[Histonet] Normal Controls for Cerb-B2

[Bryan Hewlett]

Tony,

It does indeed raise the question of detection threshold!!
If one is performing a titre to judge working antibody dilution for breast 
tumour HER2 overexpression,
the procedural control slides should contain several different samples of;
1) normal breast epithelium,
2) breast tumours known to have normal levels of expression (i.e. 
non-amplified - confirmed by FISH),
3) breast tumours known to have levels of overexpression (5-20x amplified - 
confirmed by FISH).
The working dilution should be adjusted to the HIGHEST dilution at which;
samples 1&2 are negative and sample 3 shows complete circumferential 
staining of at least 50% of tumour cells.
All of these samples should have been optimally fixed in NBF (minimum of 24 
hours at RT).
An alternative is to use optimally fixed blocks of cell lines with known 
levels of HER2 expression (these are available from couple of suppliers).
Once you have determined a working dilution, confirm with validation studies 
according to CLSI guidelines.
Locally, all 2+ positives are considered equivocal and reflexed to FISH for 
confirmation.

Check the UKNEQAS web site for insight about EQA for HER2.

Regards,

Bryan


----- Original Message ----- 
From: "Tony Henwood" <AnthonyH <@t> chw.edu.au>
To: "Bryan Hewlett" <bhewlett <@t> cogeco.ca>; 
<Histonet <@t> lists.utsouthwestern.edu>
Sent: Monday, November 20, 2006 5:14 PM
Subject: RE: [Histonet] Normal Controls for Cerb-B2


Bryan,

You are absolutely right.
This raises the question of thresholds.
At what dilution of antibody should staining reflect over-expression?
How many labs would call a borderline over-expression positive?
This would depend on the dilution of the Cerb-B2 antibody as well as the
sensitivity of the detection system.
I think I will need to peruse the external QAP figures to see if this is
an issue.

Regards

Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC)
Laboratory Manager & Senior Scientist
The Children's Hospital at Westmead,
Locked Bag 4001, Westmead, 2145, AUSTRALIA.
Tel: 612 9845 3306
Fax: 612 9845 3318




-----Original Message-----
From: Bryan Hewlett [mailto:bhewlett <@t> cogeco.ca]
Sent: Tuesday, 21 November 2006 1:10 AM
To: Tony Henwood; Histonet <@t> lists.utsouthwestern.edu
Subject: Re: [Histonet] Normal Controls for Cerb-B2


Tony,

The vast majority of normal epithelial cells express HER2 protein on
their
surface.
However, you would not want to use these as a procedural control, since
the objective is to demonstrate OVER-EXPRESSION of this normal
protein.
Detection of normal levels of HER2 would invalidate the test.

Bryan

----- Original Message ----- 
From: "Tony Henwood" <AnthonyH <@t> chw.edu.au>
To: <Histonet <@t> lists.utsouthwestern.edu>
Sent: Monday, November 20, 2006 12:15 AM
Subject: [Histonet] Normal Controls for Cerb-B2


Hi all,
Apart from Breast and Ovarian tumours (which are rare in pediatric
pathology), does anyone know what normal tissue elements contain this
protein that can be demonstrated using immunohistochemistry?

Regards

Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC) Laboratory
Manager & Senior Scientist The Children's Hospital at Westmead, Locked
Bag 4001, Westmead, 2145, AUSTRALIA.
Tel: 612 9845 3306
Fax: 612 9845 3318




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