Re: TTF-1 and CKBeta34E

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From:Kappeler Andreas <>
To:Histonet <>
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Hi Julie

I'm an old-fashioned guy from a European lab, where we still stain by hand -
so no comments on the Ventana. However we use both TTF-1 and High MW CK,
clone 34betaE12:
šTTF-1, clone 8G7G3/1, NeoMarkers MS-699-P1, working conc. 2 ug/ml. Ag
retrieval: HIER in 10 mM citrate, pH 6.0, pressure cooker, 7 min under full
pressure, then transfer to H2O at RT. Visualization is with a StrABC/AP
system and new fuchsin / Naphtol AS-BI. Lung or thyroid as positive control.
High MW CK, clone 34betaE12, Dako M 0630, working conc. 1.1 ug/ml. Ag
retrieval: HIER in 10 mM citrate, pH 6.0, microwave oven, total heating time
18 min in 3 intervals, cool down 15 min. Visualization is with a StrABC/AP
system and new fuchsin / Naphtol AS-BI. Prostate (but not prostate cancer)
is a very good positive control.

Probably you will not know what the Ab concentration is in your prediluted
antibodies, so it will be hard to compare with the above data. With me,
sales reps have a   v e r y   hard time selling kits and prediluted stuff -
I still want to control what I'm doing and I want to be able to play with
antibody concentration if a staining doesn't work the way I think it should.
So we use almost exclusively concentrated Abs that we titrate ourselves. I
also insist that I get the Ig-conc. for all the antibodies we buy. Some
companies provide this information with every vial, others not. And some of
the companies may let you know through your vendor that you don't need to
know the conc. of their antibodies, they have taken care of that for you,
and by the way you are too stupid anyway to understand that (it all happened
to me ...  I won't tell you the name of the company, though). So, with
respect to predilute antibodies, I have it with Paul E. Swanson who wrote
the following about this subject  (Paul E. Swanson, M.D. Dept of Pathology,
Washington University School of Medicine, St.Louis, MO. Am J Clin Pathol
1999; 111:445-8.):

'I have worried for some time that the marketing of predilute antibodies or
"ready-to-use" antibodies as stand-alone reagents or part of test kits was
an invitation to inexperienced laboratories to use tests that they were
qualified to neither perform nor interpret. Much of the inconsistency
between laboratories, in my opinion, has stemmed from the inevitable lack of
understanding of quality control and troubleshooting that come from reliance
on prepackaged reagents (and, as a possible corollary, automated
methodologies). The lab that is capable of working with a concentrated
antibody, developing appropriate quality assurance protocols, titering stock
reagents to optimize performance, and troubleshooting recalcitrant reagents
or detection systems is more likely to fully understand the use and
limitations of the technique and should be better able to provide consistent
diagnostically relevant results.'

As I told you, I'm old-fashioned. Good luck with TTF-1 and 34betaE12!

Andi Kappeler
Institute of Pathology, University of Bern, Switzerland

>Original message
> Good Morning All,
>    We are having problems with the TTF-1 on the Ventana.  I have spoken to
> the vendor of the antibody, and we have tried everything under the
> sun...longer antigen retrieval time, using BioCares Nuclear Decloaker,
> different TTF-1 from other vendors (all prediluted) to no avail...any
> suggestions?
>    Also, we do CKBeta34E on prostate, but prostate is not working as a
> control.  Skin works wonderfully, but our pathologists insist that we use
> prostate control...again suggestions will be welcomed.
> Thanks in advance for the responses,
> Julie
> Julie A. Sanders, BA, HT(ASCP)
> Supervisor, Anatomic Pathology/Administrative Office
> VAMC, Cincinnati, Ohio

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