[Histonet] fixed and unfixed immuno and antuibody oreps

Jasper, Thomas G. TJasper <@t> smdc.org
Fri Sep 17 12:01:28 CDT 2004


I am sorry if I seem naive but I do understand your point about unfixed in
the first place, then not having to antigen retrieve to compensate for a new
problem which has been created.
Perhaps this is more a difference in application to achieve a goal.  It's
certainly possible in a purely academic setting that the unfixed approach
could produce the best and most direct results.  What has happened in
clinical settings is that IHC has been developed for use after tissue has
been fixed.  The fixation occurs out of necessity, not for the IHC, but for
the routine Hematoxylin and Eosin work-ups.  Many times what pathologists
see on H+E staining determines if IHC is necessary to diagnose a case.  At
gross examination, before the histological work-up, the pathologist may
already want to order some antibodies prior to seeing the H+E.
Nevertheless, fixation is necessary as it is a required step in the routine
work-up for tissue specimens in a case.  Keep in mind that time and cost
effectiveness are involved. If a case can be diagnosed on H+E only or on H+E
and special stains (non-IHC) it will be done.  Most certainly pathologists
were diagnosing cases on H+Es and limited special stains long before the
development of IHC.  Routine fixation for histological work-up is basically
a clinical standard of care.
There is another practical consideration with unfixed tissue.  To my
understanding that would require freezing everything that you were
interested in, or potentially interested in for IHC.  Freezing tissue
presents a set of unique problems on it's own.  There are things such as
freezing artifact and tissues that just do not freeze well (anything fatty).
There are also specimens that cannot be practically frozen due to
calcification.  Clinically, even with exceptionally good frozen technique,
most pathologists in many instances will defer to "permanents" once the
routine work-ups are done.  In many instances surgeons will get exclusionary
information or a limited list of possible "what-ifs" from a pathologist
until further information is available.
George, I would never claim that what we do in the clinical world is the
best or most perfect way to demonstrate antigenicity in tissue specimens.
What I will say is for the most part this is what we do and why is because
this is the best way that we know how.  Again, I by no means am expert in
the field of Immunohistochemistry and please allow me the disclaimer to be
corrected about anything that I have posted.  As I've said before there are
many out in Histonetland that are have more knowledge than I do.  I guess
I'm just trying to help you understand what goes on from a standard clinical
perspective with IHC.

Thomas Jasper HT(ASCP)BAS
Anatomic Pathology Coordinator
SMDC Clinical Laboratory
Duluth, MN
tjasper <@t> smdc.org

-----Original Message-----
From: George Cole [mailto:georgecole <@t> ev1.net]
Sent: Thursday, September 16, 2004 9:47 PM
To: 'Jasper, Thomas G.'
Subject: RE: [Histonet] fixed and unfixed immuno and antuibody oreps

The note you answer so thoughfully is naïve, I admit---I mean it says
HMMM, if they are undoing the effects of fixatives on their
tissues----wouldn't NOT fixing in the first place them be an even better
way to undo fixation?  That's what it is to be retired with no lab to
fiddle with questions like that.  There is only the Histonet---may be I
can get somebody out there to wrestle with this question----sic 'em on
it, so to speak---maybe somebody will soothe this tech, retired, yes,
but still with  questions buzzing around his aged head.
georgecole <@t> ev1.net     

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Jasper,
Thomas G.
Sent: Thursday, September 16, 2004 10:45 AM
To: 'George Cole'; 'histonet <@t> lists.utsouthwestern.edu'
Subject: RE: [Histonet] fixed and unfixed immuno and antuibody oreps

Dear George,

I have read some of your postings and I have developed a soft spot for
It seems what you're after is an explanation of how immunohistochemistry
be done these days using techniques which negated your efforts in the
Also you seem to be questioning how the "best" method was determined,
why is
it OK to do IHC the way most labs do?
George, I honestly cannot answer how today's techniques were exactly
determined/developed.  I am not an expert in the field of IHC history
comparative research of the same.  Here's what I do know, I have been
Histology work for approximately 20 years (I know a babe in woods).  All
my training and experience in doing IHC has been heavily dependent upon
proper fixation.  This is for the most part formalin fixed (10% Neutral
Buffered), paraffin processed tissue specimens.  Most people working in
clinical world, as I do, base the majority of their IHC work on properly
fixed tissue specimens.  This is how we understand IHC.  My lab runs
problems occasionally when we receive referral cases (either block or
slides) that have poor/improper fixation.  This hinders our ability to
produce a good quality diagnostic stain.  Antigen retrieval techniques
employed to enhance staining and maybe that is the answer you seek as it
a post-fixation procedure.
George, I am not a researcher and there are a lot of people on the
that are better qualified than I am to speak to the finer points of IHC.
just thought I would share my thoughts with you. I consider myself to be
standard histologist with a fairly basic understanding of IHC as it
to I what I am expected to put out on a daily basis.
George, I apologize if the things I have just stated have already been
related to you or if someone with a greater understanding of IHC finds
in what I've posted.  Being a manager I don't do quite as much IHC as I
to, although I certainly have my hand in the day-to-day immunos that are
here.  I also sympathize with you in a general sense as I do like to
understand things and like clear explanations and/or reasons why.
Hope this helps.

Thomas Jasper HT(ASCP)BAS
Anatomic Pathology Coordinator
SMDC Clinical Laboratory
Duluth, MN
tjasper <@t> smdc.org

-----Original Message-----
From: George Cole [mailto:georgecole <@t> ev1.net]
Sent: Tuesday, September 14, 2004 1:00 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] fixed and unfixed immuno and antuibody oreps

I promise to drop this line---I have had an itch to find out
something--- I realize I must give it up---I have asked it repeatedly
and I don't want to keep asking the same thing---impatience is bound to
develop.  Retired as I am---I no longer have lab in which to answer such
questions. Most histotechs nowadays are brought up doing one side of the
question---indeed it is the standard procedure and my question  just
doesn't scan with  today's techs.  But if I could shuck 10 years and be
back in my lab and I was given the responsibility to do a certain class
of work I would do the following:
Antibody technique or immunofluorescence procedure---any procedure that
has a fixative in its beginning followed by some procedure to neutralize
the effects of the fixative----I would run at least 3 trials on the 2
sets of pairs: 1) Tissue unfixed 2) tissue fixed, then processed with by
whatever step was supposed to neutralize the fixation effect, rinsing
the tissues carefully afterwards.  . Then I would run tissues 1 and 2 in
the same preparation.  And I would choose as my standard procedure which
ever procedure of the pair which gave the better results. I drop that
and run.  The crowding effect of Standard Procedure Itis is out there.
But then I might just find my question was trivial and standard
procedure might win.  But has the fix-unfix procedure ever been tested
against the unfixed prep? This retired picky-compulsive tech would never
run ANY procedure as standard without testing its
effectiveness---comparing it--- with any reasonable alternative
procedure for quality. Nuff said.  Retired Tech now enters a stage of
quiet upon the subject on the histonet, but I will still brood over the
untapped quality test in ANY procedure done by rote rather than via the
search for the most excellent way to do that test. 
georgecole <@t> ev1.net
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