Laboratory markers in IBD - Free Webs



Laboratory markers in IBD

• In IBD physicians apply a combination of symptoms, clinical examination, laboratory indices, radiology, and endoscopy with histology to make the diagnosis, to assess severity, and to predict the outcome of disease.

• Clinical indices give only indirect measurement of disease activity and may not accurately predict inflammatory activity found by endoscopic and histological examination.

• Endoscopy is accurate but is invasive and expensive.

• Hence, there has been a search for biological markers in IBD for:

o Diagnostic and differential diagnostic purposes.

o Assessment of disease activity and risk of complications.

o Prediction of relapse.

o Monitoring the effect of therapy.

o Avoid invasive (endoscopic) procedures.

Characters of an ideal marker

|Table 1 Performance and qualities of an ideal marker |

|Performance |Qualities |

|Simple |Be disease specific: identify individuals at risk for IBD and differentiate IBD from non-IBD |

|Easy to perform |Able to objectively measure disease activity |

|Not or minimally invasive |Able to predict the disease course (relapse or recurrence) |

|Cheap |Able to monitor the effect of treatment |

|Rapid |Have a prognostic value in assessing morbidity/mortality |

|Reproducible between labs and individuals | |

Biomarkers that may be Used to Determine Activity in Inflammatory Bowel Disease

THE ACUTE PHASE RESPONSE AND IBD

• The presence of active gut inflammation in patients with IBD ( acute phase reaction ( migration of leucocytes to the gut ( production of several proteins ( detected in serum or stools.

|Acute phase reactants |ESR |

| |C-reactive protein |

| |Orosomucoid |

| |Thrombopoietin |

| |Platelet count |

| |Fibrinogen |

| |Lactoferrin |

| |Serum amyloid A |

| |α1-antitrypsin |

C REACTIVE PROTEIN

• Under normal circumstances: produced by hepatocytes in low quantities (200–250 mg/l.

• Characters:

• Short half life (19 hours):Rise early after the onset of inflammation and rapidly decrease after resolution of the inflammation.

• Function:

• Binds to phosphocholine containing microorganisms or particles ( complement activation.

• Plays a role in the Opsonization of infectious agents and damaged cells.

• CRP and IBD

|UC |CD |

|Diagnose and to predict the activity |Correlate with disease activity |

|Lower |Higher |

|Correlate with severe clinical activity and active disease at |CD activity index, radioactive-labelled faecal granulocyte |

|colonoscopy but not with histological inflammation |excretion and faecal calprotectin. May be low in ileitis. |

• There is remarkable heterogeneity in the CRP response between CD and UC.

o CD is associated with a strong CRP response.

o UC has only a modest to absent CRP response.

• Proposed explanation for this heterogeneity

o Serum IL-6 concentrations were significantly increased in patients with CD compared with UC.

o In UC the inflammation is confined to the mucosa whereas in CD it is transmural.

o Polymorphisms in the CRP gene, located on the long arm of chromosome 1, account for inter individual differences in baseline CRP production in humans.

ERYTHROCYTE SEDIMENTATION RATE

• Definition: rate at which erythrocytes migrate through the plasma.

• Not specific for IBD

• Depend on the plasma concentration and on the number and size of the erythrocytes.

• Peak much less rapidly and may also take several days to decrease

• May vary with age.

|UC |CD |

|Correlate with disease activity |Less accurate measure of disease activity |

|Normal in proctitis and proctosigmoiditis |Correlates more with colonic disease and does not reflect the |

| |disease activity of small bowel |

Other serum Markers

• White blood cell count.

o Increase as part of the acute phase response.

o Not specific for IBD.

▪ Seen in other inflammatory conditions.

▪ Influenced by some treatments used in IBD.

• Glucocorticoids ( increased.

• Azathioprine ( decreased.

• Platelets.

o Platelet count correlates with disease activity in IBD.

o Non specific.

• Albumin ( not specific.

• Neopterin ( synthesized and released from monocytes/macrophages upon non-specific stimulation.

o The level of neopterin in urine and serum has been shown to correlate with disease activity of UC and CD but this is not IBD specific.

|Others |Elastase |

| |Myeloperoxidase |

| |Leucocyte esterase |

| |Serum tenascin C |

| |B2-microglobulin |

| |Neutrophil elastase |

| |Soluble adhesion molecules |

| |Angiogenic proteins |

Cytokines

• The expression of proinflammatory cytokines is markedly increased in the intestinal mucosa in patients with active IBD, although not always accompanied by increased concentration of cytokines in the serum.

TNF-α and TNF-α Receptor

• Tumor necrosis factor-α is produced by activated macrophages and monocytes.

• Serum concentration of TNF-α is often not consistently elevated in IBD and are thus of limited utility as markers of disease activity.

• The same conclusion applies to the measurement of TNF-α concentration in stool.

• TNF receptors ( more studies required.

Interleukins, Interleukin Receptors and Interleukin Receptor Antagonists

|Interleukin |Action |Value in IBD |

|IL receptor antagonists (IL-1RA) |Anti-proinflammatory effects |Increased in patients with active IBD |

|IL-1RA/IL-1 ratio | |Decreases with increasing IBD activity |

|Interleukin-2 receptor (IL-R) |Shed by activated T cells into circulation |Correlates with disease activity in both UC and CD. |

| |along with IL-2. | |

|Interleukin-6 |Anti-inflammatory and proinflammatory effects |Elevated serum IL-6 concentrations are found in active CD but|

| | |not always in UC |

|Interleukin-8 |Important for neutrophil chemotaxis. |Elevated in patients with active UC but not sensitive. |

| | |Not elevated in patients with active CD. |

|Interleukin-10 |Anti-inflammatory cytokines |Elevated in active UC and CD. |

| | |May correlate with endoscopic and disease activity. |

Adhesion Molecules

• Leucocytes do not readily adhere to vascular endothelium in an unstimulated state.

• Inflammatory signals ( induce expression of proteins on the endothelial cell surface ( promote the adhesion and extravasation of activated immune cells from the circulation ( underlying tissues.

• These proteins are known as cell adhesion molecules and they are expressed by immune cell, endothelial cell and epithelial cells.

• They can be in soluble form or leucocyte-bound form.

|L-, P- and E-selectin |

|Cell adhesion molecules |Mucosal adressin-cell adhesion molecule (MAdCAM-1) |

| |Intercellular adhesion molecule-1 (sICAM-1) |

| |Vascular cell adhesion molecule-1 (sVCAM-1) |

• No support to the routine use of soluble adhesion molecules as disease activity markers in IBD.

Serum tenascin C

• Multifunctional matrix protein present in connective tissue and is induced in inflammation and repair.

• Tenascins modulates cell adhesion.

• Not specific of IBD.

Serum levels of B2-microglobulin

• Useful to assess the disease activity in CD but not in UC.

Plasma levels of neutrophil elastases

• Independent parameter for assessment of disease activity and may be more useful than other markers such as ESR and CRP in identifying the patients in remission.

Serum immune markers

• Deoxyribonuclease (DNase I) sensitive perinuclear antineutrophil cytoplasmic autoantibody (pANCA) associated with IBD. The IBD associated pANCA defines an antibody to a nuclear antigen which is sensitive to DNase I.

• ASCA (Anti-Saccharomyces cervisiae antibody). This antibody is present in the serum of up to 70 percent of Crohn's disease patients.

• Pancreatic antibody This antibody is observed in approximately 30 percent of Crohn's disease patients.

• Anti-OmpC (outer membrane porin from E. coli). An IgA response to OmpC is seen in 55 percent of Crohn's disease patients.

Genetic markers

• A significant role for genetic factors in IBD was established.

• NOD2 gene

• Nucleotide-binding oligomerization domain (NOD) proteins are cytosolic proteins that include principal regulators of apoptosis.

• There is association of Nod2 (CARD15) polymorphisms with CD.

• Mutations in the leucine-rich region of the NOD2 gene have been identified as susceptibility factors for CD

• NOD2/CARD15 genotyping is helpful in differentiating indeterminate colitis patients.

• TPMT gene

• Thiopurine methyltransferase (TPMT) catalyzes the S-methylation of azathioprine, 6-mercaptopurine and thioguanine.

• Correlations between mutations in the TPMT gene with susceptibility to leukopenia from azathioprine therapy can help select candidates for this therapy.

FAECAL MARKERS

|Faecal |Faecal excretion of 111Indium-labelled white cells |

| |Leucocytes products |

| |Serum proteins |

| |Calprotectin |

| |Lactoferrin |

• Advantages of faecal markers:

o Stools are easy accessible in IBD patients.

o Serum markers may be increased by various conditions other than gut inflammation.

o Higher specificity for IBD in the absence of gastrointestinal infection.

o Representative of mucosal inflammation in the bowel.

Calprotectin

• 36 kDa calcium and zinc binding protein

• Released from cells during cell activation or death.

• Very sensitive marker for detection of inflammation in the gastrointestinal tract.

• It is stable in faeces for several days after excretion.

• Easily measured in stool by ELISA.

• Faecal calprotectin level is 2-10 mg/L in healthy individuals.

|UC |CD |

|Correlate with endoscopic and histological activity |Correlate with endoscopic and histological activity |

|Stronger predictor of relapse |Less |

Faecal Lactoferrin

• Nature: a glycoprotein found in many body fluids as well as in granules of neutrophil granulocytes.

• Faecal lactoferrin levels quickly increase after influx of neutrophils into intestinal lumen during inflammation.

• Measured by ELISA on a single stool sample.

• Normal value is 1.45 ± 0.4 μg/g of faecal weight.

• Value in IBD:

o Increased in patients with active IBD (specificity between 85% and 90%).

o Rise significantly prior to a clinically evident relapse and may be a good marker to predict subsequent IBD flares.

o Correlates with histological inflammation.

Other Faecal Markers

|Other |Faecal α1-antitrypsin. |

| |Myeloperoxidase. |

| |Leucocyte esterase. |

| |TNF-α. |

| |111Indium-labelled white cells scan |

| |Intestinal permeability test |

| |Whole gut lavage fluid for immunoglobulin and albumin |

Intestinal Permeability

• Intestinal permeability using differential 5 h urinary excretion ratio (ratio of lactulose and l-rhamnose) and CrEDTA.

• Limited value in assessing the disease activity.

• Can predict relapse, as less than 20% of patients who have normal intestinal permeability relapse in 6 months.

White Cell Scan and 4-Day Faecal Excretion Test

• Abdominal scanning with 111-Indium white cell technique

o Visualizes segments of inflamed bowel and

o Quantitates the degree of intestinal inflammatory activity.

• When combined with 4-day faecal excretion of labelled white cells, the inflammatory activity can be quantified accurately and can be used to document the response to treatment.

• Has a good correlation with colitis but not ileitis and poor correlation with the CD activity index.

• Expensive and more technically demanding.

Whole Gut Lavage

• Gut lavage fluid proteins have been studied as marker of disease activity in IBD.

• Gut lavage IgG was found to be a more specific disease marker than albumin in the lavage fluid.

Use of laboratory markers in the diagnosis and differential diagnosis of IBD

• CRP is the most sensitive marker in detecting IBD, values range between:

o 50% and 60% for UC.

o 70% and 100% for CD.

• Faecal calprotectin has been shown to enable diagnosis of IBD.

• A cut off of 30 µg/g had 100% sensitivity in discriminating active CD from IBS.

• Increased faecal calprotectin has been described in healthy first degree relatives of patients with CD.

Use of laboratory markers to monitor disease activity in IBD

• Laboratory markers ESR, serum albumin, [pic]1 proteinase inhibitor, cholinesterase, CRP, and haematocrit have been shown to correlate with endoscopic activity.

• There is a good correlation between ESR and clinical activity, however dependent on disease location.

• ESR correlated less well with UC restricted to the rectum and with CD restricted to the upper small bowel.

• The correlation of laboratory markers with disease activity has been shown to be much stronger for CD than for UC.

• Faecal calprotectin also correlates well with endoscopic and histological activity in patients with UC and in CD, and increased calprotectin levels normalize once the inflammation is resolved.

• Good correlation exists between ß2 microglobulin and disease activity.

Role of laboratory markers for monitoring the effect of treatment

• A decrease in CRP in response to therapy is objective evidence that the drug has a beneficial effect on gut inflammation.

• Persistently raised CRP indicates failure of the therapy to control mucosal inflammation.

Role of laboratory markers in prediction of Relapse

• If relapse can be predicted in IBD, it is likely to change the approach to treatment.

A Laboratory Index for Predicting Relapse in Asymptomatic Patients with Crohn's Disease

• A prognostic index was formulated based on ESR, α2-globulin and α1-glycoprotein.

−3.5 + (ESR × 0.03) + (acid α1-glycoprotein × 0.013) + (α2-globulin × 2)

• A value more than +0.35 suggests likelihood of relapse (sensitivity 71%, specificity and positive predictive value 100%).

• Patients with CRP > 20 mg/L and an ESR > 15 mm/first hour had an eightfold increased risk of relapse in the next 6 weeks in CD.

• Serum levels of IL-6 useful to predict relapse in steroid-induced remission in patients with CD.

• High serum level of soluble IL-2 receptors in patients with CD was highly predictive of relapse.

• A significant role of faecal calprotectin and lactoferrin in prediction of relapse in IBD.

• Faecal markers and rectal mucosal IL-8 are promising biomarkers for prediction of relapse in patients with UC.

• Small intestinal permeability has sensitivity and specificity of 84% and 61%, respectively, to predict the relapse in patients with small intestinal CD.

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