Isotype "controls"

[Mario Roederer]

OK, first let me say that I put in the statement 

> Besides, "we" should all stop using isotype controls to set gates.

primarily to see how awake everyone was.  I must admit, I was (pleasantly)
surprised by the level of attention!  Of course, now I feel guilted into
actually responding.  Especially after Alice's recent posting.

First, let me thank Phil McCoy for pointing people to the published paper about
the use of isotype controls.  This is an old topic, older than FACS technology,
and it has been dealt with many times over the years.  What follows below is my
own discourse on the topic, uncolored by the rational arguments put forth over
the past decades.

Ray Hicks succinctly addressed some of the problems with isotype controls; I'll
provide a little more detail.  There are two principle issues:  (1) the concept
of isotype controls, and (2) the use of isotype controls to set gates.

First of all, let's consider the whole point of "isotype controls."  They are
meant to approximate the background binding of your conjugated antibody to cells
that wouldn't specifically bind your antibody (i.e., don't express the antigen).
But this means that we would have to use a control antibody that is (1) the
exact same isotype; (2) conjugated to exactly the same degree; (3) has the same
background binding characteristics as your antibody; and (4) is used at the same
concentration.  Rarely is more than the first criterion met.

(1) Exact same isotype.  OK, how many of you actually purchase DIFFERENT isotype
controls and use them for every different isotype in your experiment?  I would
wager a beer at the next ISAC that not a single lab on this planet does this.
While most reagents are IgG1, there are plenty of G2 (a or b), some G3, etc.
And there are some IgM's--arguably with enormous differences in background
binding compare to IgG's.

(2) Conjugation.  In trying to estimate the background, obviously the F/P (fluor
to protein) ratio is crucial.  After all, if you double the number of fluors on
your conjugate, you will double the background.  Therefore, the isotypes should
have the same conjugation ratio as the antibody you are controlling.  While this
conjugation ratio MIGHT be consistent for reagents from a single manufacturer
(and it rarely is, at that), it certainly will be different for reagents from
different manufacturers.  

(3) Furthermore, there is the problem that even at the same F/P ratio, the
conjugates could be significantly different.  It is quite possible that in one
antibody there is a fluor at a critical "background" binding site; on another
antibody, this site is unconjugated.  This could significantly change the
"stickiness" of an antibody.

For example, when you conjugate antibodies to Texas Red, you can get hugely
different "background" binding characteristics depending on the reactive form of
TR that you use--even after getting exactly the same F/P ratios.  Clearly, the
sites on an antibody that are conjugated are different by these different
reactive forms of TR, and those differences translate into different
"background" binding characteristics.

(4) Concentration.  OK, what concentration do you choose for an antibody in an
experiment?  For a regular antibody, you choose the "saturating" concentration.
For a control, there is no such thing as saturation; the more antibody you use,
the more background you get.  Therefore, the pragmatic approach is to use the
same concentration as in your original reagent.

And there's the rub!  Each conjugated reagent has been carefully titred
(hopefully) to be used at the proper minimal saturating concentration.
Therefore, every different antibody can potentially be used at a different
concentration!  Do you therefore prepare a different isotype stain for each
different concentration of conjugated antibody?  Of course not.  So how can you
claim that the isotype control is even valid as a control?

Calman Prussin and Brent Dorsett ask about isotypes having higher backgrounds.
Recently I spoke with someone who had this same question.  This researcher
contacted the manufacturer of the isotype control, who told him that he should
simply dilute the isotype control until the background was down!  This smacks of
homeopathy, doesn't it:  "Our isotype control works better the more you dilute
it!"  As Ray asserts, now one has to use a little black magic in waving ones
hands and ignoring the isotype control for these samples but not for others!

This brings me to the second major problem, the use of isotype controls to set
gates.  Unfortunately, this is not a problem that will go away when people stop
using isotype controls; most will simply use unstained cells to set gates.
(Right now, however, the isotype controls lend an inappropriate air of validity
to setting the quadrant gates).  By the way, "we" should stop using quadrant
gates!

The problem with using isotype controls (or unstained cells) to set gates
blindly is that many antigens do not should bimodal expression patterns that are
either "on" or "off".  Many are expressed even on "negative" cells; and, the
brighter your reagent is, the more off of the bacgkround these cells will be!

An excellent example of this is the expression of CD45RA on T cells.  In the CD4
population, there are 2 reasonably distinct populations, RA+ and RA-.  In the
CD8, there are also two populations, but the lower population expresses a
reasonable amount of CD45RA.  If you were to use an isotype or background
control to gate on CD45RA, you would include many of the "dim" population (which
are memory cells) in the CD45RA+ gate (with which you are trying to select naive
T cells).  Furthermore, the gate that you need to use to distinguish the CD45RA
populations is very different for CD4 cells and for CD8 cells.  

There is no easy solution to these problems.  Gating is an art, one which
requires considerable experience and knowledge of the system.  (Hence job
security for FlowJocks).  Blindly using isotype gates or background, or blindly
using quadrant gates (because you are lazy) can only lead down the path marked
"Artefact".

I do want to reiterate what Ray Hicks said:  Isotype controls can be a valuable
tool for rooting out problems.  However, it is a rare problem that will be
solved with isotype controls.

mr