Thanks to all who took the time to reply to my request for quidelines in selecting
isotype controls. My initial search of the list archives did not reveal the information
posted previously; so thanks, Ray, for directing me to the discussion. If there are
publications on this topic, it would be a service to post references to the list.
FROM THE LIST ARCHIVES
Calman Prussin (CPRUSSIN@atlas.niaid.nih.gov)
Wed, 1 Apr 1998 22:24:08 -0500
Mario Roederer noted:
> Besides, "we" should all stop using isotype controls to set gates.
>
> mr
This brings up two questions:
1.) Mario, what "should" we be using? I don't remember hearing this
one from you.
2.) How can one set statistical markers to determine the frequency
of positive staining cells in a user independent manner? Of course, much of
the time it is obvious, but when you have small frequencies, the choice of
where to put a marker can make a world of difference. A system that is
independent of user bias would be great. The positioning of markers such
that the negative controls yields a given frequency, say 0.1% makes sense,
but I would be interested in the groups comments. Let me clarify that I am
addressing data in which there is a bimodal distribution, rather than a
distribution as is seen with many adhesion molecules.
Calman
J. Philip McCoy, Jr., Ph.D. (pmccoy@UMDNJ.EDU)
Mon, 06 Apr 1998 08:35:40 -0700
I remind all that this is not a new topic of Mario's origin. The
published recommendations of the Leukemia/Lymphoma Consensus Conference
address the lack of need to use isotype controls. Refer to Cytometry
30(5), 1997 for these manuscripts.
-Phil
Brent Dorsett (brentd@nyct.net)
Sun, 05 Apr 1998 19:37:05 -0400
I don't mean to state the obvious, but when commercial suppliers choose
isotypic controls they simply evaluate a range of clone for levels of
non specific binding and choose one in the middle ( so I am told, by
them that does it ). When we apply them to everyday assays, they are at
best a compromise. Frequently it's obvious that the negative population
binds much less than the isotypic control does. It requires experience
and judgement to decide to decide how to evaluate the results, prompting
some non-flow cytometrists to suspect it's a little bit black magic.
Don't get me wrong, I think isotypic controls are a valuable tool for
rooting out problems. I just think they have to be used with common
sense. Maybe more than most topics of discussion, I welcome this one. I
think it's past due.
Brent
Mario Roederer (Roederer@Darwin.Stanford.EDU)
Fri, 10 Apr 1998 09:02:32 -0700
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
Dave Coder (dcoder@u.washington.edu)
Mon, 13 Apr 1998 10:43:09 -0700
To Mario's thoughtful response, I'd toss in a couple of ideas.
That people are thinking of using experimental controls (in larger sense) is
good and should be encouraged. But the crux of this whole discussion is the
nature of the appropriate control.
A couple of things to remember:
1. Flow cytometric measurements are measurements of light intensity.
Further, the measurements are comparative not absolute.
2. How you design the experiment depends on the question you wish to answer.
Given that measurements are comparative, you have a couple of choices: 1.
choose a good control, or 2. calibrate your instrument response with a
standard that yields biologically sensible units. As Mario pointed out,
there are number of factors make the selection of a good--i.e.,
proximal--control difficult.
(It's more a philosophical discussion as to whether you can ever get the
absolutely proper control. It may possible with an individual animal if
control serum is taken prior to immunization with the specific antigen of
interest. Of course, you would take preimmune serum only after having done a
sham immunization with any adjuvants or carriers used when challenging with
antigen; your antigen should be absolutely pure, too. Then you can do the
fluorochrome conjugation to give exact dye/protein ratios, and use precisely
the same same protein concentration for each, etc. Most antibodies, however,
are monoclonal so you really can't get the "perfect" control.)
Even if you select a proximal control, there is no guarantee that it
performs properly. (I have heard of isotype-matched control antibodies that
have different isoelectric points, perhaps explaining some nonspecific
binding differences.)
More attractive is calibrating the fluorescence intensity scale to a unit
that answers your question. Conceptually this is very attractive, and
currently there's some agreement about how to go about this, but there is a
lack of an independent standard.
Point #2: How you proceed depends on the question you want to answer.
Consider the following:
A. How many cells of a certain type are present?
B. What is the proportion of cells in the population that express an
antigen?
C. How many antigen molecules does a cell have?
D. How many antigen molecules does the "typical" cell have?
These are all different questions and different approaches may be used to
obtain answers.
The first can be very complex taxonomically, but in practice it is fairly
simple. Perhaps 4 or 5 parameters are measured, and gating may be used to
define a cell type as a subset of all cells examined. Where subsets are
clearly present (discrete not continuously variable parameters), controls
may not be needed. (A related example is setting compensation without
single-labeled controls. You can do this easily on normal peripheral
lymphocytes whose surface CD8 or CD4 are labeled with FITC or PE. You know
in advance what the population distributions are so you know what a properly
compensated bivariate distribution looks like.)
B. If you wish to know the proportion of some cell type among all cells in
the population, then the procedure is simple. But (there's always a "but"
isn't there?), if and only if, the population subset of interest is
discrete. If you can define the population using +/- classification, then
quadrant measurements of distribution are useful. All bets are off, however,
if any parameter distribution used to define the subset is continuously
variable. Take activation markers for example.
C. and D. This is like the old scholastic question: "How many angels can
dance on the head of a pin?" Quantitation of antigen expression is a bit
sticky as noted above, because the means for doing calibration and
standardization are not universally accepted. Continuously variable
distributions of antigen expression are best described if you can measure
their number. In contrast to quadrant measurements (or other proportional
measurements) that disregard the thee level of expression, quantitative
cytometry takes advantage of the instruments' capabilities and is one the
chief powers of the technique.
D. This brings us to the often discussed issue of measuring the "average"
cell. Without going into detail, I'll only point out the distribution of
cell surface antigens is not always log normal. (In fact, I have yet to see
any that are demonstrably log normal. See Coder et al. Cytometry 1994 18(2)
75-8 for discussion.) If a distribution is skewed, then the median is a
simple indicator of the "typical" cell. Some classifier range is probably
better, but how do you chose the limits to such a range? Quantiation of a
cellular property could define a range, but then how much of the
fluorescence is due to background? Hence, you need a good control.
And I'll stop the ramble here for now.
Dave
dcoder@u.washington.edu
Lucille H Kimura (lucille_h.kimura@tamc.chcs.amedd.army.mil)
Mon, 13 Apr 1998 17:56:56 -1000
Thanks to Mario Roederer, Dave Coder and others who have taken the time to
organize and put in writing some of the critical issues concerning the use of
isotype controls. As much as I feel we're wasting money running isotype
controls for peripheral blood lymphocyte immunophenotyping of immunodeficient
patient samples, there are many times that we are forced to depend upon
isotype controls to help us interpret results:
1) Many of us have had to analyze cell lines which have higher
autofluorescence and isotype control fluorescence than lymphocytes. The CAP
leukemia/lymphoma proficiency survey samples are cell lines, which make
interpretation difficult because any positive staining for an antigen
appears as one peak, with no negative population for that antigen to help
define a negative region. We are required to respond to each antigen
analyzed as negative, dimly positive, moderately positive or strongly
positive, depending upon the degree of overlap or intensity of the positive
peak compared to our isotype control. As such, our interpretations (and
misinterpretations) are unfortunately highly dependent upon the nature of the
isotype control.
2) Treatment of cells for cytoplasmic antigen detection results in overall
increased levels of fluorescence in both negative and positive cells.
Isotype controls have been helpful with leukemic samples stained for
cytoplasmic antigens but often confuse the interpretation of cytokine
analyses, especially with stimulated cells.
As biological materials are difficult to standardize, it appears that we must
continue to practice the art of flow cytometry with caution and hope that one
day someone will figure out a way to block all 'nonspecific' binding and make
isotype controls a thing of the past. I'm not holding my breath.
RECENT e-mail RESPONSES
"Pizzo,Eugene" <Pizzo@NSO1.UCHC.EDU
Re: selecting isotype control
It should be the same isotype and a similar specificity (Ag and species),
e.g. a good isotype control for an anti Mouse class II I-E/k (IgG1)
would be an anti-class II I-E/b (IgG1)
Gene/(flowcytometry.uchc.edu)
"Dennis J. Young" <djyoung@ucsd.edu
Re: selecting isotype control
It depends on what you are trying to use it for.
First, avoid using one if you can. Use an antibody that is negative for
your test antigen, but positive for a control antigen.
Second, any antibody that is of the same isotype, used at the same molar
concentration.
The assumption is that the control will have the same non-specific binding
affinity for your antigen of interest (and other components) as your test
antibody (which is highly unlikely). Unfortunately, most commercial
"control" antibodies have less non-specific binding than your test
antibodies because they were selected to have low background.
Dennis Broud 301-827-0966 FAX 301-594-3037" BROUDD@cder.fda.gov
RE: selecting isotype control
should be the same immunoglobulin subclass from the same species labelled with
the same fluorochrome as the specific antisera that you need a control for.
The key to using this control is that you must know the protein concentrations
of both your specific and isotype control serum. If the concentration is the
same as that of your specific antisera , then you would use the same dilution
of the isotype control as you do of the specific antibody. The isotype
control tube then becomes the tube on which you set your boundary for where
the cutoff is between Positive and negative would be for any specific antibody
of the same immunoglobulin subtype with the same labelling fluorochrome. You
should of course continue to run an unstained control as well. A comparison
between the unstained control and the isotype control will give an idea of the
socalled "nonspecific" binding that occurs with a specific immunoglobulin
subtype and the particular specimen that you have. One thing you should
always remember --- Antibody likes to stick to something, so if you have cells
in a protein free media/buffer, and you add antibody to the mix, it will stick
to any and all surfaces, cells tubes, etc pretty easily. If at all possible
you should keep your cells in a media, or buffer that contains some protein.
This will help you prevent a lot of nonspecific binding. The very nature of
most of the antibodies that we use today (IgG subclasses for the most part) is
that they work best in a protein rich environment. When you put them in a
strictly ionic system, neither the cells nor the antibody are working with the
normal forces that apply in vivo. So for example, I dilute all my antibodies
in Dulbecco's PBS without Ca++ and Mg++ (lack of these cations helps prevent
clumping of cells) to which I add 1.0% Bovine Albumin (the best I can afford-
usually Sigma's low endotoxin stuff so I don't stimulate the cells, and I add
a little bit of sodium azide ( 0.1%) to this to prevent capping. Of course,
the sodium azide also prevent contamination. I use this proteinated buffer as
my diluent for all antibodies, most cells, and as my wash solution after
staining. This way I keep my cells in the same buffer all the time, and I'm
not changing the tonicity of the solution that they are in. Keeps the cell
size the same basically so you don't get a lot of fuzziness on you forward/ssc
plots. Anyway got to go now, have a g'd day!!!!
JElia32589@aol.com
Re: selecting isotype control
Barbara,
your question poses an interesting set of answers. #1 the isotype control is
an irrelevant antibody of the same isotype your antibody of choice is. It
is intended to tell you if that isotype is "sticky" or not. unfortunately
some isotype controls are stickier then the antibody of interest. ( I
personally try to avoid them) #2 Isotype controls give you background color
for the fluorochrome you are staining if it is a direct conjugate. Keep in
mind that in order for an antibody to become an isotype control it is tested
to make sure that it stains nothing. So I use them for background
fluorescence of the conjugate, but not necessarily to test if that isotype is
sticky or not.
"Durett, April G." <agdurett@txccc.org>
RE: selecting isotype control
Barbara,
There are about as many opinions on the use, abuse and validity of isotype
controls as there are controls!! To be absolutely correct, each antibody
panel should have a corresponding isotype control which is protein,
fluorochrome matched, and source to the corresponding antibody. Controls
will vary by F-P ratio, but it is not a perfect world in the land of Flow
QA/QC. Since the majority of the antibodies are G1, I set up a single
control choosing the maximum protein concentration (500ng for CD34) for each
of the fluorochromes. Additional controls should be set up if (1)antibody
sources differ within the panel, ie if using a Coulter mAb conjugated to
FITC, do not use a BD isotype as the F-P ratios may differ or (2) antibody
is other than G1 isotype, ie CD5 IgG2a or CD15 IgM.
Using isotypes to set gates, ie negative populations, can be misleading, as
antigens do not show bimodal expression patterns that are wither negative or
positive. Many antigens are expressed on negative cells and the brighter
the conjugated antibody the 'less negative' these cells will be. Thus the
flourochrome choice becomes important, although many times limited to
availability.
I'm personally not a true believer in isotypes, however I run them to pacify
the various investigators. My experience with isotypes and quad stats is
that sometimes a population is positive for the isotype, however not in the
same location as the positive population and subtraction of events would not
be relevant to evaluation of the positive population. In addition, the
majority of my analysis is using sequential logical gating and this solves
the problem, as if there are non-specific events they will be in the same
location as the positive population.
Hope this is of some help,
april
April G. Durett, MSc
Kerstin <beckk@sun11.ukl.uni-freiburg.de>
Isotype <-> unstained cells
hello everybody,
what do you use as a negative controll (cells not expressing the marker) in
FACS-analyses? Cells stained with the isotype or unstained cells?
I started stainig cells for FACS-analyses for about 2 years and I always
used isotypstained cells as a negetive controll (=marker for unspecific
binding and for correcting the compensation). Someone told me, that
Isotypestaining is not a good negative controll because the Isotype is from
a different host, and can`t representate the unspecific binding. So it
would be better to use unstained cells.
What does you use/Think about this ?
Thanks
Kerstin
Ray Hicks <rh208@cam.ac.uk>
Re: Isotype <-> unstained cells
To emphasise the usefulness of the replies copied to the list:
Hi Kerstin,
There was quite a lot of discussion on the list in April 1998 on the
appropriate use of isotype controls, Mario Roederer and Dave Coder discussed
a range of issues to consider when designing a controlled experiment. You
should be able to find the discussion in the archive, although the new
search feature at Purdue doesn't seem to extend back that far - you can view
the thread(s) at:
http://www.cyto.purdue.edu/hmarchiv/98/index.html
either scroll through the messages or use your browser to search for the
text "isotyp".
Ray
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