[Histonet] p16 Pap Smears on Ventana Benchmark XT

[Gagnon, Eric]

Hi Fellow Histonetters,
 
We are currently attempting p16 immunostaining on Pap smears which have been spray-fixed with a 95% ethanol spray fixative then run on the Ventana Benchmark XT.  I should add that the slides are post-fixed in formalin, and run with cell conditioning (antigen retrieval). This protocol has worked well for most slides so far.  There are some cases in which there are some positive cells staining, but it appears that other cells in other locations on the slide show cells that should likely stain, but aren't.  
 
Eventually, ThinPreps will be used in place of Pap smears, and would provide a more uniform preparation.    For now, we are going to be attempting to add more antisera to each slide, to try to eliminate possible non-spreading of antisera on the slide.  Our other markers, including p16 on paraffin sections are working fine, so we have ruled out mechanical/instrument-related factors. Has anyone else experienced problems with (notoriously irregular) Pap smears for p16, and what worked for you?
 
Thanks for your assistance,
 
Eric Gagnon, MLT
Histology Laboratory
Kingston General Hospital
Kingston, Ontario, Canada