Re: Negative controls for immunos

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From:Jeff Silverman <> (by way of histonet)
To:histonet <>
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We, and I'm sure most of us,  usually run multiple antibodies on a given
tissue or specimen.  Is there anything wrong with using each of the other
antibodies in your diagnostic panel as negative controls for each
individual antibody in the  panel, so-called irrelevant antibodies?  I've
been doing this for years and the referees in numerous journals have
accepted this without question. It saves lots of work and kills two birds
with one stone.

Jeff Silverman

> From: Tim Morken <>
> To:
> Cc:
> Subject: Re: Negative controls for immunos
> Date: Tuesday, October 13, 1998 8:44 AM
> Tammy asked,
> <Question, a new CAP standard (08.2257) states, " Are
> negative controls used for each antibody?".>
> As usual the CAP is being as vague as possible. They want to see what
> people do in response to the question and then, after brewing a few
> potions, will come up with some kind of "standard" which will be loosly
> based on the most common response.
> This could be interpreted a number of ways:
> 1) Run a negative (normal serum or "non-sense" antibody) on the case.
> 2) Run a negative (normal serum or "non-sense" antibody) on the control.
> 3) Do both 1 and 2 above.
> 4) Use a control block which is negative for the antigen in question and
> run a "sense" antibody on it.
> 5) Use a control block which is negative for the antigen in question and
> do 3 and 4 above.
> Which of the above the CAP has in mind is anybody's guess. The worst
> case scenario is that they want to see a negative tissue control for
> each antibody (let'see, that's 15 antibodies in a run, 15 extra blocks
> to cut, 30 extra slides to stain (pos and neg on "neg" control slides)
> It seems to me, though, that what you are doing is fine. We don't
> normally run a negative on the control tissue.  We usually use normal
> serum (of the same species as the "sense antibody") for negative
> controls run on the case tissue.
> If we have background or suspected non-specific (or a surpise suspected
> specific)staining, either on the control or the case, we will often use
> one or more antibodies from the same species but directed at something
> not expected to be seen (a "non-sense" antibody). We may also try a
> different batch of normal serum to see what it shows.
> If there appears to be specific stainig we didn't expect we will run
> several blocks of similar tissue with known "non-sense" outcome using
> the antibody that gave "specific" staining. This acts as a negative
> control for the "sense" antibody (non-sense antigen).
> However, all this is only done if some confounding results pop up. If
> the control is positive, the case is cleanly positive and the results
> are consistent with the history and morphology of the case, we don't go
> through all the bother of "proving" the antibody is staining
> specifically.
> Sorry to say, however, my opinion means nothing to the CAP inspector!
> Tim Morken, B.S., EMT(MSA), HTL(ASCP)
> Infectious Disease Pathology
> Centers for Disease Control
> MS-G32
> 1600 Clifton Rd.
> Atlanta, GA 30333
> email:
> FAX:  (404)639-3043
> ----Original Message Follows----
> Date: Mon, 12 Oct 1998 09:07:00 -0500
> From: "Barnhart, Tammy" <>
> Subject: Negative controls for immunos
> To: "" <>
> Histonetters,
>                 Question, a new CAP standard (08.2257) states, " Are
> negative controls used for each antibody?".  We were wondering how this
> standard is applied at other labs.  At this time we only run negative
> controls on different tissue blocks used, both patient specimens and
> controls.  We use several multitissue control blocks ( one for CDs, one
> for
> keratins, etc) and will run different negative controls for different
> serum
> types (ie: rabbit or mouse).  If we interprete this standard literally,
> we
> will greatly increase the number of slides handled and, almost, double
> our
> reagent costs.  Which brings up a second question, how do you handle
> your
> negative controls?  Do you run them completely, using serum instead of
> primary antibody, or do an abbreviated procedure?  All reponses would be
> appreciated.  Thanks in advance,
> Tammy Barnhart
> ______________________________________________________
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