Re: [Histonet] EGFR
Andi, I loved your response to Patricia. Very informative and down to
earth... and helpful to me also, since I am working with EGFR, although, not
with the Dako Kit... Thank You.
----- Original Message -----
From: "Andi Kappeler"
To: "Traphagan, Patricia" ; "Histonet"
Sent: Friday, May 27, 2005 1:46 AM
Subject: Re: [Histonet] EGFR
> Hi Patricia
> I assume you are talking about staining of colorectal adenocarcinoma -
> that is what the DakoCytomation EGFR pharmDx kit is most frequently used
> for. A few comments:
> - Smooth muscle can stain positively for EGFR (in a low percentage of
> cases) when using the DakoCytomation kit.
> - not all "norma epithelia" are EGFR positive. If you look at colon, may
> be half of the samples will show staining of the normal epithelium.
> - I would love to see "uniform staining" of the membranes... This is
> limited to a certain percentage of tumors. The majority of cases show
> focal or heterogeneous staining of the tumor; often the majority of tumor
> cells have partial membrane reactivity, most tumors also show cytoplasmic
> - 8 hours fixation (room temperature, without microwave) may be somewhat
> short, if your samples are more than just small biopsies.
> ... and a few suggestions:
> The DakoCytomation protocol asks to place slides in water before the
> application of Proteinase K (and also beforte the peroxidase block). We
> found this to be the cause for uneven digestion and have replaced the
> water with wash buffer from the kit (contains detergents --> better
> distribution of ProtK in subsequent step) (the FDA is faaaar away from
> Switzerland, and we care more about our patients than about the FDA...).
> Comparative staining of slides with water or with wash buffer in this
> particular step revealed no difference - except for a more even staining
> in particular of the border areas of tissue samples on those slides that
> went through wash buffer.
> We do a CK8 (e.g. clone CAM 5.2) staining for all cases that are stained
> for EGFR. This clearly demonstrates the carcinoma cells and helps to
> identify small groups of cells in the invasive part of the tumor.
> Particularly useful, if you have one of those cases where smooth muscle
> and/or connective tissue show some reactivity for EGFR.
> As an in house control we use cases of liver metastasis of colorectal
> adenocarcinoma, where the adenocarcinoma is heterogeneously 2+ positive (I
> don't want a 'retina blaster' as positive control) and where the
> hepatocytes can serve as strong positive control.
> If you don't have a copy of the DakoCytomation 'EGFR pharmDx
> Interpretation Manual' (Nr. 08052), call DakoCytomation and tell them to
> send you a few copies - and then force your pathologists to put their
> noses in it, it may help them understand that not all tumors have 3+
> complete membrane staining in 110% of all tumor cells.
> Hope this helps (and let time work for you: EGFR testing will go as it has
> come, just like many other tests ...)
> Andi Kappeler
> Institute of Pathology, University of Bern, Switzerland
> ----- Original Message -----
> From: "Traphagan, Patricia"
> Sent: Wednesday, May 25, 2005 7:21 PM
> Subject: [Histonet] EGFR
>> We have been trying to achieve good staining using this antibody. We use
>> the Dako EGFR kit. Dako's control that comes with the kit, turns out
>> beautifully, which tells us that the stain procedure is correct. Our
>> problem lies with our internal control. The pathologist are just not
>> happy with it. According to them we have: Over staining of the smooth
>> muscle, normal epithelial tissue does not stain correctly, and have
>> un-uniform staining of the membranes. Can anyone tell me what this
>> means? We use 10% NBF, fixation is probably no more than 8 hours. Our
>> processing run is about 12 hours for large tissue specimens, and we use
>> an alcoholic formalin in the second station, which then goes to 70% ETOH
>> and the graded series of alcohols, xylenes, paraffin. We do not put the
>> cut tissue in the oven, we let it air-dry overnight, possibly two
>> nights. We are at a total loss. Any suggestions. We really need
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