, Rueffer A, Michalsen A, Dobos GJ. Noninvasive markers in the assessment of intestinal inflammation in inflammatory bowel diseases: performance of fecal lactoferrin, calprotectin, and PMN-elastase, CRP, and clinical indices. Am J Gastroenterol 2008; 103: 162–9.
  15. Kane SV, Sandborn WJ, Rufo PA, et al. Fecal lactoferrin is a sensitive and specific marker in identifying intestinal inflammation. Am J Gastroenterol 2003; 98: 1309–14.
  16. Dai J, Liu WZ, Zhao YP, Hu YB, Ge ZZ. Relationship between fecal lactoferrin and inflammatory bowel disease. Scand J Gastroenterol 2007; 42: 1440–4.
  17. Lamb CA, Mohiuddin MK, Gicquel J, et al. Faecal calprotectin or lactoferrin can identify postoperative recurrence in Crohn's disease. Br J Surg 2009; 96: 663–74.
  18. Silberer H, Kuppers B, Mickisch O, et al. Fecal leukocyte proteins in inflammatory bowel disease and irritable bowel syndrome. Clin Lab 2005; 51: 117–26.
  19. Schroder O, Naumann M, Shastri Y, Povse N, Stein J. Prospective evaluation of faecal neutrophil-derived proteins in identifying intestinal inflammation: combination of parameters does not improve diagnostic accuracy of calprotectin. Aliment Pharmacol Ther 2007; 26: 1035–42.
  20. Buderus S, Boone J, Lyerly D, Lentze MJ. Fecal lactoferrin: a new parameter to monitor infliximab therapy. Dig Dis Sci 2004; 49: 1036–9.



Acknowledgements
Declaration of personal interests: DSS designed the study. All authors contributed to the data collection. RS performed the initial analysis and wrote the initial draft, and all ten authors were involved in the subsequent revisions, critical analysis and final draft. David Sanders is the guarantor for this manuscript and he has served as a speaker for ScheBo Biotech UK Limited at the FOCUS meeting 2009. Declaration of funding interests: Funding assistance with provision of stool analysis kits from ScheBo Biotech UK Limited and TechLab Inc U.S.A. is acknowledged.

Alimentary Pharmacology & Therapeutics. 2010;31(12):1365-1370. © 2010 Blackwell Publishing


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## DustyKat

That's really interesting David. Once again thank you so much for all the effort you put into posting these articles. 

Dusty.


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## David in Seattle

DustyKat said:


> That's really interesting David. Once again thank you so much for all the effort you put into posting these articles.
> 
> Dusty.


It's easier than it looks  

And you're welcome


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## Mayflower537

Ditto.  Very interesting.


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## vindy

*Lactoferrin question*

Do high results of Lactoferrin (example 212mcg) indicate that you have a severe case of IBD or Chrohn's, hence inflamation?


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## David in Seattle

Vindy - The study's aim is primarily to demonstrate the test's usefullness in differentiating between IBD & IBS.  As the paper states:

This study, the largest to date on the use of LF (n = 465), has shown that LF has a high sensitivity and specificity for the discrimination of patients with active IBD against patients with IBS and healthy controls. In addition, LF levels were significantly higher in patients with inactive IBD than in patients with IBS, making it a valuable investigative tool in patients where the differentiation is difficult, based on clinical history alone. Whilst the poor correlation between symptom reporting and disease activity in IBD has been demonstrated before,[6] previous LF studies making similar comparisons have shown conflicting results with some studies failing to show a difference between inactive IBD and IBS.[13, 18, 19]

We have also demonstrated a significant difference in LF levels in patients with active IBD compared with patients with inactive IBD. This suggests that LF could be used in conjunction with other parameters (clinical and blood inflammatory markers) to determine the subset of patients who have active disease or who may require a step up of therapy. A paediatric study (n = 5) showed that LF levels is potentially useful as a biomarker for response to anti-tumour necrosis factor (anti-TNF) therapy.[20] It has also been suggested that the course of LF may be an early predictor of a relapse.[11, 20] In a recent study, LF predicted post-operative recurrence in Crohn's disease with greater accuracy than C-reactive protein, platelet count or endoscopic appearance.[17] 

Here's a table from the study, which I missed including in the original post:



Within the limits of this paper, it would probably be safe to say someone with an LF level of 212 mcg would likely have IBD, rather than IBS.  It seems conceivable there might be other causes for an elevated LF, but that goes beyond the scope of this study.


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## vindy

Thanks so much for the quick reply.  Rather than go through another series of colonoscopies, endoscopies, etc. my doctor gave me this lactoferrin test for the first time, hence the results.   I was diagnosed with Crohn's several years ago, but have been in remission for the past 3 years with no medication, no pain, no symptoms.  I am hesitant to start taking a cocktail of drugs based on this test and unfortunately do not have a previous test to base the results on.  It could be possible that my results could have been consistent for the past 3 years and still symptom free.  Therefore i was unsure if this test was just a marker (ibs vs. ibd) or true indicator of severity.


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## oldcrohn

Have been trying to find some answers. Have had Crohns for 25 years. Had full hysterectomy in October and had some mild Crohns activity on CT scan. In pain all the time and can't eat. Went to  hospital last weekend with severe pain. CT showed some mild Crohns and possible narrowing. Went home and went to Dr. next day. He says Crohns too mild to go on major drugs (on Entocort, Apriso for months) and he thinks I also have IBS...HUH? I'm so confused and now having painful BM and a lot of abdominal pain. Can anyone help?


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