Bio-Rad Bio-Plex System Validation and Calibration Tools - Manual (Page 7)

Impact on Assay Performance
It is desirable for the range of the instrument to be greater than the range of the
assay. In most experiments, the dynamic range of the assay is far less than that of
the instrument. However, in some assays, the same or different analytes may be
present in greater than 4.5 log differences in concentration. When this is the case,
the sample can be rerun at different PMT settings to extend the overall dynamic
range of the measurements.
Linearity of Reporter Channel
Definition
The reporter validation bead set is utilized to construct a plot where the reporter
channel median fluorescence intensity values are plotted against the corresponding
expected fluorescent intensity values (as determined on an independent calibrated
instrument). Instrument linearity is expressed as the coefficient of determination or
R-squared (R
2 ) value.
Impact on Assay Performance
The linearity of the instrument response may directly affect a typical standard or
calibration curve in a Bio-Plex assay, thereby impacting the unknown values
extrapolated from that curve. If the R
2 value is not within acceptable limits, it may
be necessary to realign the optics or check the response of the reporter
photomultiplier tube.
Accuracy of Reporter Channel Response
Definition
The accuracy of the reporter channel response is a more stringent measurement of
the linearity than the R
2 value. Simply stated, the accuracy of the reporter channel
response is the percent difference that the regression line is away from the
expected fluorescence intensity data point values.
Impact on Assay Performance
Since accuracy is also a measurement of the linearity of the instrument response,
the same principles that apply to linearity also apply to accuracy of the reporter
channel response. The accuracy data is evaluated in combination with optics
alignment to determine if the Bio-Plex reader will perform according to
specifications. It is possible for the accuracy value to fall out of specification before
the linearity parameter. This is expected due to the fact that the accuracy parameter
is a more sensitive measurement of linearity than the R
2 value. These data are
correlated with optics alignment data as well as assay performance to determine
when the array reader will not perform according to specifications.
Slope of the Reporter Channel Response
Definition
The slope of the regression line resulting from the plotting of reporter channel mean
fluorescent values against expected relative fluorescence is related to the dynamic
range of the instrument. The slope of the regression line is a function of the
response of the reporter channel photomultiplier tube.
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