[Histonet] RE: IHC pos. & neg. control question

Tony Henwood AnthonyH <@t> chw.edu.au
Thu May 19 21:34:37 CDT 2011

No, the best sample for negative control is NOT a section of your positive control tissue.

The positive control would have already been tested for "spurious cross-reactions". Why continue to test it?
The patient's tissue has not been tested. So a mirror section is used. 
Does the patient's tissue have brown pigments that could be confused with DAB? 
Are there unique substances (of unknown type) that might bind non-specifically to the IPX reagents? 
Is there endogenous biotin or excessive peroxidase that might give a false positive reaction? 

These are the answers I expect to obtain from my negative control. I already know these do not occur on my positive control tissue.

Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Laboratory Manager & Senior Scientist 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Jean-Martin Lapointe
Sent: Friday, 20 May 2011 3:26 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] IHC pos. & neg. control question

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 Lapointe, DMV, MS, dACVP
Vice-President, Pathologie
AccelLAB Inc
1635 Lionel-Bertrand, Boisbriand
Québec, Canada  J7H 1N8
tel:  450-435-9482 ext.247
fax: 450-435-4795
jm.lapointe <@t> accellab.com


Message: 24
Date: Thu, 19 May 2011 09:04:24 -0700
From: "Curt Tague" <c.tague <@t> pathologyarts.com>
Subject: [Histonet] IHC pos. & neg. control question
To: <histonet <@t> lists.utsouthwestern.edu>
Message-ID: <019801cc163e$6c1487d0$443d9770$@tague <@t> pathologyarts.com>
Content-Type: text/plain;	charset="us-ascii"

I got this email from a pathologist today. we have always run a positive with the patient tissue and a negative, the same patient tissue, and had no problems. Am I missing something. Is there any documented regulation dictating what needs to be used for the controls. In some cases if we get one slide of patient tissue, then we will use the pos. and neg. cont. from the same block but typically it's the pt. tissue that is used for the neg.
control. Thanks for your guidance. 



"I received slides on sentinel lymph node biopsies with a positive control on the same slide as the breast tissue, but the negative control was just the patient's lymph node and did not have the corresponding section used for the positive control.  The patient's own tissue cannot be used as a negative control.  The tissue that stained positively must serve as the negative control without the antibody.  This is critical and you need to correct that immediately."





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