[Histonet] IHC pos. & neg. control question

Fri May 20 16:24:15 CDT 2011

No problem Amos, I have a one page handout that I use for workshops and
presentations on how to do the math on this, it can be tricky, so if
anyone is interested just e-mail me.  Since we are talking about matched
isotype negative controls, essentially three things need to be taken
into consideration:

1.	The isotype of the primary antibody
2.	The protein concentration of the working dilution of the primary
antibody
3.	The antibody form - (affinity purified, culture supernatant,
ascities fluid, etc.) the isotype negative control needs to match the
primary antibody - some spec sheets list appropriate isotype negative
control reagents some do not

There is one caveat that I would like to address for those that are
working with animal tissues - you need to check the spec sheet on your
negative control reagents to make sure that it does not cross react with
the species you are working with.  If it does then you may get staining
in your negative control slide.

Here is a table that was generated from one of the older versions of the
dako handbook that addresses the antibody form and suggested negative
control reagents.

Primary Antibody Type

Suggested Negative Control Reagent

Monoclonal Mouse - produced in ascites

Antibody produced in ascites, same isotype as the primary antibody  OR

Normal nonimmune mouse serum

Monoclonal Mouse -

produced in tissue culture

Antibody produced in tissue culture, same isotype as the primary
antibody

Polyclonal Rabbit or Goat - immunoglobulin fraction

Normal rabbit or goat immunoglobulin fraction

Solid-phase absorbed Polyclonal Rabbit - immunoglobulin fraction

Rabbit immunoglobulin fraction - solid phase absorbed

Polyclonal Rabbit - whole serum 

Normal or nonimmune rabbit serum, whole serum

Hope this helps, and everyone have a great weekend

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:

1567 Skyway Drive, Unit E

Longmont, Colorado 80504

-----Original Message-----
From: Amos Brooks [mailto:amosbrooks <@t> gmail.com] 
Sent: Friday, May 20, 2011 3:05 PM
To: Liz Chlipala
Cc: histonet <@t> lists.utsouthwestern.edu
Subject: Re: [Histonet] IHC pos. & neg. control question

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 <tel:%28303%29%20682-3949> 
fax (303) 682-9060 <tel:%28303%29%20682-9060> 
www.premierlab.com

Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-----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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