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DIVA FCS3.0 linear output - Linear MFI when displayed in Kaluza?


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Dear Flowers

 

I have a question from a user below which I hope you can shed some light on. They use a BD Canto II and Kaluza for analysis. They export data from BD FACSDiva as FCS 3.0 (which is in linear format), and want to know if there is a way of obtaining the linear MFI as opposed to the log MFI which is what Kaluza is providing. Data is saved and displayed as log on DIVA and so cannot be displayed as linear. However exporting in FCS3.0 is linear. Is there a formula which can be applied to get the channel / bin number???

 

Thanks

 

Maree

 

 

 

 

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Dear Maree,

 

The best format to export BD data for use in Kaluza is FCS3.0, so what is being done is correct. The data in the FCS3.0 file is linear; Kaluza does the log transform.

 

Where I think there may be some confusion is where you say "the log MFI which is what Kaluza is providing".

 

The first thing that needs to be clarified is what is meant by the "M" in "MFI". If the "M" stands for "Median" then know that Kaluza calculates median based on the scale of the plot. If the "M" stands for "Mean" then know that Kaluza calculates that arithmetic and geometric means independent of the scale of the plot. You should use arithmetic mean for populations that are normal (gaussian) in linear space and geometric mean for populations that are normal in log space.

 

What I suspect may be going on is there may be some confusion over scaling that Kaluza does. Kaluza has a feature that scales all data to a 0 to 1024 range before it is displayed. This feature makes it easier to compare data between different instruments, but it can create confusion when comparing results to other packages. We're likely to offer a feature to disable this feature in a future release.

 

You will find that for most BD data, the values for statistics such as mean and median will be 256 times larger in Diva than Kaluza. So if Kaluza is displaying a median of 0.44, Diva will be displaying a median for the same population of about 112. You can use the information plot to do this math if you want to see un-scaled values within Kaluza. When you add statistics to an information plot it adds an expression such as '=MEDIAN("A", "488->585-A [Log]", 1)'. You can modify this expression to include math such as '=MEDIAN("A", "488->585-A [Log]", 1) * 256'. The formulas follow the same general rules as Excel formulas.

 

If you want to get "channel / bin" number, there is a way to calculate it (although you'll have to do it outside of Kaluza). That depends on how many bins you want though.

 

If you can provide some more information on what is required, with some examples of what is desired versus what Kaluza is displaying, I can be more specific.

 

Regards,

Ernie

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  • 1 year later...

Hi Ernie,

 

does this mean that flow data analysed with e.g. flowjo displays a median fluorescence intensity 256 times greater than what kaluza displays?

 

I tried to include the *256 in the formula on the information plot but I get an error message - most likely because I am doing it wrong.

 

Here is what the formula looks like in Kaluza - could you perhaps show me where to insert the *256?

 

="[" & GATENAME("Dead cells", 1) & "]" & " " & PARAMETERDESCRIPTION("FL8 INT", 1) & " " & "Median"

 

Thanks very much,

Silke

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Hi Silke,

 

It looks like you're trying to put the *256 on the name side. Each line of the information plot has a name side (left) and an information side (right). Try clicking on the cell on the right side and see if the expression makes more sense.

 

Your values in FlowJo will depend on how you have it configured, but yes, normally I would expect FlowJo to display BD values on a 0 to 262,144 scale, so these would be 256 times bigger than the values that Kaluza would display.

 

Ernie

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Sadly, no, you'll need to do this nearly every time you want it. You can do a few things with copy/paste, but unless the parameters are all exactly the same you still have work to do.

 

We're working on a different solution for this specific situation, although I can't say when it'll be released.

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  • 4 months later...

Hi Ernie,

 

another question regarding MFI.

Is there a way to calculate the mean or median for samples with different numbers of events?

 

I tried to look at MHCII expression on CD11c+ cells.

The number of CD11c+ cells varied in each sample so the median MHCII varies as well. Could I tell Kaluza to e.g. only analyse the median for let's say 20,000 events in each sample?

Or is there an easier way to do it?

 

Many thanks,

Silke

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The best recommendation I have at this point in time is to put a gate on TIME earlier in the gating hierarchy. Adjust that gate so you have exactly (or nearly exactly) the desired number of events. Unfortunately you'd have to tweak the gate for each sample. This is something we're thinking about for a future version.

 

Independent of the question about limiting the number of events, both mean and median are "central tendency" statistics. They should be largely independent of event count. Some degree of variation is to be expected just due to the nature of population statistics. The only affect that event count should have is that larger event counts should start to stabilize around a central value.

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