[Histonet] RE: Histonet Digest, Vol 77, Issue 22

Thu Apr 22 10:45:29 CDT 2010

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Subject: Histonet Digest, Vol 77, Issue 22

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Today's Topics:

   1. Re: hematoxylin-eosin saffron (Robert Richmond)
   2. Phospho antibodies and fixation (Jean-Martin Lapointe)

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Message: 1
Date: Sat, 17 Apr 2010 13:27:21 -0400
From: Robert Richmond <rsrichmond <@t> gmail.com>
Subject: [Histonet] Re: hematoxylin-eosin saffron
To: histonet <@t> lists.utsouthwestern.edu
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	<g2yabea52a61004171027g1bfd628ezbc527449dc92b576 <@t> mail.gmail.com>
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The hematoxylin-phloxine-saffron (HPS) stain is a trichrome stain that
came into use as a "general oversight" stain by surgical pathologists
in the 1930's, notably at Columbia-Presbyterian Hospital in New York
City. The Goldner-Foot trichrome stain was also used for this purpose,
on the other side of Central Park at Cornell Medical Center, probably
abandoned after Chandler Foot's retirement in 1948.

I actually saw the HPS stain in use at Columbia-Presbyterian around
1966, in place of H & E. I understand that it is still in very limited
use in a few laboratories today.

Saffron (a natural product, the stigmas of the flowers of Crocus
sativus) is extremely expensive - currently $66 for a quarter ounce
(about 7 grams) at Penzeys.com. It's used histologically as an
alcoholic extract (supposedly best done with a reflux condenser) and
can actually be purchased in that form.

You can find the HPS staining procedure in older books, such as the AFIP
manual.

As a working surgical pathologist, I find this ancient technique
intriguing but totally impractical in today's world. It's stretching
it to get a trichrome stain for your liver biopsies these days.

Bob Richmond
Samurai Pathologist
Knoxville TN

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Message: 2
Date: Sat, 17 Apr 2010 16:47:48 -0400
From: "Jean-Martin Lapointe" <jm.lapointe <@t> accellab.com>
Subject: [Histonet] Phospho antibodies and fixation
To: <histonet <@t> lists.utsouthwestern.edu>
Message-ID: <BEFD613BD39142499989F836556DDC83C43F3E <@t> ACE.accellab.lan>
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Hi Ashley,
I did a little bit of work on this a few years back. My belief is that
the main difficulty with IHC for phospho-epitopes is not formalin
fixation per se, but rather the instability of the phosphorylation. I
used to work with rodent tumor models, where we collected solid tumors
(or other tissues)  just after sacrifice, and fixed them by formalin
immersion. We found that the periphery of the tissues fixed this way
expressed various phospho-epitopes quite well, but not the center. My
interpretation was that by the time formalin had penetrated to the deep
regions of the tissue, the phosphorylation had gone away (as you know
formalin penetrates solid tissues fairly slowly). We proceeded to test
matched tissue samples, fixed either in formalin at room temperature vs.
cold formalin (formalin was at 4C at immersion, and the tissue was put
in the fridge after sampling). We found that the cold formalin samples
expressed phosphorylation sites fairly evenly, compared to only
peripheral with the room temp samples. Presumably the cold temperature
preserves the phosphorylation sites long enough to leave time for
formalin to penetrate throughout. Consequently we integrated cold
formalin fixation to all our IHC protocols for phospho-epitopes.
Hope this helps,

Jean-Martin Lapointe
Accellab

-----Original Message-----

Good day colleagues,

Does anyone have any information on phospho antibodies and fixation?  Is
there any reason to NOT fix specimens in formalin vs say, 70% EtOH? (and
then process them through formalin later?)  I can't seem to find any
information on whether or not fixation in formalin does something
strange to
a phosphorylated protien and makes it a less accessible antigen.  Also,
on
that same note, does retreival do anything to it?

I am assuming that these antibodies go through the same testing for
cross
reactivity, etc (depending on the vendor) and are reliable when used
properly (like any other antibody).  I don't, however, know if there is
enough of a difference in the epitope (the fact that it is
phosphorylated)
that would make it more susceptible to some strange cross linking with
formalin (especiallly with phosphate buffered formalin).

Any help with this topic would be greatly appreciated as I am uneducated
in
this area.

Thanks,

Ashley Troutman BS, HT(ASCP) QIHC
Vanderbilt University Histopathology
1301 Medical Center Drive TVC 4532
Nashville, TN  37232
615-343-9134
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End of Histonet Digest, Vol 77, Issue 21
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