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Serologic markers in inflammatory bowel disease (IBD). (Cover Story).

Inflammatory bowel disease (IBD) is a generic term that refers to Crohn's disease and ulcerative colitis. IBD is prevalent in children as well as adults. Crohn's disease (CD) is a granulomatous enteritis which may involve the ileum, colon, and other parts of the intestinal tract. Crohn's disease was first reported by Burrill Crohn and his colleagues in 1922 and was called regional ileitis. (1) Crohn's disease is diagnosed in at least four patients per 100,000 in the United States, and the incidence and prevalence is rising. (2)

Ulcerative colitis (UC) is a chronic disease of unknown etiology characterized by inflammation of the mucosa and submucosa of the large intestine. The inflammation usually involves the rectum down to the anal margin and extends proximally in the colon for a variable distance. Ulcerative colitis may have a prevalence of about 100 cases per 100,000 population in the United States. (3)

Overlap of Crohn's disease and ulcerative colitis

Crohn's disease (CD) and chronic ulcerative colitis (UC) are generally considered to be distinct forms of inflammatory bowel disease (IBD). However, the symptoms and clinical presentations of CD and UC commonly overlap, and the diagnostic differentiation of cases limited to the large intestine may be problematic. (4,5) There is a subgroup of cases of CD with a UC-like presentation that illustrates the similarity of CD and UC. (5-7) A patient who may have been initially diagnosed as having UC may over time be considered as a case of CD in view of extension of the disease. (8,9)

Today, new and improved therapeutic modalities are available for CD and UC. As these various cases of IBD are treated with different types of therapeutic agents, it is important to correctly diagnose IBD and to differentiate CD from UC.

What is classically termed Crohn's disease (CD) may represent a heterogeneous group of diseases manifesting similar features. (10-13) A recent study suggests that Crohn's patients may have a heterogeneous serological response to specific bacteria and bacterial related antigens. (14) The serologic responses seen in Crohn's patients include antibodies to Saccharomyces cerevisiae, mycobacteria, bacteriodes, listeria, and E. coli.

Many of the specific organisms have been proposed to directly or indirectly contribute to the pathogenesis of Crohn's disease. (14)

Irritable bowel syndrome

Irritable bowel syndrome (IBS) is the most common functional disorder of the gastrointestinal tract. The hallmark of IBS is abdominal pain or discomfort associated with a change in the consistency or frequency of stools. (15,16) IBS is differentiated from IBD, as IBD is associated with an inflammatory response. IBS occurs at a frequency of 8 percent to 23 percent in adults in the Western world. (10,16) For IBS, there is at present no specific diagnostic laboratory test and there is an absence of definite biological markers. The diagnosis of IBS is based on a constellation of symptoms. An international consensus group has developed criteria for IBS in an effort to standardize the definition of IBD. (12,18) The criteria have been termed the "Rome Criteria" in reference to the location of the meeting. The criteria have been further refined. (15)

Immune markers associated with IBD

During the past few years, several investigators have identified diagnostic serological markers for UC and CD. Drs. S. Targan and J. Braun and their respective groups of investigators have made a significant contribution. The serologic markers discussed below have been found to be useful for the diagnosis and differentiation of CD and UC. In addition, a panel of markers with use of an algorithm can identify specific subtypes of IBD that have different progressions and clinical courses. Thus, the panel of serologic markers are useful for diagnosis and management of CD and UC patients.

Classification of serum immune markers in IBD

Currently the following serum immune markers have found to be useful for giagnosis and management of IBD. (19-23) (see Chart 1)

1) 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.

2) ASCA (Anti-Saccharomyces cervisiae antibody). (21,26) This antibody is present in the serum of up to 70 percent of Crohn's disease patients.

3) Pancreatic antibody. (27-29) This antibody is observed in approximately 30 percent of Crohn's disease patients. Two distinct staining patterns have been reported.

4) Anti-OmpC (outer membrane porin from E. coli). (30-31) An IgA response to OmpC is seen in 55 percent of Crohn's disease patients.

Significance of ANCAs

(Anti neutrophilic cytoplasmic antibody)

* ANCAs associated with vascular diseases (vasculitidies). ANCAs are autoantibodies directed against the intracellular components of the neutrophils. Over the past decade, ANCA has received considerable attention as it was seen in inflammatory vasculitides. (32-35) The key antigen in the cytoplasm of neutrophils was found to be scrine proteinase 3 . (36) The staining of ANCA reaction with proteinase 3 will result in a cytoplasmic fluorescent pattern called c-ANCA. The pANCA pattern of perinuclear staining around the nuclei of neutrophils is the second type of pattern noted. The pANCA pattern is the result of positively charged molecules that migrate to the edge of the nuclei of neutrophils. This phenomenon occurs after alcohol fixation of the sub-state cells. The cytoplasmic granules redistribute around the nuclei, resulting in a pANCA pattern in case of antibodies to elastase, lactoferrin, cathepsin G, and myeloperoxidase. The pANCA pattern with myeloperoxidase is significant since antibodies to myelo-peroxi dase may be seen in vasculitis. (36) Antibodies to serine proteinase 3 and myeloperoxidase maybe specifically detected by an ELISA (enzyme linked immunosorbent assay).

* Association of ANCA with inflammatory bowel disease. IBD associated ANCA was first reported in 1966 by Faber and Elling, who described "leukocyte-specific antinuclear antibodies" in patients with Crohn's disease and ulcerative colitis. (38) It is now clear that the granulocyte specific antinuclear antibodies are in fact pANCA. (39) Many investigators have subsequently noted the association of pANCA with IBD. (5,10,25,39) The reported incidence of serum ANCA in UC patients was reported to be between 50 and 80 percent. (19-23) Serum pANCA is believed to reflect mucosal pANCA production in some instances. Studies have shown that pANCA production takes place in the colonic mucosa. (19,20) It appears that the mucosal antigens lead to local production of pANCA in the intestinal tact.

* pANCA associated with ulcerative colitis. The majority of adult patients with UC (60 percent to 80 percent) exhibit a positive test for pANCA. (19) Also pANCA has been observed in 83 percent of children. (19) Billing et al. have provided evidence that the pANCA antigen associated with UC is nuclear in location. (40) They studied the neutrophil reaction with confocal and electron microscopy and demonstrated that the UC-associated pANCA reaction was localized primarily over chromatin concentrated toward the periphery of the nuclei. (40) The UC patients' sera also did not recognize double stranded DNA. There may be multiple antigens and epitopes involved in the atypical pANCA and it has been reported as associated with histone-1, (41, 42) high mobility group nuclear protein (HMG-1 and HMG-2), (43, 44) and more recently as a 50 kilodalton nuclear envelope protein. (45)

The pANCA pattern seen in IBD is the result of nuclear antigens which are DNase I sensitive. The pANCA staining pattern is lost after the DNase I digestion of the substrate cells. In approximately 70 percent of the cases of UC, there is ablation of the pattern and antigen recogmtion, and in up to 30 percent of the cases there is conversion to homogeneous cytoplasmic staining. (19-23) In 3 percent ofUG patients evaluated displaying pANCA reactivity, the pANCA pattern was retained after DNase treatment of the substrate.(19-23) The retained pattern may represent concurrent antibodies present to cytoplasmic or nuclear antigens other than the IJC associated pANCA antigen. ANCAs are present in the sera of 60 percent to 80 percent of patients with ulcerative colitis and 10 percent to 30 percent of patients with Crobn's disease. Eighty-three percent of children and adolescents with ulcerative colitis showed the expression of ANCA in their sera. (23)

Various studies have shown that UC patients with pANCA represent subpopulations which show production of pANCA. This may be consequence of a distinct mucosal inflammatory process.

What does the expression of pANCA mean in patients with UC?

The pANCA expression allows for stratification of the TiC patients at the clinical and genetic levels. In adults, clinically distinct subsets of UC have been observed based on the presence of ANCA/pANCA as these patients have a higher probability of:

* Having more aggressive disease.

* Having left-sided ulcerative colitis which is more resistant to treatment than the usual case.

* Requiring surgery early in the course of the disease.

* Developing pouchitis in TiC following ileal pouch-anal anastomosis.

* Having specific HLA markers.

The serum pANCA in UC patients may reflect mucosal pANCA production. This suggests that recognition of mucosal antigens leads to local production of pANCA.

Targan and Braun and co-workers have recently demonstrated the presence of specific bacterial and/or bacteria-like antigens in patients with Crohn's disease and ulcerative colitis that appear to elicit antibody response in those patients. (30, 31)

With the use of phage display technology monoclonal antibodies that cross-react with antigens that are similar to those seen in sera of UC patients who are pANCA positive. These monoclonal antibodies cross react with bacterial antigens from E. coli and bacteriodes. (30)

Segregation of pANCA by DNase I treatment to differentiate ulcerative colitis from Type I autoimmune hepatitis and primary sclerosing cholangitis

UC associated ANCA yields a perinuclear staining pattern pANCA with methanol fixed neutrophils. pANCAs have been detected in the serum of patients with autoimmune hepatitis (Type I AIH), primary sclerosing cholangitis (PSC), and other autoimmune liver diseases.

The pANCA pattern has been identified in about 70 percent of ulcerative colitis (UC) patients. Also, the pANCA pattern with alcohol fixed neutrophils has been reported in 92 percent of sera from patients with well-defined Type I autoimmune hepatitis. Furthermore, the pANCA pattern was noted in up to 70 percent of PSC patients.

The pANCA associated with UC reactive antigen was associated with epitopes within the nuclei. In addition, the UC pANCA demonstrated loss of antigenic recognition after DNase I enzyme digestion of neutrophils as a dominant feature.

In direct contrast, the majority of Type I autoimmune hepatitis and PSC patients showed a pANCA pattern recognizing cytoplasmic constituents. Thus, the UC associated pANCA with epitopes within the nuclei is highly specific for inflammatory bowel disease. (45,46)

Association of serum pANCA with subgroup of Crohn's disease

The serum pANCA are seen in 10 percent to 30 percent of patients who have diagnosed as having CD. (19) In CD, expression of pANCA identifies a subgroup of CD characterized as "ulcerative colitis-like" phenotype in which patients have clinical features of left-sided colitis with histopathologic features of UC. The serum immunoglobulin IgG (immunogloulin G) of pANCA positive CD patients is similar to the pANCA seen with UC patients. The presence of pANCA in both CD and UC suggests that there is a specific type of mucosal inflammation that may be common to CD and UC. (19,27,48)

The CD patients who are pACNA positive did not respond as well as the majority of CD patients to anti-TNF (tumor necrosis factor) monoclonal antibody therapy. On the other hand, 65 percent of Crobn's disease patients responded well to anti-TNF monoclonal antibody therapy. (19)

High levels of pANCA in Crohn's disease patients were associated with later age of onset and an UC-like inflammatory response, as well as a relative decreased incidence of fibrostenosis and penetrating disease. (22)

Method of assay for serologic markers in IBD

* Indirect Immunofluorescence Assay for ANCA. (25)

Neutrophils were isolated from peripheral blood of normal persons by Ficoll-Hypaque density centrifugation followed by dextran sedimentation. (49)

The neutrophils were resuspended in phosphate buffered saline (PBS), and 100,000 cells were prepared on slides by cytocntrifugation. The slides were fixed in 100 percent methanol at 4[degrees]C for 10 minutes, air dried, and stored at -20[degrees]C. After incubation of the sera on the slides for 20 minutes, the slides were washed in PBS and stained with fluorescein-labeled F[(ab').sub.2] gammachain specific antibody. After washing, the slides were examined by fluorescence microscopy.

* Formalin fixation and pitcalls in the assay for ANCA. (50)

The British Association of Clinical Pathologists suggested that formalin acetone fixation followed by absolute ethanol may be useful to differentiate pANCA from ANA. (51) This procedure has not been confirmed by international consensus. Lee et al. (52) suggested that formaldehyde vapor fixation may be used to detect conversion of pANCA (pattern noted by alcohol fixation) to the cytoplasmic pattern when myeloperoxidase antibodies were present. However, Lee et al. found that formalin acetone fixation gave inconsistent results. (52) Other studies have shown that formalin fixation caused inconsistency; nonspecific effects and false positivity owing to enhanced fluorescence. (53)

In some cases neutrophils were incubated with the pANCA positive sera and the F[(ab').sub.2] gamma-chain specific antibody before DNase I digestion of the slides.

The pANCA which are DNase I sensitive are characteristically seen in UC patients and CD patients. When the pANCA pattern is observed in UC patients and treated with DNase I, in 70 percent there is ablation of antigen recognition, and in 30 percent there is conversion to a homogeneous pattern. (19)

* Fixed neutrophil ELISA assay for ANCA. The assay has been described by Saxon et al. (25) The microtiter plates were coated with 2.5 x [10.sup.5] meutrophils per well. These were fixed in methanol for 10 minutes and allowed to air dry. To minimize nonspecific binding, 0.25 percent bovine serum albumin was added as a blocking agent.

The control patients' sera were added to different wells at dilution of 1:100. The neutrophil bound antibody was detected by alkaline phosphatase conjugated goat anti-human IgG. After addition of p-nitrophenol, specific absorbance was read at 405 nm. The levels of ANCA are determined relative to a laboratory standard expressed as ELISA Units (EU/mL). The standard was obtained from sera of pANCA positive patients with well-characterized UC.

* ELISA assay for ASCA in CD patients. (22,48) The microtiter plates were coated with phosphopeptidomannans from the yeast Saccharomyces cerevisiae ssp uvarum. The control and patients' serum samples were added to different wells at a 1:100 dilution. Bound antibodies were detected with goat anti-human IgG and IgA labeled with alkaline phosphatase. After adding p-nitrophenol, the specific absorbance was measured at 405 nm. The absorbance of each serum sample was evaluated and assigned ELISA Units (EU/mL) values relative to the absorbance of a pool of sera collected from well-characterized patients with CD. The standard pool was arbitrarily assigned the value of 100 EU/mL. The result of the CD diagnostic system panel determines relative positivity of IgA and IgG ASCA respectively.

* IBD first step screen for ANCA, IgG ASCA IgA ASCA and IgA anti OmpC. Prometheus Laboratories developed quantitative tests that detect serum markers consistent with the presence of IBD. (54) This IBD First Step system consists of a set of four quantitative ELISA assays used together to detect ANCA, IgG ASCA IgA ASCA and IgA anti OmpG antibodies. The panel of assays shared a test sensitivity of 94 percent. The negative predictive value is greater than 95 percent when the data is modeled for an IBD prevalence commonly seen in a standard gastroenterology practice of 15 percent. The tests have a high sensitivity, but a much lower specificity. The main purpose of the test panel is to help rule out the presence of IBD. The algorithm with use of the sensitive modified assay is shown in Chart 2. The algorithm can be applied to children, adolescents and adults with IBD.

* Variability of assays for ANCA and ASCA in different clinical laboratories. Sandborn, W.J. et al. (55) conducted a study with the purpose of evaluating serological markers in a population-based cohort of patients with ulcerative colitis and Crohn's disease. Blood and sera were obtained from 162 patients who agreed to participate in the study from a group of 290 IBD patients. Of the 162 patients, 83 had ulcerative colitis and 79 had Crohn's disease. The conclusions reached by the Sandborn et al. study:

1) The sensitivity of the ANCA assays varied widely in different laboratories.

2) The prevalence of ASCA was similar in the various laboratories participating in the study.

3)The positive predictive values of the ANCA and ASCA for the diagnosis and evaluation of UC or CD are high enough to be clinically useful.

Antibodies associated with Crohn's disease

Besides the pANCA that identifies a subgroup population of Crobn's disease, there are several other antibodies that are associated with Crohn's disease. These antibodies include Saccharomyces cerevisiae antibody (ASCA), pancreatic antibody, and antibody to OmpC (outer membrane porins isolated from E. coli bacteria).

ASCA

ASCA is a serum immune marker, which has been shown to be expressed in the majority of sera of CD patients. (22,23,48) The ASCA antibodies have a high specificity for Crohn's disease. (22,48) Serum ASCA is expressed in up to 70 percent of CD patients.

Small bowel disease was present in almost all CD patients who were positive for both IgA and IgG ASCA but negative for pANCA. The majority of patients in the subgroup may have signs of small bowel obstruction and perforating disease. The CD patients with IgA ASCA and IgG ASCA appear to have a more aggressive type of CD.

The ASCA assay is performed by an ELISA method. It should be emphasized that negative tests of pANCA or serum ASCA do not rule out the presence and diagnosis of IBD. The positive tests provide evidence that the patients with IBD should be evaluated further. (48,52)

An ASCA ELISA with lower threshold was able to detect 90 percent of diagnosed Crohn's disease patients. V/hen evaluated at the lower threshold to allow exclusion of IBD as a probable diagnosis for negative samples, a positive result must be followed up with more specific test to allow probable diagnosis of IBM. (52)

Pancreatic antibodies in Crohn's disease

Pancreatic antibodies as detected by an indirect immuno-fluorescence test with human pancreas substrate occurred in 31 percent to 39 percent in Crohn's disease patients. (27) Of 212 CD patients studies, 30 patients had pancreatic antibodies characterized by a "drop-like" fluorescence in the pancreatic acini (subtype I). (28,29) Twenty-eight patients demonstrated a fine speckled staining in the acinar cells of the pancreas (subtype II). (28,29) Siebold et al. (28) concluded that pancreatic antibodies are specific markers for CD. Two subgroups were seen with different immunofluorescent patterns.

It remains to be determined whether the presence of the pancreatic, antibody is associated with a defined sub group of CD patients. The specific antigen reacting with the pancreatic antibody has not been identified. These antibodies were rarely seen to occur in family members of patients with Crohn's disease.

The relevance of pancreatic antibodies in the pathogenesis of Crohn's disease is unclear. Stocker, et al. have reported that in patients with CD diagnosed for less than 2.5 years, the prevalence of pancreatic antibodies was 25 percent. However, if the CD existed longer than 2.5 years, the incidence of pancreatic antibodies was 46 percent.

Whether the presence of pancreatic antibodies in CD identifies a subgroup of Crohn's patients remains to be determined.

The previous reports used a substrate of human type o negative pancreatic tissue. One may be able to employ primate pancreatic tissue substrate. Siebold, et al. have observed that pancreatic tissue from rats and mice showed immunofluorescent patterns similar to that observed in human. Extensive comparative data of humans and rat tissue runs waas not presented.

OmpC antibody in Crohn's disease

OmpC is an outer membrane porin antigen purified from E. coli. (30) ELISA assay with human sera demonstrated elevated IgG anti-OmpC in ulcerative colitis patients compared to healthy controls.

Cohavy et al. (30) performed experiments on the hypothesis that pANCA identifies a bacterial antigen found in the human colonic mucosa. In these experiments a monoclonal pANCA antibody was used. (30) The ANCA monoclonal antibody was cloned by a phage display method and characterized.

The pANCA monooclonal antibody was reactive with bacterioides and E. coli antigens. The E. coli protein was biochemically and genetically identified as the outer membrane porin OmpC.

In patients with Crohn's disease, IgA response to OmpG was found in 55 percent of 151 patients, 56 percent were seropositive to ASCA, and 24 percent were positive with the pANCA test.

The serological response to the OmG and panel of antigens studies by Landers et al. (31) identified more Crohn's patients. There may be patient subsets of Crohn's that demonstrate variable responses to selected bacterial antigens.

Summary

Inflammatory bowel disease (IBD) is a generic term that refers to Crohn's disease and ulcerative colitis. Crohn's disease (CD) is a granulomatous enteritis which can involve the ileum, colon, and other parts of the intestinal tract. The serologic responses seen in Crohn's disease include antibodies to Saccharomyces cercvisiae, mycobacteria, bacteroides, listeria and E. coli. Many of these organisms may be involved in the pathogenesis of the Crohn's disease.

Ulcerative colitis is characterized by inflammation of the mucosa and submucosa of the large intestine.

The CD and UC are considered to be distinct forms of IBD; however, there is a subgroup of CD with a UC-like presentation.(34)

In recent years, several serologic markers have been found to be useful for the diagnosis and differentiation of CD and UC. These markers include the following antibodies (a) pANCA, (b) ASCA, (c) pancreatic antibody, and (d) 0mpC antibody. The application of a panel of markers with the use of an algorithm can identify specific subtypes of IBD that have different clinical courses and progression of the diseases. The application of the serologic markers is useful for diagnosis and management of CD and UC patients.

Dr. Robert M. Nakamura serves as Laboratory Medical Director at Prometheus Laboratories in San Diego, CA. Mary Barry is Vice-president of Operations at Prometheus Laboratories, a small volume laboraatory serving gastrointestinal specialists throughout the U.S.

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Chart
CD COHORT

 ASCA lgA % Cumulative
 Total Panel+ OmpC+ pANCA+ Detected % Detected

Entire Cohort 175 96 54.9% 54.9%
ASCA Panel - 79 22 12.6% 67.4%
ASCA Penal -/OmpC- 57 15 8.6% 76.0%
ASCA Panel-/OmpO- 42 6.0% 76.0%
 /pAN CA-
Total % detected: 76.1%

IBD COHORT

 ASCA lgA % Cumulative
 Total Panel+ OmpC+ pANCA+ Detected % Detected

Entire Cohort 275 104 37.8% 37.8%
ASCA Panel 171 32 11.6% 49.5%
ASCA Panel -/OmpC- 139 69 25.1% 74.5%
ASCA Panel -/OmpC- 70 0.0% 74.5%
 /pANCA-
Total % detected: 74.5%

ULCERATIVE COLITIS COHORT

 ASCA lgA % Cumulative
 Total Panel+ OmpC+ pANCA+ Detected % Detected

Entire Cohort 100 68 68.0% 68.0%
pANCA neg 32 2 2.0% 70.0%
pANCA -/OmpC- 30 2 2% 72.0%
pANCA -/OmpC- 28 0.0% 72.0%
Total % detected: 72.0%

NORMAL, IBS, DISEASE CONTROLS

 ASCA lgA % Cumulative
 Total Panel+ OmpC+ pANCA+ Detected % Detected

Entire Cohort- 127 1 0.8% 0.8%
ASCA Panel - 126 3 2.4% 3.1%
ASCA Panel -/OmpC- 123 4 3.1% 6.3%
ASCA Panel -/OmpC- 119 0.0% 6.3%
 /pANCA-
Total % detected: 6.3%
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Author:Nakamura, Robert M.; Barry, Mary
Publication:Medical Laboratory Observer
Article Type:Cover Story
Geographic Code:1USA
Date:Nov 1, 2001
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