RE: [Histonet] negative IHC controls

From:"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
Hinchingbrooke Hospital
-----Original Message-----
From: []On Behalf Of Hadi Yaziji
Sent: 11 October 2003 03:07
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 []
Sent: Wednesday, October 08, 2003 3:57 PM
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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