[Histonet] IHC pos. & neg. control question

[Amos Brooks]

Thanks Liz,
     You are absolutly right there, but have you ever noticed some folks
eyes glaze over when you say that they must both be run at the same ug/mL? I
would be lying if I said it never happened to me until I forced myself to
work it out. Once you do though it isn't too bad. Unfortunately people don't
think about this much. They want to put a slide on a machine and walk away
without thinking about what they are doing. I guess I was oversimplifying
the description. Thanks for pointing that out as it is actually an important
aspect to the dilutions.

Amos

On Fri, May 20, 2011 at 4:25 PM, Liz Chlipala <liz <@t> premierlab.com> wrote:

> Amos
>
> Isotype negative controls are based upon protein concentration not
> dilution.  They must be the same protein concentration of the primary
> antibody at the dilution you are using.  Using the same dilution will
> not work unless both stock solutions (primary antibody and isotype
> control) are at the same protein concentration which is rarely the case.
>
> Liz
>
> Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
> Manager
> Premier Laboratory, LLC
> PO Box 18592
> Boulder, Colorado 80308
> office (303) 682-3949
> fax (303) 682-9060
> www.premierlab.com
>
>
> Ship to Address:
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>
> -----Original Message-----
> From: histonet-bounces <@t> lists.utsouthwestern.edu
> [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Amos
> Brooks
> Sent: Friday, May 20, 2011 2:16 PM
> To: jm.lapointe <@t> accellab.com; histonet <@t> lists.utsouthwestern.edu
> Subject: [Histonet] IHC pos. & neg. control question
>
> Hi,
>     This is simply a question of definitions. They are actually both
> right.
> If you have the patient tissue as a negative control you are not using
> the
> primary antibody on it but are replacing it (ideally) with an Ig with
> the
> same isotype AND dilution (universal negative is stupid). So if your
> primary
> is an IgG1 from a mouse and you use it at 1:100, your negative control
> would
> be a non-immune mouse serum IgG1 diluted to 1:100. Running this on
> unrelated
> material will not show you anything in this case. what it will show you
> is
> that the detection system you are using is specific to the antibody you
> put
> on the tissue and not to A) something else in the tissue or B) something
> on
> the Ig that is not the epitope itself.
>     Although, If you run the same primary antibody on tissue that you
> expect will not react with the primary antibody, this would prove that
> the
> antibody reaction is specific to the antigenin question. This does raise
> the
> question of what to do about ubiquitous antigens (like ubiquitin) that
> are
> actually everywhere. Every cell has a cell cycle so they will all at one
> point or another show proliferation or degeneration for example. Does
> this
> mean you don't run a negative control? No you need to look at it in the
> perspective of expected results.
>     There are various ways of using negative (and positive) controls.
> It
> would actually be cost prohibitive to run ALL the possible positive and
> negative controls for every test that we as histologists do. We need to
> discuss with our pathologists what they want to have done to give them
> the
> confidence in our testing to support their diagnosis. But remember that
> just
> because another lab and another pathologist does it differently doesn't
> mean
> it is wrong. It's just different.
>
> Happy Friday Folks,
> Amos
>
>
> Message: 5
> Date: Thu, 19 May 2011 13:25:49 -0400
> From: "Jean-Martin Lapointe" <jm.lapointe <@t> accellab.com>
> Subject: [Histonet] IHC pos. & neg. control question
> To: <histonet <@t> lists.utsouthwestern.edu>
> Message-ID:
>       <
> BEFD613BD39142499989F836556DDC830127283C <@t> ACE.accellab.lan>
> Content-Type: text/plain;       charset="iso-8859-1"
>
> Hi Curt,
> I agree with your pathologist. The section that you use as a negative
> control (without primary) in an IHC run should ideally be tissue that is
> a
> known positive, and of the same nature as the test sample. Therefore the
> best sample is often a section of your positive control tissue.
> The purpose of this negative control is to make sure that any positive
> staining observed in the test sample is not due to a spurious
> cross-reaction, unrelated to the primary. I don't think the issue per se
> is
> that you are using tissue from the same patient; rather, it is that you
> are
> using a sample for which the staining characteristics are not known. For
> instance, if are using a lymph node section as a negative, for a stain
> that
> targets an epithelial marker (eg Her2) in the test breast sample, then
> your
> negative tissue is not appropriate, because since lymph node contains no
> epithelial tissue, it will not stain no matter what. Therefore if your
> test
> sample shows a positive reaction in the epithelial tissue, but for some
> reason that reaction is a spurious false-positive, then the lymph node
> negative will not reveal that.
> I realize that this is all very theoretical and hypothetical, but I
> understand the pathologist wanting to be confident in the knowledge that
> all
> potential technical issues are eliminated before making his diagnosis.
>
> Jean-Martin
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