RE: [Histonet] Re: effects of elevation on immunohistochemistry

From:"Patsy Ruegg"

HIER is affected at altitude and one of the big problems we ran into in CO
was FDA compliance with the Her2 kit from DAKO which required HIER be
carried out using a waterbath and the temp requirement was 95c for 45', well
her in Denver and especially in Colorado Springs which is even higher our
water boils rapidly at 92-93C, if you leave your container in a rapidly
boiling WB for 45' all the water boils off and you still don't reach the
required temp for the required time.  We tried many solutions to compli with
FDA, people built elaborate lids to seal the WB so that at least the water
would not boil off, I tested pressure cookers extensively, and although it
was thought that with the pressure being controlled the water should not
boil violently my experience seemed to indicate that the water was boiling
violently because the sections were really damaged in the pressure cooker
compared to WB.  I am not sure what DAKO finally agreed upon for Her2, I
think they extended the time in the WB for those of us at altitude, I know
it took years for anyone to even address the issue and frankly I have moved
on and not kept up, I think it just points to the problem of physically not
being able to always comply with FDA in certain areas.  You may have noticed
that the FDA approved EGFR kit from DAKO uses enzyme digestion instead of
HIER.  It never ceases to amaze me how tests that only tested and developed
at sea level can get FDA certification without any consideration of others
at higher altitudes.  I asked the FDA this question at a CAP conference on
Her2 in Chicago and they completely blew me off as if how could it matter.
The fda made the statement that the her2 kit was tested from the east coast
of the US to the west coast and this is when I questioned them about the
higher altitude in the middle which obviously was not tested and they seemed
to just say we tested in New York and California and assumed it work the
same everywhere in between. 
On a more recent note.  I did test the Lab Vision enclosed waterbath HIER
instrument and was able to get it to 95dc and hold there for 45' without any
obvious violence done to the tissue sections.

Patsy Ruegg 

-----Original Message-----
[] On Behalf Of Rene J Buesa
Sent: Thursday, September 28, 2006 8:45 AM
To: Lisa Manning;; Sundi Readlinger
Subject: Re: [Histonet] Re: effects of elevation on immunohistochemistry

  This is a Physics issue! At higher elevation atmosferic pressure is lower
and the water boils at less than 100║C so any HIER done in an "open"
container, like heating in the MW oven or using a steamer or a water bath,
will be affected because 100║C cannot be reached.
  HIER should be done in a pressurized environment (like a pressure cooker).
There was a paper published in the Jorunal of Histotechnology not too long
ago (I do not have the exact reference) dealing with Her-2 Neu tests at high
  Any step requiring a certain temperature in an open container, will be
affected. Other steps temperature independednt will not be affected.
  Hope this will help you!
  RenÚ J.

Lisa Manning  wrote:
  Good morning, 
Does anyone have any experience or knowledge about the effects of
elevation on immunohistochemistry?
Thanks kindly, Lisa Manning

Lisa Manning R.T, B.Sc.
Histopathology Biologist
Canadian Food Inspection Agency
National Centre for Foreign Animal Disease
1015 Arlington Avenue, Suite T2300
Winnipeg, Manitoba R3E 3M4
PH. (204) 789-2087
FAX (204)789-2038
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