Re: Breast Tissue (Reply- a bit long)

From:Bryan Hewlett

The regulatory department at DAKO is correct!!
The protocol must be followed to ensure a reliable test.
We have conducted extensive testing on fixation times.
As a result of our study, with both the FDA approved test and several other non-FDA approved antibodies, we instituted a policy of fixing all breast tissue for a minimum of 24 hours some time ago. Besides, it's just good technique,"First we fix, then we process" --Histopathological techniques 101.
The significant findings were presented to DAKO at a symposium in Copenhagen 2 years ago.
We have a paper in preparation for publication.
The FDA approved test requires fixation in aqueous buffered formaldehyde for 18-24 hours.
If phosphate buffered formaldehyde(NBF) is the fixative, then false positives are not an issue.
If fixation in NBF is less than approximately 16 hours at RT, then depending upon subsequent fixation (on the tissue processor) false positives may become an issue and
false negatives ARE definitely an issue (the shorter the fixation time the more false negatives).
As an aside, ER and PR demonstration is also influenced by fixation time!
I could go on at length but will refrain!
If all samples are being sent out for FISH anyway, why do the IHC?
If you only confirm IHC positives by FISH, what about the false negatives?
It may interest your pathologist to know that the FISH protocol is also influenced by fixation, although to a much lesser extent.
Bryan R. Hewlett ART, MLT
Technical Specialist (Retired)
Anatomical Pathology,
McMaster University Medical Centre
Hamilton, Ontario,
----- Original Message -----
From: Soto, Roxanne
To: 'Tony Henwood' ; 'Patsy Ruegg' ; ;
Sent: Monday, June 02, 2003 9:07 AM
Subject: RE: Breast Tissue

I have contacted DAKO about this because this is such a great concern for clinical trial purposes.  I spoke to the regulatory department at DAKO and I was told that if the protocol is not followed than it is not a reliable test to determine eligibility for herceptin and patients must show proof of an FDA approved test.  I am waiting to hear back from them as I explained that we, like many other labs, don't necessarily fix for 18 - 24 hours in formalin.
My question is----how many people use the FDA approved test and how many people use the non-FDA approved her2neu?  And are you fixing the tissue for 18 - 24 hours?  I know other labs, that not only do not fix the tissue appropriately, but also tweak the antibody or use it on other machines besides the DAKO.  So, if anyone out there is histoland has done a study on how the fixation time either offers false positives or false negatives, I would be greatly interested.  And if they are being sent out for FISH anyway, how much does it matter? (This is the question of the Pathologist).
-----Original Message-----
From: Tony Henwood []
Sent: Sunday, June 01, 2003 6:37 PM
To: 'Patsy Ruegg';;
Subject: RE: Breast Tissue

But don't tissues pass through alcohols during processing? Since tissues, these days, are rarely completely fixed in formalin prior to processing, then ethanol probably has a secondary fixing effect.
Just my thoughts,

Tony Henwood JP, BAppSc, GradDipSysAnalys, CT(ASC)
Laboratory Manager
The Children's Hospital at  Westmead,
Locked Bag 4001, Westmead, 2145, AUSTRALIA.
Tel: (02) 9845 3306
Fax: (02) 9845 3318

-----Original Message-----
From: Patsy Ruegg []
Sent: Saturday, 31 May 2003 2:50
Subject: RE: Breast Tissue

To all who hope to do her2neu expression on breast samples.
I attended a conference presented by a pathologist from Phenopath (lg. reference lab in Seattle (Dr. Gown)) who warned that her2neu expression was adversely effected by use of alcoholic fixatives.  Phenopath does a lot of her2neu tests and they are considered experts in this field.
-----Original Message-----
From: []
Sent: Thursday, May 29, 2003 2:28 PM
Subject: Breast Tissue

Does anyone out there in histoland have any suggestions for fatty breast tissue processing?  We are having a terrible time getting some of our breast tissue fixed well.    Thanks in advance for your responses.

This email and any files transmitted with it are confidential and
intended solely for the use of the individual or entity to whom they
are addressed. If you are not the intended recipient, please
delete it and notify the sender.

Views expressed in this message and any attachments are those
of the individual sender, and are not necessarily the views of The
Children's Hospital at Westmead

This footnote also confirms that this email message has been
virus scanned and although no computer viruses were detected,
the Childrens Hospital at Westmead accepts no liability for any
consequential damage resulting from email containing computer

Privileged/Confidential information may be contained in this message. The information contained in this message is intended only for the use of the recipient(s) named above and their co-workers who are working on the same matter.

The recipient of this information is prohibited from disclosing the information to any other party unless this disclosure has been authorized in advance.

If you are not intended recipient of this message or any agent responsible for delivery of the message to the intended recipient, you are hereby notified that any disclosure, copying, distribution or action taken in reliance on the contents of this message is strictly prohibited. You should immediately destroy this message and kindly notify the sender by reply E-Mail.

Please advise immediately if you or your employer does not consent to Internet E-Mail for messages of this kind. Opinions, conclusions and other information in this message that do not relate to the official business of the firm shall be understood as neither given nor endorsed by it.

<< Previous Message | Next Message >>