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IGH Gene Clonality Assay

1. Table of Contents 1 A SAR cstseddanssscsesessdectscssbess essacsteesessssdectacssinceeceseessasssencsseodsbedactensssesesess essassdbusesossdecbsssinces sudeessass 3 2 AN AAA A RR TO 3 3 ASSAY USES ON 4 4 SPECIMEN REQUIREMENTS sisissccassscciscsvessgstienssasssctstessscsscasecsossoeecsadeatestosudoosessesscssbetse sbsauecseosecscvsesscasaseesoaedsesteavessosss 5 5 KIT CONTENTS 6 vives cnsisscsscesscsasccesiveecsssdvcbsccsesssscssecsessusasesessacestessosssncsdcosebacsevteccsacusssvcsecs secssddcedesssaestessescesetecbosesecsoendebecses 5 STATEMENT OF WARNINGS woseiesisesec cecil etecaces N ea sau tues oa aa e eaten deg ea oa Seve Sua eve ead obvea sande ea ouseputoeeeeees 5 6 STORAGE CONDITIONS vssssssiscssssscsssvcssessescossvssscosestesessestessvinseosevteesssestessevatooesenc esesdecsesesdessesnescodsvaasesbestasessentoossiestooess 5 7 REAGENTS REQUIRED BUT NOT INCLUDED 00 ssssscssssscccsscsecessscccssssccscssccccessscccssscccesssecssssscscessnecssccscsecess 6 PER AMPLIFICATION adi 6 ABTELVORESCENCE DETECTION a tee ita 6 RECOMMENDED POSITIVE CONTROLS ssscccsssssccsssssccssssccccssssccsssscccsssssccsessccccsssscccesscsccscsscccessacesesssacsecssceceeses 6 PROCEDURE NOTES sc tosssisecisssesesicsvesss seria eas sE Eare EE aN eao iera AEE eE R EEE EA E EES 6 10 REAGENT PREPARATION 6osssssscosssssccssecssccossvtecssbecsascsvaccecsssecsesesbensesestocsesnsicsseenesossesessosds sees scdeseosssdeeeodesvecesseseesssbesc
2. Diag 1999 4 2 101 117 van Dongen JJM et al Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T cell receptor gene recombinations in suspect lymphoproliferations Report of the BIOMED 2 Concerted Action BMH4 CT98 3936 Leukemia 2003 17 12 2257 2317 van Krieken JH Langerak AW Macintyre EA Kneba M Hodges E Sanz RG Morgan GJ Parreira A Molina TJ Cabe adas J Gaulard P Jasani B Garcia JF Ott M Hannsmann ML Berger F Hummel M Davi F Br ggemann M Lavender FL Schuuring E Evans PA White H Salles G Groenen PJ Gameiro P Pott Ch van Dongen JJM Improved reliability of lymphoma diagnostics via PCR based clonality testing report of the BIOMED 2 Concerted Action BHM4 CT98 3936 Leukemia 2007 21 2 201 6 IGH Gene Clonality Assay 1101002Xv7 40 FOR RESEARCH USE ONLY not for use in diagnostic procedures Page 12 of 18 17 Appendix Reagents and Special Supplies Ficoll Separation Ficoll Hypaque or Ficoll Paque Pharmacia Cat 17 0840 02 1X PBS diluted from 10X PBS Gibco BRL Cat 70011 044 RPMI 1640 Gibco BRL Cat 11875 093 DMSO Hybri Max Sigma Cat D2650 Fetal Bovine Serum Hyclone Cat SH30071 03 Gel Electrophoresis MetaPhor Agarose 125g Cambrex Cat 50180 NuSieve 3 1 Agarose 125g Cambrex Cat 50090 UltraPure 10 mg ml Ethidium Bromide Invitrogen Cat 15585 011 10X BlueJuice Gel Loading Buffer Invitrogen Cat 10816 015
3. Ready Load 100 bp Ladder Invitrogen Cat 10380 012 Novex TBE gels 6 12 well Invitrogen Cat EC62652Box Novex TBE Running Buffer 5X Invitrogen Cat LC6675 Novex Hi Density TBE Sample Buffer 5X Invitrogen Cat LC6678 Differential Fluorescence Detection HI DI Formamide with ROX size standards ABI 310 IVS Cat 6 098 0051 HI DI Formamide with ROX size standards ABI 3100 IVS Cat 6 098 0061 HI Deionized Formamide IVS Cat 6 098 0041 HI Deionized Formamide Applied Biosystems Cat 4311320 GS ROX 50 400HD Size Standard Applied Biosystems Cat 402985 18 Trouble Shooting Guide Our laboratories are located in San Diego California Technical assistance is most rapidly obtained using our Internet site http www invivoscribe com or by sending an email inquiry to support invivoscribe com Alternatively you can call 858 623 8105 for technical assistance and information on our testing kits between the hours of 8 00 AM and 5 00 PM Pacific Standard Time Questions received during business hours usually receive a response within an hour IGH Gene Clonality Assay 1101002Xv7 40 FOR RESEARCH USE ONLY not for use in diagnostic procedures Page 13 of 18 19 Sample Data VH DH JH VH FR1 primers VH FR2 primers VH FR3 primers JH primer a sp a consensus primer VH family primers Tube A 6 VH FR1 Primers JH Consensus Primer Tube B 7 VH FR2 Primers JH Consensus Primer Tube C 7 VH FR3 Primers
4. 100 IVS 0000 Y Lane 4 100 IVS 0000 gt o e rm Valid Size Range gt w ed bo om 310 360 bp gt UA ad end Valid Size Range E uo 250 295 bp wie ar s Figure 1 Figure 2 IGH Tube C IGH Tube D Lane 1 100 IVS 0013 Lane 2 100 IVS 0019 Lane 3 10 IVS 0019 Lane 4 100 IVS 0000 Valid Size Range 100 170 bp gt O Lane 1 100 IVS 0013 Lane 2 100 IVS 0024 Lane 3 10 IVS 0024 Lane 4 100 IVS 0000 Valid Size Range E 110 290 bp amp 390 420 bp _ ETIR g Figure3 Figure 4 IGH Gene Clonality Assay FOR RESEARCH USE ONLY not for use in diagnostic procedures 1101002Xv7 40 IGH Tube E Lane 1 100 IVS 0039 Lane 2 100 IVS 0008 Lane 3 10 IVS 0008 Lane 4 100 IVS 0000 E tas uy uy y C E CECE Valid Size Range gt 100 130 bp UA Figure 5 ABI Fluorescence Detection Page 15 of 18 The data shown below were generated using the master mixes indicated Amplified products were run on an ABI 3100 instrument For each IGH master mix Figures 6 10 Panel 1 displays data generated testing an alternative 100 clonal control DNA Panel 2 displays data generated testing the recommended 100 clonal control DNA Panel 3 displays data generated testing a 10 dilution of the recommended clonal control DNA Panel 4 displays data generated testing IVS 0000 Polyclonal Control DNA IGH Tube A 6FAM 100 IV
5. 50V overnight Voltage and electrophoresis time depend on the PCR amplicon size acrylamide gel thickness and type of PCR equipment Voltage and run time can be adapted accordingly Gels are stained in 0 5ug ml EtBr in water or 0 5X TBE Buffer for 5 10 minutes Gels are destained 2X in water for 5 10 minutes UV illumination is used for visualization Gel is photographed and data are interpreted ABI Fluorescence Detection with ABI 310 amp 3100 instruments 6 Ts 10 11 12 Add 1ul of reaction products from IGH Tubes A B and C IGH FR1 2 and 3 respectively in separate tubes and add 101 of HI Deionized Formamide containing ROX size standards to each tube Mix well Add 1ul of reaction products from IGH Tube D in a separate tube and add 10ul of HI Deionized Formamide containing ROX size standards Mix well Add 1ul of reaction products from IGH Tube E in a separate tube and add 10u1 of HI Deionized Formamide containing ROX size standards Mix well Add 1ul of reaction product from the template amplification control in a tube and add 10u1 of HI Deionized Formamide containing ROX size standards Mix well Reaction products are heated to 95 C for 2 minutes then snap chilled on ice for 5 minutes A sample sheet and injection list is made up for the samples As the samples are run on the machine they are fractionated detected and analyzed by the instrument Runs are 20 24 minutes in duration The 310 amp 3100 capillary ele
6. IVS 0000 Polyclonal Control DNA 4 092 0010 85 310 360 IVS 0030 Clonal Control DNA 4 088 1750 2807 325 IVS 0019 Clonal Control DNA 4 088 1090 345 IVS 0024 Clonal Control DNA 4 088 1390 342 TIVS 0008 Clonal Control DNA 4 088 0430 IGH Tube B FR2 JH Black Valid Size Range 250 295 IVS 0000 Polyclonal Control DNA 4 092 0010 250 295 IVS 0030 Clonal Control DNA 4 088 1750 260 IVS 0019 Clonal Control DNA 4 088 1090 285 IVS 0024 Clonal Control DNA 4 088 1390 277 TVS 0008 Clonal Control DNA 4 088 0430 IGH Tube C FR3 JH Green Valid Size Range 100 170 IVS 0000 Polyclonal Control DNA 4 092 0010 100 170 IVS 0030 Clonal Control DNA 4 088 1750 IVS 0019 Clonal Control DNA 4 088 1090 145 IVS 0024 Clonal Control DNA 4 088 1390 140 IVS 0008 Clonal Control DNA 4 088 0430 IGH Tube D DH JH Green Valid Size Range 110 2904 390 420 TVS 0000 Polyclonal Control DNA 4 092 0010 235 258 344 IVS 0030 Clonal Control DNA 4 088 1750 744 94 158 1764 1877 2004 344 IVS 0019 Clonal Control DNA 4 088 1090 744 94 158 1764 1877 2004 344 IVS 0024 Clonal Control DNA 4 088 1390 139 IVS 0008 Clonal Control DNA 4 088 0430 74 94 1584 176 187 2007 344 IGH Tube E DH7 JH Blue Valid Size Range 100 130 IVS 0000 Polyclonal Control DNA 4 092 0010 209 416 IVS 0030 Clonal Control DNA 4 088 1750 79 1234 IVS 0019 Clonal Control DNA 4 088 1090 79 1234 IVS 0024 Clonal Control DNA
7. JH Consensus Primer DH JH DH3 DH1 DH2 DH5 DH4 DH6 DH7 JH primer O consensus primer DH family primers Tube D 6 DH Primers JH Consensus Primer Tube E DH7 Primer JH Consensus Primer OVERVIEW This is a diagrammatic representation of the immunoglobulin heavy chain gene on chromosome 14 The locations of the variable V diversity D joining J and constant C regions are indicated The three conserved framework regions FR1 FR2 and FR3 are color coded to match the fluorescent primers that are used to identify these regions using the ABI differential fluorescence detection method IGH Gene Clonality Assay 1101002Xv7 40 FOR RESEARCH USE ONLY not for use in diagnostic procedures Page 14 of 18 Gel Detection The data shown below were generated using the master mixes indicated Amplified products were run on a 6 polyacrylamide gel For each IGH master mix Figures 1 5 Lane 1 displays data generated testing an alternative 100 clonal control DNA Lane 2 displays data generated testing the recommended 100 clonal control DNA Lane 3 displays data generated testing a 10 dilution of the recommended clonal control DNA Lane 4 displays data generated testing IVS 0000 Polyclonal Control DNA IGH Tube A tad IGH Tube B had td Lane 1 100 Ivs 0019 E Lane 1 100 IVS 0019 Lane 2 100 IVS 0030 fa Lane 2 100 IVS 0030 Lane 3 10 IVS 0030 A Lane 3 10 IVS 0030 ffs o 5 o LS Lane 4
8. in malignancy studies IGH Gene Clonality Assay 1101002Xv7 40 FOR RESEARCH USE ONLY not for use in diagnostic procedures 4 Page 5 of 18 Specimen Requirements This assay tests genomic DNA 1 5cc of peripheral blood bone marrow biopsy or bone marrow aspirate anti coagulated with heparin or EDTA Ship at ambient temperature OR 2 Minimum 5mm cube of tissue shipped frozen or at room temperature or on ice in RPMI 1640 OR 2ug of genomic DNA OR 4 Formalin fixed paraffin embedded tissue or slides Ya Kit Contents Controls and Standards IVS Catalog Concentration IVS 0030 Clonal Control DNA 4 088 1750 10011 E 200ug ml IVS 0019 Clonal Control DNA 4 088 1090 100u1 200ug ml IVS 0024 Clonal Control DNA 4 088 1390 100ul E 2001g ml IVS 0008 Clonal Control DNA 4 088 0430 10011 E 200ug ml IVS 0000 Polyclonal Control DNA 4 092 0010 100u1 200ug ml Master Mixes IVS Catalog Target IGH Tube A 2 101 001X Framework 1 JH IGH Tube B 2 101 002X Framework 2 JH IGH Tube C 2 101 003X Framework 3 JH IGH Tube D 2 101 004X DH1 6 JH IGH Tube E 2 101 005X DH7 JH Specimen Control Size Ladder 2 096 002X Multiple Genes Note X Detection format code Note MegaKits contain 10 units of each master mix and 5 units of each Controls and Standards STATEMENT OF WARNINGS The assay kit has been optimized to be used as a system Do not substitute other manufacturer s reagents Dilution reducing amplification reaction volumes or other d
9. single sized products within the smear of different sized amplicon products that form a Gaussian distribution around a statistically favored average sized rearrangement As expected primers that amplify from the different FR regions which are located at three distinct regions along the heavy chain gene produce a correspondingly different size range of V J products Gel electrophoresis is commonly used to resolve the different sized amplicon products and ethidium bromide or other DNA intercalating dyes to stain and detect these products A powerful alternative method is use of differential fluorescence detection with primers conjugated with fluorescent dyes that correspond to different targeted regions Reaction products from several different master mixes can be pooled fractionated using capillary electrophoresis and detected simultaneously This detection system results in unsurpassed sensitivity single base resolution differential product detection and relative quantification In addition the laboratory can eliminate the use of agarose and polyacrylamide gels as well as the use of carcinogens such as ethidium bromide Further differential detection allows accurate reproducible and objective interpretation of primer specific products and automatic archiving of data The limit of detection of this assay has been determined to be approximately 1 clonal cell in 100 hundred normal cells and inter assay and intra assay reproducibility in size determ
10. the interpretation of results Prepare fresh master mix and repeat amplification Negative Continue with the analysis 2 Positive Control This can also be an extraction control if positive control material is taken through extraction processes If the positive control is Positive Continue with analysis Negative Repeat assay unless specimen tests positive 3 Specimen Control Size Ladder This is run on unknown samples only If the amplification control is Positive 100 200 300 400 and 600 basepair products are seen Because smaller PCR fragments are preferentially amplified it is not unusual for the 600 basepair fragment to have a diminished signal or to be missing entirely Continue with analysis Negative Repeat assay unless specimen tests positive Sample Interpretation Following the acceptance of the controls the clinical samples are interpreted as follows 15 One or two prominent bands within the valid size range are reported as Detection of clonal immunoglobulin heavy chain gene rearrangement consistent with the presence of a clonal cell population Limitations of Procedure The assay is subject to interference by degradation of DNA or inhibition of PCR due to heparin or other agents The assay cannot reliably detect less than 1 positive cell per 100 normal cells 16 1 References Miller JE Wilson SS Jaye DJ Kronenberg M An automated semiquantitative B and T cell clonality assay Mol
11. 4 088 1390 794 1234 TVS 0008 Clonal Control DNA 4 088 0430 109 Specimen Control Multiple Blue Any Human DNA 84 96 200 300 400 600 Size Ladder Genes Note The amplicon sizes listed above were determined using an ABI 3100 platform Amplicon sizes seen on your specific CE instrument may differ 1 4bp from those listed above depending on the platform of detection and the version of the analysis software used Once identified the amplicon size as determined on your specific platform will be consistent from run to run This reproducibility is extremely useful when tracking MRD Note Under sub optimal conditions an aspecific product of 85bp can be detected in Tube A To discriminate between specific and aspecific negative control DNA should not show this band within the same experiment If a band is present we then consider the band specific Note The 280bp band is a known amplicon that lies just outside the valid size range for Tube A Note An aspecific 344bp band is the result of cross annealing of the DH2 primer to a sequence in the region upstream of JH4 In GeneScan analysis this band does not co migrate with D J products Note if These bands are known amplicons that are seen with many different cell lines These bands are absent in the presence of polyclonal backgrounds Note A 209bp PCR product represents the smallest background band derived from the germline DH7 JH1 region When the PCR amplification is very efficient a
12. Control Size Ladder master mix will ensure that DNA of sufficient quality and quantity was present to yield a valid result 12 Amplification 1 Aliquot 45ul of the master mix enzyme solutions into individual PCR wells or tubes Add 5ul of sample or control DNA to the individual tubes or wells containing the respective master mix reactions Pipette up and down several times to mix Amplify the reactions using the following PCR program We recommend the MJ Research PTC 100 PTC 200 or the PE 2600 9600 or 9700 thermocyclers using the following PCR parameters for the amplifications Note We recommend using the calculated option for temperature measurement with the PTC instruments Standard Program for AmpliTag Gold Step 1 95 C for 7 minutes Step 2 95 C for 45 seconds Step 3 60 C for 45 seconds Step 4 72 C for 90 seconds Step 5 Go to step 2 34 more times Step 6 72 C for 10 minutes Step 7 15 C forever IGH Gene Clonality Assay 1101002Xv7 40 FOR RESEARCH USE ONLY not for use in diagnostic procedures Page 8 of 18 Remove the amplification plate from the thermocycler 13 Detection Not all detection formats are available for all assays Available Template Amplification Controls The Amplification Control master mix primers are labeled with a fluorescent dye 6 FAM or Cy5 0 This label is detected as BLUE using the differential fluorescence software The amplicons produced with this master mix are at 235 basepairs Th
13. ESCENCE DETECTION WITH ABI 310 amp 3100 INSTRUMENTS ccccccccccccecccecesesesececeseseseceseseceseseseseseseseseuseeeseseseenees 18 CY5 0 FLUORESCENCE DETECTION ccccccscccccccesccccececesecsceecscceccosesescesececseecesececeeesecasscsvevevesesscacscesevavevesevesecevevevesevscasceusosecssoses 18 IGH Gene Clonality Assay 1101002Xv7 40 FOR RESEARCH USE ONLY not for use in diagnostic procedures Page 3 of 18 Thank you for purchasing our IGH Gene Clonality Assay We appreciate your business We are happy to assist you in the validation of this assay and will provide ongoing technical assistance to keep the assays performing efficiently in your laboratory Technical assistance is most rapidly obtained using our Internet site http www invivoscribe com or by sending an email inquiry to support invivoscribe com Questions received during business hours usually receive a response within an hour Alternatively you can call for technical assistance and for information on our testing kits at 858 623 8105 between the hours of 8 00 AM and 5 00 PM Pacific Standard Time L Notice This product and the methods employed are covered by United States Letters Patent Numbered 5 296 351 and 5 418 134 Australian Patent Number 626 601 and Japanese Patent Number 2 781 438 all of which are licensed exclusively to InVivoScribe Technologies TVS Purchase of this product includes a limited sublicense for non commercial practice of this technol
14. I 2 and 3 respectively in separate tubes and add 10ul of HI Deionized Formamide containing ROX size standards to the tube Mix well 2 Add 1ul of reaction products from the IGH Tube D in a separate tube and add 10ul of HI Deionized Formamide containing ROX size standards to the tube Mix well 3 Add 1ul of reaction products from the IGH Tube E in a separate tube and add 10ul of HI Deionized Formamide containing ROX size standards to the tube Mix well 4 Add 1ul of reaction product from the template amplification control in a tube and add 101 of HI Deionized Formamide containing ROX size standards Mix well 5 Reaction products are heated to 95 C for 2 minutes then snap chilled on ice for 5 minutes 6 A sample sheet and injection list is made up for the samples As the samples are run on the machine they are fractionated detected and analyzed by the instrument Runs are 20 24 minutes in duration The 310 amp 3100 capillary electrophoresis instruments routinely handle 2 runs per hour 48 and 768 samples per day respectively and automatically analyze and store data for review or comparison with other test results 7 Data are automatically displayed as size and color specific peaks Review profile and controls report results ABI Fluorescence Detection with ABI 373 amp 377 instruments 1 PCR Product Dilution Initially dilute samples 1 10 in HI Deionized Formamide or water can be altered if the fluorescence signal is outside the
15. InVivoScribe Technologies 6330 Nancy Ridge Drive Suite 106 San Diego CA 92121 USA IGH Gene Clonality Assay 858 623 8105 Phone 858 623 8109 Fax support invivoscribe com www invivoscribe com For Identification of Clonal Immunoglobulin Heavy Chain Gene Rearrangements VH DH JH VH FR1 primers VH FR2 primers VH FR3 primers JH primer Si cs cnt VH family primers Tube A 6 VH FR1 Primers JH Consensus Primer Tube B 7 VH FR2 Primers JH Consensus Primer Tube C 7 VH FR3 Primers JH Consensus Primer consensus primer DH JH IA A j lt 4 DH3 DH1 DH2 DH5 DH4 DH6 DH7 DH family primers Tube D 6 DH Primers JH Consensus Primer Tube E DH7 Primer JH Consensus Primer FOR RESEARCH USE ONLY Not for use in diagnostic procedures Storage Conditions 65 C to 85 C DNA controls may be separated from assay kits and stored at 2 C to 8 C Catalog Products 1 101 0020 1 101 0021 1 101 0024 1 101 0040 1 101 0041 1 101 0044 IGH Gene Clonality Assay for Gel Detection IGH Gene Clonality Assay for ABI Fluorescence Detection IGH Gene Clonality Assay for Cy5 0 Detection IGH Gene Clonality Assay MegaKit for Gel Detection IGH Gene Clonality Assay MegaKit for ABI Fluorescence Detection IGH Gene Clonality Assay MegaKit for Cy5 0 Detection JH primer consensus primer uantit 33 Reactions 33 Reactions 33 Reactions 330 Reactions 330 Reactions 330 Reactions Page 2 of 18
16. S 0019 Panel 1 345 bp SR AN ARE POS AS VI iia 100 IVS 0030 l l Panel 2 325 bp sM aoe A EOE ESE A CEL aiiin 10 IVS 0030 Panel 3 325 bp AA IS Valid Size Range 310 360 bp 7 Panel 4 Ala Urb rl tr A rat AA AA ns IAA Ano Figure 6 IGH Gene Clonality Assay 1101002Xv7 40 FOR RESEARCH USE ONLY not for use in diagnostic procedures Page 16 of 18 IGH Tube B NED 100 IVS 0019 EJ a 100 IVS 0030 Panel 2 ws 10 IVS 0030 Panel 3 Valid Size Range 250 295 bp Panel 4 Figure 7 IGH Tube C HEX mw EJ 210 bp 100 IVS 0030 Panel 1 we EJ w 0 so 100 IVS 0019 Panel 2 10 IVS 0019 Panel 3 Valid Size Range 100 170 bp Panel 4 Figure 8 IGH Gene Clonality Assay FOR RESEARCH USE ONLY not for use in diagnostic procedures 1101002Xv7 40 IGH Tube D HEX w 100 IVS 0013 Panel 1 wl 100 IVS 0024 Panel 2 Panel 3 se m w anl ol Figure 9 IGH Tube E 6FAM 100 IVS 0039 100 IVS 0008 Panel 2 10 IVS 0008 Panel 3 Figure 10 IGH Gene Clonality Assay Valid Size Range 100 130 bp FOR RESEARCH USE ONLY not for use in diagnostic procedures Panel 4 Page 17 of 18 1101002Xv7 40 Page 18 of 18 20 1 Ze Single Page Flow Chart Using gloved hands remove the master mixes from the freez
17. age and run time can be adapted accordingly Gels are stained in 0 5ug ml EtBr in water or 0 5X TBE Buffer for 5 10 minutes Gels are destained 2X in water for 5 10 minutes UV illumination is used for visualization Gel is photographed and data are interpreted Gel Detection Heteroduplex Analysis RECOMMENDED 1 2 3 4 DU moo Denature 20ul of PCR products at 94 C for 5 minutes Re anneal PCR products at 4 C for 60 minutes Assemble electrophoresis unit using a 6 non denaturing polyacrylamide TBE gel made with 1X TBE Invitrogen Cat EC62652Box and 0 5X TBE running buffer Invitrogen 5X TBE Cat LC6675 Add 5 1 of ice cold non denaturing bromophenol blue loading buffer to samples Load 20ul of mixture into wells of the gel Run gel at 110V for 2 3 hours or 40 50V overnight Voltage and electrophoresis time depend on the PCR amplicon size acrylamide gel thickness and type of PCR equipment Voltage and run time can be adapted accordingly Gels are stained in 0 5ug ml EtBr in water or 0 5X TBE Buffer for 5 10 minutes Gels are destained 2X in water for 5 10 minutes UV illumination is used for visualization 0 Gel is photographed and data are interpreted IGH Gene Clonality Assay 1101002Xv7 40 FOR RESEARCH USE ONLY not for use in diagnostic procedures Page 9 of 18 ABI Fluorescence Detection with ABI 310 amp 3100 instruments 1 Add lul of reaction products from the IGH Tubes A B and C IGH FR
18. ctrophoresis instruments routinely handle 2 runs per hour 48 and 768 samples per day respectively and automatically analyze and store data for review or comparison with other test results Data are automatically displayed as size and color specific peaks Review profile and controls report results Cy5 0 Fluorescence Detection The Cy5 0 fluorochrome is capable of being detected by many different detection platforms Please see your instruments user manual for instructions on the fractionation and detection of your samples IGH Gene Clonality Assay 1101002Xv7 40 FOR RESEARCH USE ONLY not for use in diagnostic procedures
19. e products of this master mix should be run separately The Specimen Control Size Ladder master mix primers are labeled with a fluorescent dye 6 FAM or Cy5 0 This label is detected as BLUE using the differential fluorescence software The amplicons produced with this master mix are at 100 200 300 400 and 600 basepairs Please note that the 100bp band is comprised of a 84bp and 96bp bands Both of these bands co migrate on a gel The products of this master mix should be run separately Gel Detection Agarose TBE Gels 1 2 3 A 2 MetaPhor or NuSieve 3 1 agarose TBE gel is prepared 20ul from each of the amplification reactions are individually mixed with 4ul of 6X gel loading buffer 20ul of this mixture is loaded into separate wells of the gel flanked by DNA size standards Products are detected using ethidium bromide or an equivalent dye Gel is photographed and data are interpreted Gel Detection Polyacrylamide TBE Gels 1 ep e ON A Assemble electrophoresis unit using a 6 non denaturing polyacrylamide TBE gel made with 1X TBE Invitrogen Cat EC62652Box and 0 5X TBE running buffer Invitrogen 5X TBE Cat LC6675 Add 5 1 of ice cold non denaturing bromophenol blue loading buffer to samples Load 20ul of mixture into wells of the gel Run gel at 110V for 2 3 hours or 40 50V overnight Voltage and electrophoresis time depend on the PCR amplicon size acrylamide gel thickness and type of PCR equipment Volt
20. en Valid Size Range gt 110 290 390 420 IVS 0024 Clonal Control DNA 4 088 1390 139 IGH Tube E DH7 JH Blue Valid Size Range 100 1307 IVS 0008 Clonal Control DNA 4 088 0430 109 Specimen Control Multiple Blue Valid Size Range 84 96 200 300 400 600 Size Ladder Genes IVS 0000 Polyclonal Control DNA 4 092 0010 84 96 200 300 400 600 Note The amplicon sizes listed above were determined using an ABI 3100 platform Amplicon sizes seen on your specific CE instrument may differ 1 4bp from those listed above depending on the platform of detection ABI and the version of the analysis software used Once identified the amplicon size as determined on your specific platform will be consistent from run to run This reproducibility is extremely useful when tracking MRD Note A 280bp band may also be present and is a known amplicon that lies just outside the valid size range for IGH Tube A Note A 209bp PCR product represents the smallest background band derived from the germline DH7 JH1 region When the PCR amplification is very efficient longer products might also be obtained because of primer annealing to downstream JH gene rearrangements e g 416bp DH7 JH2 1028bp DH7 JH3 etc Note Because smaller PCR fragments are preferentially amplified it is not unusual for the 600bp fragment to have a diminished signal or to be missing entirely 9 Procedure Notes Autoclaving does not eliminate DNA contamination Work
21. er Allow the tubes to thaw then gently vortex to mix In a containment hood or dead air box remove an appropriate aliquot to clean sterile microfuge tube one tube for each of the master mixes Aliquot volumes should be 45 1 for each sample 135 1 for the positive negative and no template controls We recommend adding an additional 2011 to correct for pipetting errors Add the appropriate amount of either AmpliTaq Gold or AmpliTaq DNA polymerase 0 2511 of either AmpliTaq Gold or AmpliTaq E 5U ul per SOul total PCR reaction volume to each of the master mixes and gently mix by inverting several times or gently vortexing Aliquot 45ul of master mix to individual wells of a PCR plate Add 5ul of DNA from the unknown and control samples to individual tubes or wells containing the respective master mix reactions and pipette up and down several times to mix Amplify target DNA using the universal thermocycler program Gel Detection Heteroduplex Analysis 6 7 8 9 10 11 12 13 14 15 Denature 20ul of PCR products at 94 C for 5 minutes Re anneal PCR products at 4 C for 60 minutes Assemble electrophoresis unit using a 6 non denaturing polyacrylamide TBE gel made with 1X TBE Invitrogen Cat EC62652Box and 0 5X TBE running buffer Invitrogen 5X TBE Cat LC6675 Add 5ul of ice cold non denaturing bromophenol blue loading buffer to samples Load 20ul of mixture into wells of the gel Run gel at 110V for 2 3 hours or 40
22. ese products The assays described herein are not approved by any regulatory agency for clinical use These assays are not for diagnostic or therapeutic use This product is sold FOR RESEARCH USE ONLY not for use in diagnostic procedures Principle NOTICE InVivoScribe Technologies Gene Rearrangement and Translocation Assays represent a new approach to PCR based clonality testing These standardized assays were carefully optimized testing positive and negative control samples using multiplex master mixes Assay development was followed by extensive validation testing more than 400 clinical samples using Revised European American Lymphoma REAL Classification Testing was done at more than thirty prominent independent testing centers throughout Europe in a collaborative study known as the BIOMED 2 Concerted Action Results from this BIOMED 2 study appear in a leading peer reviewed journal Leukemia 2003 Dec 17 12 2257 2317 Nature Publishing Group BACKGROUND Polymerase chain reaction PCR assays are routinely used for the identification of clonal B cell populations These tests amplify the DNA between primers that target the conserved framework FR and joining J regions Tubes A C and the diversity D and joining regions Tubes D amp E These conserved regions lie on either side of an area within the V J region where programmed genetic rearrangements occur during maturation of all B and T lymphocytes The antigen receptor genes that u
23. eviation in this protocol may affect the performance of this test and or nullify any limited sublicense that comes with the purchase of this testing kit Close adherence to the protocol will assure optimal performance and reproducibility It is recommended that glass distilled de ionized molecular biology grade water be used with the preparation of specimen DNA This can be purchased from several manufacturers In addition laboratory personnel are reminded to wear appropriate personal protective equipment and follow good laboratory practices and universal precautions when working with specimens Specimens should be handled in approved biological safety containment facilities and opened only in certified biological safety cabinets Please see Section 9 for further details Storage Conditions PCR master mixes are sensitive to freeze thaw cycles Therefore for any duration other than immediate use our master mixes and assay kits should be stored at 65 C to 85 C The reason for this is quite straightforward Due to the high salt concentrations in our master mixes the effective freezing and thawing temperature of the master mixes is approximately 10 C The temperature in a standard laboratory 20 C freezer can easily reach 10 C or warmer during the day when these freezers are opened on a regular basis At these temperatures PCR master mixes may go through multiple freeze thaw cycles resulting in precipitation of the primers Accordingly to min
24. flow in the PCR laboratory should always be in a one way direction between separate work areas beginning in Master Mix Preparation moving to the Specimen Preparation then to the Amplification and finally to Detection 1 Do not bring amplified DNA into the areas designated for master mix or specimen preparation Due to the analytical sensitivity of this test extreme care should be taken to avoid the contamination of reagents or amplification mixtures with samples controls or amplified materials All reagents should be closely monitored for signs of contamination e g negative controls giving positive signals Discard reagents suspected of contamination 3 All pipettes pipet tips and any equipment used in a particular area should be dedicated to and kept to that area of the laboratory IGH Gene Clonality Assay 1101002Xv7 40 FOR RESEARCH USE ONLY not for use in diagnostic procedures Page 7 of 18 4 PCR trays bases and retainers must to be decontaminated in 10 bleach and rinsed with distilled water two separate times before returning them to the starting areas 5 Sterile disposable plastic ware should be used whenever possible to avoid RNase or cross contamination 10 Reagent Preparation All unknown samples should be tested using the template amplification control Amplification Control or Specimen Control Size ladder master mix This is to ensure that no inhibitors of amplification are present and there is DNA of suffic
25. ient quality and quantity to generate a valid result All samples should be tested in singlicate Positive negative and no template controls should be tested for each of the master mixes 1 Using gloved hands remove the master mixes from the freezer Allow the tubes to thaw then gently vortex to mix 2 In containment hood or dead air box remove an appropriate aliquot to clean sterile microfuge tube one tube for each of the master mixes Aliquot volumes should be 45ul for each sample 135ul 3 x 45u1 for the positive negative and no template controls We recommend adding an additional 20ul to correct for pipetting errors 3 Add the appropriate amount of either AmpliTaq Gold or AmpliTaq DNA polymerase 0 25ul of either AmpliTaq Gold or AmpliTaq 5U ul per 50ul total PCR reaction volume to each of the master mixes and gently mix by inverting several times or gently vortexing The master mixes are now ready for distribution to reaction tubes or plate and addition of sample 11 Sample Preparation Using any method of DNA extraction extract the genomic DNA from unknown samples Resuspend DNA to final concentration of 100ug 400ug per ml in TE 10 mM Tris HCl 1mM EDTA pH 8 0 or distilled water This is a robust assay system A wide range of DNA concentrations will generate a valid result Therefore quantifying and adjusting DNA concentrations is generally not necessary Testing sample DNAs with the Amplification Control or Specimen
26. imize the exposure of your master mixes to freeze thaw cycles VS recommends that master mixes be stored at 65 C to 85 C IGH Gene Clonality Assay 1101002Xv7 40 FOR RESEARCH USE ONLY not for use in diagnostic procedures Page 6 of 18 Please note that our DNA standards are best stored at 2 C to 8 C However these standards can be stored at any lower temperature as long as they are vortexed after thawing and before use to ensure that they are re suspended completely If you have any questions please contact our technical staff We are happy to help you determine your optimal storage needs 7 Reagents Required But Not Included PCR Amplification AmpliTaq Gold DNA Polymerase RECOMMENDED Applied Biosystems Cat N808 0241 AmpliTaq DNA Polymerase Applied Biosystems Cat N808 0161 ABI Fluorescence Detection HI DI Formamide with ROX size standards ABI 310 IVS Cat 6 098 005 1 HI DI Formamide with ROX size standards ABI 3100 IVS Cat 6 098 0061 8 Recommended Positive Controls Master Mix Target Color Control DNA Catalog Product Size in Basepairs IGH Tube A FR1 JH Blue Valid Size Range 310 360 IVS 0030 Clonal Control DNA 4 088 1750 280 326 IGH Tube B FR2 JH Black Valid Size Range 250 295 IVS 0030 Clonal Control DNA 4 088 1750 260 IGH Tube C FR3 JH Green Valid Size Range sss 100 170 IVS 0019 Clonal Control DNA 4 088 1090 145 IGH Tube D DH JH Gre
27. ination using capillary electrophoresis is approximately 1 2 basepairs This reproducibility and sensitivity allows monitoring and tracking of individual tumors during research or methods development The automatic archiving of specimen data allows comparison of data collected at different times This test kit includes 6 master mixes Tubes A B and C target the framework 1 2 and 3 regions respectively within the variable region and the joining region of the Ig heavy chain locus Tubes D amp E target the diversity and joining regions The last master mix the Specimen Control Size Ladder targets multiple genes and generates a series of amplicons of 100 200 300 400 and 600 base pairs to ensure that the quality and quantity of input DNA is adequate to yield a valid result A single thermocycler program and similar detection methodologies are used with all of the BIOMED tests Many of our customers have remarked that this improves consistency and facilitates cross training on a broad range of different assays These robust InVivoScribe assays can be used to test DNA extracted from virtually any source Assay Uses Immunoglobulin Heavy Chain Gene Rearrangement Assays are useful for Identifying clonal B cell populations highly suggestive of B cell malignancies Lineage determination of leukemias and lymphomas Monitoring and evaluation of disease recurrence Detection and assessment of residual disease Evaluation of new research and methods
28. lso longer products might be obtained because of primer annealing to downstream JH gene rearrangements e g 416bp DH7 JH2 1028bp DH7 JH3 etc Note A non specific peak may be seen at 227bp Results can be reported as Positive or Negative for Detection of clonal immunoglobulin heavy chain gene rearrangement consistent with the presence of a clonal cell population 1 Samples that fail to amplify following repeat testing should be reported as A result cannot be reported on this specimen because there was DNA of insufficient quantity or quality for analysis 2 It is acceptable to call a sample Positive when a product is generated in the valid size range yet the positive control for that master mix fails 3 Samples that test negative should be repeated if the positive control reaction failed IGH Gene Clonality Assay FOR RESEARCH USE ONLY not for use in diagnostic procedures 1101002Xv7 40 Page 11 of 18 4 All assay controls must be examined prior to interpretation of sample results If the controls do not yield the correct results the assay is not valid and the samples should not be interpreted The following describes the analysis of each of the controls and the decisions necessary based upon the results 1 Negative Control Polyclonal control water or no template blank If the negative control is Positive Possible contamination of all PCR amplification reactions Do not continue with
29. ndergo rearrangement are the immunoglobulin heavy IGH Gene Clonality Assay 1101002Xv7 40 FOR RESEARCH USE ONLY not for use in diagnostic procedures Page 4 of 18 chain amp light chains genes in B cells and the T cell receptor genes in T cells Each B and T cell has a single productive V J rearrangement that is unique in both length and sequence Therefore when this region is amplified using DNA primers that flank this region a clonal population of cells yields one or two prominent amplified products amplicons within the expected size range Two products are produced in cases when the initial rearrangement was non productive and was followed by rearrangement of the other homologous chromosome In contrast DNA from a normal or polyclonal many clones population produces a bell shaped curve of amplicon products Gaussian distribution that reflect the heterogeneous population of V J region rearrangements Since the antigen receptor genes are polymorphic consisting of a heterogeneous population of related DNA sequences it is difficult to employ a single set of DNA primer sequences to target all of the conserved flanking regions around the V J rearrangement N region diversity and somatic mutation further scramble the DNA sequences in these regions Therefore multiplex master mixes which target several FR regions are required to identify the majority of clonal rearrangements As indicated clonal rearrangements are identified as prominent
30. ocsss 7 11 SAMPLEPREPARATION issiicsiciscccecatesscastasessactacctcavessdssonddooscanscccecatessduasesscosscddevonssessauedecausbesseasesessudensonsesecssdetesstsavecsosss 7 ANAIS AR O 7 13 AR ssuiustvosebbesdssusuess bucussstessessevenehesvususseeseccdbestesbisudeeseste 8 AVAILABLE TEMPLATE AMPLIFICATION CONTROLS ccccccccccecececececesescsecececesecesesesesesecesesececesesececesessesssessssssesesesesesssessseususeaeaes 8 GEL DETECTION AGAROSETBE GEES 0 a EAEE EERTE 8 GEL DETECTION POLYACRYLAMIDE TBE GELS occccccccnncncncnonononononononononononononononononononononononononononononononenonononenenenenenenenenenenenenes 8 GEL DETECTION HETERODUPLEX ANALYSIS RECOMMENDED cccccccessescecseseeeessnececeeaaececseeeecessaseeceeaaeceseaaeeesneeeeenas 8 ABI FLUORESCENCE DETECTION WITH ABI 310 amp 3100 INSTRUMENTS ccccccccccccccececeseceseceseseseseseseseseseseseseseseseseeesseseeeseseaeaes 9 ABI FLUORESCENCE DETECTION WITH ABI 373 amp 377 INSTRUMENTS ccccccccccccccccececececeseseseseseseseseseseseceseseseseseseseesssesesesueeaeaes 9 CX3 0 FLUORESCENCE DETECTION a aia 9 14 INTERPRETATION AND REPORTING cssssscssssscsssscccsssccccesssccccssssccccssssccscnsscccssacccessnessenscecesssecccsessssececssceesess 9 EXPECTED SIZE OF AMPLIFIED PRODUCT Sireen enaena ar a aa iea E a E E aK EEEE 10 SAMPLE INTERPRETATI N ii id die 11 15 LIMITATIONS OF PROCEDURE vssccssissssecsssssssccssvsscssoosssossvectsosstecsi
31. ogy for use within or with respect to data or product that are transmitted to the United States Japan or Australia only when the purchaser is registered with IVS as an exclusively non commercial user of IVS products No sublicense is granted simply by purchase of these products Non commercial practice of the technology means sample testing done for teaching and basic research Non Commercial practice excludes testing if any of the following apply a test results products or information derived from the tests are used for or in support of patient care or are transferred to a healthcare professional involved in patient care b test results are clinically utilized to determine cause of death c compensation in any form or manner is received for performing the tests To request a form for registration as an exclusively non commercial product user to discuss terms for a potential sublicense for broader practice of these methods or for any questions concerning the scope or content of the non commercial sublicense please contact our legal department by email at legal invivoscribe com or by telephone at 858 623 8105 These methods also require nucleic acid amplification methods such as Polymerase Chain Reaction PCR which is covered by patents owned by Hoffmann LaRoche Inc and F Hoffmann LaRoche Ltd No license under these patents to use the PCR process is conveyed expressly or by implication to the purchaser by the purchase of th
32. optimal range 2 Add 2ul of diluted reaction product from IGH Tube A in a tube 2ul of HI Deionized Formamide containing 0 5ul of ROX size standards 0 5ul of blue Dextran loading dye Mix well 3 Add 2ul of diluted reaction product from IGH Tube B in a tube 2ul of HI Deionized Formamide containing 0 5ul of ROX size standards 0 51 of blue Dextran loading dye Mix well 4 Add 2ul of diluted reaction product from IGH Tube C in a tube 2ul of HI Deionized Formamide containing 0 5ul of ROX size standards 0 51 of blue Dextran loading dye Mix well 5 Add 2ul of diluted reaction product from IGH Tube D in a tube 2ul of HI Deionized Formamide containing 0 5ul of ROX size standards 0 5ul of blue Dextran loading dye Mix well 6 Add 2ul of diluted reaction product from IGH Tube E in a tube 2ul of HI Deionized Formamide containing 0 5ul of ROX size standards 0 51 of blue Dextran loading dye Mix well 7 Add 2ul of diluted reaction product from the template amplification control in a tube 2ul of HI Deionized Formamide containing ROX size standards 0 5ul of blue Dextran loading dye Mix well 8 Reaction products are heated to 94 C for 2 minutes then snap chilled on ice for 5 minutes 9 Load the 5ul of each of these preparations in separate wells of a preheated gel and run using the standard sequencing protocol Cy5 0 Fluorescence Detection The Cy5 0 fluorochrome is capable of being detected by many different detec
33. sssecsssossansseessbcsseenssesssnctsesessdsassdencssevesestdesssasseuseeess 11 16 REFERENCES tcvoiossi iia cientos cranin sesso cbsbexeses soetos Noain Acida RARA oncideatbvesscoveuces ebweeebssevescsuavasessesucbdessesesshdeaesete 11 ANA TO 12 REAGENTS AND SPECIAL SUPPLIES occccccccncncononononononononononononononnnnnnnnn nn nn nn nn nn nn nn r E nro nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnncnnnnnnnnncnonononininons 12 ELCOLES para cai iii ocd vis cadees ius adaaades vucdaags EE EE Ea OOE a a ES Oa TREE EEE SAE EEE E OME nds 12 Gel ElectrOphoresis ui E E E E dde 12 Differential Fluorescence Detection cecccssccesnceessecsenceessecsenceessecsencecssecsencecssecseaeeessecseneeesaecssaeecsaecseaeecsaeceeaeecsaeeeeaeeeseensaes 12 18 TROUBLE SHOOTING GUIDE wuu ssscccssscscssssccccssssccscssccccsssscccessseccesssccccensccscsssaccscnscscessnaecscssccccsessacecesssaccscssssecees 12 CAS IIA O 13 OVER VTE NM eee ee ET in cognac 13 GEL DETECTION AA ose ad EEEE 14 ABT FLUORESCENCE DETECTION ernennen innne i n cocesenstecetecsegces ous coccveite cocesoastoacgedbepeus ob cue cwvits docwseustoentecsapeelens teense 15 20 SINGLE PAGE FLOW CHART iiisssssssiscsoccsvecssssssecsecvevensesnssosioveatessvnsasecsescedssoesssdectaseobenteosssestscesdesiessoonssosseveasesseasesessess 18 GEL DETECTION HETERODUPLEX ANALYSIS occcccccccocononononononononononononononononnnononononnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnonononnnnonananenanannninnnos 18 ABI FLUOR
34. tion platforms Please see your instruments user manual for instructions on the fractionation and detection of your samples 14 Interpretation and Reporting Note This assay is for research use only Although positive results are highly suggestive of malignancy these assays are designed for Research Use Only and if used in a clinical setting should only be used in support of diagnosis Positive and negative results should be interpreted in the context of all clinical information and laboratory test results PCR based testing does not identify 100 of clonal cell populations therefore repeat testing by Southern blot may be advisable to rule out clonality The size range for each of the master mixes has been determined testing positive control samples For accurate and meaningful interpretation it is important to ignore peaks that occur outside of the proscribed valid size range for each of the master mixes Peaks that are outside of the range cannot be assumed to be valid IGH Gene Clonality Assay 1101002Xv7 40 FOR RESEARCH USE ONLY not for use in diagnostic procedures Page 10 of 18 Note Color indicates the color of products generated with the master mix when using differential fluorescence detection format e g ABI instruments Expected Size of Amplified Products Master Mix Target Color Control DNA Cati Product Size in basepairs IGH Tube A FR1 JH Blue Valid Size Range 310 360

IGH Gene Clonality Assay

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