[Histonet] negative IHC controls

[Nick Kirk]

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
  -----Original Message-----
  From: histonet-admin <@t> lists.utsouthwestern.edu
[mailto:histonet-admin <@t> lists.utsouthwestern.edu]On Behalf Of Hadi Yaziji
  Sent: 11 October 2003 03:07
  To: histonet <@t> pathology.swmed.edu
  Subject: Re: [Histonet] negative IHC controls


  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 <@t> rogers.com]
    Sent: Wednesday, October 08, 2003 3:57 PM
    To: histonet <@t> 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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