Instruction Manual - Bio-Rad
Contents
1. 19 Troubleshooting Guide 21 Ordering Information 21 Reference 2 lt s 2 sua 2 09 oy Pe esis es ee ae je 22 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 suspension 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 suspension array system 1 optics alignment 2 integrity of fluidics 8 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 For research use only Not for diagnostic procedures Section 2 Product Description The following reagents are included in the Reagent Optics validation bead set Optics beads 1 and 2 1x105 bead2. Parameter Specification Measured Value Carryover lt or 4 0 1 35 EK Create Report Fig 7 Fluidics validation results 9 If value is not within range repeat the procedure If value again is not within the specification contact Bio Rad technical support for assistance 10 4 Validation of Reporter Channel Performance Procedure 1 If not already done follow the procedure for start up and calibration of the Bio Plex system Note Be sure to calibrate immediately before validation Use either the High or Low RP1 Target value for the CAL2 calibration depending on the type of assay being performed 2 Remove the reporter validation bead set from 4 C storage and vortex each bottle for 30 sec 3 Place 5 drops of each reporter bead into the corresponding reporter well labeled as Blank 1 2 3 4 5 and 6 in the MCV plate Ill see Figure 8 4 Store reporter beads at 4 C as soon as possible after use Protect the beads from light 5 Fill the DI H2O and 70 isopropanol reservoirs 6 Select Instrument from the main menu Select Validation from the pull down menu A dialog will appear Enter user name and control number Select Reporter Validation Select OK The following dialog appears Figure 8 15 Reporter Validation 11 This procedure performs Reporter validation 1 Fill DI H20 and 70 isopropanol reservoirs 2 Vortex each Reporter vial for 30 seconds 3 Load 5 drops of each vial int
3. 999999 Low RP1 Target Value 3885 Reader Serial _LX10000266002 Optics Validation Date Time 04 Aug 04 11 55 AM Expiration Date 15 Oct 06 RP1 PMT Voltage 571 67 Access Level Unrestricted Result Passed Parameter Specification Measured Value Pass Fail DD Median 4774 6593 6133 Pass CL1 Median 3383 4135 3615 Pass CL1 CV 2 00 7 00 3 52 Pass CL2 Median 3520 4302 3883 Pass CL2 CV 3 00 8 00 6 93 Pass RP1 Median 3509 4290 3878 Pass RP1 CV 4 00 10 00 8 89 Pass Il Fluidics Validation Result Passed Parameter Specification Measured Value Pass Fail Carryover lt or 4 0 1 3 Pass Ill Reporter Validation Result Passed A Calculated Values B Raw Values Parameter Specification Measured Value Pass Fail Bead Median FI Dynamic Range 4 33 4 45 4 40 Pass Blank 2 Linearity gt 0 995 1 000 Pass 1 8 Slope 33 94 39 42 37 57 Pass 2 54 Accuracy gt 90 00 96 94 Pass 3 656 Threshold lt 6 MFI 2 Pass 4 1576 5 4453 6 25059 IV Classify Validation Result Passed A Classify Efficiency Classify Bead Specification Measured Value Pass Fail Bead A 1 gt 80 0 98 1 Pass Bead B 4 gt 80 0 93 5 Pass Bead C 40 gt 80 0 90 5 Pass Bead D 54 gt 80 0 95 3 Pass Bead E 100 gt 80 0 93 0 Pass B DD Efficiency Parameter Specificatio
4. which are set during calibration of the instrument Turning up the PMT voltage High RP1 Target will allow for the ability to quantitate lower concentrations of analyte while turning down the PMT voltage Low RP1 Target allows for the ability to quantitate higher concentrations of analyte A new feature with the Bio Plex Validation Kit 4 0 is the ability to perform validation at either of these two instrument PMT settings rather than just at the High RP1 Target as was previously the case 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 and the blank bead gives a reading that is greater than zero intensity units Impact on Assay Performance It is desirable for the range of the instrument to be greater than the range of the assay In most expe
5. Measured Value 6165 3668 3 71 3824 6 78 3860 8 92 PR Create Report Fig 5 Optics validation results 13 10 3 Validation of Fluidics Integrity Procedure 1 If not already done follow the procedure for start up and calibration of the Bio Plex system Note Be sure to calibrate immediately before validation Use either the High or Low RP1 Target value for the CAL2 calibration depending on the type of assay being performed 2 Oo NOM Select instrument then validation from the main menu and select fluidics in the dialog box Enter user name and control number Select OK the following dialog box appears Figure 10 Fluidics Validation This procedure performs Fluidics validation 1 Vortex each Fluidics vial for 30 seconds 2 Load 5 drops of each vial into the specified well 3 Click Eject then load MCV plate MCY Plate Ill 4 Select OK to start AA Eject Retract Plate Cancel Fig 6 Fluidics validation dialog Add 5 drops each of fluidics beads 1 and 2 to the designated wells on the MCV plate Ill Select the Eject button in the dialog box to eject the plate holder Place the MCV plate Ill in the microplate platform Select OK to begin the fluidics validation procedure When the procedure has been completed results will appear in a dialog box as shown in Figure 7 14 Validation Results Fluidics Control Number 999999 Low Date amp Time 04 Aug 04 10 37 AM Result Passed
6. the same manner as a low classify efficiency Section 8 Principle of Fluidics Validation Principle The fluidics system of the Bio Plex suspension array reader reguires 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 Fl 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
7. 0 96 2 Bead B 4 gt 80 0 87 5 Bead C 40 gt 80 0 80 6 Bead D 54 gt 80 0 93 9 Bead E 100 gt 80 0 90 4 PR Create Report Fig 11 Classify validation results 11 The results will also be logged into a Validation Log in Bio Plex Manager 4 0 software To access this log select view from the main menu then Validation Log Each type of validation is stored in a separate log for the purpose of tracking data over time See Bio Plex Manager 4 0 software user guide for more information on using the validation log 12 If any values do not meet specifications repeat the procedure If values are again not within specifications contact Bio Rad technical service for assistance 10 6 Generating a Validation Report Procedure The results from each validation procedure are sent to a validation log in Bio Plex Manager 4 0 software This log may be used to create individual reports as well as track multiple validation results over time Each type of validation is logged into a separate view optics validation fluidics validation reporter validation and classify validation You may maneuver through each of the views using either the main menu items or the toolbar icons The specifications for each control number of the validation kit are also shown in a separate window below the results If All validation was selected an entry matching the specific date and time will appear in each of the validation logs All of the va
8. Bio Plex Validation Kit 4 0 Instruction Manual Catalog 171 203001 For use with Bio Plex Manager Software Version 4 0 and MCV plate III 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 Section 2 Section 3 Section 4 Section 5 Section 6 Section 7 Section 8 Section 9 Section 10 10 1 10 2 10 3 10 4 10 5 10 6 10 7 Section 11 Section 12 Section 13 Introduction 1 Product Description 2 Specifications 3 Storage and Handling 3 Principle of Optics Validation 3 Principle of Reporter Channel Validation 4 Principle of Classify Validation 7 Principle of Fluidics Validation 8 Software Utility Installation 8 Procedure for Bio Plex Manager 4 0 MCV plate Ill 9 One Step Procedure for all Validation Parameters 9 Validation of Optics Alignment 12 Validation of Fluidics Integrity 14 Validation of Reporter Channel Performance 15 Validation of Classify Efficiency 17 Generating a Validation Report 18 Validation Report Example
9. 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 The accuracy data is evaluated in combination with optics alignment to determine if the Bio Plex 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 R2 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 Slope of the Reporter Channel Response Definition The slope of the regression line resulting from the plotting of reporter channel mean fluorescent values against expected relative fluorescence 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 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 i
10. by the introduction of a population of beads with a significantly different median FI Section 9 Software Utility Installation Introduction This section provides instructions for installing the control number information from the BizCard CD contained in the Validation Kit into the Bio Plex Manager database The BizCard CD contains the control number expiration date and specifications for your validation kit Procedure 1 Exit out of Bio Plex Manager prior to proceeding 2 Insert the BizCard CD into the CD Rom drive of your computer The installation program should start automatically The application can also be launched through Windows Explorer by double clicking on the file InstallControlNumbers exe 3 If the control numbers on the BizCard CD already exist in the database you will receive the message that the control numbers are already installed and the program will exit Click on the OK button and remove the BizCard CD 4 Ifthe control numbers on the BizCard CD are not found in the database you will see a dialog asking you to press OK to install the control numbers with their specifications Click on the OK button 5 A dialog box will appear informing you that control numbers installation is complete You may now proceed with validation in Bio Plex Manager using the current control number Section 10 Procedure for Bio Plex Manager 4 0 and MCV plate Ill Introduction This section provides instructions for use of th
11. e Expiration Date 15 Oct 06 15 Show Specifications Note The Control Number is located on the Validation Kit box Validation Type All Optics Fluidics Reporter Classify C Optics Fluidics C Reporter C Classify Cancel Fig 1A Main validation dialog 6 Enter user name and control number Select All Select OK The following dialog appears Figure 1B All Validation This procedure performs Optics Fluidics Reporter and Classify validation 1 Vortex each Validation vial for 30 seconds 2 Load 5 drops of each vial into the specified well OGAG AGBA 3 Fill DI H20 and 70 isopropanol reservoirs MCY Plate Ill 4 Click Eject then load MCY plate 5 Select OK to start Ah Fject Retract Plate Cancel Fig 1B All validation dialog 10 7 Place 5 drops each of bead into the respective wells on the MCV plate Ill see Figure 2 below or Figure 1B Store beads at 4 C as soon as possible after use Protect the beads from light Bio Plex MCV Plate Ill OPTICS REPORTER CLASSIFY E Blank a f y FLUIDICS 70 Isopropanol e 1 ce i mer P N 171 203032 10 Bleach Fig 2 MCV plate III 8 Select the Eject button in the dialog box to eject the plate holder 9 Place the MCV plate Ill in the microplate platform 10 Select OK to begin all validation procedures 11 When the procedure has completed the results will be displayed in a dialog box as shown i
12. e Bio Plex Validation Kit 4 0 with Bio Plex Manager version 4 0 and MCV plate Ill Bio Plex Manager now provides a fully automated validation routine that sequentially performs all the validation tests without further user intervention To perform all validations in a single step follow instructions in Section 10 1 If you wish to perform an individual validation routine these instructions are provided in Sections 10 2 10 5 10 1 One Step Procedure for all Validation Parameters Procedure 1 Turn on the Bio Plex array reader microplate platform and computer as specified in the Bio Plex hardware and Bio Plex Manager user manuals 2 Perform start up procedure as directed 3 Calibrate Bio Plex array reader using CAL1 and CAL2 beads found in the Bio Plex Calibration Kit according to the Bio Plex Manager software manual Note Be sure to calibrate immediately before validation Use either the High or Low RPI Target for CAL2 calibration depending on the type of assay being performed 4 Remove all validation bead sets from 4 C storage and vortex each and every bottle for 30 sec This is very important for proper validation 5 In Bio Plex Manager Software select Instrument from the main menu Select Validation from the pull down menu The following dialog box appears Figure 1A Validation User Name Last CAL2 Calibration O Pocekaj Date amp Time O4 Aug 04 10 21 AM RPI Target Low Control Number 999999 Se Add Remov
13. ed on the Bio Plex array reader and the efficiency of multiplexing is quantitated A classify efficiency of gt 80 is required for optimal results DD Efficiency is a measure of the percentage of the Classify beads that fall within the DD Gates The DD gates Doublet Discriminator 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 value less than 75 may increase the read time of the assay and affect results in
14. formed 4 Remove the optics validation bead set from 4 C storage and vortex each bottle for 30 sec 5 Place 5 drops each of optics beads 1 and 2 into the respective wells on the MCV plate Ill 6 Store optics beads at 4 C as soon as possible after use Protect the beads from light 7 In Bio Plex Manager Software select Instrument from the main menu Select Validation from the pull down menu 8 Enter user name and control number Select Optics Select OK The following dialog appears Figure 4 12 10 11 12 Optics Validation MCY Plate III This procedure performs Optics validation 1 Vortex each Optics vial for 30 seconds 2 Load 5 drops of each vial into the specified well 3 Click Eject then load MCY plate 4 Select OK to start A Fject Retract Plate Cancel Fig 4 Optics validation dialog Select the Eject button in the dialog box to eject the plate holder Place the MCV plate Ill in the microplate platform Select OK to begin the optics validation procedure When the procedure has been completed the results will be displayed in a dialog box as shown in Figure 5 Validation Results Optics Control Number Date amp Time Result Parameter DD Median CL1 Median CLI CY CL2 Median CL2CV RP1 Median RP1 CV 999999 Low 04 Aug 04 10 31 AM Passed Specification 4774 6593 3383 4135 2 00 7 00 3520 4302 3 00 8 00 3509 4290 4 00 10 00
15. lidation results for a specific date and time will be included in a created report The entire log may be printed by selecting Print then Results from the main menu A general procedure for creating a report from the Validation log is shown below For more detailed instructions on the use of the validation log consult the Bio Plex Manager 4 0 user guide 18 1 Open Bio Plex Manager 4 0 software by clicking on the application icon on the desktop Ev 2 Select View from the main menu then select Validation Log from the pulldown menu The validation log will open 3 Choose the desired validation log by using the main menu or the toolbar icons for optics fluidics reporter and classify validation 4 Click on the desired entry in the validation log The selected row will be highlighted in black 5 Select the create report icon A report will automatically be generated in Microsoft Excel ER 6 Print the report in Excel by selecting File then Print from the main menu Alternatively you may use the print button in Excel 10 7 Validation Kit Worksheet and Report Form Examples The following is a sample validation report from Bio Plex Manager 4 0 software Note that the values included here are for demonstration purposes only Consult your product insert for values specific to your product control number 19 V Bio Plex Suspension Array System Validation Report Test Performed by JPocekay Validation Kit Control
16. ly 50 daily calibration routines 171 203032 Validation kit 4 0 21 Bio Plex MCV Plate Ill for use with Bio Plex Manager 4 0 and Section 13 Reference Alder Henry Introduction to Probability and Statistics Alder HL and Roessler EB eds W H Freeman San Francisco p118 1968 By purchasing this kit which contains fluorescent labeled microsphere beads authorized by Luminex you the customer acquire the rights under Luminex s patent rights to use certain portions of this kit including without limitation the microsphere beads contained herein only with Luminex s laser based fluorescent analytical test instrumentation known under the name of Luminex 100 for example as marketed by Bio Rad Laboratories Inc in the Bio Plex system Including but not limited to US patent 5 981 180 6 046 807 6 057 107 Certain Bio Plex validation kit components are licensed under US patent 5 723 218 22 Bio Rad Laboratories 2000 Alfred Nobel Dr Hercules CA 94547 1 800 424 6723 4410185 RevA
17. matically loaded into Bio Plex Manager 4 0 upon installation of the disc 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 18 months from the date of manufacture when stored as specified in this manual 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 Sectio
18. n Measured Value Pass Fail DD Efficiency gt or 75 0 96 7 Pass Comments Reviewed by Date 20 Section 11 Troubleshooting Guide Problem Optics validation procedure shows value outside of acceptable range Reporter validation procedure shows value outside of acceptable range Classify validation procedure shows value outside of acceptable range Fluidics validation procedure shows value outside of acceptable range Section 12 Cause Problem with the optical component of the array reader Problem with the optical component of the array reader Problem with the calibration or optical component of the array reader Problem with fluidics lines valves or sample needle of array reader Ordering Information Catalog H 171 203001 Description Solution Repeat the procedure If value is still out of range contact Bio Rad technical support Repeat the procedure If value is still out of range contact Bio Rad technical support Repeat the procedure If values are still out of range contact Bio Rad technical support Repeat procedure If value is still out of range contact Bio Rad technical support Bio Plex Validation Kit 4 0 includes optics validation fluidics validation reporter validation and classify validation bead sets for approximately 50 validation routines 171 203060 Bio Plex Calibration Kit includes CAL1 and CAL2 calibration beads for approximate
19. n 6 Principle of Reporter Validation Principle The reporter RP1 channel is the fluorescence channel used for assay guantitation 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 gualification R phycoerythrin R PE is the primary reporter molecule used in Bio Plex assays A series of beads dyed with verying 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
20. n Figure 3 Validation Results Optics Fluidics Reporter Classify Control Number 999999 High Date amp Time 20 Aug 04 01 15 PM Result Passed Parameter Specification Measured Value DD Median 4774 6593 5600 CL1 Median 3383 4135 3514 CLI CV 2 00 7 00 4 03 CL2 Median 3520 4302 3893 CL2 CV 3 00 8 00 7 03 RP1 Median 15205 18583 18496 RPI CV 4 00 10 00 8 39 EK Create Report Fig 3 Validation results 11 12 Check the results for each validation If specifications are not met for any validation repeat that validation If the value again is not within specification contact Bio Rad technical support for assistance 13 The following sections describe the steps for performing the individual validations optics fluidics reporter or classify 14 Validation results are also logged into a validation log in Bio Plex Manager To access this log select View from the main menu then Validation Log Each type of validation is stored in a separate log for the purpose of tracking data over time See the Bio Plex Manager 4 0 user manual for more information on using the validation log 10 2 Validation of Optics Alignment Procedure 1 If not already done follow the procedure for start up and calibration of the Bio Plex system Note Be sure to calibrate immediately before validation Use either the High or Low RP1 Target for CAL2 calibration depending on the type of assay being per
21. n turn impacts the quantifiable range of an assay Instrument Threshold Sensitivity Definition Every instrument has an inherent level of background noise Noise can be attributed to the laser the PMT the amplification electronics or the fluidics The instrument threshold sensitivity of the Bio Plex array reader is one way to demonstrate noise of the system It is defined as the reporter channel signal of a blank bead which contains no reporter dye Impact on Assay Performance The instrument threshold level demonstrated using the Bio Plex validation kit is a low fluorescence intensity value The typical background or zero standard of a Bio Plex cytokine assay falls at a median fluorescence intensity of 100 This is a desired result as the instrument threshold should not limit the assay sensitivity If the instrument threshold is too close to zero then a low assay signal may be masked if too high then the linearity and dynamic range may be compromised 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 analyz
22. o the specified well MCV Plate III 4 Click Eject then load MCY plate 5 Select OK to start A Eject Retract Plate Cancel Fig 8 Reporter validation dialog Select the Eject icon in the dialog box to eject the plate holder Place the MCV plate Ill in the microplate platform Select OK to start the reporter validation procedure When the procedure is completed values will be displayed in a dialog box as shown below Figure 9 Validation Results Reporter Control Number 999999 Low Date amp Time 04 Aug 04 10 44 AM Result Passed C Raw Values Parameter Specification Measured Value Dynamic Range 4 33 4 45 4 41 Linearity gt 0 995 1 000 Slope 33 94 39 42 38 17 Accuracy gt 90 00 97 59 Threshold lt 6MFI 2 PR Create Report Fig 9 Reporter validation results The results will also be logged into a validation log in Bio Plex Manager 4 0 software See Bio Plex Manager 4 0 user manual for more information on using the validation log 16 12 Repeat procedure if values are not within specifications If any values are again not within acceptable ranges contact Bio Rad technical service for assistance 10 5 Validation of Classify Efficiency Procedure 1 If not already done follow the procedure for start up and calibration of the Bio Plex system Note Be sure to calibrate immediately before validation Use either the High or Low RP1 Target value for the CAL2 calibration depending
23. on the type of assay being performed 2 Remove the classify validation bead set from 4 C storage and vortex each bottle for 30 sec 8 Place 5 drops of each classify bead into the corresponding classify well labeled as A B C D and E in the MCV plate III see Figure 10 4 Store stock vials at 4 C as soon as possible after use Protect beads from light 5 Select Instrument from the main menu Select Validation from the pull down menu 6 Enter the User name and select control number Select classify validation Select OK The following dialog box appears Figure 10 7 Select the Eject button to eject the plate holder Classify Validation This procedure performs Classify validation 1 Vortex each Classify bottle for 30 seconds 2 Load 5 drops of each vial into the specified well 3 Click Eject then load MCV plate MCV Plate Ill 4 Select OK to start A Eject Retract Plats Cancel Fig 10 Classify validation dialog 8 Place the MCV plate Ill on the microplate platform 9 Select OK to start the classify validation procedure 10 When the procedure is completed values will be displayed in a dialog box as shown below Figure 11 The classify efficiency and DD efficiency results may be accessed in this view 17 Validation Results Classify Control Number 999999 Low Date amp Time 04 Aug 04 10 59 AM Result Passed C DD Efficiency Region Specification Measured Value Bead 4 1 gt 80
24. riments the dynamic range of the assay is far less than that of the instrument However in some assays the same or different analytes may be present in greater than 4 5 log differences in concentration When this is the case the sample can be rerun at different PMT settings to extend the overall dynamic range of the measurements 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 expected fluorescent intensity values as determined on an independent calibrated instrument Instrument linearity is expressed as the coefficient of determination or R squared R2 value Impact on Assay Performance 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 R2 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 R2 value Simply stated the accuracy of the reporter channel response is the percent difference that the regression line is away from the expected fluorescence intensity data point values Impact on Assay
25. s ml Fluidics validation bead set Fluidics bead 1 1x105 beads ml Fluidics bead 2 Reporter validation bead set Reporter blank 1 2 3 4 5 and 6 1x105 beads ml Classify validation bead set Bio Plex Validation Kit 4 0 Quantity 2 x 10 ml black bottles 1 x 10 ml black bottle 1 x 10 ml black bottle 6 x 10 ml white bottles Classify bead A 1 B 4 C 40 D 54 E 100 5 x 10 ml black bottles 1x105 beads ml NOTE Validation should be performed immediately following calibration BizCard CD A CD with the lot specific product specifications The following materials are reguired but not supplied Bio Plex MCV plate Bio Rad catalog 171 203032 MCV plate Ill use with Bio Plex Manager 4 0 171 001010 Bio Plex Suspension array System Bio Rad catalog 171 000001 171 000002 171 000003 171 00004 171 000005 171 000006 171 000007 or 171 000009 Bio Plex Calibration Kit Bio Rad catalog 171 203060 mini vortexer sterile distilled water 70 isopropanol 10 bleach bulb pipets Section 3 Specifications Specifications for the Bio Plex validation kit may differ from lot to lot Please refer to the package insert provided with your validation kit for specifications Section for Software Installation The Bio Plex Validation Kit 4 0 includes a BizCard CD containing files with the specifications for the validation beads along with additional information The file information is auto
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