Getting Diva and Flowjo to play nice

Different export formats in Diva work differently in Flowjo, and Diva 8 has some changes from Diva 6 that I thought it was worth addressing…

We have Diva 6 running on our LSRs and Diva 8 on our SORP Aria. So in Diva 6, when you export as “FCS”, the file name is “Specimen_tube.fcs”, so when you load the file into Flowjo the file is identified by the filename (which is specimen and tube), and all parameters map correctly.

When you export as “Experiment” in Diva 6, the file name for the fcs files is a 5 digit number associated with the database in Diva (“34567.fcs”), and if you use these files in Flowjo the file name in the workspace is just the number (which you can get around by having the workspace display the tube name for the file so you can identify them) and if you display H and W parameters, they are switched—FSC-H is showing FSC-W and vice versa. Area is displayed normally.

NOW, in Diva 8 (not sure about 7, I didnt have it long enough to know), BOTH exporting formats (experiment or fcs) label the file “specimen_tube_###.fcs” where the number is the order of the files in the experiment layout, so you can’t easily tell the different export formats apart…

The standards for field labels for FCS 3.1 can be found here on page 10 of 34:
http://isac-net.org/PDFS/90/9090600d-19be-460d-83fc-f8a8b004e0f9.pdf
but this can get confusing because different software platforms can use different fields for different things, such as the $SRC field, or “source”, is used to store the “Specimen name” in Diva.

$FIL is for the filename.  For EXPERIMENT exported files in BOTH Diva 6 and 8, this is the number assigned to the file in the database, which you can also see in the D>BDDatabase>BDData folder.  For FCS exported files, $FIL is “Specimen_Tube name” in Diva 6, or “Specimen_Tube_###” in Diva 8. When you load a file that was exported from Diva 8 as an experiment, Flowjo will go to the $FIL field to display in the workspace, which in this case is just a number even though the name of the actual file is from the specimen and tube.

In the Flowjo preferences, workspace tab, “How should data files be named” you can choose to specify keywords instead of the default which uses the $FIL field. Choose “$SRC” (specimen) and “Tube Name” and as you choose each it will add it to the list below the drop down menu.  There is an option to “clear keywords” in the pulldown menu to clear chosen keywords. That will set the “name” in flowjo to display as specimen tube instead of filename, and FCS or experiment exported files will show up the same way, but keep in mind that Experiment exported files will STILL switch H and W signals.

Exporting in Diva
So this brings us to data management and exporting in Diva.  In Diva 6 on our LSRs, if the experiment isn’t that large I typically export as experiment, which creates a folder with the name of the experiment, and in that folder creates an xml file named after the experiment and then the fcs files which are all just numbered. Then I’ll export as fcs, which if done on its own will create a folder named after the experiment, but since that already exists from the experiment export, it just sticks the fcs files in that same folder.  The “experiment” exported files are numbers, and the “fcs” exported files are all named Specimen_Tube. You cant do this in Diva 8 because the file names for FCS or Experiment are the same, so you have to segregate them into separate folders and keep track of which is which.

Another local core facility (thanks Laura, who also helped me figure out some of the details of what was going on) will actually set the experiments to export as zip for all new accounts, so that the folders are kept separate that way by default (Diva will remember your choices from the previous export, so directory path, zipped, and in Diva 8 whether you want FCS 3.0 or 3.1).  You really only want to use the Experiment export in Diva, so zipping it is a good way to keep it apart, and when you export as FCS it will make a new, unzipped folder named after the experiment for your fcs files.

If anyone has any other solutions you would like to share, or clarifications if I’ve gotten some details wrong, please comment or email me!

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Einstein Express

We have a request form that our sort users have to submit to request sorting time, and they often have a copy that they have saved to re-submit, and often forget to update the date, it made me think of the old SNL skit:

einsteinexpress

if you haven’t seen it:
http://tinyurl.com/facsexpress

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Tracking settings

When a researcher is using the same panel for experiments run on different days, the temptation is to just use the same voltages as the first day.  Due to instrument variations (fluctuation in laser power, variation in detector sensitivity due to heat and humidity,  how long the instrument has been on, etc), this likely will not give you the same results.  The only way to compare samples run on different days is to make sure the instrument is providing the same output for the same fluorescence signal on both days, to confirm that differences between data files are due to actual sample differences and not to instrument variation.

Keep in mind that staining consistency is also critical, as variation in your technique can lead to differences measured on the cytometer, and a good control for this is to run the same sample on each day and make sure they are consistent– so have a bank of PBMC aliquots from the same patient, and stain one with each experiment.  If the instrument is consistent, and the sample is matched, then any variation would be due to changes in the staining and this will give you an idea of the consistency of your staining on different days, and how much of a change is actually significant.

With BD instruments, CST (Cytometer Setup and Tracking) in Diva can adjust voltages based on daily CST results (“Application Settings”), but you have to re-synchronize the settings for each new configuration, batch of CST or each new baseline for EACH user, which becomes challenging for a core facility with many users.

Another benefit of monitoring your instrument response for your experiment is that if someone has swapped filters on the instrument and neglected to restore them to “default”, it will usually be readily apparent in a large shift in your bead peak and hopefully shorten any troubleshooting you might have to do (“Gee, my pacific blue single stain has no signal, let me re-make the comp tube.  Still didn’t work, oh wait someone swapped the filter…”).

Here is the writeup:

https://newenglandcytometry.files.wordpress.com/2015/02/rainbowtracksettings.pdf

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2014 NECyto presentations posted

I’ve posted PDF’s of the talks that were presented at NECyto 2014 on October 2, 2014 in Boston, MA.  They can be found here:
http://newenglandcytometry.com/meetings/2014-new-england-cytometry-user-group-meeting/

I would also like to announce a Business Skills Workshop for core managers that will be hosted by the University of Virginia, view the flyer here.  Joanne Lannigan announced it during her presentation on successful core management, I’m sure it would be a useful tool in developing a successful core if you are able to attend.

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Thank you, and upcoming seminars…

Thank you to all of the attendees, vendors, and speakers at the 2014 NECyto meeting. We had over 120 participants and a great series of talks which we plan to post as PDFs in the coming week.

I’d like to announce an upcoming event tour presented by BD Biosciences, on October 28th in Farmington, CT, October 29th at the Ragon Institute in Cambridge MA, and November 4th in Houston, TX. There will be three seminars (“Introduction to Flow Cytometry”, “BD Accuri: Applications of Flow Cytometry in Cancer Research and Cell Biology”, and “Flow Cytometry as a Tool for Microbial Analysis”), followed by an instrument demo of the Accuri C6. Please view the flyer here.

and a flow related comic added to the Humor page…

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Three fluors one PMT

There persists a misconception that if things are measured in the same channel, they are the same color or “close enough”. The example that I’ll use to illustrate the potential problems with this was a panel that used BV510 and V500 as the labels for the dump channel markers, and Live/Dead Aqua for viability, which are all measured off of 405 excitation and a bandpass around 525/50.  You can see in the image below that they are all slightly different:

3Fluors1

http://www.biolegend.com/spectraanalyzer  A quick aside:

thankyou

On our system, we have the following detectors on our violet laser.  Shown are the uncompensated signals from the 3 different dyes on single stained beads.

3Fluors2

If you compensate based on V500, you can see the other parameters are not properly compensated in all channels:

3Fluors3

Compensating based on BV510:

3Fluors4

And Live/Dead Aqua:

3Fluors5

The cytometer has no way to tell WHICH dye is generating the signal, so it will not be able to correct the signals properly, but will just apply whatever values your matrix contains.  The actual matrix values for the different dyes:

3Fluors6

If you still decide to mix fluors, in this case (viability combined with a dump channel) I would use the Live/Dead dye to determine compensation. ALL cells will be stained with the L/D dye (dead cells will be brightest) so errors in compensation will affect your populations of interest. For a dump channel you are keeping the negative cells so even if compensation isn’t quite right, it should not affect your cells of interest.

Note that a similar situation can occur if you use a tandem dye for your dump channel–the tandem for each different antibody may be slightly different, so your compensation may not be correct for all markers.

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Talk titles posted for NECyto

We have posted the talk titles for our speakers at this years NECyto Fall Meeting being held on October 2, 2014:
http://newenglandcytometry.com/meetings/2014-new-england-cytometry-user-group-meeting/
Registration deadline is September 26th.

And if you are in town the day before, come across the river to join the Ragon Institute for a talk presented by Biolegend’s Kelly Lundsten (view flyer):
“Characterization of Chemokine Receptor Antibody Function and Human Chemokine Receptor Expression”

And another new entry to the humor page, in a galaxy far far away…
http://newenglandcytometry.com/humor/

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