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

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Section 5
Principle of Optics Validation
Principle
The Bio-Plex array reader is a laser-based fluorescence detection system containing
sensitive optics components. Alignment of the laser/optics system is critical for
optimal instrument performance. A method for the assessment of the optics
alignment is included in the validation kit. Acceptable specifications for the
alignment procedure are listed in the product insert.
Impact on Assay Performance
The alignment of the optics bench of the Bio-Plex array reader is critical for proper
assay performance. Misalignment of the reporter optics path can result in 1)
reduced assay sensitivity or 2) poor well-to-well assay precision. Misalignment of
the classification optics path can lead to 1) increased read times or 2)
misclassification of one assay into another, leading to false positive or negative
results. Correlation studies have been performed to determine the direct effect of
misalignment on assay performance.
Section 6
Principle of Reporter Validation
Principle
The reporter (RP1) channel is the fluorescence channel used for assay quantitation
(See Bio-Plex system hardware manual for more information regarding the principle
of Bio-Plex technology). Therefore, validation of this component of the Bio-Plex
system is a critical part of operational qualification. R-phycoerythrin (R-PE) is the
primary reporter molecule used in Bio-Plex assays. A series of beads dyed with
varying intensities of a fluorochrome spectrally matched to R-phycoerythrin are used
for this procedure. Each of the reporter beads has been assigned a specific
intensity value corresponding to the number of fluorescent R-PE molecules. These
units of fluorescent measure are known as molecules of equivalent soluble
fluorescence (MESF). MESF units allow direct correlation of instrument performance
to a typical assay using R-PE as the indicator molecule. The primary reporter
channel performance parameters are as follows: dynamic range, linearity, accuracy
of reporter channel response, sensitivity, and slope of the response. Each of these
parameters is related directly to the performance of the Bio-Plex array reader and
has defined acceptable specifications. Definitions for the parameters and the
applicability to a typical assay performed on the Bio-Plex array reader are listed
below. If any of the parameters are not within the specified range, contact Bio-Rad
Technical support for assistance.
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