How to separate cells in urine for FISH? - (Oct/26/2007 )

We are doing FISH testing of urine using Abbott's Urovysion with computer assisted image analysis from BioView. We frequently get cohesive groups of cells, which may be true papillary clusters or sheets of exfoliated urinary epithelial cells. The BioView has difficulties with these, and a monolayer of individual cells would work much better. Can anyone suggest a protocol (as specific as possible) for disaggregating cells. I would assume a trypsin or Dispase treatment would be along the right lines, but have no experience doing this, and everything I can find on line relates to cells in cell culture, not in urine.

Thanks,

Lester J. Raff, MD

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The only thing I can think of is to spin the cells down out of the urine, wash in PBS, trypsinize. You can spin cells down at about 2000rpm for about 5-10 mins and have them stay alive. I've done this for cells that grow in suspension and tend to clump. The only thing is I had millions upon millions of cells so it was easy to see a cell pellet after spinning. I'm not sure if you have enough cells in the urine samples to begin with. Another idea, but I've never actually done this, is to filter the urine (gravity, not vacuum) so the cells remain on filter paper. Treat this with trypsin and recover the cells in a buffer of your choice.

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