College of American Pathologists
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The matter of methodology

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August 2000
William Check, PhD

Some of the controversy about the value of serologic tests in inflammatory bowel disease focuses on methodology. Says Dr. Jonathan Braun, chairman of pathology and laboratory medicine at UCLA and a co-founder of Prometheus, which offers testing for pANCA and ASCA, "The definition and titer of pANCA differs among laboratories. We have made an effort to define the technique that best distinguishes the ulcerative colitis type of autoantibody from other antibodies in the ANCA family."

Prometheus uses a three-step approach for pANCA. Reactivity of serum against fixed neutrophils in an ELISA, a high-sensitivity procedure, is followed by immunofluorescence staining on ANCA-ELISA-positive samples, a more specific procedure. Specificity is confirmed by disappearance of staining after DNase treatment of neutrophils.

For the ASCA assay, too, there are technical issues. "At Prometheus," Dr. Braun says, "the method for purifying polysaccharide and the measurement of both IgG and IgA binding activity distinguish this test from what would be offered by most other laboratories."

Dr. Charles Elson, director of the division of gastroenterology and hepatology at the University of Alabama, Birmingham, has obtained intriguing data about the Prometheus technique for pANCA staining, in which antibody detects pANCA on human neutrophils only if the neutrophils are fixed with methanol on a slide. "It has always been mysterious as to why you would have reactivity to something you don’t have in the body," Dr. Elson says. He considered the possibility of cross-reactivity, in which methanol changes some neutrophil antigen so that it looks like a bacterial antigen.

Dr. Elson and his colleagues were able to absorb pANCA reactivity from human serum using bacteria from the gut of a mouse with an experimental model of colitis. Whether these results are reproducible and what they imply about the meaning of the pANCA test and the etiology of ulcerative colitis are under investigation.

William Check, PhD