REGULATORY COMPLIANCE AND METHOD VALIDATION - Flow Cytometry in Drug Discovery and Development - page 246

evident when monocytes/macrophages or activated cells are used. Compar-
ison of negative control autofluorescence (unstained cells) with a positive
control signal (stained cell) in each fluorescence detector will confirm that
the stained sample cells will be on scale for each parameter. In addition, the
unstained cell controls are used for light scatter gating and defining the
threshold signal, above which true positives are determined.
(ii) Compensation controls are positive controls to remove spectral overlap in
multicolor flow cytometry analysis. Beads (i.e., Comp Beads) or cellular
samples can serve as compensation controls when labeled with single-color
monoclonal antibody reagents used in the method [42]. Fluorochrome
emission cross talk into other detectors must be compensated for to achieve
an accurate fluorescence signal. A contemporary practice is to exercise
the fluorescence-minus-one (FMO) paradigm, where each antibody-
fluorochrome conjugate is dropped in succession. This allows one to identify
the culprit of fluorescence spillover by deduction; this is also a very useful
gating control as discussed below.
(iii) Specificity or gating controls are reagent controls used to verify antibody
reactivity and to verify method staining is specific for the target marker and
cell type. Labeling with a species relevant immunoglobulin of the same
isotype is used to distinguish nonspecific binding (due to Fc receptors on cells
of interest) that is gated out to measure specific binding. Isotype controls are
usually used for experiments using “sticky” cells such as monocytes and
dendritic cells. The isotype control antibody should be of the same con-
centration and have the same fluorescence-to-protein ratio as the antibody of
interest. Another gating control to determine positive staining is fluorescence
minus control (FMO), which is a control labeled with all but one antibody
reagent used in the actual sample panel. FMO controls are applicable for
experiments using four or more colors and are usually used to set discrimi-
nate gating for positive/negative expression of the missing antibody [49].
Events that display fluorescence above these controls are considered positive
for the marker of interest. For indirect staining techniques, specificity
controls include samples with just primary antibody or secondary antibody.
An unstimulated sample is an ideal control for setting the gate for positive
events in a stimulated sample.
(iv) Sample controls are used as an internal control of the assay method. A
stabilized positive sample can be commercially obtained (CD-Chex from
Streck, Cyto-trol fromBeckman Coulter) and labeled with the same reagents
as the test samples. These method controls should be considered for use both
in assay development and in validation [49]. Unstimulated controls may also
be used as negative method controls.
12.3.1.8 Data Reporting The method of reporting signal readout is established
during assay development. Besides reporting cell percentages, the use of mean or
median fluorescence intensity (MFI) can be used to estimate expression level on a per