RE: [Histonet] negative IHC controls
A
cautionary note
I
agree that false positives are rare but they do happen. We had a couple a while
ago when it turned out that the Teflon coat on the probe of our DAKO Autostainer
had perished and was carrying over reagents from one slide to another and also
contaminating our negative control solution with primary
antibody.
Also
they are very useful in checking that there is no endogenous biotin activity
(more common than people think) and also that your hydrogen peroxide solution
hasn't gone off when quenching endogenous peroxidase
activity.
As for
the argument about only one positive control per antibody per run rather than
per patient -
Well
the question I would pose is this. If in 5 years time you wish to review the
immuno for a particular case, how do you ensure that the staining quality was
adequate at the time if there aren't any positive controls for that
case?
On
medico-legal grounds alone you should be doing positive controls for each case,
especially if those results end up with the patient having a particularly
aggressive treatment like chemotherapy or a particularly invasive surgical
procedure such as a colectomy or a mastectomy. If someone wants to sue your
organisation at a later date for inappropriate treatment you need every bit of
proof that your part of the investigation was above reproach and controls is one
thing that will definitely be asked about. I certainly wouldn't like to stand up
in court and try and defend myself against that one!
Incidentally, we use Surgipath's Control slides which
allow you (most of the time depending on the size of the test section) to have
the positive control and test section on the same slide, which saves a lot of
space on the immunostainer and neatly solves the problem of where do you store
the positive control if you use the single control per run model and you have
multiple cases with the same antibody. It also acts as a check that the slide
has received the correct primary antibody and that someone hasn't loaded the
immunostainer wrongly.
At the
end of the day I still go by the analogy someone else made here earlier about
airbags and seat belts in cars. Would you drive in a car without them? They may
never be needed in your entire driving life, but you would be a dam fool not to
have them wouldn't you?
And
I think we will have to agree to disagree Hadi, with your last
statement, it is poor quality control by any definition of the term, not to use
both negative and positive controls for each case.
Nick
Kirk
Histopathology
Hinchingbrooke Hospital
Huntingdon
England
Theoretically, I agree with you that (a) running
multiple negative control sections to cover the different
antibodies/pretreatments is ideal and (b) money should be well spent on QC (I
can't emphasize that enough). However, I honestly don't remember when was the
last time when I had a false positive staining on a patient's slide that the
negative control was the only way that allowed me to recognize the false
positive signal on the real slide. And we look at 100s of IHC stains every
day.
Experienced pathologists and technologists can still recognize in
> 99% of the times a false positive signal from a real signal on the tissue
section of a given antibody without even having to look at negative controls.
I don't think it should be a CAP requirement at all. In fact, I'd say the same
thing on positive controls. All you need is one positive control per antibody,
but not one control/per antibody/per patient. In many cases positive internal
controls are present on the same slide, so you can tell whether your antibody
worked simply by evaluating the expected positive internal controls. In fact,
if your positive 'external' control worked and internal controls didn't, then
you need to repeat your antibody test regardless of the positive external
control. External controls are different tissues, fixed differently, processed
differently and the tissue ages differently (in terms of its antigenicity).
Individuals who perform and interpret IHC studies must have the
knowledge to recognize the expected sub-cellular localization of every
antibody on every type of tissue, including aberrant signals. For instance,
you can easily dismiss a granular cytoplasmic TTF-1 signal as not positive,
because TTF-1 is a nuclear transcription factor and we know it's expressed in
the nuclei. However, hepatocellular carcinomas can give you a consistent and
reproducible granular and cytoplasmic TTF-1 signal, and if you subsequently
run confirmatory markers (HepPar1, polyclonal CEA, CD10, etc.) they will be
positive. Such (granular cytoplasmic) signal should be recognized by the
interpreter (tech or pathologist) as a specific signal for tumors with
hepatoid phenotypes. This is just one of many examples..
The bottom
line: in real life, one negative control per case is more than
sufficient. And to say the least, it's inaccurate to state this is poor
patient care and lousy quality control.
I look forward to any
constructive criticism.
Hadi Yaziji, M.D.
PhenoPath
Laboratories
On Thursday, October 9, 2003, at 07:03 AM, Horn, Hazel V
wrote:
Not
only should you be running a negative control for each patient
slide. That negative control should be treated just as your
antibody is. If the antibody is rabbit and antigen retrieved, so
should your control. If another antibody on the same patient is
mouse and not retrieved another negative control should be run with this
same protocol. In the United States, labs that are inspected by
the CAP, are required to run these controls. MONEY should
never be considered as a reason to stop doing a part of a
procedure. It's poor patient care and lousy quality
control.
IMHO.
Hazel Horn, HT/HTL
(ASCP)
Histology Supervisor
Arkansas Children's Hospital
Phone
- 501.364.4240
Fax - 501.364.3912
-----Original
Message-----
From: vermast
[mailto:vermast@rogers.com]
Sent: Wednesday, October 08, 2003 3:57
PM
To: histonet@lists.utsouthwestern.edu
Subject:
[Histonet] negative IHC controls
I
would like to get a feel for how many out there are running negative control
slides for IHC.
In
our lab we do just a handful of antibodies and initially I had been running
a negative control slide with each patient slide. After much
discussion with our pathologists, we decided to omit these negatives (which
were conistently negative) and continue to just run a positive control
with each primary antibody for the run. We use the Dako autostainer
and prediluted primaries. The decision to stop running negatives also
coincided with Dako's decision to sell the negative control sera separately
from the primaries (they used to come packaged together). Perhaps I
assumed that discontinuing to pair these reagents together meant that few
labs were using the negatives.
Anyhow,
after having reviewed the last QMPLS (Canada) survey committee comments, I
believe the committe would like a negative control run with each patient
tissue slide in order to evaluate background (they have used NCCLS
guide pages as reference). Incidentally we weren't a part of the
survey due to a technicality.
Any
help or advice would be appreciated.
L.
Vermast
Stratford,
Ont.
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