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