Bio-Rad Bio-Plex System Validation and Calibration Tools - Manual (Page 6)

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.
The primary reporter channel performance parameters are as follows: dynamic
range, linearity, slope, accuracy, and instrument threshold (sensitivity) of the reporter
channel 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.
High Versus Low PMT Calibration and Validation
Some assays may require quantitating a range of analyte concentrations greater
than what can be achieved running the instrument at a single PMT setting. In these
cases, the samples may be run with different detector channel PMT settings: a high
RP1 target setting and a low RP1 target setting. They are set differently depending
on your version of Bio-Plex Manager™ software.
For Bio-Plex Manager 4.0/4.1, the targets are set during calibration of the
instrument. Using the high RP1 target will allow for the ability to quantitate lower
concentrations of analyte, while using the low RP1 target allows for the ability to
quantitate higher concentrations of analyte. For Bio-Plex Manager 5.0 and later,
calibration is performed using the low RP1 target and the high RP1 target is then
selected during the validation setup.
Dynamic Range of Reporter Channel
Definition
The theoretical maximum dynamic range of the Bio-Plex array reader can be
represented by the maximum number of channels in the A/D converter. Currently,
the available range of channels is from 0 to 32,767, or about 4.5 decades on a log
scale. These channels are represented by FI, or Fluorescence Intensity units. The
dynamic range of the Bio-Plex array reader is measured by taking the log of the
fluorescence intensity value of the highest reporter bead minus the fluorescence
intensity of the blank bead. This value is the calibrated dynamic range of the
instrument and is always less than 4.5 log decades because the highest intensity
bead in the validation kit that is read is less than 32,767 FI units, while the blank
bead gives a reading that is greater than zero FI units.
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