Bio-Rad Bio-Plex System Validation and Calibration Tools User Manual
Bio-Rad Laboratories
2000 Alfred Nobel Dr.
Hercules, CA 94547
1-800-424-6723
4110007 Rev C
Instruction Manual
Catalog # 171-203000
For use with Bio-Plex Manager Software Version 3.0 and MCV plate II
or
For use with Bio-Plex Manager Software Version 2.0 and MCV plate
For technical service, call your local Bio-Rad office, or
in the US, call 1-800-4BIORAD (1-800-424-6723)
For research use only. Not for diagnostic procedures.
Table of Contents
Section 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1
Section 2 Product Description . . . . . . . . . . . . . . . . . . . . . . . . . . . .2
Section 3 Specifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3
Section 4 Storage and Handling . . . . . . . . . . . . . . . . . . . . . . . . . . .3
Section 5 Principle of Optics Validation . . . . . . . . . . . . . . . . . . . .4
Section 6 Principle of Reporter Channel Validation . . . . . . . . . . .4
Section 7 Principle of Classify Validation . . . . . . . . . . . . . . . . . . .7
Section 8 Principle of Fluidics Validation . . . . . . . . . . . . . . . . . .8
Section 9 Procedure for Bio-Plex Manager 3.0/MCV plate II . . .9
9.1 One-Step Procedure for all Validation Parameters . . . . . . . . .9
9.2 Validation of Optics Alignment . . . . . . . . . . . . . . . . . . . . . .11
9.3 Validation of Fluidics Integrity . . . . . . . . . . . . . . . . . . . . . . .13
9.4 Validation of Reporter Channel Performance . . . . . . . . . . . .15
9.5 Validation of Classify Efficiency . . . . . . . . . . . . . . . . . . . . . .16
9.6 Generating a Validation Report . . . . . . . . . . . . . . . . . . . . . .18
9.7 Validation Report Example . . . . . . . . . . . . . . . . . . . . . . . . .19
Section 10 Procedure for Bio-Plex Manager 2.0/MCV plate . . . .21
10.1 Validation of Optics Alignment . . . . . . . . . . . . . . . . . . . . . .21
10.2 Validation of Reporter Channel Performance . . . . . . . . . . . .23
10.3 Validation of Classify Efficiency . . . . . . . . . . . . . . . . . . . . . .25
10.4 Validation of Fluidics Integrity . . . . . . . . . . . . . . . . . . . . . . .26
10.5 Generating a Validation Report . . . . . . . . . . . . . . . . . . . . . .28
10.6 Validation Kit Worksheet and Report
Section 11 Troubleshooting Guide . . . . . . . . . . . . . . . . . . . . . . . . .32
Section 12 Ordering Information . . . . . . . . . . . . . . . . . . . . . . . . . .32
Section 13 Reference . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .33
Form Examples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .28
Section 1
Introduction
Qualification of analytical instruments is a formal process of documenting that an
instrument is fit for its intended use and that it is kept maintained and calibrated.
The Bio-Plex validation kit is used for operational qualification (OQ) of the Bio-Plex
protein array system. The validation kit is designed to validate the operation of all of
the primary components of the system and is a valuable tool that allows the user to
discriminate between assay and instrumentation problems.
The Bio-Plex validation kit consists of beads to evaluate the following components
of the Bio-Plex protein array system: 1) optics alignment, 2) integrity of fluidics, 3)
reporter channel performance, and 4) classify efficiency. A brief definition of the
parameter and the principle of each procedure is described, along with complete
procedures for evaluating each of the primary components. An explanation of the
potential impact of each process on a typical Bio-Plex cytokine assay is included to
assist the user in assay troubleshooting and development.
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BBiioo--PPlleexx MMaannaaggeerr 22..00 rreeqquuiirreess MMCCVV ppllaattee ((CCaatt## 117711--220033003300)) ffoorr uussee wwiitthh tthhiiss kkiitt,,
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For research use only. Not for diagnostic procedures.
1
Section 2
Product Description
The following reagents are included in the Bio-Plex validation kit:
Reagent Quantity
Optics validation bead set:
Optics beads 1 and 2 2 x 10 ml black bottles
5
(1x10
beads/ml)
Fluidics validation bead set:
Fluidics bead 1 1 x 10 ml black bottle
5
(1x10
beads/ml)
Fluidics bead 2 1 x 10 ml black bottle
Reporter validation bead set:
Reporter blank, 1, 2, 3, 4, and 5 6 x 10 ml white bottles
5
(1x10
beads/ml)
Classify validation bead set:
Classify bead 34, 38, 54, 73, 77 5 x 10 ml black bottles
5
(1x10
beads/ml)
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The following materials are required but not supplied:
Bio-Plex MCV plate
Bio-Rad catalog #171-203031 MCV plate II, use with Bio-Plex Manager 3.0,
Bio-Rad catalog #171-203030 MCV plate, use with Bio-Plex Manager 2.0
Bio-Plex Protein Array System
Bio-Rad catalog #171-000001, 171-000003, or 171-000005
Bio-Plex Calibration Kit
Bio-Rad catalog #171-203060
mini vortexer
sterile distilled water
70% isopropanol
10% bleach
bulb pipets
2
Section 3
Specifications
General specifications for the validation kit are listed below. Certain specifications
for the Bio-Plex validation kit may differ from lot to lot. For a complete listing of the
current specifications, please refer to the package insert provided with your
validation kit.
Parameter Specification
Optics Validation
DD mean See insert
CL1 mean See insert
CL1 CV% (Coefficient of Variable) 3–7%
CL2 mean See insert
CL2 CV% 4–8%
RP1 mean See insert
RP1 CV% 5–10%
Fluidics Validation
% carryover < or = 4.0%
Reporter Validation
Dynamic range 4.15–4.28
Linearity >0.995
Slope of response 0.0593–0.0799
Accuracy of response >90%
Sensitivity <200 MESF
Classify Validation
Classify Bead 34, 38, 54, 73, 77 >80.0%
DD Efficiency
% Efficiency > or = 75%
Section 4
Storage and Handling
The Bio-Plex validation kit beads are stable if stored at 4°C protected from light.
When using the Bio-Plex validation kit, remove beads from 4°C storage and
dispense into the MCV plate. Return to 4°C storage immediately following use to
preserve shelf life. All components are guaranteed for 6 months from the date of
purchase when stored as specified in this manual.
3
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.
4
Dynamic Range of Reporter Channel
Definition
The dynamic range is the calculated number of decades covered by the log
amplifier from the slope and the histogram scale. The available range of channels
on the Bio-Plex array reader is 4.5 log amp decades or 32,767 relative linear
channels. The acceptable dynamic range of fluorescence measured by the Bio-Plex
array reader using the Bio-Plex reporter beads is 4.15–4.28.
Impact on Assay Performance
The dynamic range of the Bio-Plex array reader is 4.5 log amp decades or 32,767
relative linear channels. It is desirable for the range of the instrument to be greater
than the range of an assay. If the dynamic range of the instrument is less than that
of an assay, the range of quantitatable analyte may be limited. This parameter will
guide the user in defining the instrument versus the assay dynamic range
limitations.
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
assigned MESF values. Instrument linearity is expressed as the coefficient of
determination or R-squared (R
Impact on Assay Performance
2
) value. The R2value must be >0.995.
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 actual
MESF value data points. The desired accuracy value is >90%.
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. Accuracy values <90% could impact assay performance. The
accuracy data is evaluated in combination with optics alignment to determine if the
Bio-Plex array 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.
5
Slope of the Reporter Channel Response
Definition
The slope of the regression line resulting from the plotting of reporter channel
mean fluorescent values against assigned reporter channel validation bead MESF
values 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. The acceptable range for the slope is 0.0593–0.0799.
Impact on Assay Performance
The slope of the regression line is directly related to the dynamic range of the
instrument. The slope yields direct information about the response of the
photomultiplier tube. If the photomultiplier tube signal saturates at low
fluorescence values, the dynamic range of the instrument is affected. The slope
of the line impacts the dynamic range and the range in turn impacts the
quantitatable range of an assay. If the validation kit yields a value for the slope
that is not within specifications, assay results could be adversely affected.
Sensitivity of Reporter Channel
Definition
Every instrument has an inherent level of noise due primarily to the electronics.
The sensitivity of the Bio-Plex array reader is defined as the lowest detectable
signal above instrument noise. Noise can be attributed to the laser, the
photomultiplier tube, the amplification electronics or the fluidics. The acceptable
sensitivity using the Bio-Plex array reader is <200 MESF.
Impact on Assay Performance
The sensitivity using the Bio-Plex validation kit is expressed in terms of MESF.
The fluorescence is traceable to R-PE, the primary molecule used in Bio-Plex
assays. The typical background or zero standard of a Bio-Plex cytokine assay
falls at a median fluorescence intensity of 100. The background of a "blank" bead
from the validation kit exhibits a median fluorescence intensity of 8. A plot of the
median RP1 fluorescence intensity versus the MESF units illustrates that the
instrument is approximately 10 times more sensitive than a Bio-Plex cytokine
assay (See Figure 1). This is a desired result, as the sensitivity of the instrument
should not directly limit assay sensitivity.
6
Assay MESF
Reader sensitivity
MESF (R-PE)
RP1 Channel
Fig. 1. Assay vs. instrument sensitivity.
Assay Background
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
Section 8
Principle of Fluidics Validation
Principle
The fluidics system of the Bio-Plex suspension array reader requires routine
maintenance to prevent clogging and other malfunctions. Strict adherence to the
maintenance procedures is mandatory for optimal instrument performance. An
assessment of the integrity of the fluidics is automatically performed in the
Fluidics Validation procedure. In the fluidics validation test, a sample of beads is
analyzed followed by a sample of buffer to assess the carryover of beads from
one well to another. This procedure should be performed once per week to
ensure that assay results are not adversely affected. The fluidics path, including
the sample needle must be completely free of debris and excess beads for
optimal array reader performance.
Impact on Assay Performance
If a system is exhibiting a high level of carryover, due to valve malfunction or
partially clogged sample needle, a significant percentage of beads may be carried
over from one well to another. This phenomenon may adversely affect the median
fluorescent intensity values. For example, if a well with a high median fluorescent
intensity (FI) is read immediately prior to a well with a low median FI, the signal in
the well with the low fluorescent intensity may shift upward. This phenomenon
only occurs in extreme cases since the median fluorescent intensity statistic is
robust and is not easily shifted by the introduction of a population of beads with a
significantly different median FI.
8
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