Re: Microwave Processing

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From:"Gary R. Login" <> (by way of histonet)
To:histonet <>
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>We recently purchased a Energy Beam microwave for processing our
>STAT biopsies on our transplant specimens.   The current
>protocol is as follows:
>Zenkers (with zinc not mercury) minimum 1 hour
>100% ethyl alcohol 8 min at 67 degrees
>Isopropyl alcohol 8 min at 74 degrees
>Paraffin 3 min at 64 degrees, same container reset for 5 min at 82
>All reagents are replaced each run. Sections are cut at 2 microns and
>the hematoxylin time at 12 minutes (routine time is at 7). Has anyone in
>HistoNet land experienced a similar problem or used Zenker in the
>microwave?  We have had to discontinue the microwave processing on
>Zenker specimens and go back to processing on our VIP, with all times
>set a 5 minute. With this VIP procedure the sections look great, but it
>takes almost 2 hours (our customer need the results faster than that).
>Please help!

Pamela, thanks for your detailed description of your microwave and routine
procedures.  I have the following comments-

The microwave device and fixation steps are OK.

I think all of the artefacts you report seeing in your specimens are
related to problems with microwave heating.

1) I recommend that you do all steps of tissue processing in a flat tray
that has a solution depth of no more than 1 cm (microwaves do not penetrate
beyond 1 cm in most aqueous solutions). Solution volumes between 50 ml and
200 ml are OK if the solution tray is less than 1 cm deep.

2) Do not stack tissue cassettes on top of each other.  Most of the
microwave energy is absorbed the specimen in the top-most cassette.

3) I recommend heating your specimens to a final temperature no higher than
60 C.  It is OK to then use the microwave to hold the temperature at ~60 C.
In my experience, the endpoint temperature at 67-74 C that you are using
for the alcohol steps is too hot and is causing shrinkage of tissue,
melting of collagen (which has a transition temperature @~ 60 C), pyknosis
of nuclei, and ghosting of red blood cells.

4) I recommend using a broader ethanol gradient for dehydration (i.e., two
changes of 70% ethanol before absolute).  Microwave heating is only
speeding up diffusion of solutions into the specimens.  Therefore, by using
too abrupt a transition between dehydrating solutions artefacts will be
more pronounced in microwave processing than in room temperature
processing.  (Solid tissue has extracellular elements that support the
cells- therefore, it is not an unexpected result that the bone marrow
biopsies and endometrial currettings show more artefacts than the solid
tissue biopsies you process by the schedule you reported in your e-mail.)

5) Only after trying recommendations 1-4, would I recommend decreasing your
processing times to further reduce heat damage.

Following recommendations 1 and 2 will result in more reproducible
microwave heating of samples.
Following recommendations 3 and 4 will result in better tissue architecture.

The Microwave Tool Book has a worksheet for trouble shooting microwave
procedures and for calibrating the microwave oven.  Most likely, you can
get hold of the Microwave Tool Book from your hospital library, local NSH
chapter, Histology supplies distributor or me. Login GR, Dvorak AM. The
Microwave Toolbook.  A Practical Guide for Microscopists.   (Beth Israel
Hospital, Boston, 1994):pp. 184.

Please keep me informed

Gary R. Login, D.M.D., D.M.Sc.
Dept. Pathology
Beth Israel Deaconess Medical Center
330 Brookline Avenue
Boston, MA 02215

phone: 617-667-2034
fax: 617-667-8676


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