In our laboratory, we have two BD FACSCanto II flow cytometers equipped with 3 lasers and 8 fluorescence detectors, and over the last several years, we have developed 8-color flow cytometry for use in our routine clinical work. During the course of our developmental and validation work, we experimented with a multitude of different fluorochromes, including tandem dyes and relatively new fluorochromes with which our lab had no direct experience. Trying to find optimal (or even acceptable!) antibody-fluorochrome combinations, and then trying to arrange them into clinically purposeful 8-color “tubes” was no small feat.  

To complicate the process further, we observed some peculiar, but reproducible, compensation artifacts with one of the violet-excited dyes that we used initially, AmCyan. Eventually, we discovered that we could only compensate AmCyan appropriately if we used cells, rather than antibody-capture beads, as compensation controls (DiGiuseppe JA, Cardinali J. Improved compensation of the fluorochrome AmCyan using cellular controls. Cytometry Part B 2011; 80B:191–194.)  Shortly after we learned this, a new fluorochrome (BD Horizon V500) became available, whose spillover into FITC was significantly less than that of AmCyan.  Nonetheless, we still saw inconsistent compensation results with BD Horizon V500 when we used beads, as opposed to cells, as compensation controls.

Of course, beads are convenient compensation controls, and are particularly useful when tandem-dye conjugates of antibodies recognizing rare antigens must be compensated. Indeed, some experts have suggested that antibody-capture beads are ideal for use as compensation controls for anti-human antibody conjugates (Mahnke YD, Roederer M. Optimizing a multicolor immunophenotyping assay. Clin Lab Med 2007; 27:469-485). As a result of these considerations, we have developed a hybrid approach to compensation, in which cells are used to compensate BD Horizon V500, and beads are used to compensate all other antibody-fluorochrome combinations.  A key concept in implementing such a hybrid approach, though, is that “positive/labeled” and “negative/unlabeled” controls for each fluorochrome must have identical autofluorescence properties (Mahnke and Roederer). What this means for our practice is that when we have a fluorochrome that is being compensated with beads, we make sure the negative is a bead and when we are testing a fluorochrome that is being compensated with cells, we use cells as our negative.  This is our hybrid approach and it is briefly summarized below.

In order to generate a compensation matrix, we create a compensation control tube for each specific antibody-tandem-dye conjugate that we use (e.g., CD19-PE-Cy7); for every other (i.e., non-tandem) fluorochrome used in our antibody panels, we create a compensation control tube using CD45 conjugates (e.g., CD45-FITC).  For all fluorochromes other than BD Horizon V500, each compensation control tube contains both “negative/unlabeled” beads and “positive/labeled” beads (Figure 1A and 1B). In the case of BD Horizon V500, we prepare a “negative” tube that contains washed, unstained blood cells, and a “positive” tube that contains washed blood cells stained with CD45-BD Horizon V500.  These two tubes are processed through our standard lyse/wash procedure; the unlabeled and labeled cells are then mixed together to generate the compensation control tube for BD Horizon V500 (Figure 1C and 1D).  In order to compensate all of the various fluorochromes and tandem-dye conjugates used currently in our panels, we prepare and acquire a total of 25 compensation control tubes. Compensation values are calculated after gating on the negative and positive peaks in each tube.

Figure 1.  Example of hybrid approach to compensation using both beads and cells.
A: Gating on beads using FSC and SSC.
B: Gating unlabeled beads (using gate P3) and CD19 Pe-Cy7 labeled beads (using gate P2).
C: Gating on lymphs using SSC and FSC.
D: Gating on unlabeled lymphs (using gate P3) and CD45 V500 positive lymphs (using gate P2).

We have been using this approach for about a year now, with remarkably consistent results. We have also learned a few “tricks” that simplify interaction with our acquisition software (BD FACS Diva). For instance, with appropriate manipulation of our laser configuration, we can toggle among the settings for different flourochromes used for a given detector.  If anyone is interested in trying this hybrid approach to compensation, or hearing about software “tricks” in more detail, I would be happy to discuss these with you in further detail.

Jolene Cardinali, MT(ASCP)
Hartford Hospital Special Hematology Laboratory
(860) 545-3813
E-mail: jcardin@clpct.com