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In a tube that combines CD2(FITC)/CD4(PE)/CD8(PCP) mAb's, which values
are most appropriate to use to calculate the CD4:CD8 ratio
correctly.Would one use:
(i) the total %CD4 and the total %CD8 derived from single parameter
histograms?
or
(ii) the %CD4+CD8- and the %CD8+CD4- derived from the CD4 vs CD8 dot
plot quadrants?
or
(iii) Should one be using the %CD2+CD4+ and the %CD2+CD8+ values
(Something that was just recently suggested to me)?
RESPONSES Rec'd Included:
(1) From <April_G._Durett/MDACC%MDACC@notes.mdacc.tmc.edu>
We are using a similar combination in evaluating our human
blood-marrow transplants, using CD4FITC/CD8PE/CD3PerCP.
Our evaluation is performed within the lymphocyte region and we use
the double positives of CD3+CD4bright and CD3+CD8bright, the
corresponding dim populations usually appear on monocytes or early
lymphocytes. My experience with CD2 in human is that it is also
expressed on rosetting lymphocytes, Tcell subsets, and NK cells. We
are using a logical gating and color definition technique that gives
us results for all possible permutations and can be visualized on a
two-axis dot-plot . It makes life a lot easier than trying to
calculate from multiple two-axis plots.
(2) From Simon Monard FACS Lab Manager
I would suggest you use CD3 instead, there is a Macaque CD3 available
from Biosource (ph 1 800 242 0607) works fine. Using CD2 can lead to
misleading results as NKs which can make up a considerable and
variable proportion of your cells stain for CD2 but not CD3.
As for ratios I would use %CD3+CD4+ : %CD3+CD8+
Dunno what to do about the double positives
(3) From Karen Henell, Oregon Health Sciences University
Portland, Oregon 97201 USA, <henellk@ohsu.edu>
>Diane: There is an antibody called FN18, not available commercially
>as far as I know, which might be available by contacting Margreet
>Jonkers in Holland, which reacts nicely and cleanly with macaque CD3.
>I've used it myself on cynomolgus monkeys (M. fascicularis).
P.S NB/ It is now available through BIOSOURCE (DTL)
If it were my study and I couldn't add an anti-CD3 or anti-NK, I'd use
the %CD2+CD4+CD8- corrected for monocyte contamination divided by the
%CD4-CD8+bright (to eliminate the NKs which are generally dimmer for
CD8) for a T4:T8 ratio in cynos. You already realize that without an
anti-CD3 or an NK marker, you will have a difficult time keeping NKs
out of your ratio. Monos will screw up your ratio if you're not
careful, and I've had high CD2 backgrounds in monkey monos.
(4) From Henk Niphuis <niphuis@bprc.nl>
Our mouse anti CD3 antibody (FN18-FITC, BIOTIN and unlabelled) is now
available. In the USA contact Biosource (fax (805-987-3385),
in Europe Dr P v/d Meiden (fax:+31-15-284-3999 or
e-mail:meiden@bprc.nl) at the BPRC in the Netherlands.
For CD4/CD8 ratio you should take CD4+CD3+/CD8+CD3+, and only the
cells within a lymphocyte gate.
CD8+CD3- cells are "NK-like cells" and are sometimes more than 50% of
your total CD8+ population in macaques.
(5) From Ana K. Stankovic, MD, PhD Department of Pathology
University of Alabama at Birmingham
The best way to calculate the CD4/CD8 ratio is to gate on the CD2+
cells (FL1) and then display FL2 vs. FL3 (namely CD4 vs. CD8) of
the gated cell population, and get the actual ratio from that plot.
This is done because some non-T cells (CD2-) can express CD4 or CD8
antigens.
(6) From Dr. P.J.D. Bouic, Head: Immunology
Tygerberg Hospital, SOUTH AFRICA, <pjdb@maties.sun.ac.za>
You would have to use the CD2+CD4+ vs CD2+CD8+ values to work out the
ratio since not all CD4+ cells are true helper cells and similarly,
not all CD8+ cells are T suppressor/cytotoxic cells.
(7) From Jan Nicholson CDC Atlanta, GA <jkn1@CIDDAS1.EM.CDC.GOV>
In humans, the correct way is to use data only from the T cells and
not monocytes or NK cells. CD3 does not normally label either of
these populations. CD2 (in humans) does label the NK cells, so unless
the monkeys are different from humans, use of CD3 to distinguish T
cells from NK cells is not useful. CD2 should not label the
monocytes, so you are OK there. I believe, in general, for monkeys,
the CD4 and CD8 ratio are determined based on CD4 and CD8 only so that
the double-positives would be included for both determinations.
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