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Correct diagnosis of inflammatory bowel disease (IBD), especially the differentiation between Crohn's disease (CD) and ulcerative colitis (UC), is highly important toward treatment and prognosis. Serological markers are noninvasive diagnostic tools that could be of value in differentiating CD from UC, in cases of indeterminate colitis, and in the identification of subgroups in IBD. The aim of this study was to evaluate the diagnostic accuracy of perinuclear antineutrophil cytoplasmic (pANCA) and anti-Saccharomyces cerevisiae antibodies (ASCA) for IBD.ASCA and pANCA were studied in a large cohort of consecutive IBD patients (n = 582: 407 CD, 147 UC, and 28 indeterminate colitis), patients with non-IBD diarrheal illnesses (n = 74), and healthy controls (n = 157). An indirect immunofluorescence technique and a standardized ELISA were performed for detection of pANCA and ASCA, respectively.Prevalence of ASCA and pANCA was high in CD patients (59.7%) and UC (49.7%) patients, respectively. Positivity for both markers was significantly lower in healthy and non-IBD controls. Accuracy data (sensitivity, specificity, PPV, and NPV, respectively) for differentiating IBD from controls are as follows: ASCA+: 60% (243/407), 91% (345/378), 88% (243/276), and 68% (345/509); pANCA+: 50% (73/147), 95% (605/638), 69% (73/106), and 89% (605/679); ASCA+/pANCA−: 56% (229/407), 94% (355/378), 91% (229/252), and 67% (355/533); and pANCA+/ASCA−: 44% (65/147), 97% (620/638), 78% (65/83), and 88% (620/702).Specificity of serological markers for IBD is high, but low sensitivity makes them less useful as diagnostic tests. The combination of tests is probably more powerful, although, clinical subgroups still need to be defined. The usefulness of these markers in indeterminate colitis needs to be studied prospectively.