Determination of Anti-Neutrophil Cytoplasmic Antibody (ANCA) and Anti-Saccharomyces Cerevisiae Antibody (ASCA) is considered experimental, investigational and unproven for workup and monitoring of patients with inflammatory bowel disease (IBD).
Fecal measurement of neutrophil-derived proteins (i.e., Calprotectin [Cal], Lactoferrin [Lf], PMN-Elastase (PMN-e and Lysozyme [Lys]), for the diagnosis and management of IBD, Crohn’s disease (CD), and ulcerative colitis (UC) are considered experimental, investigational and unproven.
CPT codes 83516 and 88347 are not specific to ANCA, ASCA, Calprotectin, Lactoferrin, PMN-e and Lysozyme. 83993 is specific to Calprotectin, 83630 and 83631 are specific to lactoferrin.
A serology panel including anti-neutrophil cytoplasmic antibodies (ANCA), anti-Saccharomyces cerevisae IgG and IgA antibodies (ASCA), and anti-OmpC antibodies (outer membrane porin from E. coli) are marketed by Prometheus Laboratories as the IBD First Step. This panel has not been shown to have levels of specificity sufficient to distinguish ulcerative colitis from CD in indeterminate cases.
Research into the pathogenesis of inflammatory bowel disease in the areas of mucosal immunology, genetics, the role of bacterial products, and mediators of tissue damage has identified new sets of “subclinical” serological markers known as ANCA. ANCA have also been found to be associated with Wegener's granulomatosis and other forms of systemic vasculitides, and more recently with sclerosing cholangitis and other autoimmune liver diseases.
“Atypical” ANCA yielding a perinuclear staining pattern (pANCA) with alcohol-fixed neutrophils is primarily found in patients with ulcerative colitis; pANCA has been found to be detectable in 50-80% of patients with ulcerative colitis, and 10-40% of patients with Crohn's disease. Anti-Saccharomyces cerevisae antibody (ASCA) is primarily detected in patients with Crohn's disease; ASCA has been found to be detectable in 46% to 70% of patients with Crohn's disease and 6-12% of patients with ulcerative colitis.
These tests, however, have insufficient sensitivity to diagnose ulcerative colitis or Crohn's disease. In a paper for the North American Society for Pediatric Gastroenterology and Nutrition, Griffith's concluded that “the relatively low sensitivities of serology for Crohn's disease and ulcerative colitis as documented in all studies argue against there being any greater value of ASCA/ANCA as routine or first-line screening tests for inflammatory bowel disease in comparison to clinical acumen and the equally sensitive measurement of acute phase reactants. Moreover, the need for performance of definitive radiologic and endoscopic studies to guide therapy by defining the extent and nature of IBD will not be averted by positive serologic tests.” Gupta, et al. examined the concordance of serologic testing for inflammatory bowel disease with clinical diagnosis established by traditional testing in children. The investigators found that the sensitivity of serologic testing is insufficient to replace traditional studies when evaluating children for inflammatory bowel disease. The investigators evaluated the results of ANCA and ASCA testing in 107 children who had serologic testing for inflammatory bowel disease at their center, and compared these results with their clinical diagnosis. The investigators calculated that the sensitivity, specificity, positive and negative predictive values of serologic testing for UC were 69.2%, 95.1%, 90.0% and 87.1%, respectively, and for CD were 54.1%, 96.8%, 90.9% and 80.8% respectively. The investigators concluded that “the low sensitivity, especially for CD, precludes the possibility that the IBD Diagnostic System can replace traditional studies when evaluating for inflammatory bowel disease.”
Some investigators have proposed using these serologic tests to differentiate Crohn's disease from ulcerative colitis (Kornbluth & Sachar, 2004). Differentiation of Crohn's disease from ulcerative colitis is clinically problematic only when inflammation is confined to the colon. A number of studies have reported that IgA and IgG ASCA titers are significantly greater and highly specific for CD, and that pANCA positivity is highly specific for ulcerative colitis. However, there is much less published information concerning the subgroup of IBD patients with colitis only, where differentiation of ulcerative colitis from Crohn's disease is clinically problematic. One investigator reported ASCA positivity in only 47% of 17 patients with Crohn's colitis. Another investigator found only 32% of 37 patients with Crohn's colitis were ASCA positive and pANCA negative. Conversely, studies have found that the majority of Crohn's patients positive for pANCA have ulcerative colitis-like presentations. Griffiths explained, “hence, the usefulness of serology is less (where it is needed most), given the higher prevalence of pANCA positivity and the lower prevalence of ASCA positivity in CD confined to the colon.”
“Similarly, whether or not ASCA/ANCA measurement may be helpful in classifying otherwise 'indeterminate' colitis cannot as yet be ascertained. Only a few patients have been studied, and follow-up is too limited.” In the only prospective study of serologic testing in indeterminate colitis, Joossens et al. examined the results of serologic testing for ASCA or ANCA and final diagnosis of Crohn's disease or ulcerative colitis after a six-year follow-up of 97 patients with indeterminate colitis. The largest group of 47 subjects was negative for both ANCA and ASCA; three of these patients had a final diagnosis of Crohn's disease, four patients had a final diagnosis of ulcerative colitis and 40 patients had a final diagnosis of indeterminate colitis. Of 26 subjects who were ASCA positive and ANCA negative, eight had a final diagnosis of Crohn's disease after six years follow up, two had a final diagnosis of ulcerative colitis, and 16 remained with a diagnosis of indeterminate colitis. Of 20 subjects who were ASCA negative and ASCA positive, four had a final diagnosis of Crohn's disease, seven had a final diagnosis of ulcerative colitis, and nine had a final diagnosis of indeterminate colitis. Only four of the 97 subjects were positive for both ANCA and ASCA; two of these had a final diagnosis of Crohn's disease, one had a final diagnosis of ulcerative colitis, and one remained with a final diagnosis of indeterminate colitis. Thus, about one-third (31%) of subjects who were ASCA positive and ANCA negative progressed to Crohn's disease during the six-year follow-up period, and about one third (35%) of subjects who were ASCA negative and ANCA positive progressed to ulcerative colitis during the six-year follow-up period. Joossens, et al. (2002) calculated that, thus far, the sensitivity of ASCA+/ANCA- for Crohn's disease was 66.7% and the specificity is 77.8%, and the sensitivity of ASCA-/ASCA+ for ulcerative colitis is 77.8% and the specificity is 66.7%. Noting that these calculations exclude subjects who remained with the diagnosis of indeterminate colitis, a technology assessment of serologic testing for inflammatory bowel disease by the Institute for Clinical Systems Improvement noted that only a “small number” (21) of subjects were included in this analysis of sensitivity and specificity. Based on their analysis of this prospective study and other published studies of serologic testing in indeterminate colitis, the Institute for Clinical Systems Improvement (ICSI) concluded that the clinical utility of serologic testing in indeterminate colitis has not been established. It has also been noted that this study does not provide direct evidence of improvement in clinical outcomes by basing the management of persons with indeterminate colitis on serologic testing.
ANCA and ASCA testing has not been proven to be useful in selecting therapeutic interventions. “Although this would be desirable, there is no evidence as yet that serological test results can be used to predict the likelihood of therapeutic response to specific interventions,” Griffiths explained. In a prospective clinical study of Crohn's disease patients, Esters,et al. (2002) found no significant relationship between these serologic markers and response to anti-tumor necrosis factor (TNF) therapy.
In addition, studies have not demonstrated correlation of ANCA or ASCA with disease activity, duration of illness, extent of disease, extra-intestinal complications, or surgical or medical treatment in patients with IBD. The Institute for Clinical Systems Improvement (ICSI) technology assessment of serologic testing for IBD concluded “the clinical utility of serological testing is not yet established for the diagnosis of inflammatory bowel disease in patients presenting with symptoms suggestive of IBD” and “the clinical utility of serological testing is not yet established for differentiating between UC and CD in patients with inflammatory bowel disease”.
Additional assays have been developed to use in conjunction with ANCA and ASCA in an effort to improve the diagnostic capabilities of serologic testing. OmpC IgA is an autoantibody to outer membrane porin to Escherichia coli (E. coli) included in the Prometheus serology panel to enhance detection of Crohn's disease (Landers et al). I2 is an IgA antibody that has been detected in patients with Crohn's disease. The I2 serologic response recognizes a novel homologue of the bacterial transcription-factor families from a Pseudomonas fluoresceins associated sequence (Sandborn 2004). However, there are no studies of the clinical utility of anti-OmpC and I2 IgA antibodies in distinguishing CD from UC colitis in persons with inflammatory bowel disease whose diagnosis cannot be established by standard methods.
Landers et al. (2002) reported on serologic test results of a referral center population of 151 patients with Crohn's disease. This study found that immune responses to specific antigens (ASCA, pANCA, OmpC, and I2) are not uniform among Crohn's disease patients. ASCA was detected in 56% of patients, OmpC in 55% of patients, I2 in 50% of patients, and ANCA in 23% of patients. The investigators reported that 85% of patients responded to at least one antigen and that only 4% responded to all four. This study did not demonstrate, however, how these serologic test results relate to clinical behavior and response to therapy. The authors stated that “the relationship of these different patterns of immune responses to clinical behavior is not yet clear.” The authors concluded “defining how these antibody re-activities relate to clinical behavior and response to therapeutic modalities will require larger numbers of phenotypically well-characterized patients.”
Mow et al. (2004) reported on the results of a hypothesis generating study, the aim of which was to determine whether Crohn's patients with predominant serum antibody reactivity toward bacterial antigens OmpC and/or I2 were more likely to achieve remission with antibiotics. Study subjects were patients with moderately active right-sided colonic and/or small bowel Crohn's disease who were participating in an 8-week randomized clinical trial comparing the steroid budesonide with or without the antibiotics metronidazole and ciprofloxacin. Subject's serum was analyzed for ASCA, pANCA, anti-OmpC, and anti-I2 antibodies, and subjects were put into one of four “profile groups” (ASCA, pANCA, anti-OmpC/I2, and no or little antibody) depending upon the subjects' levels of antibody response. Twenty-five of 121 subjects were excluded from the analysis because their level of antibody response did not fit the four predominant profile groups. Only two subjects had an ANCA predominant profile, and these subjects were excluded from the analysis. In the steroid plus antibiotic group, 5 of 11 subjects (45.5%) with predominant OmpC and I2 antibodies achieved remission, 5 of 16 subjects with predominantly ASCA antibody (31.3%) achieved remission (similar to the overall remission rate), and 5 of 21 subjects (23.8%) with little or no antibodies achieved remission. In the steroid only group, 7 of 16 subjects (43.8%) with little or no antibodies achieved remission, 8 of 20 (40%) with predominantly ASCA antibody achieved remission, and 3 of 10 (30%) subjects with predominant OmpC and I2 antibodies achieved remission. Although there was a trend toward greater responsiveness to therapy that includes antibiotics in subjects with predominant OmpC and I2 antibodies, and a trend toward less responsiveness in subjects with little or no antibodies, these trends did not achieve statistical significance. The authors concluded that this hypothesis-generating study provides preliminary evidence to suggest that serologic information about Crohn's disease patients may be helpful in defining patients who would best respond to therapy. The authors noted, however, that, “although these trends are provocative, they lack statistical significance.” The authors concluded that “prospective randomized placebo controlled trials that do not limit patient selection by disease location and do not have concomitant therapy are warranted to test this hypothesis.”
Mow et al. (2004) evaluated the sera of 303 patients with Crohn's disease to determine whether expression of certain antibodies is associated with phenotypic manifestations. The investigators found that patients expressing I2 were significantly more likely to have fibrostenosing Crohn's disease (64.4% versus 40.7%), and to require small bowel surgery (62.2% versus 37.4%). Patients with anti-OmpC were more likely to have internal perforating disease (50% versus 30.7%) and to require small bowel surgery (61.4% versus 44.2%). The investigators stated that these findings suggest an association between these immune responses and Crohn's disease complications. The investigators concluded that “in the future, knowledge of serological response may help the clinician determine the risk for more severe disease characteristics and predict disease behaviors. As a result, it may be possible to tailor therapy more effectively on the basis of specific serological responses. However, these findings must be confirmed by prospective studies that evaluate the presence of these antibody responses and the development of complicated small bowel disease phenotypes.” An editorial accompanying this study explained that this study is limited by its retrospective nature (Vermeire & Rutgeerts, 2004): “Therefore, it is important that these findings first be confirmed in independent series and more importantly, that prospective studies with these markers be conducted to assess the risk of microbial responses on the development of strictures and perforations and subsequent need for surgery.”
Calprotectin is a calcium- and zinc-binding protein of the S100 family derived mainly from neutrophils and monocytes. It is excreted in excess in stools during IBD. Fecal calprotectin level has been reported to parallel intestinal inflammation and can predict relapse of ulcerative colitis (Hanai et al., 2004).
Fecal measurement of calprotectin is emerging as a tool for the differential diagnosis of inflammatory (e.g., Crohn's disease, ulcerative colitis) from non-inflammatory gastrointestinal disease (e.g., IBS), for monitoring patients' response to therapy and for predicting recurrence of IBD.
Berni et al (2004) examined fecal calprotectin values in different pediatric gastrointestinal diseases (n = 281; age ranging from 13 to 216 months) comparing them with those obtained in healthy children (n = 76; age ranging from 13 to 209 months). These investigators concluded that fecal calprotectin is a sensitive, but not disease specific, marker to easily detect inflammation throughout the whole gastrointestinal tract. It may help in identifying an organic disease characterized by intestinal mucosa inflammation and in the differential diagnosis of functional bowel disorders. Wassell et al (2004) posited that a single measurement of fecal calprotectin may help gastroenterologists in the differential diagnosis of CD and IBS.
Tibble et al (2000) examined if measurement of intestinal permeability and inflammation could predict relapse of IBD. Forty-three patients with CD and 37 with UC in clinical remission provided a stool sample to be assayed for calprotectin and patients with CD additionally underwent a small intestinal permeability test. Relapse was defined using clinical disease activity indices. Twenty-five (58 %) patients with CD and 19 (51 %) with UC had a relapse over the 12-month period. Median calprotectin levels in the relapse groups (122 mg/L for CD, 123 mg/L for UC; normal < 10 mg/L) differed significantly (p < 0.0001) from those of the non-relapse groups (41.5 mg/L for CD, 29.0 mg/L for UC). At 50 mg/L, the sensitivity and specificity of calprotectin for predicting relapse in all patients with IBD were 90 % and 83 %, respectively. Permeability in the CD patients who relapsed (median, 0.075; normal < 0.04) differed significantly (p = 0. 004) from that in the non-relapse group (median, 0.038). At the level of 0.05, the sensitivity and specificity of permeability in predicting relapse were 84 % and 61 %, respectively. The authors concluded that fecal calprotectin may be useful in predicting clinical relapse of disease activity in patients with CD and UC, whereas small intestinal permeability may be a useful predictor of relapse in patients with small intestinal CD.
Although the study by Tibble et al (2000) suggested that high fecal calprotectin levels may identify IBD patients in remission who are at risk for early relapse, there are reports that there may be differences in relapse prediction in patients with CD compared to those with UC.
Costa et al (2005) examined if the predictive value of fecal calprotectin is different in CD and UC. Seventy-nine consecutive patients with a diagnosis of clinically quiescent IBD (38 CD and 41 UC) were followed for 12 months, undergoing regular clinical evaluations and blood tests. A single stool sample was collected at the beginning of the study from each patient and the calprotectin concentration was measured by a commercially available enzyme linked immunoassay. In CD, median calprotectin values were 220.1 microg/g (micrograms per gram) of stool in those patients who relapsed during follow-up, and 220.5 microg/g in non-relapsing patients (p = 0.395). In UC, median calprotectin values were 220.6 microg/g and 67 microg/g in relapsing and non-relapsing patients, respectively (p < 0.0001). The multivariate Cox regression model, after adjustment for possible confounding variables, showed a 2- and 14-fold increase in the relapse risk, respectively, in those patients with CD and UC in clinical remission who had a fecal calprotectin concentration higher than 150 microg/g. The authors concluded that fecal calprotectin proved to be an even stronger predictor of clinical relapse in UC than in CD, which makes the test a promising non-invasive tool for monitoring and optimizing therapy.
In a commentary on the role of biomarkers for predicting relapse in patients with IBD, Pardi and Sandborn (2005) stated that based on the studies of Tibble et al (2000) as well as Costa et al (2005), fecal calprotectin appears to be a relatively sensitive and specific marker of the risk of relapse for UC. It also appears to be a sensitive marker of relapse risk in CD but the data on specificity are conflicting at this juncture. However, these data need to be interpreted carefully since the number of studies is small, and in both studies using calprotectin to predict relapse risk, most patients were on medical therapy. Calprotectin may behave differently in patients who are not on therapy. Thus, before fecal calprotectin or any other biomarker of inflammatory activity in the gastrointestinal tract can be incorporated into routine clinical practice, other studies in larger and diverse groups of patients will be needed to further clarify its role.
In the guidelines for chronic diarrhea compiled at the request of the British Society of Gastroenterology, Thomas et al (2003) stated that stool markers of gastrointestinal inflammation such as calprotectin are of considerable research interest. However, these tests have not been introduced into clinical practice. Moreover, in a review on the diagnostic and therapeutic strategies in the IBD, Cremonini and Talley (2004) stated that the usefulness of fecal tests such as calprotectin to exclude organic bowel disease is not adequately established. Furthermore, the use of calprotectin is not mentioned in the practice parameters on the management of Crohn's disease in adults by the American College of Gastroenterology (Hanauer et al 2001) and the practice guideline on the management of ulcerative colitis by the Society of Surgery of the Alimentary Tract (2001).
Several articles attempted to correlate titers of ANCA and/or ASCA with disease activity, but did not generally find such a correlation. Mow and colleagues investigated whether serologic antibodies were associated with disease complications. In this case series of 303 patients with Crohn’s disease, certain antibodies were associated with fibrostenosis or perforating disease. However, it is unclear how this information would be used in the management of the patient. Other studies evaluated the presence of serum markers in unaffected relatives of patients with IBD, reporting positive results in approximately 25%–50% of family members. However, these studies did not report on the incidence of IBD in these relatives with positive antibodies. Two additional antibodies have been also been studied, E.coli anti-OmpC and I2 antibody. However, the same limitations in the published literature apply to these antibodies.
No studies demonstrated the use of these markers in lieu of a standard workup for IBD. A number of authors claim that these markers can be used to avoid invasive testing, but no studies demonstrated an actual decrease in the number of invasive tests through use of serum markers.
Schoepfer and colleagues studied the results of various testing in 64 patients to compare the accuracy of fecal markers (i.e., PhiCal Test, IBD-SCAN) , C-reactive protein, blood leukocytes, and antibody panels (ASCA and pANCA) for discriminating IBD from IBS and to define a "best test." The authors concluded PhiCal Test and IBD-SCAN are highly accurate for discriminating IBD from IBS. Additional diagnostic accuracy is only marginal when the PhiCal Test and IBD-SCAN are combined with ASCA and pANCA. ASCA and pANCA have a high specificity for IBD; however, they should not be primarily measured for discriminating IBD from irritable bowel syndrome as their additional value to fecal leukocyte markers in this issue is only marginal.
A review article discussed the expansion of the panel of serologic markers for IBD. An increasing amount of data are available on newly discovered antibodies (i.e., Anti-OmpC, Anti-12, Anti-CBir1, and antiglycan antibodies) directed against various microbial antigens. However, ASCA and P-ANCA remain the most widely investigated. The authors noted that the role of the assessment of various antibodies in the current IBD diagnostic algorithm is often questionable due to limited sensitivity. They concluded that further prospective clinical studies are needed to establish the clinical role of serologic tests in IBD.
A literature search through September 2010 did not identify any high quality clinical trials regarding the clinical utility of fecal testing of neutrophil-derived proteins, such as calprotectin and lactoferrin. These technologies are considered investigational for the diagnosis and monitoring of Crohn’s disease and inflammatory bowel disease. There is insufficient evidence in the scientific literature that these tests are effective in evaluating impact on net health outcome.
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