What is Detector Setting Independent (DSI) Scaling
BD FACSDiscoverTM A7 cytometers now include scaling factors to normalize parameters across machines, removing the impact of varied detector gains, and making per parameter measurements directly comparable. These scaling factors are referred to as DSI.
DSI data are created by calculating a scaling factor on BD FACSChorusTM software during acquisition that is written to the resulting FCS file for application to unmixed data.
The overall impact is that scaling factors are applied automatically to both the hardware and software unmixed parameters in FlowJo, allowing essentially all operations in FlowJo to be performed normally, but equivalent data will align even in the presence of varied detector gains, making direct comparisons much easier.
Operation in FlowJo
In FlowJo, DSI data with a matrix applied will appear in the sample list with a gray acquisition defined matrix:
If you double click the matrix to open the matrix editor, you’ll notice that the matrix includes the notation “DSI / BD SpectralFXTM”, as shown in Figure 1. This indicator informs you that the matrix is compatible with both default scaled and DSI scaled FACSDiscover data.
NOTE: This assumes BD SpectralFXTM has been applied, which is the default as of 10.10.2. If you choose to switch to OLS, the annotation on the matrix will drop the “BD SpectralFXTM”.
The 
matrix icon can then be dragged to a sample or group of samples to apply the selected matrix. You can drag a DSI matrix to any FACSDiscover sample (DSI scaled or default scaled) that shares a common set of detectors and the gain settings embedded in that FCS file will be used.
Figure 1 Annotation on a gain normalized matrix
Applying a matrix that was created without gain-aware samples produces one of two error messages depending on whether the target sample is DSI scaled, or simply gain aware as all FACSDiscover data are:
Figure 2 Error messages when applying a matrix that was created from data that were not gain aware to either DSI scaled samples (top) or gain aware samples (bottom)
Creating New Matrices
New unmixing matrices can be created from gain aware controls with no differences due to DSI. If these matrices are applied to DSI scaled data with matching parameters, DSI scaling factors will be applied.
Additionally, identity matrices can be created and applied to DSI data. When creating the identity matrix we recommend selecting the unmixed parameters. This will create an editable matrix of just parameter-parameter interactions and will preserve the parameter names.
Annotation Convention for DSI data
You can check your files’ keywords to see if they are DSI scaled. DSI scaling information is contained in several keywords
- DSI Scaling – either true or false
- BDSPECTRAL SCALE FACTORS – DSI factors expressed per unmixed fluorochrome
- PnTARGETVALUE – factors used to determine per-detector gain adjustments
When DSI Scaling = True, FlowJo applies the DSI scaling factors in BDSPECTRAL SCALE FACTORS to the events, per parameter.
When DSI Scaling = False, FlowJo still adjusts unmixing matrices to the gains of individual samples using information embedded in “gain aware” FCS files but does not apply DSI scaling factors. Gain aware means that a series of keywords pertaining to the gain settings at acquisition are present in the file. All BD FACSDiscover instruments create gain aware outputs.
For additional information see the DSI Technical Bulletin.