Gates obtained by define.gate.landmarks() with X-coordinates outside of the allowed range

[pbMCs]

Hello! I am using a dataset acquired on a FACSDiscover A8 and am trying to define a monocyte landmark gate with define.gate.landmarks().

My understanding is that the function identifies the events expressing the marker used to define the population (in my case TLR2 in the YG2 (590)-A channel), selects the brightest events according to n.cells, and then uses their FSC/SSC distribution to define a scatter gate. This gate is then used to identify the corresponding population in the other single-colour controls that have monocyte specified as their gate. The gates are defined correctly but when I run define.flow.control () it gives me this error:

Error:
! Gate 'monocyte' has X-coordinates outside the allowed range
[0.000000, 24140237.000000].

The ranges in the scatter for my actual fcs files are much larger than the range defined in asp, is that normal? Or did I do something weird during the export?

I am using:

asp <- get.autospectral.param(
cytometer = "a8",
figures = TRUE
)

gate.monocyte <- define.gate.landmarks(
control.file = UpdatedName,
control.dir = RCLocation_cells,
asp = asp,
n.cells = 2000,
percentile = 70,
gate.name = "monocyte"
)

If I change the scatter.data.max.y and scatter.data.max.x in the asp object the solution works, but I don't know if that is a correct approach.

Thank you,
Mar

[DrCytometer]

Yes, this is an issue that is patched in the next release. What you've done to change the scatter.data.max.y and scatter.data.max.x in the asp object is exactly correct, and is what the patch does. The problem with many of the new cytometers is that they have huge ranges for scatter, and it's not easy to find a good default viewing section. I'll see if I can figure out a smarter way of handling it.

[pbMCs]

Thank you so much!

[DrCytometer]

The patch for this is now released as part of version 1.8.0. You'll want to update AutoSpectralRcpp as well if you use that. There are several performance improvements in this version, so you should see processing times come down.