ethanol/xylene processing

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From:"Wayne D H" <>
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Mark Schrenzel wrote asking for an alternate fixative for DNA/RNA.

For six years we have been using Kerry Beebe's Ethanol/Xylene processing technique on our VIP 2000 processor ( see Journal of Histotechnology, March 2000, page 45). The application to Mark's question is that we frequently do the gross dissection on breast biopsies sent for frozen section and put them in the "70/30" alcohol/xylene without the tissue ever touching formalin. We have never have a block come out the processor incompletely processed or infiltrated with paraffin. They cut beautifully.

Switching from the standard formalin/ graded alcohols processing to "70/30" alcohol/xylene  (7 parts 100% alcohol/ 3 parts xylene) eliminated a number of processing and cutting problems in our lab. We never have an "unfixed" block come out of the processor. Gone are the days of blocks where the edges cut and the center smudges into mush. Sound familiar? The problem
isn't fixation, it's incomplete dehydration. The larger, denser pieces of tissue need more time in alcohol to remove the water than is available in most processing schedules. If the water doesn't come out, the wax can't go in. With "70/30" processing the tissues have six hours ( 1 hour in each of six stations) to completely dehydrate and be completely fixed by the alcohol, if they weren't previously fixed. It would be possible to reduce the times for biopsies, but we only have one processor.

The xylene in the "70/30" eliminates the shrinkage and hardening one would expect with that much time in alcohol. As well it removes the fat from the "greasy" stuff like breast, brain, and bowel permitting complete processing and infiltration. These blocks are a breeze to cut, even brain and spinal cord; anyone with basic skills can cut them. In fact we often put brain in
with other tissue like liver, kidney or heart in the same cassette (autopsy tissues) and have NO problem cutting it.

We have not found a single negative thing about "70/30" after six years. Kelowna General Hospital, where it was developed, has used it for over fifteen years. You can call them at (250) 862-4000 ext 7510, ask for Kathy Schaeffer ( Kerri just retired for those who noticed the "unsubscribe" from three weeks ago-(( not as it says in the article in
the Journal)) but I'm sure he wouldn't mind answering questions if you contact him directly, just remember to tell the rest what you find out).
Enough already! I could spout off about the virtues of this stuff for hours. Read the article in the Journal and:

                           TRY IT, YOU'LL LOVE IT!!!

p.s. We use 10% nbf  for fixation, tried the BB's fixative mentioned in the article but the pathologist didn't like it, penetration was too slow if I remember correctly. 

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

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