RE: [Histonet] fixed and unfixed immuno and antuibody oreps
I have read some of your postings and I have developed a soft spot for you.
It seems what you're after is an explanation of how immunohistochemistry can
be done these days using techniques which negated your efforts in the past.
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 and
comparative research of the same. Here's what I do know, I have been doing
Histology work for approximately 20 years (I know a babe in woods). All of
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 the
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 into
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 are
employed to enhance staining and maybe that is the answer you seek as it is
a post-fixation procedure.
George, I am not a researcher and there are a lot of people on the histonet
that are better qualified than I am to speak to the finer points of IHC. I
just thought I would share my thoughts with you. I consider myself to be a
standard histologist with a fairly basic understanding of IHC as it relates
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 flaws
in what I've posted. Being a manager I don't do quite as much IHC as I used
to, although I certainly have my hand in the day-to-day immunos that are run
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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