"70/30" alcohol/xylene processing

From:Wayne D H <whohn@lycos.com>

I've had a number of requests for information on this, so I'll answer everyone at once.
     "70/30" alcohol/xylene processing was developed by Kerry Beebe, A.R.T., at Kelowna General Hospital, Kelowna, B.C., Canada about 15 years ago. His article was published by the N.S.H. in the March 2000 Journal of Histotechnology (page 45).
     The 70/30 is 7 parts of reagent alcohol (90% ethanol, 5% methanol, 5% isopropanol) and 3 parts xylene. I don't know if Kerry tried any xylene substitutes when he developed the technique, but they use xylene now.
     Kelowna now has a fairly new VIP and I have VIP 2000 about 11 years old.
     I'm using the published method with 2 exceptions. Our prosecting schedule is irregular, so our delay is station 1. The other difference that I use the pressure\vaccuum cycle on the VIP and have no problem with tissue disruption or other artifact. The only problem I have is boiling away a bit of the reagent under vaccuum, so it is necessary to change the activated charcoal in the filter fairly often. I just moved into a new well ventilated lab with the processor in a seperate room and in a custom designed hood so the problem of xylene fumes is gone.

     The published method is:

Station    Solution     Time(min)   Temp    Vacuum 
1          70/30        60          ambient off
2          70/30        60          "       off
3          70/30        60          "       off
4          70/30        60          "       off
5          70/30        60          "       off
6          70/30        delay       "       off
7          xylene       30          "       off
8          xylene       60          "       off
9          xylene       90          "       off
10         xylene       30          45      off
11         wax          30          60      5 mmHg
12         wax          60          60      10mmHg
13         wax          90          60      15mmHg
14         wax          30          60      off    

     We only have one processor so all surgical specimens, from the smallest to the largest, get the same treatment. The small biopsies are overprocessed,
so I have to soak on ice water to soften them prior to cutting.
    We tried BB's fixative but the pathologist didn't like it, so we use 10% NBF.
    I'll try to answer any questions, but don't be afraid to try this, it really is great. No more half processed specimens and much easier cutting.

Wayne Hohn
Kootenay Lake Reg. Hosp.
Nelson, B.C.     

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