IdentiClone™ T Cell Receptor Gamma Gene
Contents
1. TM Technical Service support invivoscribe com Customer Service US sales invivoscribe com Customer Service EU sales eu invivoscribe com IdentiClone T Cell Receptor Gamma Gene Rearrangement Assay 2 0 CE For Identification of T Cell Clonality For in vitro Diagnostic Use Key to Symbols Used For In Vitro Diagnostic Use Catalog Number Reagent Volume Lot Number Storage Conditions Expiration Date Authorized Representative in the European Community Manufacturer SERosGEEE Consult Instructions for Use 4 Storage Conditions 65 C to 85 C o ES DNA controls may be separated from assay kits and stored at 2 C to 8 C AG Nn V i VO S C r b e Catalog Products uantit 9 207 0101 IdentiClone T Cell Receptor Gamma Gene Rearrangement Assay 2 0 33 Reactions 9 207 0111 IdentiClone T Cell Receptor Gamma Gene Rearrangement Assay 2 0 MegaKit 330 Reactions Page 2 of 19 Table of Contents 1 PROPRIETARY NAME asecorcirs soos tes enan TANEET ANNEE tninaeweada aaa 3 Ze INTENDED USE 0 A aa ae ca oa ean ia 3 3 SUMMARY AND EXPLANATION OF THE TEST u cccccsccccccccccccccccccccccccccccccccccccccccccsccccccccccccccccccccceccccecs 3 4 PRINCIPLES OF THE PROCEDURE Guiada adenda 3 4 1 POLYMERASE CHAIN REACTIONS AA Aa 3 4 2 FEUORESCENCENDETES BON e ee e ol de e ee NO le oa e eo e o e a 4 5 REAGENTS ook Seen ca A case ae cunteuc ds 4 5 1 REAGENT COMPONENTS o o ee Sen eat ks ee ae 4 D2 WAR2. 14 TECHNICAL AND CUSTOMERSERVICE 0 dd 17 T5 PDEGAL NOTICE osmoro e ds 17 16 END USER LICENSE AGREEMENT iii ia iS riera 18 1 2 Page 3 of 19 Proprietary Name IdentiClone T Cell Receptor Gamma Gene Rearrangement Assay 2 0 IdentiClone T Cell Receptor Gamma Gene Rearrangement Assay 2 0 MegaKit Intended Use The IdentiClone T Cell Receptor Gamma Gene Rearrangement Assay 2 0 is an in vitro diagnostic product intended for PCR based detection of clonal T cell receptor gamma chain gene rearrangements in patients with suspect lymphoproliferations Specifically the T Cell Receptor Gamma Gene Rearrangement Assay 2 0 can be used to identify clonality in suspect lymphoproliferations Summary and Explanation of the Test Rearrangements of the antigen receptor genes occur during ontogeny in B and T lymphocytes These gene rearrangements generate products that are unique in length and sequence Polymerase chain reaction PCR assays can be used to identify lymphocyte populations derived from a single cell by detecting the unique V J gene rearrangements present within these antigen receptor loci This IdentiClone PCR assay employs multiple consensus DNA primers that target conserved genetic regions within the T cell receptor gamma chain gene Amplifying the region with fluorescently labeled primers is followed by fractionation by capillary electrophoresis and analysis by instrument software This DNA based test is used to dete
3. Storage Genomic DNA should be stored at 2 C to 8 C or at 65 C to 85 C until use Assay Procedure 8 1 Materials Provided Table 2 2 207 0091 TCRG 6FAM 2 096 0021 Specimen Control Size Ladder 6FAM 4 088 3320 5 TCRG Positive Control DNA 4 092 0020 TCRG Negative Control DNA 8 2 Materials Required But Not Provided Table 3 Reagent Material Recommended Reagents Materials and Suppliers Applied Biosystems PNA KOy merase AmpliTaq Gold DNA Polymerase Cat N808 0241 Glass Distilled De ionized Mol lar Biol Grad N A Water must be sterile and free of A AS DNase and RNase or USP Water Must be able to accurately measure A o ee O omame JR O Page 7 of 19 Reagent Material Recommended Reagents Materials and Suppliers ABI Capillary Electrophoresis Instrument Applied Biosystems ABI 3100 3130 or 3500 series Invivoscribe Technologies HI Deionized Formamide Cat 6 098 0041 Applied Biosystems Hi Di Formamide Cat 4311320 Invivoscribe Technologies Hi Di Formamide w ROX size standards for ABI 3100 Cat 6 098 0061 Applied Biosystems For ABI 3100 or 3130 instruments GeneScan 400HD ROX Cat 402985 For ABI 3500 instruments GeneScan 600 LIZ v2 0 Cat 4408399 Applied Biosystems For ABI 3100 and 3130 instruments Dye set used to spectrally calibrate DS 30 Matrix Standard Kit Dye Set D Cat 4345827 ABI instrument for use with 6FAM Sets For ABI 3500 instrument
4. control samples However valid clonal TCR gamma rearrangements can occur outside the specified size range Product s that are suspect TCR gamma gene rearrangement s that lie outside the specified size range should be sequenced to confirm their identity 9 1 Analysis 1 Samples that fail to amplify following repeat testing should be reported as A result cannot be reported on this specimen because the DNA was of insufficient quantity or quality for analysis 2 Ifthe positive or negative control reactions fail testing must be repeated 3 If samples run in duplicate yield differing results the samples should be re tested and or re evaluated for sample switching 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 must not be interpreted The following describes the analysis of each of the controls and the decisions necessary based upon the results Table 5 Type of Control Expected Result Aberrant Result No Template Control No amplification present continue with Amplification present check for analysis contamination and repeat the assay TCRG Negative Control Normal Gaussian distribution from 159 bp to Algorithm worksheet flags one or more peaks 207 bp No clonal peaks are flagged by as Significant Repeat the assay Algorithm worksheet Continue with analysis Page 11 of 19 Type of Control Expecte
5. or provision shall be enforced to the maximum extent permitted by law so as to affect the intent of the parties and the remainder of the EULA shall continue in full force and effect P N 280267 Revision B 2012 05 IdentiClone T Cell Receptor Gamma Gene Rearrangement Assay 2 0 ABI Fluorescence Detection
6. 020 AA a lation 50 uL 1 5 ot Control DNA HE E E 65 C to 85 C Note There are no preservatives used in the manufacture of this kit 5 2 Warnings and Precautions 1 IVD This Product is for in vitro Diagnostic Use 2 The assay kit should be used as a system Do not substitute other manufacturer s reagents Dilution reduction of amplification reaction volumes or other deviation 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 10 11 Page 5 of 19 Materials are stable until the labeled expiration date when stored and handled as directed Do not use kits beyond their expiration date Close adherence to the protocol will assure optimal performance and reproducibility Care should be taken to ensure use of correct thermocycler program as other programs may provide inaccurate faulty data such as false positive and false negative results Do not mix or combine reagents from kits with different lot numbers 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 Itis recommended that glass distilled de ionized molecular biology grade water be used with the preparation of specimen DNA
7. 858 224 6601 Fax 33 0 4 88 56 22 89 Technical Service support invivoscribe com Technical Service support invivoscribe com Customer Service sales invivoscribe com Customer Service sales eu invivoscribe com Website www invivoscribe com Website www invivoscribe com Business Hours 7 00AM 5 00PM PST PDT Business Hours 9 00AM 5 00PM CET CEST Technical and Customer Service Representatives are available Monday through Friday to answer phone e mail or website inquiries 15 Legal Notice This is an in vitro diagnostic product and is not available for sale or use within the United States This product is covered by issued United States Patent Number 7 785 783 and other pending patent applications originating from International Patent Application PCT AU2004 000625 European Patent Application Number 04732551 9 16 countries Brazil Patent Application Number PI0410283 5 Canadian Patent Application Number 2525122 Indian Patent Application Number 5792 DELNP 2005 Japanese Patent Application Number 2006 529437 Mexican Patent Application Number PA a 2005 012102 Chinese Patent Application Number 200480016603 5 and Korean Patent Application Number 10 2005 7021561 all of which are licensed exclusively to Invivoscribe Technologies Inc Use of this product may require nucleic acid amplification methods such as Polymerase Chain Reaction PCR No patent license to use amplification processes or enzymes is conveyed expressly or by implication to
8. A Licensee may not modify adapt translate reverse engineer decompile disassemble or create derivative works based on any portion of the Product including any documents or other content produced using the software 3 OWNERSHIP OF PRODUCT Title to ownership of and all rights and interests in the Product all copies thereof and Intellectual Property relating thereto remains at all times vested in Licensor 4 COPY RESTRICTIONS The Product is copyrighted Unauthorized copying or modification of the Product including software that has been modified merged or included with other software is expressly forbidden Licensee may be held legally responsible for any copyright infringement that is caused or incurred by Licensee s failure to abide by the terms of this EULA 5 TRANSFER RESTRICTIONS This Product is licensed to Licensee and may not be transferred to anyone without the prior written consent of Licensor In no event may Licensee transfer assign rent lease sell or otherwise dispose of the Product or any portion thereof on a temporary basis except as expressly provided herein 6 TERMINATION This license will be terminated automatically without notice if Licensee fails to comply with any provision of this license 7 LIMITED WARRANTY THE PRODUCT INCLUDING SOFTWARE AND ACCOMPANYING WRITTEN MATERIALS INCLUDING INSTRUCTIONS FOR USE ARE PROVIDED AS IS WITHOUT WARRANTY OF ANY KIND EITHER EXPRESS OR IMPLIED INC
9. Due to the high 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 To minimize contamination wear clean gloves when handling samples and reagents and routinely clean work areas and pipettes prior to doing PCR Autoclaving does not eliminate DNA contamination Work 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 Do not bring amplified DNA into the areas designated for master mix or specimen preparation All pipettes pipette tips and any equipment used in a particular area should be dedicated to and kept to that area of the laboratory Sterile disposable plastic ware should be used whenever possible to avoid RNase DNase or cross contamination 5 3 Storage and Handling For any duration other than immediate use assay kits should be stored at 65 C to 85 C The optimum storage temperature for the DNA controls is 2 C to 8 C but DNA controls can also be stored at 65 C to 85 C All reagents and controls must be thawed and vortexed or mixed thoroughly prior to use to ensure that t
10. LUDING BUT NOT LIMITED TO THE IMPLIED WARRANTIES OF MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE LICENSEE BEARS THE ENTIRE RISK OF THE QUALITY AND PERFORMANCE OF THE PRODUCT 8 LIMITED LIABILITY IN NO EVENT SHALL LICENSOR BE LIABLE FOR ANY DIRECT INDIRECT CONSEQUENTIAL OR INCIDENTAL DAMAGES INCLUDING DAMAGES FOR LOSS OF PROFITS LOSS OF DATA BUSINESS INTERRUPTION OR LOSS OF GOODWILL AND THE LIKE ARISING OUT OF THE USE OF OR INABILITY TO USE THE PRODUCT EVEN IF LICENSOR HAS BEEN ADVISED OF THE POSSIBILITY OF SUCH DAMAGES Because some states do not allow the exclusion or limitation of liability for consequential or incidental damages the above limitations may not apply to Licensee 9 GOVERNING LAW This EULA shall be governed by the laws of the State of California and Licensee consents to jurisdiction by the state and federal courts sitting in the State of California 10 COMPLETE AGREEMENT This EULA is the entire agreement between Licensor and Licensee with respect to the specific terms set forth herein concerning license and warranties of the Product and any other included term or obligation Page 19 of 19 This EULA replaces all prior understandings and agreements whether written or oral This EULA may not be modified unless Licensor and Licensee both assent in writing 11 SEVERABILITY If for any reason a court of competent jurisdiction finds any provision or part of any provision of this EULA unenforceable that part
11. Ladder master mix the number of reactions m will be m of samples Run each sample in singlicate 1 TCRG Negative Control DNA 1 No Template Control water 1 Additional Reaction m of samples 3 Total The total aliquot volume for the Specimen Control Size Ladder master mix will be m x 45ul 3 Add 1 25 units or 0 25ul at 5 units ul of AmpliTaq Gold DNA polymerase per reaction to each master mix The total AmpliTaq Gold DNA polymerase added to the TCRG master mix will be n x 0 25ul and m x 0 25ul for the Specimen Control Size Ladder master mix Gently vortex to mix 4 For each reaction aliquot 45ul of the appropriate master mix DNA polymerase solution into individual wells in a PCR plate or tube IdentiClone T Cell Receptor Gamma Gene Rearrangement Assay 2 0 ABI Fluorescence Detection Page 8 of 19 5 Add 5ul of appropriate template sample DNA positive control DNA negative control DNA or water to the individual wells containing the respective master mix solutions Pipette up and down several times to mix 6 Cap or cover the PCR plate 7 Samples are now ready to be amplified on a thermocycler Quick Guide For each master mix and n reactions mix n x 45 ul Master Mix n x 0 25 ul AmpliTaq Gold DNA polymerase Vortex gently to mix Aliquot 45ul of master mix DNA polymerase solution into each reaction well Add 5ul of appropriate sample to each well Total reaction volume 50ul 8 4 Amplificati
12. NINGS AND PRECAUTION oe eos neae ies 4 5 3 pid AQ PG Sieg WN MM a OY Ui GB 1 INI E ee heel er el lin dl la oe Sa ode 5 6 INSTRUMENT Sonar ida 5 6 1 THERMOCYELE ui dl dd e a ll dl a 5 6 2 ABI CAPILLARY ELECTROPHORESIS INSTRUMENTS id 5 Te SPECIMEN COLLECTION AND PREPARATION 22 ccccccccccccccccccccccccccccccccccccccccccccccccccccccccccceccecccccccccceccecoes 6 Pele PPE TIN St albedo ias 6 AA INTERFERING SUBSTANCES ad Meee 6 7 3 SPECIMEN REQUIREMENTS AND HANDLING i 6 7 4 AS A II A A nce ete eae ee A 6 7 5 SAMPLES TORMO Brea a el e e ie a a e S A 6 8 ASSAY PROCEDURE a aS ANE 6 8 1 MATERIA ES PROVIDE D sips te hirsuta areas ae do o pane aaa do eo eine ase asta 6 8 2 MATERIALS REQUIRED BUT NOT PROVIDED iaa 6 8 3 REAGENT PREPARA TN e e laces 7 8 4 AMPLIFICATION E E e e teense 8 8 5 ABI FLUORESCENCE DETECTION i cses ese etic deca adactbs a a a a a i a 8 8 6 MAT APN AY SIS roo as OPA yu aaa Sette cash aah ne Ses oa bata ds alas aati ae cata eaeea inc dae a aaa a eats 9 8 7 OUALTEY CONTROL iio 10 8 8 ASS EOE E O ia oO 10 9 INTERPRETATION OFRESUE TS 10 9 1 ANAE A A A A A RnR ee Poe 10 9 2 SAMPLE INTERPRETA TION dale do ea eS 11 10 LINMILEXTIONSOBPROCEDUR Bo A A A tons 11 PL EXPECTED VALUES lt lt ui isa 12 11 1 EXPECTED SIZE OF AMPLIELED PRODU TS el SE dd O coo cua SS 12 11 2 A A A A eid Se eae bet ea Oe ae a ae cc a RN Bg a Se Sat ae eae 13 2 PERFORMANCE CHARACTERISTICS cuina ais 14 13 BERBLIOGR APE Y i 17
13. P N 4 088 0230 5 IVS 0016 is P N 4 088 0950 5 IVS 0021 is P N 4 088 1250 and 5 IVS 0039 is P N 4 088 2330 Using clinical samples the TCRG V2 assay results were compared to Roche 454 sequencing for the identification T cell receptor gamma gene rearrangements For the 454 sequencing any DNA sequence that was present at levels greater than 5 of the total sequences detected was considered a clonal event If more than 2 sequences exceeded the 5 threshold that sample was defined as oligoclonal The TCRG V2 assay had 100 concordance for the 7 samples that were identified Page 17 of 19 as clonal by sequencing There was 75 concordance for the 12 samples that were either negative for a clonal event or were oligoclonal Sample types included peripheral blood bone marrow and FFPE samples It is important to note that the presence or absence of clonal peaks in a clinical sample does not always correlate with actual clinical outcomes 13 Bibliography 1 Miller JE et al An automated semiquantitative B and T cell clonality assay Mol Diag 1999 4 2 101 117 14 Technical and Customer Service Manufacturer Authorized Representative and EU Technical Assistance Invivoscribe Technologies Inc Invivoscribe Technologies SARL sad 6330 Nancy Ridge Drive Suite 106 Le Forum Bat B San Diego California 92121 3230 515 Avenue de la Tramontane USA ZI Ath lialV 13600 La Ciotat France Phone 1 858 224 6600 Phone 33 0 4 42 01 78 10 Fax 1
14. ROX size standards 11 2 Sample Data Page 13 of 19 The data shown below was generated using the TCRG 6FAM Master Mix Amplified products were run on an ABI 3130 instrument Panel 1 displays data generated testing the 100 TCRG Positive Control DNA Panel 2 displays data generated testing the 5 TCRG Positive Control DNA Panel 3 displays data generated testing the TCRG Negative Control DNA 100 120 140 160 180 200 220 240 260 280 6000 1 100 TCRG Positive Control DNA Figure 2 Panel 2 coa AN pl 100 120 140 160 180 200 220 240 260 280 Specified Size Range 159 207 bp Panel 3 TCRG Negative Control DNA ili IA For the Specimen Control Size Ladder master mix Panel 1 displays data generated testing a negative water control Panel 2 displays data generated testing the IVS 0000 Polyclonal Control DNA Panels 3 and 4 display data generated testing two different 100 clonal control DNAs Figure 3 Specimen Control Size Ladder 6FAM Panel 1 l Water Panel IVS 0000 sl l l Panel 3 IVS 0030 Panel 4 lo j 96 bp 198 bp 298 bp 399 bp IVS 0029 581 bp Ai seba rr lr ioc IdentiClone T Cell Receptor Gamma Gene Rearrangement Assay 2 0 ABI Fluorescence Detection Page 14 of 19 12 Performance Characteristics The assay was able to detect clonal rearrangement
15. Required But Not Provided Use the default settings for your polymer and capillary type IdentiClone T Cell Receptor Gamma Gene Rearrangement Assay 2 0 ABI Fluorescence Detection Page 6 of 19 7 See section 8 5 ABI Fluorescence Detection for sample preparation Specimen Collection and Preparation 7 1 Precautions Biological specimens from humans may contain potentially infectious materials All specimens should be handled in accordance with the OSHA Standard on Bloodborne Pathogens or Biosafety Level 2 7 2 Interfering Substances The following substances are known to interfere with PCR e Divalent cation chelators e Low retention pipette tips e EDTA e Heparin 7 3 Specimen Requirements and Handling This assay tests genomic DNA extracted and purified from peripheral blood bone marrow aspirates or paraffin embedded tissue 7 4 Sample Preparation Extract the genomic DNA from patient specimens as soon as possible Resuspend DNA to a final concentration of 10 ug to 200 ug per ml in 1 10 TE 1 mM Tris HCl pH 8 0 0 1 mM EDTA or in molecular biology grade or USP 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 DNA with the Specimen Control Size Ladder master mix will ensure that DNA of sufficient quality and quantity was present to yield a valid result 7 5 Sample
16. a clonal population the yield is one or two prominent amplified products amplicons within the valid size range IdentiClone T Cell Receptor Gamma Gene Rearrangement Assay 2 0 ABI Fluorescence Detection Page 4 of 19 Gr Gr Il Gr Ill Gr IV Vy2 Vy3 Vy4 Vy5 Vy8 Vy9 Vy10 Vy11 Jy1 Jy2 JyP JyP1 JyP2 Figure 1 This diagram of the T cell receptor gamma gene shows the approximate placement of the upstream and downstream DNA primers 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 a multiplex master mix which targets multiple V and J regions Figure 1 is required to detect the majority of clonal rearrangements As indicated clonal rearrangements are identified as one or two prominent single sized products within the background of different sized amplicon products that form the Gaussian distribution around a statistically favored average sized rearrangement 4 2 Fluorescence Detection Fluorescence detection is commonly used to resolve the different sized amplicon products using a capillary electrophoresis instrument Primers are conjugated with a 6FAM fluorescent dye fluorophore so that they can be detected after
17. aks flagged by the Algorithm worksheet within the valid size range are reported as T cell receptor gamma chain gene rearrangements are consistent with the detection of biclonality or oligoclonality e An absence of significant peaks flagged by the Algorithm worksheet within the valid size range is reported as Negative for the detection of clonal T cell receptor gamma chain gene rearrangement s Note A visual confirmation should be conducted the electropherogram should be visually reviewed to ensure that the algorithm interpreted the pattern correctly Limitations of Procedure This assay does not identify 100 of clonal cell populations This assay cannot reliably detect below 5 positive cells per 100 total cells The results of molecular clonality tests should always be interpreted in the context of clinical histological and immunophenotypic data The algorithm requires a reasonably consistent signal background and that the data is entered correctly Gaps in the background can cause the algorithm to call a sample incorrectly All electropherograms should be reviewed to confirm the validity of the interpretation PCR based assays are subject to interference by degradation of DNA or to inhibition of PCR due to EDTA heparin or other agents IdentiClone T Cell Receptor Gamma Gene Rearrangement Assay 2 0 ABI Fluorescence Detection Page 12 of 19 11 Expected Values 11 1 Expected Size of Amplified Produc
18. al peak compared to the smallest adjacent peak must be gt 4X e The RFU of the suspected clonal peak must be gt 20 of the RFU of the highest peak in that sample If a peak is present 1 bp from a clonal peak 1t can be ignored if RFU Pk 1 lt RFU Pk e There must be gt 2 bp difference between two positive peaks IdentiClone T Cell Receptor Gamma Gene Rearrangement Assay 2 0 ABI Fluorescence Detection Page 10 of 19 e Samples must be run in duplicate and both replicates must show positive results for a suspected peak e The size of the suspected clonal peaks in both replicates must be within 1 bp from each other 8 7 Quality Control Positive and negative controls are provided with the kit and must be included with each assay run In addition a no template control e g water must also be included A buffer control may also be added to ensure that no contamination of the buffer used to resuspend the samples has occurred The values for the positive controls are provided under section Expected Size of Amplified Products Additional controls and sensitivity controls dilutions of positive controls into our negative control are available from Invivoscribe Technologies 8 8 Assay Control The amplicon sizes listed in Table 4 were determined using an ABI 3130 platform The amplicon sizes measured on your specific capillary electrophoresis instrument may differ by 1 to 4 base pairs from those listed depending on th
19. aller of its neighboring peaks and it must exceed a cutoff of 4 0 The D x value is based on a variation of the Kolmogorov Smirnov test which compares two empirical distributions and determines whether they are statistically different and its value must be greater than 0 0419 Principles of the Procedure 4 1 Polymerase Chain Reaction PCR PCR assays are routinely used for the identification of clonal T cell populations This test amplifies the DNA between primers that target conserved regions within the variable V and the joining J regions that flank the unique hypervariable antigen binding region 3 CDR3 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 undergo rearrangement are the immunoglobulin heavy chain and light chains 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 DNA from a normal or polyclonal population is amplified using DNA primers that flank the V J region a Gaussian distribution bell shaped curve of amplicon products is produced within an expected size range This Gaussian distribution reflects the heterogeneous population of V J rearrangements In certain cases where lymphocyte DNA is not present no product is detected For DNA from samples containing
20. ct the vast majority of clonal T cell populations Presence or absence of clonality can support the differential diagnosis of reactive lesions and certain T and B cell malignancies This assay cannot reliably detect clonality present at less than 5 of the total lymphocyte population It should be emphasized that the results of molecular clonality testing should always be interpreted in the context of all available clinical histological and immunophenotypic data This test kit consists of a single master mix that contains primers that target the Vy2 3 4 5 8 9 10 amp 11 and Jy1 Jy2 JyP and JyP1 JyP2 regions The PCR amplicons have an expected size range between 159 and 207 base pairs The Specimen Control Size Ladder master mix targets multiple genes and generates a series of amplicons of approximately 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 methodology is used with all of our Gene Clonality Assays This improves consistency and facilitates cross training on a broad range of our different assays A software based algorithm has been developed for analyzing the peaks that were measured on the capillary electrophoresis instrument The algorithm calculates the relative peak height ratio RPR and a statistical parameter D x value for each peak The RPR is calculated by dividing the height of each peak to the sm
21. d Result Aberrant Result 5 TCRG Positive Control This can also be an extraction control if positive control material is taken through extraction processes Specimen Control Size Ladder Peaks present at 194 bp 196 bp Algorithm worksheet flags at least the196 bp as Significant The 194 bp peak may or may not be identified as Significant Continue with analysis If the 100 200 300 and 400 bp peaks are seen continue with analysis Because smaller PCR fragments are preferentially amplified it is not unusual for the 600 bp fragment to have a diminished signal or to be missing entirely Algorithm worksheet does not flag peak at 196 bp as Significant Repeat the assay If no peaks are detected repeat the assay unless specimen tests positive If only the 100 200 or 300 bp peaks are present re evaluate sample for DNA degradation unless specimen tests positive 9 2 Sample Interpretation Given that the controls produce expected results the clinical samples should be interpreted as follows e One or two significant peaks flagged by the Algorithm worksheet within the valid size range are reported as Positive for the detection of clonal T cell receptor gamma chain gene rearrangement s consistent with the presence of a clonal cell population In the context of overall diagnostic criteria clonal cell populations can indicate the presence of hematologic malignancy e Three or more significant pe
22. dye In the Analysis menu select Analyze For each analyzed sample file open the associated display plot To ensure that only the Blue Dye is shown in the display plot go to the View menu select Dyes gt Blue Dye Next in the View menu choose Tables gt Sizing Table Highlight the display plot peaks in the valid size range from 159 bp to 207 bp In the Sizing Table Copy the Size bp and Height RFU column data for the highlighted peaks within the valid size range Paste the peak size and height data into the unlocked portion of the TCRG Algorithm worksheet cells are highlighted in grey Note for GeneMapper version 3 5 and lower this data must be entered into the worksheet manually The worksheet will output a summary of RPR D x and RFU max for the five peaks that are the most significant outliers from a normal Gaussian distribution If a peak in the summary table meets the criteria for a clonal peak as defined in the TCRG Algorithm worksheet it will read Yes in the column titled Significant If a peak in the summary table does not meet the criteria for a clonal peak as defined in the TCRG Algorithm worksheet it will read No in the column titled Significant The following criteria are implemented in the worksheet to define peaks as Positive for Clonality e The D x value of the suspected clonal peak as calculated within the locked portion of the worksheet must be gt 0 0419 e The RPR of the suspected clon
23. e 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 Note Color indicates the color of products generated with the master mix when using the default color assignment on ABI fluorescence detection systems Table 4 Master Mix Target Color Control DNA Cat Product Size in Base Expected Algorithm Pairs Results Vy1 Vy11 Specified Size Range 159 207 One peak at 196 bp TCRG 6FAM 5 TCRG A Control 4 088 3320 194 196 flagged as Significant Jy1 Jy2 JyP JyP 1 JyP2 Specimen Control Multiple Genes TCRG Negative Control 4 092 0020 84 96 200 300 400 Size Ladder DNA 600 Note Because smaller PCR fragments are preferentially amplified it is not unusual for the 600 bp fragment to have a diminished signal or to be missing entirely For ABI fluorescence detection the 600 bp peak may not appear during normal run times In addition the size of this peak may differ by over 30 bp when fragment size is extrapolated using the GeneScan 400HD ROX size standards Interpretation of Results Although positive results are highly suggestive of malignancy both positive and negative results should be interpreted in the context of all clinical information and laboratory test results The size range for the TCRG 6FAM master mix has been determined to be 159 bp to 207 bp by testing positive and negative
24. excitation by a laser in the capillary electrophoresis instrument This highly sensitive detection system provides single base pair size resolution and relative quantification Inter and intra assay reproducibility in size determination using capillary electrophoresis is approximately 1 to 2 base pairs This reproducibility and sensitivity coupled with the automatic archiving of specimen data allows for the monitoring tracking and comparison of data from individual patients over time S5 Reagents 5 1 Reagent Components 9 207 0101 IdentiClone T Cell Receptor Gamma Gene Rearrangement Assay 2 0 33 Reactions 9 207 0111 IdentiClone T Cell Receptor Gamma Gene Rearrangement Assay 2 0 MegaKit 330 Reactions Table 1 Reactii Calot Reagent Components Unit 9 207 0101 9 207 0111 Storage 8 8 active ingredients Quantity of Units of Units Temp TCRG 6FAM Multiple oligonucleotides targeting the Master Mixes 2 207 0091 Vy2 3 4 5 8 9 10 amp 11 and Jy1 Jy2 JyP 1500 uL l 10 65 C to 85 C and JyP1 JyP2 regions of the T cell receptor gamma gene in a buffered salt solution Manel Specimen Control Size Ladder 6FAM e trol 2 096 0021 Multiple oligonucleotides targeting 1500 uL 1 10 65 C to 85 C Master Mix housekeeping genes Positive 4 088 3320 5 TCRG Positive Control DNA 50 uL 1 5 a ie Ene Ei E th Control DNA 50 ug mL of DNA in 1 10 TE solution 65 C to 85 C Negative 2 C to 8 C Normal 4 092 0
25. hey are mixed completely Excessive vortexing may cause labeled primers to lose their fluorophores Materials are stable until the labeled expiration date when stored and handled as directed Do not use kits beyond their expiration date The PCR master mixes and controls have been validated for 6 freeze thaw cycles with no loss in performance Aliquot reagents into sterile o ring screw cap tubes if more freeze thaw cycles are necessary Instruments 6 1 Thermocycler Use or Function Amplification of DNA samples Performance Characteristics and Specification Minimum Thermal Range 15 C to 96 C Minimum Ramping Speed 0 8 C sec Follow manufacturer s installation operation calibration and maintenance procedures See section 8 4 Amplification for thermocycler program 6 2 ABI Capillary Electrophoresis Instruments Use or Function Fragment detection and analysis Performance Characteristics and Specification The following capillary electrophoresis instruments will meet the performance needs for this assay ABI 3100 Avant Genetic Analyzer 4 capillaries ABI 3100 Genetic Analyzer 16 capillaries ABI 3130 Genetic Analyzer 4 capillaries ABI 3130XL Genetic Analyzer 16 capillaries ABI 3500XL Genetic Analyzer 24 capillaries Follow manufacturer s installation operation calibration and maintenance procedures The ABI instrument used must be calibrated with appropriate Matrix Standards as outlined in section 8 2 Materials
26. nt Assay 2 0 ABI Fluorescence Detection Page 16 of 19 The assay when performed in combination with the TCRG Algorithm worksheet was capable of detecting DNA from 6 control cell lines 200 ng ul diluted into tonsil DNA 200 ng ul at 5 v v and the results are shown in Table 9 Table 9 Peak One Peak Two Product Product Size po RFR significant Size Do BER significant Ratio Ratio bp bp Yes rept 17887 02964 4250 Yes 18469 0 1322 5 61 _ 5 IVS 0004 Sas Prep 18436 onos 2759 Yes 17839 ooog 2118 ves rep 3 23 93 epa 18069 0 1200 1497 ves 17878 ox009 2982 Yes eps 18036 ox342 1212 ves 17840 onis 3207 sept 16038 onos 1159 ves isan ono 232 no o rep 224 No 1vS 0016 rep4 16938 0 1028 1009 Yes 193 77 o104 245 yo Yes E reps 169 00 0 0957 sso ves 19353 oo944 2 50 _ rep 1 7 rep 2 187 58 0 1003 182 53 0 1298 14 23 0 1vS 0021 rep4 18792 om 699 Yes temor 0 1238 1268 rep EEE AE E E rep1 19597 3 60 Paz vem Poza on E ne 1vS 0039 2 84 faa oan ee ae ee ek ee EPT reps 19571 ozs rs ve 19365 ormo 336 no gt gt eS A E E 5 PF 382 ES as 48 Yes e as 1151 a st sa 02822 sos ves 15839 ozs nss ves a as 53 930 Yes rept 190 74 ome sor vo sro fons oss ve MOLT 13 190 46 0 1983 187 57 0 1114 Yes Note 5 IVS 0004 is
27. on 1 Amplify the samples using the following PCR program Note We recommend using the calculated option for temperature measurement with the BioRad MJ Research PTC thermocyclers PCR Program 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 2 Remove the amplification plate or tubes from the thermocycler Although amplified DNA is stable at room temperature for extended periods of time PCR products should be stored at 2 C to 8 C until detection Detection must be done within 30 days of amplification 8 5 ABI Fluorescence Detection Please note that for ABI fluorescence detection a preceding peak is often seen and is an artifact due to the detection method the ABI platforms use Preceding peaks are sometimes skewed and have bases that slope on the right side towards the real peak This is especially evident in the Specimen Control Size Ladder master mix where the 96 base pair peak has a preceding peak that shows up at 84 base pairs ABI 3100 and 3130 Platforms 1 In anew microcentrifuge tube mix an appropriate amount 10 ul per PCR reaction of Hi Di Formamide with ROX Size Standards Vortex well 2 Inanew 96 well PCR plate add 10 ul of Hi D1 Formamide with ROX size standards to individual wells for each PCR reaction 3 Transfer 1 ul of each PCR reaction
28. right to use the software in any manner shall be controlled by this EULA and that such use shall be strictly in accordance with the terms and conditions of this EULA 1 GRANT OF LICENSE Licensor grants to Licensee a non exclusive non transferable limited license without right to sublicense to use the software and any accompanying written materials and any documents or other content embodying information contained within the software collectively the Product only for Licensee s own use Licensor reserves all rights not expressly granted to Licensee or to Licensee The limited license granted by this EULA and Licensee s payment of the license fee give Licensee the right to use the Product only in accordance with the terms of this EULA This license is not a sale of the original software or any copy 2 CONFIDENTIALITY Licensee agrees that the Product is based on and includes one or more proprietary trade secrets copyrights patent applications and or granted patents Intellectual Property of Licensor Possession and use of the Product shall be strictly in accordance with this EULA and receipt or possession does not convey any rights to divulge reproduce or allow others to use the Product outside the terms of this EULA without specific written authorization of Licensor Licensee agrees not to disclose publish translate release or distribute copies of the Product or any portion thereof to others outside the terms of this EUL
29. s HEX NED and ROX DS 33 Matrix Standard Kit Dye Set G5 Cat 4345833 Applied Biosystems Polymer POP 7 Polymer POP 7 for 3130 3130XL 3500XL Genetic Analyzers Cat 4352759 8 3 Reagent Preparation All samples should be tested using the Specimen Control Size Ladder master mix This is to ensure that no inhibitors of amplification are present and there is DNA of sufficient quality and quantity to generate a valid result All samples must be tested in duplicate If duplicate testing provides inconsistent results re testing or re evaluation of the sample is necessary Positive negative and no template controls must be tested Hi Di Formamide Size Standards Spectral Calibration Dye 1 Using gloved hands remove the master mixes from the freezer Allow the tubes to thaw completely then gently vortex to mix 2 Remove the calculated volume of each master mix to individual microcentrifuge tubes The aliquot volume is 45 ul for each reaction We recommend adding an additional reaction for every 15 reactions to ensure an adequate volume is available For the TCRG 6FAM master mix the number of reactions n should be n 2x of samples Run each sample in duplicate 1 5 TCRG Positive Control DNA 1 TCRG Negative Control DNA 1 No Template Control water 1 Additional Reaction n 2x of samples 4 Total The total aliquot volume for the TCRG 6FAM master mix will be n x 45ul For the Specimen Control Size
30. s in 11 positive control cell lines The following well characterized T cell leukemia cell lines known to be positive for TCRG rearrangements were tested with TCRG 6FAM master mix and the results are shown in Table 7 Two prominent peaks were detected with each of the cell lines Table 7 Cell Line IVS Part Number Peak One bp Peak Two bp 100 IVS 0004 4 088 0190 178 8 187 9 Page 15 of 19 The assay showed robust results Table 8 when tested with IVS 0039 DNA 200 ng ul diluted into tonsil DNA 200 ng ul at 5 10 25 50 and 75 v v Table 8 Peak One Peak Two Product py RPR Product px RPR Size i Significant Size E Value Ratio Value Ratio bp bp 196 20 Significant rep 1 Lp 19581 rvs oo39 rep3 19571 2 45 rep4 196 18 195 79 2 70 196 24 196 15 0 3382 193 95 0 1877 4 03 No No Yes 195 66 0 2790 196 15 0 3382 195 80 0 2983 196 02 03947 196 11 03216 195 72 04059 196 11 03212 rep 5 rep 1 rep 2 10 IVS 0039 tep3 rep 4 rep 5 rep 1 rep 2 25 IVS 0039 Tep3 rep 4 rep 5 196 15 0 2939 0 3817 0 4503 0 3586 rep 195 67 195 66 196 07 195 67 196 11 19572 195 62 196 15 195 71 rep 2 50 IVS 0039 rep 3 rep 4 0 4404 0 2387 03154 0 4520 0 2911 XS 0 3301 i rep 5 rep 195 71 04316 rep 2 75 Ivs 0039 rep 3 rep 4 rep 5 IdentiClone T Cell Receptor Gamma Gene Rearrangeme
31. sults See sections 9 Interpretation of Results and 11 Expected Values Note Please see Applied Biosystems accompanying product insert for mixing Hi Di Formamide with size standards for different ABI instruments Note 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 ABI capillary electrophoresis instruments routinely handle 2 runs per hour for the 16 and 24 capillary instruments this is equal to 768 and 1152 samples per day respectively and automatically analyze and store data for review or comparison with other test results 8 6 Data Analysis The TCRG Algorithm worksheet has been developed to analyze the TCRG V2 data output k 2 So ea 11 12 3 14 15 Open the TCRG Algorithm Worksheet the worksheet requires Microsoft Excel Add raw data files derived from CE analysis to a new project in GeneMapper software Verify that Analysis Method selected is Microsatellite Default and that the appropriate Size Standard is selected It may be necessary to lower the Minimum Peak Height threshold in order to detect all peaks in a Gaussian distribution In order to do this select GeneMapper Manager from the Tools Menu go to the Analysis Methods tab open the Microsatellite Default Analysis Method Editor Go to the Peak Detector tab select User Specified rfu toggle and input the desired peak height for the Blue
32. the purchaser by the purchase of these products Purchase of this product includes a limited sublicense for non commercial uncompensated practice of this technology if and only if the purchaser is registered with IVS as an exclusively non commercial user of IVS products No sublicense for such use is granted simply by purchase of these products 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 other questions please contact IVS by email at legal invivoscribe com or by telephone at 1 858 224 6600 IdentiClone is a trademark of Invivoscribe Technologies Inc IdentiClone T Cell Receptor Gamma Gene Rearrangement Assay 2 0 ABI Fluorescence Detection Page 18 of 19 16 End User License Agreement THIS AGREEMENT MUST BE ACCEPTED BY AN AUTHORIZED REPRESENTATIVE OF THE END USER OF THIS PRODUCT PRIOR TO USING THE TCRG ALGORITHM WORKSHEET EXCEL FILE BY USING THE TCRG ALGORITHM WORKSHEET EXCEL FILE YOU ASSERT THAT YOU ARE AN AUTHORIZED REPRESENTATIVE OF THE END USER WITH AUTHORITY TO ENTER INTO THIS AGREEMENT This End User License Agreement EULA is made and entered into by and between INVIVOSCRIBE INC Licensor and You either an individual or a legal entity the Licensee as defined herein for the licensing and usage of the Licensor s software Licensee acknowledges and agrees that Licensee s
33. to the wells containing Hi Di Formamide with ROX size standards Add only one sample per well Pipette up and down to mix Cap or cover the PCR plate Heat denature the samples at 95 C for 2 minutes then snap chill on ice for 5 minutes Prepare a sample sheet and injection list for the samples Run the samples on an ABI 3100 3130 capillary electrophoresis instrument according to its user manual Data are automatically displayed as size and color specific peaks Review profile and controls report results See sections 9 Interpretation of Results and 11 Expected Values below Po ere Page 9 of 19 ABI 3500 Platforms 1 In anew microcentrifuge tube mix an appropriate amount 9 5 ul per PCR reaction of Hi Di Formamide with LIZ Size Standards Vortex well 2 Inanew 96 well PCR plate add 9 5 ul of Hi Di Formamide with LIZ size standards to individual wells for each PCR reaction 3 Transfer 0 5 ul of each PCR reaction to the wells containing Hi Di Formamide with LIZ size standards Add only one sample per well Pipette up and down to mix 4 Cap or cover the PCR plate 5 Heat denature the samples at 95 C for 3 minutes then snap chill on ice for 5 minutes 6 Prepare a sample sheet and injection list for the samples 7 Run the samples on an ABI 3500 capillary electrophoresis instrument according to its user manual 8 Data are automatically displayed as size and color specific peaks Review profile and controls report re
34. ts The amplicon sizes listed were determined using an ABI 3130 platform Amplicon sizes seen on your specific capillary electrophoresis instrument may differ 1 to 4 bp from those listed 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 monitoring disease recurrence Note Color indicates the color of products generated with the master mix when using the default color assignment on ABI fluorescence detection systems Table 6 Master Mix Target Color Control DNA Cat Product Expected Size in Algorithm Results Base Pairs Specified Size Range 159 207 TCRG 6FAM All V and J genes TCRG Negative Control DNA 4 092 0020 159 207 No Significant Peaks Vy9 Vy10 Jy1 Jy2 5 TCRG Positive Control DNA 4 088 3320 194 amp 196 Significant Peak at 196 bp and possibly second peak at 194 bp Specimen Control Multiple Genes Any Human DNA 84 96 200 Size Ladder 300 400 600 Note Because smaller PCR fragments are preferentially amplified it is not unusual for the 600nt fragment to have a diminished signal or to be missing entirely For ABI fluorescence detection the 600 bp peak may not appear during normal run times In addition the size of this peak may differ by over 30 bp when fragment size is extrapolated using the GeneScan 400HD
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