The matter of methodology
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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