[Histonet] RE:Fixed etc. long response
Thank you George and Thomas for raising and discussing this topic.
I think that there is always somewhat of a disconnect between biologic
and biochemical procedures. In biochemical work an experiment was
usually carried out three times and if it worked all three times was
regarded as valid. In the biologic literature some things if they work
once are published as rock hard truths. Sometimes such results should be
labeled "observations" and be indicators rather than hard facts but once
published are usually taken as hard facts. Part of this is the tendency
to examine the most attractive field rather than all fields, and it
seems to me that this is especially true of electron microscopy.
IHC is really following in the footsteps of histochemistry. Initially
there were relatively few techniques and several positive and negative
controls were always used. If enzymes were studied, their location in
frozen or freeze dried sections was used as the gold standard although
this was usually a cumbersome process and not always possible. Sometimes
fixatives were used but the results were compared to the gold standard.
As with general histology, the standard for looking at tissues was fixed
tissues processed to paraffin wax. For those not used to examining
frozen sections, The image in frozen sections was sometimes difficult to
compare to that of fixed tissues due largely to the smaller degree of
shrinkage and the more open appearance of tissues.
I agree with George's general sentiment about fixation as the tissue is
not in its natural state and even if antigen retrieval is used it
doesn't mean that the original state has been restored.
Another problems is that in some cases the claims of manufacturers
regarding their antibodies has been taken as the gospel truth. One of
the best examples I know concerns claims for "pan keratin" antibodies.
We would assume from some of the claims that virtually all cytokeratins
can be demonstrated with this antibody. In most cases nothing could be
further from the truth. Before I use an antibody I would like know how
it binds to epitopes, preferably in the native state.
Most labs use buffered formalin, and specific times etc. However, common
sense tells me that the sections produced by one lab will differ, albeit
in small measure from another lab. It would therefore be logical to have
a standard technique for each lab based on testing a particular antibody
on their processed tissue and then comparing this to results using
frozen section. Once this comparison has been made you can standardize
to your procedure. Individual labs fix, process, section and stain in
slightly differently. Sometimes IHC is tolerant of variation, sometimes
not. It would be optimal if a standardized image was available to all
labs so that results could be compared and techniques in individual labs
changed to obtain this optimal result.
In answer to you question, no I have not been drinking, its only 3 pm!!
I know that this is pie in the sky but I am worried about the different
responses that we get on Histonet regarding the same technique and often
using the same antibody. Perhaps we could have a set of IHC images using
fluorescence and another using peroxides so that we could get some
[mailto:email@example.com] On Behalf Of Jasper,
Sent: Thursday, September 16, 2004 12:45 PM
To: 'George Cole'; 'firstname.lastname@example.org'
Subject: RE: [Histonet] fixed and unfixed immuno and antuibody oreps
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,
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
From: George Cole [mailto:email@example.com]
Sent: Tuesday, September 14, 2004 1:00 PM
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.
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