Standard brush cytology sampling is performed on patients undergoing endoscopic retrograde cholangiopancreatography (ERCP) for suspicious biliary tract strictures. Brushes are placed in a ThinPrep vial containing PreservCyt or CytoLyt solution. The specimen is sent in a single vial with or without the brush. If brush is present, it is removed and cells are collected from it by scraping them into a single vial containing 20 mL of PreservCyt solution. Two aliquots are prepared and used for each portion of the test. The cytology specimen is processed using the ThinPrep 2000 processor. Specimens are stained using a Papanicolaou stain and analyzed microscopically by a cytotechnologist and pathologist.
Biliary cells are harvested, fixed, and placed on a slide. Fluorescently labeled DNA probes to 1q21 (MCL1), 7p12 (EGFR), 8q24 (MYC), and 9p21 (CDKN2A) (Abbott Molecular, Inc, Des Plaines, IL) are hybridized to the cells on the slide. The slide is then washed and stained with DAPI (a nuclear counterstain). Fluorescence microscopy with unique band filters is used to assess 100 consecutive epithelial cells for gains and losses of probe signals (ie, chromosomal loci). Specimens are considered abnormal if cell counts exceed predetermined cutoff values for one or more of the following abnormalities: polysomy, homozygous 9p21 loss, single locus gain, single locus gain with 9p21 loss in the same cells, and/or tetrasomy. If the cutoff for polysomy is not attained in the 100-cell enumeration, then the remainder of the slide is assessed for polysomy until the cutoff is reached or the slide is exhausted.(Unpublished Mayo method)