[Histonet] RE: negative IHC controls

[Nick Kirk]

Hadi

I am sorry but I must take issue with your very condescending and if I may
say so insulting tone.

For your information, I have over 18 years of experience doing IHC and my
lab does a considerable number of slides per day.
I have set up the IHC service in two labs and have received much praise for
doing it.
I have also done extensive lab based trials work for both DAKO and
Novocastra here in the UK as well, so I think my credentials are better than
most.
I'm not at issue with the interpretation issue at all, that is indeed a very
skilled task that takes many years of study and experience to become
competent in.
What I am taking issue with is the issues around clinical audit and best
practice.
Now I don't know what clinical audit systems you have in your establishment,
but in ours it is regarded as poor clinical governance i.e. bad practice by
both myself, the Head Biomedical Scientist of the department and all of my
Pathologist colleagues not to have separate controls per case.
This is especially the case when you are reviewing tumour antigenicity on
samples that may come out of the archive.
All our slides are stored in case order so to have positive controls for
each case actually makes it much easier the extract all the relevant slides
per case for later review. Having slides for the same case filed in separate
places introduces the potential for error, especially when being initially
filed.
I know this to be true because it has happened to me on several occasions
where slides filed in different places have been mis-filed and become
difficult to retrieve.
In the UK we use a system of evidence based good practice and evidence
suggests that is precisely what we are doing.

I hope that now explains it to you.

Nick Kirk BSc (Hons) FIBMS (I've got letters too)
Head Biomedical Scientist
Histopathology
Hinchingbrooke Hospital
Huntingdon
Cambridgeshire
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 16:22
  To: histonet <@t> pathology.swmed.edu
  Subject: Re: [Histonet] negative IHC controls


  Dear Nick,

  Thanks for the email. You definitely misread my email. First, where in my
message did I advocate not to use negative and positive controls per case?
Of course it's poor patient's care not to do that. Is it necessary to run +
and - controls on every antibody? I don't think so provided you have the
necessary experience to interpret them. To address your other point about
reviewing cases, we keep our positive and negative controls indefinitely.
You can always pull the controls that pertain to the run of the particular
day of the case in question.

  You will be 'dam fool' (I am copying your words) to run IHC if you have no
experience. That's much more dangerous legally and ethically. I've seen
slides misinterpreted where the positive control is present on the same
slide.

  Bottom line: one + control per run is adequate (provided you have the
experience, which I'm not sure you have based on your reply). One - control
per case is more than sufficient.

  I hope this addresses your concerns.

  Hadi Yaziji, M.D.
  PhenoPath Laboratories


  On Saturday, October 11, 2003, at 12:16 AM, Nick Kirk wrote:


    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.





    <image.tiff>


    The information contained in this message may be privileged and
confidential and protected from disclosure. If the reader of this message is
not the intended recipient, or an employee or agent responsible for
delivering this message to the intended recipient, you are hereby notified
that any dissemination, distribution or copying of this communication is
strictly prohibited. If you have received this communication in error,
please notify us immediately by replying to the message and deleting it from
your computer. Thank you. Arkansas Children's Hospital



-------------- next part --------------
An HTML attachment was scrubbed...
URL: http://lists.utsouthwestern.edu/pipermail/histonet/attachments/20031013/981c9fe4/attachment.htm