RE: Paraffin temp for immuno's

From:"Bartlett, Jeanine"

Before I came to work at CDC I worked in a small lab and we sent immunos to
an outside lab.  They always asked that I send the slides "unmelted" because
they said often labs would overheat them.  They told me anything over 55°C
was too hot.  Funny thing is that here at CDC we routinely heat at 60=C2C and
things work just fine

Go figure.

Jeanine Bartlett, HT(ASCP)
Centers for Disease Control
Infectious Disease Pathology Activity
1600 Clifton Rd., N.E.  MS-G32
Atlanta, GA  30333 

-----Original Message-----
From: Ross Stapf []
Sent: Thursday, October 17, 2002 12:05 PM
Subject: Re: Paraffin temp for immuno's

Thank you to those who have replied so far.  

The reason for this post is that in the troubleshooting section of Dabbs'
Diagnostic Immunohistochemistry it mentions that if the control stains
positive and a patient who is expected to stain positive does not stain
positive, then one of the possibilities is that the paraffin is over 56

While this is not very common, we do about 100 immuno's a day and we have 1
or 2 cases like this every month that the Pathologists have us repeat the
stains over and over on with no luck.  While most are content that this is
just a freak thing with certain patients, they are not content in less
everything works like they think it should work.

I know that our main immuno pathologist will eventually see this comment in
this book and I want to get more information prior to getting pressured from
him.  Lately perfecting every detail of Immuno quality has been a big push.

Ross Stapf 
Histology Supervisor
Washington Adventist Hospital

>>> "Vinnie Della Speranza"  10/17/02 11:12AM >>>
it is always dangerous to generalize especially because we use ihc to
stain for a wide variety of cellular and tissue antigens and something
is always likely to be the exception however I'm going to risk it.=20
The rule of thumb is to try to prevent overheating of tissues during
exposure to processing chemicals. There are certainly proponents of low
metling point paraffins but we have a very successful
immunohistochemistry laboratory that routinely utilizes a 58 degrees
metling point paraffin. I do not recommend that you change paraffin to
resolve your quality issues as I personally don't believe that this is
likely to make the difference. Lastly I would add that in my opinion, if
your tissues are well fixed, most if not all antigens will survive
exposure to 60 degrees. Its interesting to note that we frequently
expose tissues to very high heat (90-100 degrees C) to achieve epitope
retrieval which seems to contradict this notion that temperatures must
be kept as low as possible.
Vinnie Della Speranza
Manager for Anatomic Pathology Services
Medical University of South Carolina
165 Ashley Avenue  Suite 309
Charleston, SC 29425
Ph: 843-792-6353
fax: 843-792-8974

>>> Ross Stapf  10/16/02 04:04PM >>>
Hi everyone:

I was reading through Diagnostic Immunohistochemistry by Dabbs and came
across a comment that paraffin temperatures should not exceed 56 degrees
when doing immuno's on the tissue later.

We have been trying to standardize our methods for optimal immuno
results.  We have the occasional case where the staining is not as
expected.  We are trying to eliminate as many of these problems as
possible.  Is the paraffin temp really that important?  I am beginning
to wonder if this is one of the reasons for those cases that should, but
just don't stain positive. 

  We have been using Paraplast and Paraplast xtra for over 10 years at
60 degrees.  If I do experiment with a paraffin with a lower melting
point, what can I expect?  Will my techs have a harder time cutting the
blocks?  Will I need to increase my infiltration times?

Turn around time is very important, I don't want techs complaining that
they can't get good routine sections just to possibly fix an immuno
variable.  Also from my research so far the lower melting point paraffin
is more expensive.

Basically has anybody made a change in paraffin for this reason, and
was it worth it?

Ross Stapf
Histology Supervisor
Washington Adventist Hospital
Takoma Park MD

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