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Composites and logicle scales in Kaluza


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Hi.

 

I got a problem regarding the analysis of flow cytometry data in a logicle scale within composites in Kaluza. We are measuring 15 different stainings in whole blood of the same person. So we created a composite out of 15 different single protocols. The composite was necessary for linking gates between between the 15 stainings. For analysis we like to use the logicle scale to adjust cell populations for compensation.

 

My problem: when I define a setting for a logicle scale ("decade" and "negative percentage") for one fluorescence channel in one staining (e.g. staining 1: FL4 - CD4) than Kaluza defines the same settings for the same fluorescence channel in all stainings (e.g. staining 2: FL4 - CD86 and staining 3: FL4 - CD14) within this composite. Modifying the settings for logicle scale in one staining (or single protocol) leads to changing the settings for all stainings. Is it possible to create settings for every single staining or protocol within the same composite? Because it is possible to define different compensations for every staining or protocol within the same composite, so there should be a possibility to also adjust the logicle scale for this different compensations.

 

I hope someone could help me.

Thx paul.

 

 

additional info: we are currently using Kaluza version 1.2

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Hi Paul,

 

First, I'd like to apologize for the delay in your post appearing in the forum. To prevent spam, we have to approve posts before they will appear.

 

There is a solution available for this. The logicle settings for a parameter are tied across data sets when the NAME of the parameters match. So if you have three data sets and they all have a parameter named "FL2" then the logicle settings for each one will be tied together. However, if each one has its own name (say "CD4-PE", "CD8-PE", and "CD19-PE") then the logicle settings will be independent for each one. The description of the parameters is not taken into account.

 

If you make the names of your parameters meaningful before you acquire your data then you will automatically avoid this problem. Obviously, you already have some data acquired so you'll need to have a way to work with that. You can change the names in the parameters pane of each data set. In a composite there's a data set drop down at the top of the parameters pane so you can see each one. If you want to make the same changes to a series of analyses, you can copy a line in the analysis list and then right click on the destination analyses and choose "Parameters" from the "Paste Special" menu. It may be easier to do what you want to do if you get the parameters named correctly before you create the composites.

 

Ernie

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Hi Ernie,

 

thank you so much for this solution. I just tried it out with same data and it is just working fine. This is great! However, now this brings me to a new question. Could you may explain me how to change the parameter names when aquiring data with the Galios cytometer. Until now, I only changed the parameter descriptions which are also got imported into Kaluza. For the already measured data I' m going to change the parameter names as you explained. But for future measurements (which are a lot) it woult be great to already adjust the parameter names in measurement. I just tried to figure out how to change the parameter names but couldn't find a way.

 

Best regards,

Paul

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Hi Paul -- There are a couple of ways to assign parameter stain names (PnS) in Gallios. This should be done prior to acquisition.

 

To save a protocol with parameter stain names:

  1. Go to File >> Edit FCS Header Attributes.
  2. Select the parameter you want to assigne on the left side of the dialog and enter the desired name.
  3. Repeat Step 2 for all parameters you want to assign a name to.
  4. Select OK to close the dialog and save the protocol.

If you use one protocol and you run multiple tubes with different antibodies, then you may consider creating a panel in Gallios and enter the parameter stain names in Acquisition Manager.

  1. Open the desired protocol into the CXP workspace.
  2. To view the parameter name field in Acq Manager, select View >> Customize Worklist Columns.
  3. On the Customize Worklist Colums dialg, scroll down to see the P1 (Parameter 1) check box and check it, then select OK to close the dialog. Acquisition Manager will now display a column for each parameter in your protocol.
  4. Use the "Insert Test" button (the button icon looks like a single test tube with a black arrow) to add tests for this panel. It will duplicate the protocol selected in step 1).
  5. For each test in this panel, click in the parameter cells and enter the parameter stain names.
  6. Right click anywhere in the green and white space of Acquisition Manager and save the panel. When you run this panel, the plots will update with the parameter stain names when acquisition commences for each tube.

 

With either method, having the parameter stain name present before you acquire the tube assures that the data file will have the parameter stain name included and you will be able to use Kaluza composites and the Logical Scale as Ernie describes.

 

I hope this helps! Thank you --Karen


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Dear Karen,

 

thank you very much for your respond. But I think what you just described was how to change the stain names for every channel. This I already did, but it seems to have no influence on availability of manipulating the logicle scales. Ernie wrote that the parameter names (not the stain names) have to be changed. But I couldn't manage this because the field for changing the parameter names in "Edit FCS Header Atrributes" is greyed out. Nevertheless I want to thank you for your explanation how to change the parameter names directly in the acquisition manager because its much faster than changing them in the FCS Header Attributes.

 

It would be really great, if you or one of your colleagues could explain me how to directly change the parameter names ($P1N, $P2N, $P3N) in the gallios software.

 

 

Thanks! Paul

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Hi! It's me again.

 

I just tried to rename the parameter names of a series of analyses. First I manaully renamed a complete series and tried to copy all parameter names to the next series like Ernie described. But Kaluza gives me an error ("Mismatched parameter names between xxx1 and xxx2. Error code: 11051" with xxx are the names of the datasets). This error also occours when I copy the renamed parameters to the same data-file with the old parameter names.

 

Does that mean I have to change all datasets manually or is there another way to copy parameter names (copying the "description" of the parameters was never a problem.)

 

Thank you again. Best regards - Paul.

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Paul,

 

I did a little digging into this and I'm afraid that I gave you some bad advice. In general, copying and pasting parameter information is a good way to save time when you're making similar changes to a set of files. Unfortunately there's one exception to that: the parameter names themselves. We use the names to match up parameters from one data set to another, so if they get changed there won't be a match.

 

I'm afraid that I can't think of a quick and easy way to change a large group of names.

 

Sorry,

Ernie

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Ernie,

 

thank you very much for your effort! It would be possible for me to change all already aquired data sets manually because as PhD student I am used to night shifts...hihi. But is there any way to change the parameter names for acquirement of future datasets in the gallios software? We 're are going to measure about 500 samples with 18 protocols (we added 3 control stainings).This 18 protocols will be analysed within one composite. But I really don't want to change all 9000 parameter names manually.

 

I hope that anyone could tell me a way how to change parameter names in the gallios software. If not I need to learn to live with adjusting the logicle scales for all staining at once and make the best out of it. Hopefully it would be possible to change logicle scales independently for each data set within a composite in future versions of Kaluza. I am very much in favour of it.

 

Best Regards!

Paul

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Hi Ernie,

 

I tried following the insctructions of Karen Fischers reply but this just works for renaming the staining names or parameter descriptions not for changing the parameter names. So I am at my starting point again. Does anybody have another idea how to change the parameter names and not only the parameter descriptions in the gallios software?

 

Best Regards,

Paul

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Paul,

 

I apologize for the delay in responding, I've been looking into this issue in more detail. It would appear that there are few good options currently. It isn't possible to make the Gallios software to save parameters with different names. Without this, under the current implementation of Kaluza the logicle settings for matching parameters are going to be linked.

 

I'm sympathetic to your plight, although I hope that it won't be necessary to un-link EVERY parameter. The Gallios is a very stable instrument and I would expect that for the most part you should be able to use identical logicle settings across samples with good results. There will inevitably be a few cases where you need to make adjustments, but I hope that in those cases there won't be too many names that have to be changed. In fact, I would hope that this feature could become a benefit to you. If all of the settings were unlinked by default, then you'd have a lot of logicle adjustments to make.

 

Your issue has made it clear to us that simply linking based on names, given that names are often inflexible, is not sufficient. We'll try to address this in a future release with settings that better address your need.

 

Best regards,

Ernie

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Hi Ernie,

 

thanks a lot for your effort. I tried to adjust the settings best for the logicle scales so that most of the dot plots are looking good. And most of them are fine. Some look a bit compressed but an analysis is possible for all stainings. After renaming four parameters for independent adjustment of the logicle scale I have no more negative populations stuck to the edge of a dot plot. So the settings are fine and the composite settings are suitable for future analysis of whole blood stained the same way.

 

Perhaps a future version of Kaluza will bring some new adjustment possibilities for logicle scale. Thanks again!

 

 

Best regards,

Paul

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  • 1 year later...

Hi Ernie,

 

thanks for the report. And this is just in time. I started to prepare new analysis protocols for a new panel. I am looking forward to benefit from the different logicle scales and to try out the new Kaluza software. I hope this works well.

 

Thanks Paul

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