Diagnosis of Inflammatory bowel disease have challenges including differentiating from Irritable bowel disease using noninvasive biomarkers. Fecal calprotectin is a novel fecal marker which meets the diagnostic & monitoring requirements for IBD.

1. Biomarker for
Inflammatory Bowel
Disease (IBD)
DR RAJESH V BENDRE
Md(Path), DNB(Path), DPB
Chief Pathologist

2. INTRODUCTION
• Inflammatory bowel diseases (IBD) are a group of conditions that cause a
pathological inflammation of the bowel wall with a spectrum of chronic
disorders affecting the gastrointestinal tract, with Crohn disease (CD) and
ulcerative colitis (UC) as the major entities. Neutrophils influx into the bowel
lumen as a result of the inflammatory process.
• Incidence - UC – 11/100,000 ; CD – 7-8/100,000
• Age – Initial & common peak –15-30 years, Second-smaller peak at >60
years 10-25% of IBD cases in childhood- increase incidence over past 4
yrs(CD >UC)
• Sex CD – M>F, 1.8:1 ; UC – M:F, equal
• Ethnicity – incidence highest in Ashkenazi Jews and lowest in African
Americans and Hispanics
• Complications
• CD – fistulas, abscesses
• UC – massive hemorrhage, toxic megacolon, marked increase in
incidence of colon cancer

3. IBD versus IBS
• Diagnosis - Rome Criteria 3
3m of Abdominal Pain /
Discomfort
Associated with 2 of 3
• Altered frequency
• Altered consistency
• Improves with defaecation
• Other symptoms -
• Tiredness / lethargy
• Poor sleep
• Backpain
• Fybromyalgia
• Urinary urgency and frequency
• Dysguesia - Unpleasant taste in
mouth
IBS - What does it mean to
me?
• Non-organic disorder
• Functional bowel
symptoms (FBS)
• Talk to your patients about
their life and their bowel
habits
“Don’t treat the symptoms
Treat the cause”
• Anyone with chronic
diarrhoea need full
Investigation

4. Diagnosis
Radiology and imaging
Ultrasound & CT
scanning: Can help
define thickness of the
bowel and mesentery
and can be useful to
evaluate disease progress
& chart fistula
formation.
Barium follow-through –
findings consistent with
Crohn’s include an
asymmetrical alteration
in mucosal pattern with
deep ulceration and areas
of narrowing or
stricturing.
Colonoscopy, terminal ileoscopy &
biopsy: These allow direct
visualisation and allows for a
biopsy of the mucosa to be taken.
This is central to macroscopic and
microscopic diagnosis.

5. Blood tests
Haematology: results suggesting anaemia,  platelet count &  ESR suggests an
inflammatory bowel condition.
Serum Markers:  C-reactive protein and  serum albumin suggests active CD.
Liver biochemistry may be abnormal.
Stool cultures Stool cultures should be done to rule out infection as a differential
diagnosis
Faecal Markers
Faecal calprotectin: provides accurate marker of inflammatory activity. Calprotectin is
a neutrophil-derived cytosolic protein that is resistant to bacterial degradation and if
present in the stools provides an accurate index of intestinal inflammatory activity. It
has the potential to be a simple, cheap, non-invasive marker.
.
Diagnosis

6. Correlated expression of high-sensitivity C-reactive
protein in relation to disease activity in
inflammatory bowel disease
Pearson coefficients of correlation between log hs-CRP and the disease
severity score in the 2 IBD groups.
(a) CD (r = 0.365, p = 0.001), (b) UC (r= 0.262, p = 0.03)
Digestion 2006; 73:205-209

7. Guidelines for the
investigation of chronic
diarrhoea, Gut 2003
• “Stool markers of
gastrointestinal
inflammationsuch as
lactoferrin and, more
recently, calprotectin, are
of considerable research
interest but, as yet, these
have notbeen introduced
into clinical practice.”
NICE (2008) for IBS in Primary Care
• Positive [exclusion] diagnosis of IBS if
appropriate symptoms
• Refer to secondary care for further
investigation people with possible IBS
symptoms who have any of the following
‘red flag’ indicators:
- Unintentional or unexplained weight loss
- Rectal bleeding
- Abdominal or rectal masses
- Anaemia
- Family history of bowel or ovarian cancer
- Change in bowel habit lasting more than 6
weeks in person aged over 50 yrs, with
looser and/or more frequent stools
- Raised inflammatory markers for
inflammatory bowel disease

8. Recommended Primary Care guidelines
Patient <45 years with symptoms of
(change in bowel habit, abd pain, bloating) for the past 3 months
Measure TTG + FBC, TSH and
stool for calprotectin at same time
TTG positive,
treat as coeliac
disease.
If TTG, FBC,
TSH & FC - NAD
Likely IBS
Measure faecal
calprotectin
FC <50 ug/g, no evidence of active bowel
inflammation. Symptoms highly likely to be due
to IBS. If diarrhoea persists, or there remains
clinical concern, consider referral for further
investigations
FC >50 ug/g, Raised calprotectin
consistent with active bowel
inflammation,further
Gastroenterology investigations.
Follow anaemia
pathway.

9. • Fecal calprotectin
• 35 KDa Calcium and Zinc binding protein found in neutrophils,
monocytes, and macrophages. Up to 60% of the total cytosolic protein
content of neutrophils
• First Described in 1980
FUNCTIONS-
• Binds to calcium and has antimicrobial & antitumoural properties.
• Reduces local zinc concentrations, and inhibits zinc dependent
metalloproteinases
• Released from cells during cell activation or death
• Stable in faeces for several days (upto 5 days) after excretion
Fecal Markers

10. • Measurement of Faecal Calprotectin has been shown to be strongly
correlated with 111-indium-labelled leucocytes - considered the gold
standard measurement of intestinal inflammation.
• The main diseases that cause an increased excretion of Faecal Calprotectin
are Crohn's disease, ulcerative colitis and neoplasms . Levels of faecal
calprotectin are normal in patients with irritable bowel syndrome (IBS).
• Specific indications for measuring Faecal Calprotectin are in:
• Identify organic bowel disease against functional bowel disease (IBS), and thus
avoid the need for invasive tests such as colonoscopy.
• Assessing efficacy of IBD treatments.
• Predicting relapses or flares of IBD.
Fecal Markers

11. Fecal calprotectin correlate with Disease activity in
UC CD
Int J Colorectal Dis 2007; 22:429-437

12. Sensitivity and specificity
%
(95% C.I.)
N = Sensitivity Specificity Positive
predictive value
Negative
predictive
value
Raised CRP or
ESR
33 40
(14-72)
91
(70-98)
67
(24-94)
78
(57-91)
Rome II
criteria
45 91
(78-97)
10
(5-43)
76
(59-87)
25
(13-78)
Imm’ik cut off
50 ug/g
28 78
(40-96)
95
(72-100)
88
(47-99)
90
(68-98)
Combined data 48 85
(54-97)
86
(69-95)
69
(41-89)
94
(79-99)
In diagnostic meta-analysis, the pooled results (adults):
sensitivity 0.93 (0.85-0.97) specificity 0.96 (0.79-0.99)

13. • UC, p value = 0.0000. CD , p value = 0.0432
Fecal calprotectin predict relapse
Kaplan-Meier survivor curves
Gut 2005; 54:364-368

14. Calprotectin Levels in the Different Diagnostic
Groups

15. Fecal Calprotectin levels in a high risk population for colorectal
neoplasia. Kronberg et al Gut 2000 (46)795-800

16. Sensitivity/Specificity for Organic and Non-Organic
Disease
Sensitivity Specificity
Calprotecin >
10mg/L
89 79
Positive Rome
Criteria
85 71
CRP > 5.0 mg/L 50 81
ESR > 10
mm/Hr
58 72
Use of surrogate markers of inflammation and Rome criteria to distinguish organic from non-
organic intestinal disease Tibble J. Gastro. 2002; (123): 450-460

17. Fecal Calprotectin as an aid to Diagnosis in intestinal
inflammation. Dolwani et al DDW 2003
• 65 patients with abdo pain + diarrhoea
• All referred for Barium follow through
• 15 false negatives: 6 IBD, 4 IBS, 5 uncertain
Ba FTNormal Ba FTabnormal
Calprotectin < 60 33 1
Calprotectin > 60 15 16

18. Faecal calprotectin- Test
• Non-invasive test
• Suitable for Primary Care
• Also useful for monitoring known IBD patients, and investigating symptom
flares non-invasively
• More sensitive and specific than current inflammatory markers (ESR &
CRP)
• Easily measured in stool by commercially available ELISA
• The median faecal calprotectin level in healthy individuals
• 2 mg/L with an upper limit of 10 mg/L
• Pooled sensitivity 93% and specificity 96% (diagnostic meta-analysis)
Sensitivity (%) Specificity (%)
CRP & ESR 35 73
Faecal Calprotectin 90 80

19. Results
1.0
10.0
100.0
1000.0
10000.0
0 1 2 3 4 5 6 7 8 9 10
calprotectinmg/l
Buhlmann kit Immundiagnostik kit
IBDin
remission
IBS
IBD
Otherorganic
disease
IBDin
remission
IBS
IBD
Otherorganic
disease

20. • Extraction of the stool sample -
• Use of a stool sample preparation kit for dosing 100 mg of stool sample The stool sample must be
suspended in 5 ml extraction buffer.
• Constant buffer volume: 5 ml , Constant dilution factor: 1:50
• Afterwards, mix stool sample and buffer; vortex for at least 30 sec. depending on the stool consistency.
• Transfer 1 ml stool suspension (dilution step I) to an Eppendorf-tube and centrifuge for 5 minutes at
13000 g.
• 20 μl supernatant (dilution I) + 980 μl wash buffer = 1:50 (dilution step II)
• For Analysis, pipette 100 μl of the supernatant of dilution step II per ELISA well.
• Principle of the ELISA -
• The assay utilizes the two-site “sandwich” technique with two selected monoclonal anti-bodies that bind
to human Calprotectin. During the first incubation step, Calprotectin in the samples is bound by the
immobilized antibody. Then a peroxidase labeled conjugate is added to each well and the following
complex is formed: capture antibody - human Calprotectin –Peroxidase conjugate. Tetramethyl-
benzidine (TMB) is used as a substrate for peroxida-se. Finally, an acidic stop solution is added to
terminate the reaction. The color changes from blue to yellow. The intensity of the yellow color is
directly proportional to the Cal-protectin concentration of sample. A dose response curve of the
absorbance unit (optical density, OD at 450 nm) vs. concentration is generated, using the values
obtained from standard.
• Results – To obtain the calprotectin concentration in stool samples, multiply the estimated value by the
dilution factor according to the sample preparation, that is, 2500 (dilution step I x dilution step II) to get the
final concentration.
Faecal calprotectin- Test

21. • Expected values- Calprotectin in stool < 10mg/l, Grey area: 10–15 mg/l ,
Significant levels > 15 mg/l
• The original ELISA method for calprotectin determination was first
described in 1992 by Roseth et al. , and the results were provided in “per
liter of faecal homogenate”. In 2000, a new assay for faecal calprotectin
became widely available, which was five times as sensitive as the original
assay and measured this faecal biomarker in µg/g rather than mg/L . A
number of authors have asserted that results obtained from the old assay
method may be directly compared with results obtained from the new
method through simply multiplying the former by a factor of 5.
• Recently suggested upper limit of normal has been changed to 50 µg/g,
while 100 µg/g cut off for better accuracy in IBD
Faecal calprotectin- Test

22. LIMITATIONS-
• A main disadvantage of faecal calprotectin is that it increases after the
use of non-steroidal anti-inflammatory drugs (aspirin included),
probably due to the associated induced enteropathy. Therefore
termination of these medications’ treatment should be recommended
before taking samples for analysis
• It has been estimated that a bleeding volume of at least 100mL daily
may cause an elevated faecal calprotectin concentration . Therefore, it
has been suggested that patients with menstrual or nasal bleeding be
excluded to avoid measuring calprotectin from neutrophils in blood
that could contamine the faecal sample
Faecal calprotectin- Test

23. Conclusion
• Extensively validated, showing consistent abnormalities in patients
with IBD, colorectal carcinoma, and nonsteroidal enteropathy
• Sensitivity >95% for detecting patients with IBD
• Failure to lower Calprotectin predicts those patients with steroid
refractory disease (even if the patient has had a good symptomatic
response to steroids)
• Asymptomatic patients with IBD with Calprotectin > 50µg/g have a
90% probability of relapse in the next 12 months
• Calprotectin reduction in IBD treated patients appears to correlate
with endoscopic mucosal healing
• Calprotectin levels much more clinically useful in IBD than any of
the currently used systemic immune tests (CRP, ESR, Igs, Plts)

24. THANK YOU