Bio-Rad Bio-Plex System Validation and Calibration Tools - Manual (Page 10)

Fig. 1. Assay vs. instrument sensitivity.
Section 7
Principle of Classify Validation
Principle
Bio-Plex technology relies on the ability of the Bio-Plex array reader to
discriminate between assay beads impregnated with varying ratios of 2
fluorescent dyes. This is the concept whereby multiplexing within a single well
may occur. The periodic evaluation of the classify efficiency is necessary to
complete the Bio-Plex array reader qualification process. A series of beads with
varying ratios of the classification dyes are analyzed on the Bio-Plex array reader
and the efficiency of multiplexing is quantitated. A classify efficiency of >80% is
required for optimal results. DD Efficiency is a measure of the percentage of the
Classify beads that fall within the DD Gates. Greater than 75% of the beads
should fall within the gates for optimal results.
Impact on Assay Performance
Inefficient classification of beads may have several potential effects on an assay. If
a bead region exhibits a classify efficiency of less than 80%, the read time of a
96-well plate may be increased. The Bio-Plex array reader tabulates a specified
number of defined events in each region for each well sampled. If the percentage
of beads within a specific region is low, the time required to count is increased,
therefore the total time to read an entire plate is prolonged. Extremely prolonged
assay read times could impact well-to-well precision, since the kinetics of a
sandwich assay, for example, are not 100% stable over a period of 3–5 hrs.
Another potential impact of inefficient classification is the misclassification of one
assay bead into another bead region. This could yield false positive or negative
results for a particular assay. A DD efficiency value less than 75% may increase
the read time of the assay and affect results in the same manner as a low classify
efficiency.
7
RP1 Channel
Assay Background
Assay MESF
Reader sensitivity
MESF (R-PE)
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