OpenCyto -- proper gating of CD45
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@ulrik-stervbo-1723
Last seen 5.6 years ago
Germany

Hello,

I am trying to analyse monocytes, lymphocytes and granulocytes by CD45/SSC. I am not sure how to do the gating with openCyto. Attached is an immage of how I'd do it by hand, and what I can get so far with open cyto.

Some cells are not lysed and must be removed - they are low in CD45 and side scatter (they cannot be removed by Forward/Side scater gate). The problem is that immature granulocytes are also low in CD45, but high in side scatter. For now I have a boundary gate to remove the lysed cells, but that also takes a big chunk of immature granulocytes.

Basophils, lymphocytes and monocytes are high in CD45 and low in side scatter, though monocytes have slightly higher side scatter than lymphocytes, but much lower than eosinophils, which have high CD45 and side scatter. 

We want to know the proportion of granulocytes including the immature and go on to analyse eosinophils and neutrophils. We are also interested in basophils, lymphocytes and monocytes which we extract in later gates.

What is the best way of going about this with opencyto? Is there way of handling the diagonal between granulocytes and lymphocytes? Will polygons be adjusted per sample?

Thanks

Ulrik

opencyto gating flowworkspace • 1.7k views
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Jiang, Mike ★ 1.3k
@jiang-mike-4886
Last seen 7 weeks ago
(Private Address)

Maybe you can use flowClust to gate out the granulocytes first. Then separating the unlysed from lymph can be easily achieved by any 1d gating method.

Here is the example of how to set target for flowClust 

http://rpubs.com/wjiang2/176974

which can be easily translated into your template.

 

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I am using flowclust.2d to identify granulocytes, but it always sems the gate is either too narrow and excludes too many immature and eosinophils, or becomes far too long and includes - depending on sample - to many lymphocytes.

flowClust.2d: quantile=0.83, K = 2, target = c(5,600), with the prior_flowClust, K=2.

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