REGULATORY COMPLIANCE AND METHOD VALIDATION - Flow Cytometry in Drug Discovery and Development - page 242

Another parameter that affects antibody specificity arises from their ability to bind
many cell types by their nonspecific (Fc) ends. This is especially true of monocytes.
In this setting, the use of Fab or F(ab 0 )2 fragments (antibodies without their Fc ends)
is recommended, whenever possible, to help alleviate this problem. Addition of an
Fc-receptor blocking agent may also be included to minimize nonspecific binding.
Finally, dead cells, with compromised membrane integrity, tend to be sticky and bind
reagents nonspecifically. Careful cell preparation and “gating out” dead cells in data
analysis - either by propidium iodide staining to positively identify dead cells by their
membrane permeability or by avoiding cells with low FSC intensity - will exclude the
dead cell population from analysis.

12.3.1.2 Compensation In a multicolor flow cytometry assay, the spectral overlap
of fluorochromes contributes to unwanted fluorescence spillover into adjacent
channels. The process of compensation shall be followed to subtract the spillover
of fluorescent signal to the detectors that are not assigned to the particular
fluorochrome. Compensation has historically been manually performed by visual
comparison of positive and negative stained populations, when assay panels were
limited to three or four fluorescent-conjugated mAb. With the advance of digital
cytometers providing multiple lasers/detectors and the availability of additional
fluorochromes, it is now possible to design an assay panel to assess detailed cell
phenotypes with up to 18 colors. Meanwhile, application of correct compensation
becomes a much more complex process, even with the implementation of computer
software to set up compensation automatically [40, 41]. To minimize undesired
spectral spillover and resolve the compensation issues, it is important to choose the
best fluorochrome and mAb combination to ensure assay sensitivity and
specificity [42]. Usually, FITC, PE, and APC are reserved for conjugating mAb to
detect protein with low expression level (i.e., intracellular cytokine, phosphorylated
protein, etc.). Tandem dyes (i.e., PE-Cy5, APC-Cy7, etc.) are chosen for the detection
of well-characterized markers (i.e., CD3, CD4, etc.). Although the intricacies of
compensation are not fully addressed here, the end game is to use fluors with strong
emission profiles to detect antigens with low expression level (and vice versa). The
alternative possibility to apply strong fluors to highly expressed antigens can create
instances where excessive compensation is necessary to rectify fluorescence spillover;
insomecases, evenahighdegreeof compensationcannot remove theartifactual signal.

12.3.1.3 Staining Conditions Appropriate staining conditions are important for
optimal sample acquisition and for clearly distinguishing positive from negative
staining. In general, cells should be at the highest concentration to use the lowest
fluidics pressure possible to pass through the cytometer while minimizing doublets/
cell aggregates to achieve a suitable flow rate (typically about 1000 cells per second).
Using high pressure to achieve high flow rates will affect resolution of staining
intensity. Also, cell staining suspension volume should be as low as possible
(50-100

L is ideal). Smaller staining volumes require less antibody reagent to
achieve saturating concentration that is most important. Staining volumes can be
adjusted at acquisition time to get optimal flow rate. Multiple samples of controls will

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