BD Horizon™ Guided Panel Solution (GPS) tool
Panel design can be a difficult and time-consuming process but is essential to obtaining good data. The BD Horizon™ Guided Panel Solution (GPS) provides a guided workflow for reagent selection based on the principles of panel design. This tool will help you streamline the panel design process and avoid reagent selections that may negatively affect population resolution.
The panel design process has been broken into six steps. At each step, you will be prompted to input information that is pertinent to your panel. The information you will be prompted to enter is information that you likely use when designing panels currently. The tool has information about fluorochromes brightness, instrument configurations and resolution impact, so you do not have to enter that information. The tool takes all of this information into account to provide alerts for reagent selections that may negatively affect population resolution.
The more accurate the inputs are, the more likely the tool will be able to provide relevant outputs. It is also important to note that the tool will not be able to detect every possible reagent selection that may negatively affect resolution, so we cannot guarantee that the panel designed will perform optimally. Additionally, even good panels can have alert messages. This is especially true with higher order panels for which it may be difficult to avoid spectral overlap between reagents. In this case, the tool will allow you to see which populations are most likely to be negatively affected by the reagent selections and trade offs can be assessed.
If you would like to get expert advice prior to running your panel or if you have questions about the data obtained from your panel, our Technical Support team is readily available, just email ResearchApplications@bd.com.
|Panel Iteration 1||Panel Iteration 2||Panel Iteration 3|
|CD127 Alexa Fluor® 647
|BD Horizon GPS notifications of reagent selections that may affect population resolution|
Mismatch of fluorochrome brightness and antigen resolution affects population resolution
Fluorochrome brightness/antigen resolution rules applied, but spectral overlap on co-expressed markers prevents optimal resolution
Principles taken into account and population is well resolved
We welcome feedback on the tool so we can make improvements in later versions.
Name your panel and provide any important notes.
Define Markers: Here you will enter the target species, panel markers and their "type." The reagent type denotes the expression pattern of each marker as defined here. The tool takes this information into account when determining how fluorochrome brightness and spectral overlap may affect resolution. If you have a specific clone that you would like to use, you may select it from the clone drop down menu. If you select a clone, only reagents available for that clone will be displayed.
Classification of Antigens
|Primary||Well characterized, easily classified as positive or negative, typically describe large subsets or lineages
Examples: CD3, CD4, CD19
|Secondary||Well characterized, typically expressed at a higher density, often over a continuum
Examples: CD27, CD28, CD19, CD45RA, CD45RO
|Tertiary||Expressed at low levels, variable upon activation unknown, critical.
Examples: CD25, STAT5, FoxP3
In this section you will define your gating strategy and critical populations. Critical populations are the populations that you are most interested in studying.
1. Create a population tree which will outline your gating strategy. Drag the markers from the box on the left to the space on the right to form the population tree. To create more than one branch off a tree, drag the markers on top of the node.
2. Next you will define the critical populations. Once you click the node of the critical population, you will be prompted to name the population, select the box indicating that it is a critical population and enter the expression level of the markers expressed in this population. The expression level should represent the expression level of that antigen on this critical population.
You may select your cytometer from the menu or create a custom configuration. Most standard configurations are available from the configuration drop down menu. Once you select an instrument and configuration, you can modify the configuration by adding or subtracting fluorochromes. If adding a fluorochrome, you also need to note the relative brightness.
If you have a custom configuration or instrument, click “Create Cytometer” and name your instrument. You will then select “Create Configuration” and name the configuration. In the same window, you will select the lasers on your instrument. A configuration will be displayed and can be modified to match your configuration. Once you click “Continue”, your custom configuration will be saved to the Cytometer drop down menu and can be selected for future panels.
You may also create a Resolution Impact Matrix for your instrument by clicking “Create RIM” under “Resolution Impact” and following the instructions. You will need to collect data for each channel on your instrument and upload into the tool. The tool will transform the data into a heat map that can be used to note fluorochrome combinations that may cause resolution issues. Additionally, the tool will use the RIM data to provide alerts when fluorochrome combinations have been chosen that may negatively affect population resolution. Data for each fluorochrome should be collected in order to create the RIM. This data can be generated using the CD4 Fluorochrome Evaluation Kits (Human: Cat No. 566352, Mouse: 566644) which contain CD4 conjugates in >25 dyes.
In this step, you will make reagent selections by matching markers with fluorochromes. To do so, drag a marker over to the fluorochrome list. You will notice that many of the flourochromes are highlighted in blue. This designates that the marker/fluorochrome combination is available in the BD catalog.
There are features on this page to help with reagent selection:
The co-expression table: This table summarizes the information from the previous tabs to denote which markers are co-expressed and to what level. In general, a highly expressed marker should be matched with a dim fluorochrome and vice versa. The relative fluorochrome brightness is noted next to the fluorochromes (Dim-1, Moderate-2, Bright-3, Very Bright-4). If there is a mismatch between fluorochrome brightness and antigen resolution for a reagent selection, the tool will note it with a red exclamation mark. Additionally, if more than one fluorochrome for a particular detector is selected, the tool will note it with a red exclamation mark (e.g. FITC and BB515).
Resolution Impact: A key factor in good panel design is maximizing the resolution of populations, in particular double-positive populations. As shown below, the resolution for a given antigen (fluorescence parameter) is decreased by the spread due to spillover from other fluorochromes. That is, the addition of a reagent may reduce the resolution of another reagent, potentially affecting overall population resolution and data quality. Spread is most important when considering reagents for co-expressed antigens.
The Resolution Impact is the percent loss of resolution when a new co-expressed marker/fluorochrome is added. In the example above, the addition of the co-expressed PE marker has reduced the resolution of the PE-CF594 marker by 93% [1-(50/700)x100%]. The resolution impact value is based upon markers of equal density and is a way to measure the loss of resolution of a reagent by the addition of another reagent. This Resolution Impact Matrix (RIM) table allows the assessment of the spread introduced to the cells stained with the primary fluorochrome after the addition of the secondary fluorochrome. The resolution is most likely to be impacted when the antigen on the secondary fluorochrome is highly expressed and the antigen on the primary fluorochrome is lowly expressed. The color coding in the table makes it easy to visualize which fluorochromes have the greatest impact on each other. Use this table to decide which fluorochrome combinations are least likely to negatively impact the resolution of the population of interest.
The RIM that is provided in the tool is based on generic instrument platforms and is used for guidance only. The resolution impact of fluorochromes on your specific instrument may differ due to configuration, filters, laser power etc. Therefore, you may want to create a RIM specifically for your instrument. Since the RIM is used to characterize, rather than optimize your instrument, it should be created after the instrument settings have been optimized.
Analyze Resolution Impact: When you click the "Analyze Resolution Impact" button, the tool will perform an assessment of the selected fluorochromes based on the Resolution Impact Matrix and provide alert messages for fluorochrome selections that may reduce critical population resolution due to spread. If an alert is displayed, you may choose different reagent combinations and then click the "Analyze Resolution Impact" button again to see if an improvement was achieved.
As the number of markers increases, it may become more difficult to design a panel that has no alert messages. The goal is to maximize resolution of the critical population by minimizing fluorochrome selections that may reduce population resolution. Once you are satisfied with the reagent selections, you can move to the next step.
Example of a Population Resolution Alert
This step displays all of the reagents in the BD catalog that correspond to your panel reagent selections. You can select the reagents you want and then export them to an Excel file by clicking the Export button. You may also add the products to a Shopping List or directly into the Shopping Cart for easy purchase.
For Research Use Only. Not for use in diagnostic or therapeutic procedures
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