- Bio-Rad
Contents
1. 16 Generating a Validation Report 18 Validation Report Example 19 Procedure for Bio Plex Manager 2 0 MCV plate 21 Validation of Optics Alignment 21 Validation of Reporter Channel Performance 23 Validation of Classify Efficiency 25 Validation of Fluidics Integrity 26 Generating a Validation Report 28 Validation Kit Worksheet and Report Form EXaplOS snare abt oh ove Boe eek eae ch hake dong Se ES 28 Troubleshooting Guide 32 Ordering Information 32 Reference sacs sad pulta dns x Haba a eed 33 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 classify2. 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 lt 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
3. o o No GM If the unclog procedure was successful the following message will appear Bio Plex Manager x The unclog procedure was successful f Fluidics integrky has been vakdated A 10 Check yes in Section IV of the validation kit worksheet 11 If the procedure is unsuccessful the following message will appear If this is the case repeat steps 4 11 x x The unclog procedure was not successful Please repeat procedure If procedure fak a second time please contact Technical Service For assistance Ce 12 If the procedure is again unsuccessful contact Bio Rad technical support for assistance Bio Plex Manager 2 0 and MCV Plate Instructions 27 10 5 Generating a Validation Report Procedure li 2 3 O o o N o li 12 Insert the Bio Plex Manager CD into your computer s CD drive Open Microsoft Excel Select Open and using your browser choose Bio Plex validation report from the Bio Plex CD Click on the Worksheet tab Enter the values from the worksheet used during the validation procedure A series of calculations will be automatically performed Click on the Report Tab to view the Validation report Values out of specifications will be shown in red Select File then Print to print the report Select File then Save Enter the desired file name and location then select OK to save the file If any values are not within acceptable specifications repeat that specific
4. CL2 Median 3520 4302 4035 CL2 CY 4 8 5 62 RP1 Median 15205 18583 18148 RP1 CV 5 10 8 34 Record the values in the worksheet provided with the Validation Kit Follow Kit instructions to complete the Validation Procedure If any results are not within acceptable specifications shown under specifications in list please contact Bio Rad Technical service Fig 5 Optics validation results 14 Acceptable optics validation specifications Parameter Specification DD median See insert CL1 median See insert CL1 CV 3 7 CL2 median See insert CL2 CV 4 8 RP1 median See insert RP1 CV 5 10 If any values do meet specification repeat the procedure If values are again not within specifications contact Bio Rad technical support for assistance 9 3 Validation of Fluidics Integrity Procedure 1 If not already done follow the procedure for start up and calibration of the Bio Plex system 2 Select instrument then validation from the main menu and select fluidics in the dialog box 3 Enter user name and control number Select OK the following dialog box appears Figure 10 Bio Plex Manager 3 0 and MCV Plate II Instructions 13 Fluidics Validation x 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 MCY plate 4 Select OK to start Eject Retract P
5. To Generate Report 1 Open Excel and open the Val idationReport xls file 2 Click on Worksheet tab and enter values from this worksheet 3 Click on Report and select Fi Bio Plex Manager 2 0 and MCV Plate Instructions e then Print to print report 29 4106178 Rev A n Protein Array Reader Bio Pi Validation Report Performed by Serial Number Kit Lot number Optics Validation Parameter Specification Measured Value 4774 6593 6150 CL1 median 3383 4135 CL1 CV CL2 CV 4 8 6 43 RP1 median 15205 18583 17201 ll Reporter Validation Slopa Accuracy Region 38 Region 54 Note Values in red indicate parameters not within specifications If values are not within acceptable specifications please repeat procedure If values are still not within specifications contact Bio Rad technical service for assistance See Bio Plex validation kit manual for specific definitions of each parameter and the impact on Bio Plex assay performance Bio Plex Manager 2 0 and MCV Plate Instructions 30 Pa Protein Array Reader Bio Plex Validation Report IV Validation of Fluidics Integrity not within specifications If values are m thin acceptable are stil not within specifications conta in red in e paramete adure If val p se 8 5 of sach parameter and the impact on Bio Plex ww ance Comments Reviewed by Date Bio Plex
6. validation procedure If values are not within acceptable specifications after repeating a second time contact Bio Rad technical support for assistance 10 6 Validation Kit Worksheet and Report Form Examples The following is a sample validation kit worksheet used to record values obtained during the various validation procedures A full size version suitable for photocopying is included with the kit Also included is an example of the validation kit report output from Bio Plex Manager see Section 13 Note that the values included here are for demonstration purposes only Consult your product insert for values specific to your product control number Bio Plex Manager 2 0 and MCV Plate Instructions 28 Bio PI Protein Array Reader Validation it Worksheet Performed by l Optical Validation Parameter Specification Measured Value DD median 4774 6593 CL1 median 3383 4135 CL1 CV 3 7 CL2 median 3520 4302 CL2 CV 4 8 RP1 median 15205 18583 RP1 CV 5 10 Il Reporter Validation Bead Median Channel B Blank 1 Om o p Ill Classify Validation Region Specification Measured Value Region 34 gt 80 Region 38 gt 80 Region 54 gt 80 Region 73 gt 80 Region 77 gt 80 IV Validation of Fluidics Integrity Fluidics Integrity Validated Place X in appropriate box YES NO
7. Classify from the pop up menu The following dialog box appears Classify validation x This procedye validates the performance of the Classify channels of the Bio Plex Reader 1 Place 5 drops pf each of the Classify beads 34 38 54 73 77 in the wels spectied on the MCY plate 2 Click eject to eject plate carrer Load MCY plate on reader 3 Press OK to start operation Ejeci Retract Plate Cancel Fig 17 Classify validation dialog Select the Eject button to eject the plate holder Bio Plex Manager 2 0 and MOV Plate Instructions 25 10 11 Place the MCV plate on the Bio Plex microplate platform Select OK to start the classify validation procedure When the procedure is completed values will be displayed in a dialog box as shown below Classify Validation x Classify efficiency Acceptable values are shown under spectication column Region 34 gt 80 31 60 Region 38 gt 80 36 30 Region 54 gt 80 34 49 Region 73 gt 80 1 60 Region 77 gt 80 87 94 Record the values in the worksheet provided with the Validation Kit Follow Kit instructions to complete the Validation Procedure If any resuks are less than 80 contact technical support for further assistance Fig 18 Classify Validation dialog Record the classify efficiency values in a copy of the validation kit worksheet provided in this manual Acceptable classify efficiency values Classify Bead Specificat
8. been completed values will be displayed in a dialog box as shown on the next page 13 Record the values for each optics validation parameter in the validation kit worksheet provided with the validation kit Bio Plex Manager 2 0 and MCV Plate Instructions 22 Optics Yalidation Results xj DD Median 4774 6593 6318 CL1 Median 3383 4135 3892 CL1 Cy 3 7 4 67 CL2 Median 3520 4302 4035 CL2 CY 4 8 5 62 RP1 Median 15205 18583 18148 RP1 CY 5 10 8 34 Record the values in the worksheet provided with the Validation Kit Follow Kit instructions to complete the Validation Procedure If any results are not within acceptable specifications shown under specifications in list please contact Bio Rad Technical service Fig 14 Optics validation results 14 Acceptable optics validation specifications Parameter Specification DD median See insert CL1 median See insert CL1 CV 3 7 CL2 median See insert CL2 CV 4 8 RP1 median See insert RP1 CV 5 10 If any results are not within the acceptable specifications please contact Bio Rad technical service If any values do meet specification repeat the procedure If values are again not within specifications contact Bio Rad technical service for assistance 10 2 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 im
9. 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 NOTE Bio Plex Manager 3 0 requires MCV plate II Cat 171 203031 for use with this Validation Kit see Section 9 Bio Plex Manager 2 0 requires MCV plate Cat 171 203030 for use with this kit see Section 10 Bio Plex Manager 2 0 does not allow use of fluidics 1 and fluidics 2 beads This step is omitted follow Section 10 instructions For research use only Not for diagnostic procedures 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 1x105 beads ml Fluidics validation bead set Fluidics bead 1 1 x 10 ml black bottle 1x105 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 1x105 beads ml Classify validation bead set Classify bead 34 38 54 73 77 5 x 10 ml black bottles 1x105 beads ml NOTE The validation kit must be used following CAL2 calibration using the RP1 high target value Validation should be performed immediately following calibration T
10. 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 actual MESF value data points The desired accuracy value is gt 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 lt 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 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 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 M 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 ESF
11. within specifications contact Bio Rad technical service for assistance 9 6 Generating a Validation Report Procedure The results from each validation procedure are sent to a validation log in Bio Plex Manager 3 0 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 validation 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 3 0 user manual 1 Open Bio Plex Manager 3 0 software by clicking on the application icon on the desktop Al 2 Select View from the main menu then select Validation Log from the pulldown menu The validation log will open Bio Plex Manager 3 0 and MCV Plate II Instructions 18 8 Choose the des
12. Bio Rad Laboratories 2000 Alfred Nobel Dr Hercules CA 94547 1 800 424 6723 4110007 Rev C Validation Kit 3 0 Instruction Manual Catalog 171 203000 For use with Bio Plex Manager Software Version 3 0 and MCV plate I 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 Section 2 Section 3 Section 4 Section 5 Section 6 Section 7 Section 8 Section 9 9 4 9 2 9 3 9 4 9 5 9 6 9 7 Section 10 10 1 10 2 10 3 10 4 10 5 10 6 Section 11 Section 12 Section 13 Introduction 1 Product Description 2 Specifications 3 Storage and Handling 3 Principle of Optics Validati on 4 Principle of Reporter Channel Validation 4 Principle of Classify Validation 7 Principle of Fluidics Validation 8 Procedure for Bio Plex Manager 3 0 MCV plate Il 9 One Step Procedure for all Validation Parameters 9 Validation of Optics Alignment 11 Validation of Fluidics Integrity 13 Validation of Reporter Channel Performance 15 Validation of Classify Efficiency
13. Il Fluidics Validation Result Completed Parameter Specification Measured Value Pass Fail Carryover lt or 3 5 2 Pass Ill Reporter Validation Result Completed A Calculated Values B Raw Values Parameter Specification Measured Value Pass Fail Bead Median FI Dynamic Range 4 15 4 28 4 1936 Linearity gt 0 995 1 000 1 19 Slope 0 0593 0 0799 0 0664 Accuracy gt 90 00 98 Sensitivity lt 200 MESF 120 4 7006 IV Classify Validation Result Completed A Classify Efficiency Measured Value gt 80 0 99 Pass Bead 38 gt 80 0 97 Pass Bead 54 gt 80 0 96 Pass Bead 73 gt 800 Bead 77 gt 800 97 Pass B DD Efficiency Parameter Comments Specification Measured Value Pass Fail DD Efficiency gt or 750 91 Pass Reviewed by Bio Plex Manager 3 0 and MCV Plates Il Instructions 20 Date Section 10 Procedure for Bio Plex Manager 2 0 and MCV Plate 10 1 Validation of Optics Alignment 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 Note Be sure to calibrate immediately before validation Use the High RP1 Target value for CAL2 calibration Perform start up procedure as directed Calibrate Bio Plex array reader using CAL1 and CAL2 beads found in the Bio Plex calibration kit according to the Bio Plex Manager software man
14. Manager 2 0 and MCV Plate Instructions 31 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 171 203000 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 3 0 includes optics validation fluidics validation reporter validation and classify validation bead sets for approximately 50 validation routines 171 203060 171 203031 171 203030 Validation kit 3 0 32 Bio Plex Calibration Kit includes CAL1 and CAL2 calibration beads for approximately 50
15. 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 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 a
16. a dialog box as shown below Reporter Validation Results x Reporter Median plot values Record the values in the worksheet provided with the Validation Kit Follow Kit instructions to complete the Validation Procedure Fig 16 Reporter validation results Bio Plex Manager 2 0 and MCV Plate Instructions 24 11 Record the median channel values in a copy of the worksheet provided with the validation kit 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 13 Acceptable reporter validation specifications Parameter Specification Dynamic Range 4 15 4 28 Linearity gt 0 995 Slope 0 0593 0 0799 Accuracy gt 90 Sensitivity lt 200 MESF 10 3 Validation of Classify Efficiency Procedure 1 6 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 the High RP1 Target value for CAL2 calibration Remove the classify validation bead set from 4 C storage and vortex each bottle for 30 sec Place 5 drops of each classify bead into the corresponding classify well labeled as 34 38 54 73 and 77 in the MCV plate see Figure 2 Store stock vials at 4 C as soon as possible after use Protect beads from light Select Instrument from the main menu Select Validation from the pull down menu Select
17. daily calibration routines Bio Plex MCV Plate Il for use with Bio Plex Manager 3 0 and Validation kit 3 0 Bio Plex MCV Plate for use with Bio Plex Manager 2 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 33
18. e Report Fig 9 Reporter validation results 11 The results will also be logged into a validation log in Bio Plex Manager See Bio Plex Manager user manual for more information on using the validation log 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 13 Acceptable reporter validation specifications Parameter Specification Dynamic Range 4 15 4 28 Linearity gt 0 995 Slope 0 0593 0 0799 Accuracy gt 90 Sensttivity lt 200 MESF 9 5 Validation of Classify Efficiency Procedure 1 If not already done follow the procedure for start up and calibration of the Bio Plex system 2 Remove the classify validation bead set from 4 C storage and vortex each bottle for 30 sec 3 Place 5 drops of each classify bead into the corresponding classify well labeled as 34 38 54 73 and 77 in the MCV plate Il see Figure 2 4 Store stock vials at 4 C as soon as possible after use Protect beads from light Bio Plex Manager 3 0 and MCV Plate Il Instructions 16 Select Instrument from the main menu Select Validation from the pull down menu Enter the User name and control number Select classify validation Select OK The following dialog box appears Figure 8 Select the Eject button to eject the plate holder This procedure performs Classify validation 1 Vortex each Classify bottle for 30
19. ers 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 CAL 1 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 the High RP1 Target for CAL2 calibration 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 Bio Plex Manager 3 0 and MCV Plate II Instructions 5 Select Instrument from the main menu Select Validation from the pull down menu The following dialog box appears Figure 2A Validation x User Name DDavis r Control Number Defaut z Add Delete tS Show Specifications Close ji Select Validation Type All Optics Fluidics Reporter Classify C Optics Fluidics Reporter C Classify Note The Control Number is located on the Validation Kit box Fig 2A Main validation dialog 6 Enter user name and control number Select All Select OK The following dialog appears Fibure 2B All Validation X This procedure performs Optics Fluidics Reporter and Classify validation 1 Vortex each Validation vial for 30 seconds s eeee6 2 Load 5 drops
20. ffected 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 by the introduction of a population of beads with a significantly different median FI Section 9 Procedure for Bio Plex Manager 3 0 and MCV plate ll Introduction This section provides instructions for use of the Bio Plex Validation Kit 3 0 with Bio Plex Manager version 3 0 and MCV plate II 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 9 1 If you wish to perform an individual validation routine these instructions are provided in Sections 9 2 9 5 9 1 One Step Procedure for all Validation Paramet
21. gure 2 4 Store reporter beads at 4 C as soon as possible after use Protect the beads from light Fill the DI H2O reservoir with water 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 5 Reporter Validation x 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 into the specified well MCY Plate Il 4 Click Eject then load MCY plate 5 Select OK to start A Eject Retract Plate Cancel Fig 8 Reporter validation dialog 7 Select the Eject icon in the dialog box to eject the plate holder 8 Place the MCV plate Il in the microplate platform 9 Select OK to start the reporter validation procedure 10 When the procedure is completed values will be displayed in a dialog box as shown below Figure 7 Bio Plex Manager 3 0 and MCV Plates II Instructions 15 Validation Results x Optics Fluidics Reporter Classity Date amp Time 19 Nov 02 10 26 AM Result Completed Calculated Results C Raw Values Parameter Specification Measured Value Dynamic Range 4 15 4 28 4 20 Accuracy gt 90 94 94 Linearity gt 0 995 0 999 Sensitivity lt 200 MESF 103 16 Slope 0 0593 0 0799 0 0679 Creat
22. he 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 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 lt or 4 0 Reporter Validation Dynamic range 4 15 4 28 Linearity gt 0 995 Slope of response 0 0593 0 0799 Accuracy of response gt 90 Sensttivity lt 200 MESF Classify Validation Classify Bead 34 38 54 73 77 gt 80 0 DD Efficiency Efficiency gt or lt 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
23. 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 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
24. ion 34 gt 80 0 38 gt 80 0 54 gt 80 0 73 gt 80 0 77 gt 80 0 Note 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 4 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 the High RP1 Target value for CAL2 calibration Remove the calibration bead set from 4 C storage and vortex each bottle for 30 sec Bio Plex Manager 2 0 and MCV Plate Instructions 26 2 Select the Unclog icon from the toolbar 3 The following dialog box appears xi This operation removes cbsteuctions fom the tkidica lines 1 Plsca 3 0 ml of 10 blesch in the blesch reservoir of the MOV plate 2 Place 3 0 ml of DI water in the DI H2O reservoir of the MCV plate 3Plece 5 chops of CALI beads in CALI well of the MCV plate 4 Cick Eject to eject plate carnier Load the MCV plate on reader 5 Press OK to stat the operation Enc eine Pale Cene Fig 19 Unclog dialog Add 5 drops of CAL1 beads to the CAL1 well on the MCV plate Fill the DI H2O and 70 isopropanol reservoirs Select the Eject button in the dialog box to eject the plate holder Place the MCV plate in the Bio Plex microplate platform Select OK to begin the validation of fluidics integrity procedure
25. ired 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 mij 6 Print the report in Excel by selecting File then Print from the main menu Alternatively you may use the print button in Excel 9 7 Validation Kit Worksheet and Report Form Examples The following is a sample validation report from Bio Plex Manager Note that the values included here are for demonstration purposes only Consult your product insert for values specific to your product control number Bio Plex Manager 3 0 and MCV Plates Il Instructions 19 Bio PI Test Performed by Dano Validation Kit Control 90610 RP1 Target Value 17677 Optics Validation Result Completed Template ET 873 01 BETA 10 Suspension Array System Validation Report Date Time 01 13 2003 12 20 Reader Serial 4 Ix10099092008 RP1 PMT Voltage 808 79 Parameter Specification Measured Value Pass Fail DD Median 4774 6593 5799 Pass CL1 Median 3383 4135 3732 Pass CLI CY 3 00 7 00 6 Pass CL2 Median 3520 4302 3892 Pass CL2 CY 4 00 8 00 7 Pass RP1 Median 16205 18583 17332 Pass RP1 CV 5 00 10 00 8 Pass
26. late Cancel Fig 6 Fluidics validation dialog 4 Add 5 drops each of fluidics beads 1 and 2 to the designated wells on the MCV plate Il Select the Eject button in the dialog box to eject the plate holder Place the MCV plate II 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 11 Oo NOM alidation Results x Fluidics Date Time O9Jan 03 11 52AM Result Completed Parameter Specification Measured Value Carryover lt or 4 0 2 00 PR Create Report Fig 7 Fluidics validation results 9 Acceptable fluidics validation value carryover lt or 4 0 Note If value is not within range repeat the procedure If value again is not within the specification contact Bio Rad technical support for assistance Bio Plex Manager 3 0 and MCV Plates II Instructions 14 9 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 the High RP1 Target value for the CAL2 calibration 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 B blank 1 2 3 4 and 5 in the MCV plate Il see Fi
27. mediately before validation Use the High RP1 Target value for CAL2 calibration 2 Remove the reporter validation bead set from 4 C storage and vortex each bottle for 30 sec Bio Plex Manager 2 0 and MCV Plate Instructions 23 3 Place 5 drops of each reporter bead into the corresponding reporter well labeled as B blank 1 2 3 4 and 5 in the MCV plate see Figure 2 4 Store reporter beads at 4 C as soon as possible after use Protect the beads from light Fill the DI H2O reservoir with water Select Instrument from the main menu Select Validation from the pull down menu A dialog box will appear Select Reporter from the pop up menu The following dialog box appears Reporter validation xj es procedure valideles the perloemance cf the Reporter Channel s the DiorPtex tager 1 FLD H29 reserves with datod mas 2 Vortex each baltie on high setong ot 30 seconds A Placa 5 crops each of the Feporar Validwinn heads fB 1 2 3 4 Spin tie zpectiad was of tha Feportar esiumn in the MEV plete 4 Click eject to elect plate camer Load MCV plate on izads Hi Press UK to stat operation Eject Retct ate Cancel Fig 15 Reporter validation dialog 7 Select the Eject icon in the dialog box to eject the plate holder Place the MCV plate in the Bio Plex microplate platform 9 Select OK to start the reporter validation procedure 10 When the procedure is completed values will be displayed in
28. near 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 R2 value The R2 value must be gt 0 995 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
29. of each vial into the specified well 3 Fill DI H20 and 70 isopropanol reservoirs MCY Plate Il 4 Click Eject then load MCY plate 5 Select OK to start A Eject Retract Plate Cancel Fig 2B Bio Plex Manager 3 0 and MCV Plate Il Instructions 10 9 10 Place 5 drops each of bead into the respective wells on the MCV plate II see Figure 3 below or Figure 2B Store beads at 4 C as soon as possible after use Protect the beads from light Bio Plex MCV Plate II OPTICS REPORTER CLASSIFY RINSE WELLS BEFORE AND AFTER USE P N 171 203031 Fig 3 MCV plate Il Select the Eject button in the dialog box to eject the plate holder Place the MCV plate II in the microplate platform Select OK to begin all validation procedures 9 2 Validation of Optics Alignment 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 Perform start up procedure as directed 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 the High RP1 Target for CAL2 calibration 4 Remove the optics validation bead set from 4 C storage and vortex each bottle for 30 sec Place 5 drops each of optics beads 1 and 2 into the respective wells on the MCV
30. plate Il Store optics beads at 4 C as soon as possible after use Protect the beads from light Select Instrument from the main menu Select Validation from the pull down menu Bio Plex Manager 3 0 and MCV Plate II Instructions 11 8 10 11 12 13 Enter user name and control number Select Optics Select OK The following dialog appears Figure 4 Optics Validation x 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 MCY Plate II 4 Select OK to start Ah Eject Retract Plate Cancel Fig 4 Select the Eject button in the dialog box to eject the plate holder Place the MCV plate Il 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 The results will also be 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 Bio Plex Manager 3 0 user manual for more information on using the validation log Bio Plex Manager 3 0 and MCV Plate Il Instructions 12 Optics Yalidation Results x DD Median 4774 6593 6318 CL1 Median 3383 4135 3892 CLI CY 3 7 4 67
31. seconds 2 Load 5 drops of each vial into the specified well 3 Click Eject then load MCV plate 4 Select OK to start Eject Retract Plate Cancel Fig 10 Classify validation Place the MCV plate Il on the microplate platform Select OK to start the classify validation procedure When the procedure is completed values will be displayed in a dialog box as shown below Figure 9 The classify efficiency and DD efficiency results may be accessed in this view Validation Results x Optics Fluidics Reporter Classify Date Time 19 Nov 02 10 26 AM Result Completed Classify Efficiency C DD Efficiency Specification Measured Value gt 80 90 4 gt 80 85 3 gt 80 82 4 gt 80 92 2 gt 80 94 0 Create Report Fig 11 Classify validation results Bio Plex Manager 3 0 and MCV Plate Il Instructions 17 11 The results will also be 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 Bio Plex Manager 3 0 user manual for more information on using the validation log 12 Acceptable classify efficiency values Classify Bead Specification 34 gt 80 0 38 gt 80 0 54 gt 80 0 73 gt 80 0 77 gt 80 0 Note If any values do not meet specifications repeat the procedure If values are again not
32. 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 gt 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 li
33. ual Remove the optics validation bead set from 4 C storage and vortex each bottle for 30 sec Place 5 drops of optics bead 1 into the first of the 3 optics wells labeled with 1 on the MCV plate see Figure 2 below Leave the 2 remaining wells labeled with 1 empty 12 MCV plate Place 5 drops of optics bead 2 into the first of the 3 optics wells labeled with 2 on the MCV plate Leave the 2 remaining wells labeled with 2 empty Store optics beads at 4 C as soon as possible after use Protect the beads from light Bio Plex Manager 2 0 and MCV Plate Instructions 21 8 Select Instrument from the main menu Select Validation from the pull down menu Select Optics from the pop up menu The following dialog box appears Optics Validation x This procedure validates the alignment of the Bio Plex Reader optics 1 Place 4 drops of Optics bead 1 in the first red well labeled 1 in the Optics column of the MCV plate 2 Place 4 drops of Optics bead 2 in the first green well labeled 2 in the Optics column of the MCV plate 3 Click Eject to eject the plate carries Place MCY plate in Reader 4 Press OK to start operation Eject Retract Prate Cancel Fig 13 Optics validation dialog 9 Select the Eject button in the dialog box to eject the plate holder 10 Place the MCV plate in the Bio Plex microplate platform 11 Select OK to begin the optics assessment procedure 12 When the procedure has
34. 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 100000 10000 Assay MESF 1000 a 100 Re ae a o a a ia E u 10 Reader sensitivity Assay Background i 1 1 10 100 1000 10000 RP1 Channel 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 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 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
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