[Cytometry] Isotype controls: non specific staining controls
Menendez, Fred A.
fmenende at jhsph.edu
Fri May 15 11:23:23 EDT 2009
I'd like to add my 2 cents to this thread.
The quote isn't complete, but as Dr. Varmus writes so nicely in his book The Art and Politics of Science (p103): "I encountered a kind of "two cultures" problems. Most clinicians, even academic ones, and basic scientists, even those trained originally as physicians, do not converse easily. They know different things, seem to speak different dialects, have different goals and standards. Furthermore, the time from biological discovery to clinical advance can be extrordinarily long, and the process may be encumbered with administrative and regulatory hurdles."
This seems to hit the nail on the head. I'm not a doc, PhD or MD, but a med tech. But I've been in the laboratory, clinical and research, long enough to appreciate the complexities of attempted standarization in flow cytometry specifically, and medical arts/sciences generally. Who's standard is best seems to be the $64K question. I'm not an apologist for the CAP, but they do the best they can and attemp to bring some kind of standardization to the world of the clinical laboratory, where, I think, we all can agree there is a need for standardization.
Isotype controls are but one tool in the tool box and used correctly they can be beneficial. We use them routinely, along with other controls, as needed for a specific experiment.
Johns Hopkins Bloomberg School of Public Health
Flow Cytometry Core Laboratory
From: cytometry-bounces at lists.purdue.edu [cytometry-bounces at lists.purdue.edu] On Behalf Of Ruud Hulspas [ruud-hulspas at Cytonome.com]
Sent: Friday, May 15, 2009 10:28 AM
To: SIMON MONARD
Cc: Mario Roederer; cytometry at lists.purdue.edu
Subject: Re: [Cytometry] Isotype controls: non specific staining controls
Reviewers is one thing, but as Carl mentioned, regulatory agencies for
the medical world is another.
(by the way, does a group of pathologists, like CAP, really represent
clinical flow cytometrists best these days ?)
It seems logic that research is ahead in applying the latest knowledge
in flow cytometry and that the clinic is therefore always 'catching up'.
However, it is not clear (to me) how (or even if) the
adaptation/incorporation of improved procedures and technology in
clinical flow cytometry is managed efficiently. If this process is based
simply on the degree of demand from clinical cytometrists, it may not be
the most efficient way. I'm under the impression that regulatory
agencies are significantly understaffed and thus may simply not be able
to catch up as fast as we like. Can we help ?
SIMON MONARD wrote:
> Quoting Mario Roederer <roederer at drmr.com>:
> (PS, the excuse that they keep uneducated reviewers happy makes me
> really cringe. Don't do bad science because that helps get it
> published. Take pride in your work and do it the right way.)
> Mario, I don't think doing unnecessary isotype controls implies you
> are doing bad science, if you are aware that they can't be used to
> determine positivity. You are in a position to argue the case against
> them most eloquently but PhD students defending their theses will find
> it more difficult. Many reviewers have been out of the lab for a while
> but I think the message is getting though.
> FMO controls and using antibody capture beads for compensation seems
> to have really taken off, thanks to you for that! For work we do at
> our place on tissue and embryonic stem cells both FMOs and beads are
> essential, data is just junk without them. We have to couple our own
> beads though as the commercial ones have too low a background. I find
> myself harping on about controls a lot these days.
>> I agree completely, Simon. Isotype controls have their use -- they
>> can indicate IF there is a problem with nonspecific binding.
>> The problem is that many people will rely on them like a crutch -- and
>> use them to indicate HOW MUCH of a problem nonspecific binding is.
>> And that's where they are potentially very misleading.
>> There are no perfect controls -- we should strive to use as many
>> controls as possible to understand our experiments. I generally
>> suggest not using isotype controls because that forces people to think
>> more carefully about the problem and perhaps come up with more
>> reasonable solutions. By taking the crutch away, I think people can
>> learn to walk again! And that gets them ready to run. And then fly!
>> Broken metaphors aside -- we all know that the most common use of
>> isotype controls is to set gates for positivity. And this is the
>> WRONG use for isotype controls. They can help -- they can identify
>> problems -- but you cannot rely on them blindly for this.
>> (PS, the excuse that they keep uneducated reviewers happy makes me
>> really cringe. Don't do bad science because that helps get it
>> published. Take pride in your work and do it the right way.)
>> On May 15, 2009, at 6:19 AM, SIMON MONARD wrote:
>>> I know isotype controls are unfashionable but how else can one control
>>> for "non specific" staining? Some types of cells can show dramatic non
>>> specific staining, M5 leukaemias for instance. Isotype controls may be
>>> far from perfect but are surely better than nothing. Another reason to
>>> use them is to keep reviewers happy who don't know any better. Its
>>> never going to harm your experiment doing them it is just the expense
>>> of buying them and using a few more cells. We all know that they are
>>> not useful for determining what is positive but some sort of control
>>> to see if antibodies are adhering to cells non-specifically would be
>>> useful sometimes. If you work with lymphocytes then this is not an
>>> issue but for some other types of cells it is.
>>> There, I said it!
>>> Simon Monard
>>> FACS Facility Manager
>>> MRC Centre for Regenerative Medicine
>>> Institute for Stem Cell Research
>>> University of Edinburgh
>>> Roger Land Building
>>> West Mains Road
>>> EH9 3JQ
>>> Tel. Lab 0131 6505876
>>> Tel Office 0131 6517265
>>> The University of Edinburgh is a charitable body, registered in
>>> Scotland, with registration number SC005336.
>>> Cytometry mailing list
>>> Cytometry at lists.purdue.edu
>> Cytometry mailing list
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> Simon Monard
> FACS Facility Manager
> MRC Centre for Regenerative Medicine
> Institute for Stem Cell Research
> University of Edinburgh
> Roger Land Building
> West Mains Road
> EH9 3JQ
> Tel. Lab 0131 6505876
> Tel Office 0131 6517265
*Dr. Ruud Hulspas, Ph.D.*
Director of Cytometry
*/Cytonome/ST, LLC/* <http://www.cytonome.com>*//*
27 Drydock Avenue
Boston, MA 02210
phone: 617-330-5030 x226
RHulspas at cytonomest.com <mailto:RHulspas at cytonomest.com>
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