Re: Negative Reagent Controls For Immuno

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From:r weimer <>
To:"Mackinnon, John" <>
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As regards controls for imunohistochemistry - everyone has a viewpoint and a
practice.  After reading/watching the net and discussions about
positive/negative controls I believe that many applications are inappropriate
or just plain wrong. Not many labs do an adequate job to control their studies
because so much practice is based upon inadequate information  and
misinformation perpetuated from one source to the next.

My comments are not to discuss controls, but to relate a story about how some
people handle them (history).

Long ago I was teaching my course at the Given Institute of PathoBiology in
Aspen. As a guest I had one of the very, VERY biggest names /personalities (a
founding father) of immunohistochemistry in the workshop. Also, in the workshop
was a graduate student of this BigWig (both were/are wonderful, dedicated
research people). We all discussed positive/negative controls and then, as a
lab assignment, each was to design a system to control an immunohistochemical
experiment. The group consisted of all levels of expertise from neophyte to Lab
Tech to MD/pH D's to Research Professionals. What a diverse array of control
solutions were produced! Discussion after the experiment were almost chaotic.
Everyone had a viewpoint, a reason, a necessity, a demand for any particular
control. As a group we considered all viewpoints and options and ended up with
an acceptable control system. To control the immunohistochemical staining of
five slides (IgG, IgA, IgM, Kappa, and Lambda) utilizing poly and monoclonal
antibodies, required five patient slides and 105 control slides (of patient,
positive and negative tissue)!!!

I knew then that the problem of adequate/appropriate controls was one of the
major issues facing this new science. I believe it still has to be resolved.

Other than the realization of the magnitude of the problem was an exchange
between the BigWig MD/PhD and the GradStudent. 0/0. The GradStudent had more
than 45 controls
(a reasonable number at the time) and the BigWig had six (6!). BigWig to the
GradStudent - "you will waste your life doing controls" (waste of time, money,
confusion in interpretation, undesired results, etc., etc.). The GradStudent
responded immediately to his 'superior'; "you will waste YOUR life for NOT
doing controls!"

My last communications with both was that each has been very successful in
research/clinical sciences doing 'the appropriate controls'.

Upshot: we can only do the best we can under the circumstances.
Any discussions/comments welcome.

Bob Weimer

P.S. (is this an email term?) I essentially agree with  "Mackinnon, John" , but
a major confusion exists as regards what exactly is 'negative reagent', a
'negative for each block', a 'negative monoclonal/polyclonal', 'negative HIER
slide, etc.  Also substitute 'positive'. These are particular statements that
do not convey sufficient information to the people one is trying to reach. On
the other hand, the statement "All of your controls must be treated the same
way that your test sections are treated, if you don't do this you are not
really controlling the procedure" Right On!!.

> When running negative reagent controls you should be running a negative for
> each block, if you are running both monoclonal and polyclonal antibodies you
> should run a negative for each of these.  If one of your antibodies requires
> HIER another enzyme and one no pretreatment you should be running a negative
> for each of these.  All of your controls must be treated the same way that
> your test sections are treated, if you don't do this you are not really
> controlling the procedure.  Expense is no excuse for not running proper QC.
> I can't remember ever hearing a biochemist saying that they were not going
> to run all of the appropriate QC because it seemed excessive and expensive.
> John MacKinnon MLT, ART
> Senior Technologist, Pathology
> Lakeridge Health Oshawa
> Ontario, Canada

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