Bio-Rad Bio-Plex System Validation and Calibration Tools - Manual (Page 9)

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. Doublet Discriminator (DD) efficiency is a measure of
the percentage of the classify beads that fall within the DD gates. The DD gates
are used to distinguish single beads from bead clusters, bead fragments or other
small particles passing in front of the detector. 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 several hours.
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 of less than 75% may increase the
read time of the assay and affect results in the same manner as a low classify
efficiency.
7
4110185B.qxp 9/25/2007 11:55 AM Page 7