Biomedical Engineering Reference
In-Depth Information
High-quality flowcytometry assays are important to all stages of drug development
and are essential for appropriate decision making. A high-quality assay is well
controlled, reproducible, well documented, and characterized to meet the intended
use. The same assay can be utilized as a drug advances from the exploratory discovery
stage to early clinical studies and late-stage trials; however, assay validation con-
siderations may differ to meet intended objectives. Development of a versatile flow
cytometric assay for clinical utility requires identification of (i) the appropriate
method; (ii) intended use of the results from the assay; and (iii) an optimization,
validation, and implementation strategy. An assay development plan is highly
recommended for all stages of drug development to ensure appropriate execution
of flow cytometry experiments in a timely manner. The development plan is a useful
tool to obtain relevant well-characterized reagents, assay controls, and samples (cell
lines or primary cells or tissue); to establish time lines; and to identify critical steps in
the assay optimization process to further plan validation experiments.
12.3.1 Method Development and Optimization
(Method Feasibility/Prevalidation)
During assay development and optimization, conditions for all aspects in perform-
ing the assay should be established (specimen collection/processing, reagent
selection, gating/data reporting, instrument setup and monitoring). The following
sections discuss various parameters of a flow cytometry assay that are important to
evaluate in generating a good-quality flow-based assay that is reproducible and
reliable.
12.3.1.1 Specificity and Reagent Selection The strict definition of accuracy is
defined as the closeness of agreement between the averages of the test results when
compared to a well-defined official reference standard. Accuracy assessment is not
possible in flow cytometry assays in this regard, as reference standards are not readily
available. In practice, many of the biomarker-type assays supported by the flow
cytometry laboratory report results relative to a study subject's baseline sample. In this
vein, assay precision, and the reliability of ameasurement under test-retest conditions,
steps forward as a very important parameter.
Specificity is another key assay parameter that can be addressed during assay
development. Most flow cytometric methods use well-characterized monoclonal
antibodies or nucleic acid binding dyes. It is important to examine appropriate
antibody reagents that identify markers of interest, whether used in single-color
analysis or as part of a multicolor panel. Reagents that are determined to be specific
and that show optimal (good signal-to-noise) staining intensity should be selected.
Prior to selecting the reagent source, consideration should be given to whether it will
be able to support validation and subsequent clinical testing of samples. Although
the use of antibodies that have not received a cluster designation (CD) assignment is
often necessary, such reagents are a liability to the flow cytometry laboratory because
the weight of responsibility to understand specificity rests squarely upon the
investigator's shoulders.
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