Advances in clinical laboratory tests for inflammatory bowel disease

www.elsevier.com/locate/clinchim

Clinica Chimica Acta 335 (2003) 9–20

Review

Advances in clinical laboratory tests for inflammatory

bowel disease

Robert M. Nakamuraa,b,*, Mariko Matsutanib, Mary Barryc

aDepartment of Pathology, Scripps Clinic, La Jolla, CA, USAbPrometheus Clinical Laboratories, San Diego, CA, USAcPrometheus Laboratory Operations, San Diego, CA, USA

Received 4 April 2003; received in revised form 30 May 2003; accepted 30 May 2003

Abstract

Inflammatory bowel disease (IBD) is a generic term that refers to Crohn’s disease and chronic ulcerative colitis (UC). The

CD and UC are considered to be distinct forms of IBD; but there is a subgroup of CD with a UC-like presentation.

The genetic factors play a significant role in IBD. IBD is associated with a strong familial pattern. Recent studies support the

hypothesis that IBD patients have a dysregulated immune response to endogenous bacteria in the gastrointestinal tract. The

serologic responses seen in Crohn’s disease include antibodies to Saccharomyces cerevisiae, mycobacteria, bacteroides andE. coli.

The pANCA antibody seen in UC and CD has been demonstrated to react with epitopes of H1 histone, Bacteroides caccae (Ton-B

linked outer membrane protein), Pseudomonas fluorescens-associated bacterial protein I-2, mycobacterial histone 1 homologue

called Hup B.

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) anti-pancreatic antibody, (d) OmpC antibody and

(e) I-2 antibody and antibodies to anaerobic coccoid rods.

The application of a panel of markers with the use of an algorithm (i.e. IBD First Step) can identify specific subtypes of IBD

that have different clinical courses and progression of the diseases. The serologic markers are useful for the diagnosis and

management of CD and UC patients.

D 2003 Elsevier B.V. All rights reserved.

Keywords: IBD (inflammatory bowel disease); CD (Crohn’s disease); UC (ulcerative colitis); Serologic antibody markers; pANCA in IBD

1. Introduction and definitions

1.1. What is inflammatory bowel disease (IBD)?

Chronic inflammatory bowel disease (IBD) refers

to two diseases: (a) Crohn’s disease (CD), formerly

0009-8981/03/$ - see front matter D 2003 Elsevier B.V. All rights reserve

doi:10.1016/S0009-8981(03)00286-9

* Corresponding author. Department of Pathology, Scripps

Clinic, La Jolla, CA, USA.

E-mail address: [email protected] (R.M. Nakamura).

known as Regional Enteritis, and (b) ulcerative colitis

(UC), which are generally considered to be distinct

forms of IBD [1].

Crohn’s disease (CD) is a nonspecific granuloma-

tous inflammatory disease affecting the lower end of

the ileum and often involving the colon and other

parts of the intestinal tract [1]. Crohn’s disease was

first reported by Burill Crohn and his colleagues in

1922 and was called Regional Enteritis [2]. Crohn’s

disease is diagnosed in four patients per 100,000

d.

R.M. Nakamura et al. / Clinica Chimica Acta 335 (2003) 9–2010

population in the United States and the incidence and

prevalence is rising [3].

Ulcerative colitis (UC) is a chronic disease of

unknown etiology characterized by inflammation of

the mucosa and submucosa of the large intestine. In

UC, inflammation may involve the rectum down to

the anal margin and often characterized with a bloody

diarrhea [1]. UC may have a prevalence of 100 cases

per 100,000 population in the United States [4].

In IBD, the first clinical signs of disease typically

begin between adolescence and the third decade of life

[5,6].

1.2. 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 prob-

lematic [7,8]. There is a subgroup of cases of CD with

a UC-like presentation that illustrates the similarity of

CD and UC [8–10]. 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 [11,12].

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 [13].

1.3. What is irritable bowel syndrome (IBS)—how

does it differ from IBD?

IBS is the most common functional disorder of the

gastrointestinal tract and is not associated with an

inflammatory response; thus, IBS is differentiated

from IBD.

IBS is characterized by abdominal pain and dis-

comfort associated with a change in the consistency or

frequency of stools [14,15]. IBS is found in a fre-

quency of 8% to 23% of adults who are mostly

females in the Western world [13,15]. At the present

time, there is no specific diagnostic laboratory test or

biological marker to diagnose IBS. The diagnosis is

based on a constellation of a group of symptoms.

[16–18]. An international group has made an effort to

standardize the definition and diagnosis of IBS

[14,15,18]. The criteria have been termed the Rome

Criteria in reference to the location of the meeting.

Since the first meeting, the criteria have been further

defined [18,19].

1.4. Progress in studies on IBD

During the past decade, there has been consider-

able progress in the study of IBD [13,20]. It has

become important to specifically identify CD and

UC, as the therapeutic regimens for the individual

patients with either CD or UC will vary.

The diagnosis of IBD can be difficult in any age

group and particularly difficult in the elderly age

group [21]. Older patients may have other medical

conditions, such as advanced vascular disease or

diverticulosis with inflammation [20]. CD may be

difficult to diagnose in older patients and conditions,

such as infections, ischemic colitis, diverticulitis,

collagenous colitis, and lymphomas and radiation

colitis may mimic IBD.

Significant advances have been made in identifi-

cation of serological antibody markers for diagnosis

and management of CD and UC. This review will

discuss the use of serological markers for the diagno-

sis and differentiation of CD and UC. Recent studies

have shown that a panel of serological markers with

use of an algorithm can (a) help identify specific

subtypes of IBD that have different progressions and

clinical courses and are (b) useful for the diagnosis

and management of CD and UC patients.

2. Genetic factors in IBD

There is strong evidence that genetic factors are

important in the pathogenesis of Crohn’s disease and

ulcerative colitis [22,23]. Some of the most compel-

ling evidence are from three studies of concordance

rates in twin pairs [24]. A total of 322 twin pairs have

been reported. After combining the data from the

European studies, the concordance rate in Crohn’s

disease is 37% in monozygotic twin pairs and 7% in

dizygotic twin pairs. In ulcerative colitis, the concor-

dance rate in monozygotic twins is 10% and in

dizygotic is 3% [24].

R.M. Nakamura et al. / Clinica Chimica Acta 335 (2003) 9–20 11

IBD is associated with a strong familial pattern and

a number of genetic loci have been implicated in

disease susceptibility [22,23]. Approximately 15% of

patients with IBD have first-degree relatives who also

have IBD [22,25]. The incidence of IBD among first

degree relatives of IBD is 30 to 100 times greater than

the general population [25].

The genetic factors play a significant role in IBD

pathogenesis [24,26–28]. It has been suggested that

there may be 10 to 15 genes which may account for

susceptibility to IBD as well as relationship between

CD and UC. There is a definite complex pattern of

inheritance.

Linkage studies have implicated several genomic

regions as likely containing IBD susceptibility genes

[24,29,30].

The best replicated linkage region, IBD1 on chro-

mosomes 16q, contains CD susceptibility gene NOD2.

Other IBD genomic regions include IBD2 on chro-

mosome 12q (observed more in UC) and IBD3,

containing the major histocompatibility complex re-

gion [24,29–31]. Also, a short genomic region has

been associated with CD in chromosome 5q [32].

A putative locus on chromosome 12 has been shown

to be associated with a predisposition to CD in British

and United States Caucasian families [24,33]. Toyoda

et al. [34] studied the association of HLA class 11 genes

in IBD. They showed that DR andDQmolecules firmly

separate UC and CD on genetic grounds and the

contributions of HLA class II genes to the disease

susceptibility are different for UC and CD.

However, there are several variations in pene-

trance and expression of the genes as well as demo-

graphics and epidemiologic factors, indicating a

prominent role for environmental factors in the path-

ogenesis of IBD.

2.1. NOD2 gene and relationship to Crohn’s disease

Ogura et al. [35,36] and Hugot et al. [37] described

the NOD2 gene and found that a genetic variant was

highly associated with Crohn’s disease in about 15%

of patients with CD. The NOD2 gene maps to the IBD1

region of chromosome 16 is a susceptibility locus for

CD. The NOD2 gene belongs to a class of genes

whose products have a role in recognizing bacterial

components in the gut and coordinating the immune

system’s response. It appears that Crohn’s patients

produce a modified protein that lacks control of the

immune system. Thus, an abnormal inflammatory

response to enteric bacteria has been proposed as a

pathogenic mechanism in Crohn’s disease. Abreu et

al. [38] have reported that mutations in NOD2 are

associated with fibrostenosing disease in patients with

Crohn’s disease.

2.2. Cytokine genes as determinants of disease

susceptibility in IBD

Cytokine genes are important candidate genes in

IBD. Allelic association studies has shown that the

genes that encode the interleukin-1 receptor antagonist

(IL-IRA) and tumor necrosis factor-alpha are important

determinants of disease susceptibility in IBD [39–41].

The interleukin-1 receptor antagonist (IL-IRA) gene

has been demonstrated to be important for prediction of

a severe and extensive course of UC [39,40].

With a greater understanding of genetic differences

underlying IBD susceptibility and response to therapy,

the IBD patients may receive more specific and

effective therapeutic treatments [31].

3. Pathophysiology and pathogenesis of IBD

The exact etiology and cause of CD and UC still

remain unknown and elusive. However, many recent

studies support the hypothesis that patients with IBD

have a dysregulated immune response to endogenous

bacteria in the gastrointestinal tract. [6,22,23,42].

Also, the inflammatory bowel diseases (IBD) consist

of a heterogeneous overlapping subgroup of inflam-

matory intestinal disorders as described below.

3.1. Possible relationship of IBD to enteric bacteria

Several investigators have provided evidence that

certain enteric bacteria may be involved in the path-

ogenesis of IBD and particularly Crohn’s disease

[43–47]. Several bacterial species and viruses have

been implicated in human CD by direct detection or

by disease associated antimicrobial immune responses.

Analyses of rodent IBD model systems have demon-

strated a pathologic role for enteric bacteria [45]. The

rodents rendered germ-free were protected from dis-

ease onset [46].


Crohn’s disease (CD) may consist of a heteroge-

neous group of diseases with similar features [8,13].

Abreu et al. [38] have reported that patients with CD

demonstrate a heterogeneous and serologic response

to specific bacterial and bacterial related antigens. The

serologic response seen in CD patients includes anti-

bodies to Saccharomyces cerevisiae, mycobacteria,

bacteroides and E. coli. Many of the organisms may

contribute directly or indirectly to the pathogenesis of

CD.

Perinuclear anti-neutrophilic antibody (pANCA) in

IBD as described below has been shown to be reactive

with determinants of E. coli [46], B. caccae [46,48],

mycobacteria [43] and other enteric bacteria [44].

These findings are consistent with the hypothesis that

IBD patients have antibodies to bacterial proteins and

the immune dysregulation may contribute directly or

indirectly to the pathogenesis of IBD.

Table 1

Patient groups tested for IBD serologic markers (a) IgG and IgA

ASCA, (b) IgA OmpC antibodies and (c) pANCA with DNase I

sensitivity

Number of

patients

Percent

detected

Group I

IBD patients 275 76.1

Group II

CD patients 175 76.1

Group III

UC patients 100 72

Group IV

Normal and other

disease controls

127 6.3

4. Serum immune markers in IBD

(A) The following serum immune markers in IBD

have been reported. Several of them are currently

being used in the clinical laboratory and have been

useful for the diagnosis and management of IBD

[49–53].

(1) Deoxyribonuclease (DNase I)-sensitive perinu-

clear anti-neutrophil cytoplasmic autoantibody

(pANCA). The IBD-associated pANCA defines

an antibody to a nuclear antigen which is

sensitive to DNase I [51,53,55,59].

(2) Anti-S. cerevisiae antibody (ASCA) [48,56]. This

antibody is present in the serum of up to 70% of

Crohn’s disease patients.

(3) Pancreatic antibody [57–59]. This antibody is

observed in approximately 30% of Crohn’s

disease patients. Two distinct staining patterns

have been reported.

(4) Anti-OmpC (outer membrane porin from E. coli)

[45,46]. An IgA response to OmpC is seen in

55% of Crohn’s disease patients.

(5) Antibody to P. fluorescens-associated sequence I-

2 [44,45]. A prevalence of 54% in CD and 10%

in UC was reported.

(6) HupB (32-kDa protein iron regulated protein and

mycobacterial histone HI homogue). HupB is

reactive with pANCA and monoclonal antibody

and serum IgA from CD patients [43]. The

association of HupB-binding serum IgA from CD

patients supports evidence for the association of a

mycobacterial species with Crohn’s disease.

(7) Antibodies to anaerobic coccoid rods [60,61].

Linskens et al. [60] reported that in the evaluation

of CD, the sensitivity was 52% and the specificity

was 90%.

(B) Currently the most practical and commonly

used serologic antibody markers for the diagnosis and

management of IBD are [20,51–53].

(1) DNase sensitive pANCA

(2) ASCA, IgA and IgG antibodies

(3) Anti-Omp C IgA antibody

Usefulness of the panel of markers is shown in

Table 1. The patients in each group were diagnosed as

IBD, UC or CD patients by standard methods of

gastrointestinal biopsy, clinical and history evaluation

as well as by laboratory test. The patients were all

tested by Prometheus Laboratories, San Diego, CA.

In Table 1, the testing panel of clinical laboratory

tests are:

(a) ASCA panel-IgG, IgA antibodies to S. cerevisiae

(b) OmpC-IgA antibodies to outer membrane porin

isolated from E. coli.


(c) pANCA-immunofluorescence analysis with

DNase I sensitivity.

� In Group I (IBD patients), the test profile detected

74.5% of the 275 patients.� In Group II (CD patients), 76.1% of the patients

were identified and detected.� In UC patients in Group III, 72% of 100 patients

were identified.� Group IV, consisting of normal patients and

patients with other diseases than IBD, showed a

positive detection rate of 6.3%.

In the analysis of Table 1, the test panel can

successfully identify 76% of Crohn’s patients, 72%

of UC patients. It should be noted that a ‘‘negative

result with above three serologic antibody markers

does not rule out the possibility of IBD.’’

5. Anti-neutrophilic cytoplasmic antibody (ANCA)

5.1. ANCAS associated with vascular diseases

ANCAs are antibodies directed against the intra-

cellular components of the neutrophils. ANCA has

received considerable attention as it was seen in

inflammatory vasculitides [62–66]. The main antigen

in the cytoplasm of neutrophils associated with vas-

culitis was found to be serine proteinase [67]. The

staining of ANCA reaction with proteinase 3 will

result in a cytoplasmic fluorescent pattern called c-

ANCA.

Perinuclear anti-neutrophilic cytoplasmic antibody

(pANCA) is the second type of staining pattern which

was observed. In the regular clinical laboratory testing

for ANCA, a pANCA perinuclear pattern may be

detected. The pANCA pattern is the result of posi-

tively charged protein molecules that migrate to the

edge of the nuclei of neutrophils. This phenomenon

occurs after alcohol fixation of the substrate cells, as

the protein molecules are soluble in alcohol. The

cytoplasmic granules redistribute around the nuclei,

resulting in a pANCA pattern in case of antibodies to

elastase, lactoferrin, cathepsin G and myeloperoxidase

[68]. The pANCA pattern with myeloperoxidase is

significant since antibodies to myeloperoxidase are

seen in vasculitis [67]. Savige et al. [68] have reported

that antimyeloperoxidase antibodies were common in

microscopic polyarteritis (6/14, 43%) and systemic

vasculitis (5/10, 31%).

In cases of patients with suspected vasculitis, the

antibodies to serine proteinase 3 and myeloperoxidase

may be specifically detected and identified by en-

zyme-linked immunosorbent assay (ELISA).

5.2. pANCA and association with IBD

IBD-associated ANCA was first reported in 1966

by Faber and Elling [69], who described ‘‘leukocyte-

specific antinuclear antibodies’’ in patients with

Crohn’s disease and ulcerative colitis. It is now clear

that the granulocyte specific antinuclear antibodies are

in fact pANCA [70].

Several investigators have subsequently noted the

association of pANCA with IBD [8,13,53,54]. The

incidence of serum pANCA in UC patients has been

reported to be between 50% and 80% [49–53]. Serum

pANCA is believed to reflect mucosal pANCA pro-

duction in some instances. Studies have shown that

pANCA production takes place in the colonic mucosa

[49,50]. It appears that the mucosal antigens lead to

local production of pANCA in the intestinal tract.

Muller-Ladner et al. [71] have reported that the

reactivity of the immunoglobulin classes and IgG

subclasses of antibodies (ANCAs) in UC and CD

differ from each other and from the distribution of

antibodies observed in vascular diseases, such as

Wegener’s granulomatosus and microscopic vasculitis.

5.3. DNase-sensitive pANCA associated with

ulcerative colitis (UC)

The majority of adult patients with UC (60% to

80%) exhibit a positive test for pANCA [49]. Also,

pANCA has been observed in 83% of children [49].

Billing et al. [72] have provided evidence that the

pANCA antigen associated with UC is nuclear in

location and differs from other types of pANCAs.

They studied the neutrophil reaction with confocal

and electron microscopy and demonstrated that the

UC-associated pANCA reaction was localized pri-

marily over chromatin concentrated toward the pe-

riphery of the nuclei [72]. 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 associ-

ated with histone H-1 [73], high mobility group

nuclear protein (HMG-1 and HMG-2)[74,75] and

more recently as a 50-kilodalton (kDa) nuclear enve-

lope protein [76].

Sobajima et al. [75] reported that 89% (25/28)

of patients with autoimmune hepatitis (AIH) dem-

onstrated antibodies which reacted with HMG1/2

non-histone chromosomal proteins. Terjung et al.

[76] reported that the pANCA antibodies in pro-

gressive sclerosing cholangitis (PSC) and AIH

reacted with the 50-kDa nuclear envelope protein

seen in UC.

It appears that the pANCA-associated antigen

associated with IBD located in the nuclei may be a

complex conformational epitope associated with the

histone H-1I, HMG-1, HMG-2 and a nuclear envelope

protein. The procedure of DNase-sensitive pANCA as

studied by Vidrich [79] appears to be more specific to

IBD as there is differentiation of IBD from AIH and

PSC. Vidrich [79] reported, by their assay for DNase-

sensitive pANCA, that the majority of Type I AIH and

PSC patients showed a pANCA pattern recognizing

cytoplasmic constituents.

The pANCA pattern seen in IBD is the result of

nuclear antigens which are DNase-sensitive and have

been called ‘‘atypical pANCA’’. The pANCA stain-

ing pattern is lost after the DNase digestion of the

substrate cells. In approximately 70% of the cases of

UC, there is ablation of the pattern and antigen

recognition, and in up to 30% of the cases, there is

conversion to homogeneous cytoplasmic staining

[49–53]. In 3% of UC patients evaluated displaying

pANCA reactivity, the pANCA pattern was retained

after DNase treatment of the substrate [49–53]. The

retained pattern may represent concurrent antibodies

present to cytoplasmic or nuclear antigens other than

the UC-associated pANCA antigen.

ANCAs are present in the sera of 60% to 80%

of patients with ulcerative colitis and 10% to 30%

of patients with Crohn’s disease. Eighty-three per-

cent of children and adolescents with ulcerative

colitis showed the expression of ANCA in their

sera [53]. Various studies have shown that UC

patients with pANCA represent subpopulations

which show production of pANCA. This may be

a consequence of a distinct mucosal inflammatory

process.

5.4. Reactivity of UC pANCA human monoclonal

antibody with various bacterial antigens and histone

H-1 homologue

UC pANCA human monoclonal antibodies, Fab 5-

3 and 5-2 were developed by a phage display cloning

technique from B-cells obtained from UC patients

[77]. These human monoclonal antibodies (Fab 5-3

and 5-2) cross-reacted with antigens that were similar

to those seen with sera of UC patients who were

pANCA-positive.

Eggena et al. [77] have that demonstrated histone

H1 bearing a recurring COOH-terminal epitope reacted

with UC-associated pANCA marker antibodies.

Perinuclear anti-neutrophil antibody (pANCA)

seen in patients with UC and a subset of CD have

been demonstrated to react with antigens of microbial

agents which may be involved in the pathogenesis of

IBD. Supporting evidence which have been reported

are:

(1) Cohavy et al. [43] identified a novel mycobacte-

rial histone H1 homologue (HupB) as an antigenic

target of pANCA monoclonal antibody and serum

immunoglobulin A from patients with Crohn’s

disease. The association of HupB binding serum

IgA from Crohn’s patients provides evidence for

the association of mycobacterial species with

Crohn’s disease.

(2) Sutton et al. [44] have identified a novel bacterial

sequence called I2 which is related to 1D

pathogenesis.

(3) Wei et al. [48] also demonstrated that a pANCA

monoclonal antibody reacted with a 100-kDa

protein of IBD clinical isolate of B. caccae. The

reactive protein was B. coccae Ton-B linked outer

membrane protein. The gene was termed ompW.

The monoclonal pANCA was reactive with

recombinant ompW.

5.5. pANCA association with CD subgroup

pANCA is detected in 10% to 30% of patients who

have been diagnosed as having CD [49]. In CD,

expression of pANCA identifies a subgroup of CD

characterized as ‘‘ulcerative colitis-like’’ phenotype in

which patients have clinical features of UC. The serum

immunoglobulin IgG 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 [49,52,78].

The CD patients who are pANCA-positive did not

respond as well as the majority of CD patients to anti-

tumor necrosis factor (TNF) monoclonal antibody

therapy. On the other hand, 65% of Crohn’s disease

patients responded well to anti-TNF monoclonal an-

tibody therapy [49].

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 [52].

5.6. pANCA in IBD vs. pANCA in 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 [79,80].

The pANCA pattern has been identified in about

70% of ulcerative colitis (UC) patients. Also, the

pANCA pattern with alcohol-fixed neutrophils has

been reported in 92% of sera from patients with

well-defined Type I autoimmune hepatitis [79]. Fur-

thermore, the pANCA pattern was noted in up to 70%

of PSC patients [79].

The pANCA associated with UC reactive antigen

was associated with epitopes within the nuclei. In

addition, the UC pANCA demonstrated loss of anti-

genic recognition after DNase I enzyme digestion of

neutrophils as a dominant feature.

In direct contrast, ‘‘the majority of Type I autoim-

mune 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 [79].

5.7. Meaning of pANCA in UC

The pANCA expression allows for stratification of

the UC 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 [9,13,23]:

(a) Left-sided ulcerative colitis which is more re-

sistant to treatment than the usual case.

(b) More aggressive disease.

(c) Requiring surgery early in the course of the

disease.

(d) Developing pouchitis in UC following ileal

pouch–anal anastomosis.

(e) Having specific HLA markers.

6. Serum antibodies for the evaluation of Crohn’s

disease

Besides the pANCA that identifies a subgroup

population of Crohn’s disease, there are several other

antibodies that are associated with Crohn’s disease. As

stated above, these antibodies include S. cerevisiae

antibody (ASCA), pancreatic antibody and antibody

to OmpC (outer membrane porins isolated from E.

coli bacteria).

6.1. ASCA

ASCA is a serum immune marker, which has been

shown to be expressed in the majority of sera of CD

patients [52,86]. The ASCA antibodies have a high

specificity for Crohn’s disease [52,81]. Serum ASCA

is expressed in up to 70% of CD patients. The anti-

bodies to S. cerevisiae seen in CD are associated with

the oligomannosidic epitopes on the S. cerevisiae

[82–84].

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 [81].


An ASCA ELISAwith lower threshold was able to

detect 90% of diagnosed Crohn’s disease patients.

When evaluated at the lower threshold to allow exclu-

sion of IBD as a probable diagnosis for negative

samples, a positive result must be followed up with

more specific tests to allow probable diagnosis of IBD

[53,85].

6.2. Pancreatic antibodies in Crohn’s disease

Pancreatic antibodies as detected by an indirect

immunofluorescence test with human pancreas sub-

strate occurred in 31% to 39% in Crohn’s disease

patients [58,59]. Of 212 CD patients studied, 30

patients had pancreatic antibodies characterized by a

‘‘drop-like’’ fluorescence in the pancreatic acini (sub-

type I) [58,59]. Twenty-eight patients demonstrated a

fine speckled staining in the acinar cells of the

pancreas (subtype II) [58,59].

Seibold et al. [58] concluded that pancreatic anti-

bodies 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

subgroup of CD patients. The specific antigen react-

ing with the pancreatic antibody has not been identi-

fied. 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. [57] have reported that in patients with CD

diagnosed for less than 2.5 years, the prevalence of

pancreatic antibodies was 25% with use of an immu-

nofluorescence tissue assay. However, if the CD

existed longer than 2.5 years, the incidence of pan-

creatic antibodies was 46%.

Whether the presence of pancreatic antibodies in

CD identifies a subgroup of Crohn’s disease 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. Seibold et

al. have made a comment in the paper that pancreatic

tissue from rats and mice showed immunofluorescent

patterns similar to that observed in humans. Extensive

comparative data of humans and rat tissue runs were

not presented. However, autoantibodies to endocrine

organ tissue often demonstrated species specificity.

One should use human or primate tissues for detection

of pancreatic antibodies.

This test may become helpful if the pancreatic

antibodies will identify a specific subset of Crohn’s

disease or will help increase the laboratory diagnostic

detection percentage for Crohn’s disease.

6.3. OmpC antibody in Crohn’s disease

OmpC is an outer membrane porin antigen purified

from E. coli [45,46]. The E. coli protein was bio-

chemically and genetically identified as the outer

membrane porin OmpC.

The ELISA assay with human sera demonstrated

elevated IgG anti-OmpC in ulcerative colitis patients

compared to healthy controls.

In patients with Crohn’s disease, IgA response to

OmpC was found in 55% of 151 patients, 56% were

seropositive to ASCA, and 24% were positive with

pANCA test [20,45,46].

6.4. Coccoid agglutination test for anaerobic cocci in

CD [60]

Wensinck and Van de Merwe [61] isolated anaero-

bic bacteria from the feces of patients with CD and

developed a serum agglutination test. Other investiga-

tors have also reported on the use of similar agglutina-

tion tests. Agglutinating antibodies were found in 58%

of CD patients with very high specificity.

Linskens et al. [60] used several strains of Gram-

positive anaerobic coccoid rods in their agglutination

test. The agglutinating antibodies to coccoid rods are

mostly of IgG isotype and less frequently of IgM

isotype. The test format is the mixing of two drops of

patient’s serumwith one drop of bacterial suspension on

a slide placed on a rotary mixer platform. Results were

read after 5 min by two observers. Titers were deter-

mined with doubling dilutions of serum with phy-

siologic saline (0.85%). Linskens et al. [60] reported a

sensitivity of 52% and specificity of 90% for CD.

7. 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 [83]. 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 OmpC antibodies. The panel of

assays shared a test sensitivity of 94%. The negative

predictive value is greater than 95% when the data are

modeled for an IBD prevalence commonly seen in a

standard gastroenterology practice of 15%. 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.

After the initial screening, patients who may have

IBD are reflexed to a more extensive quantitative panel

of tests, including IgG ASCA, IgA ASCA, IgA anti-

OmpC and DNase I-sensitive pANCA assays.

On a study of 128 pediatric patients and tested by

the first step screen test for IBD, it was demonstrated

that unnecessary work can be avoided in 70 true

negatives. Only 10% of false positives would have

been subjected to unnecessary work [53].

8. Variability of assays for ANCA and ASCA in

different clinical laboratories

Sandborn et al. [85] conducted a study with the

purpose of evaluating serological markers in a popula-

tion-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 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.

9. Summary

Inflammatory bowel disease (IBD) is a generic

term that refers to Crohn’s disease and chronic ulcer-

ative colitis. Crohn’s disease is a granulomatous enter-

itis which can involve the ileum, colon and other parts

of the intestinal tract. Chronic ulcerative colitis (UC)

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; but there is a subgroup of CD with a UC-like

presentation.

The genetic factors play a significant role in IBD.

IBD is associated with a strong familial pattern.

Recent studies support the hypothesis that IBD

patients have a dysregulated immune response to

endogenous bacteria in the gastrointestinal tract.

The serologic responses seen in Crohn’s disease

include antibodies to S. cerevisiae, myobacteria, bac-

teroides and E. coli. The pANCA antibody seen in UC

and CD has been demonstrated to react with epitopes

of HI histone, B. caccae (Ton-B linked outer mem-

brane protein), P. fluorescens-associated bacterial pro-

tein I-2, mycobacterial histone 1 homologue called

HupB.

In recent years, several serologic markers have

been found to be useful for the diagnosis and differ-

entiation of CD and UC. These markers include the

following antibodies: (a) pANCA, (b) ASCA, (c)

pancreatic antibody, (d) OmpC antibody and (e) I-2

antibody and (f) antibodies to anaerobic coccoid rods.

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 these serologic

markers is useful for the diagnosis and management

of CD and UC patients.

10. Uncited reference

[74]

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Advances in clinical laboratory tests for inflammatory bowel disease