How to determine percentage of T-cell subsets by flow. Basic math help. - (Apr/17/2009 )

Hello Everyone,

I am starting a new project. Though I understand the concept I have never worked with flowcytometry before and I am very confused with how to calculate the final percentages of cells.

I have been given several flowcytometry reports and have been asked to determine the percentages of:

1. gamma-delta/ CD8 +
2. apha-beta/CD8+
3. gamma-delta/ CD8 -
4. CD4-/ CD8- / gamma-delta
5. CD3-/CD4-/CD8-


For some reason when I start subtracting one population from another that sounds logical to me I get results not so logical. The acquisition was done years ago and repeating it with other Ab pairings is not an option. These were the Ab pairings used:

a. CD4/CD8
b. Gamma-delta/alpha-beta
c. CD8/CD3
d. CD4/CD3
e. CD4/CD8
f. CD16/56 /CD3
g. CD19/ CD3
h. CD19/CD16/56
i. CD3/CD103

If anyone can help me with basic math I would appreciate it.
Thanks.

What do you mean by your results are "not so logical"? You should be able to use the analysis software to gate the cells based on a threshold and calculate the percentages in each population automatically. What program are you using for analysis?

gfischer on Apr 17 2009, 01:27 PM said:

Thank you for your reply, gfischer!

I do not have any software. Just a bunch of printed papers with dot plots. My PI gave me an example of how to calculate CD8-/gammadelta. He suggested subtracting gamma-delta % from the CD8-/CD4- %.

Example:

CD4+ 6
CD8+ 9
ab 15
gd 6
No TCR 78
CD4-/CD8- 82
CD3- 69
CD3+/CD8- 10
CD3-/CD8- 80
CD3-/CD4- 78

In the following case if I do that it will give me:

Gamma-delta/CD8- = 82% -6% = 76%

But how can there be 76% of gamma-delta/CD8- cells if 78% have no TCR?
This is what I do not find logical.

Thanks again if you can help me calculate all those subsets!