b"Reference | SupportCommon Technical Support Questions1.7.11.What are the differences between the TCRG Gene 15.What are the recommended settings for my ABI instrument?What sample types may be suitable for analysis with What are the differences between our IGH Gene Invivoscribe Gel and Capillary assays?Rearrangement Assays and the IGH Gene Clonality Assays? Clonality Assay and the T-Cell Receptor Gamma GeneInstruments should be calibrated with the DS-30 matrix We recommend high-quality DNA for clonality testing withThe IGH Gene Rearrangement Assay was designed byRearrangement Assay 2.0? standards (Dye set D) for the ABI 310, 3100, or 3130 instrument our assays. This can be extracted from frozen or fresh tissue,Invivoscribe; whereas, the IGH Gene Clonality Assay wasThe TCRG Gene Clonality Assay was designed by theseries. For the ABI 3500 sequencer series, we advise that you peripheral blood, bone marrow, skin biopsies, etc.designed by the EuroClonality/BIOMED-2 Group. Both assaysEuroClonality/ BIOMED-2 Group and consists of two mastercalibrate the instrument with DS-33 matrix standards. We also target the conserved IGH framework regions, Framework 1,mixes. For polyclonal populations, four Gaussian distributions arerecommend using either POP-4 or POP-7 depending on which 2.When should the recommended controls be run with our assays? Framework 2, and Framework 3. The IGH Gene Clonality Assaygenerated. The T-Cell Receptor Gamma Gene RearrangementABI instrument you are using. If your equipment supports POP-7,The no template, positive, and negative controls should bealso targets incomplete D H -J Hrearrangements. The IGH GeneAssay 2.0 was designed by Invivoscribe and it's performancewe recommend using this polymer as it can be utilized for both included in every run for each target, per the productClonality Assay includes 33 reactions per master mix and thewas subsequently reviewed and validated by the EuroClonality/ fragment analysis and sequencing; whereas, POP-4 can only be insert or instructions for use. IGH Gene Rearrangement Assay includes 30 reactions perBIOMED-2 Group. It targets all functional V H- J Hrearrangementsutilized for fragment analysis.master mix. in a single master mix and produces smaller amplicons 3.What is the purpose of the Specimen Control Size LadderHow should peaks outside the valid size range be interpreted grouped under a single Gaussian distribution. This allows for16. 8.What do IGH Tubes D and E target do and why are they easier interpretation and makes the assay more suitable forwhen using assay kits?and Amplification Control master mix? What is the difference between these master mixes? challenging to interpret? DNA extracted from FFPE tissue, which may consist of partiallyYou should not interpret peaks outside of the valid size range; Tubes D and E of our IGH Gene Clonality Assays targetdegraded DNA that would not amplify well with the larger validalthough, in theory, it is possible to have a true rearrangement The Specimen Control Size Ladder and Amplification Controlincomplete IGHD H- J Hrearrangements. It is common to seesize range of the TCRG Gene Clonality Assay. fall outside this region. If you are concerned about a suspect master mixes are used as troubleshooting tools that allow youknown amplicons listed in the instructions for use in casespeak, you may sequence your product for confirmation. Please to determine if the quality and quantity of your DNA sample12.What are the differences between the IGH-BCL2 Translocation where a polyclonal background is absent (this is likely becausenote that samples should always be interpreted within the is suitable for use with our assays. The Specimen Control Sizethese rearrangements are rare). Some of our customers areAssay and the IGH-BCL2 t(14;18) Translocation Assay? context of all available clinical information.Ladder amplifies DNA at approximately 100, 200, 300, 400, andconcerned by this, especially because there may be someThe IGH-BCL2 Translocation Assay was designed by the 600 base pairs; whereas, the Amplification Control amplifiessamples that have robust germline amplification greaterEuroClonality/BIOMED-2 Group and is available as either a17. s cell-free DNA (cfDNA) a suitable sample type for Invivoscribe DNA at 235 bp. Ithan the valid size range. We do not expect the germlineCE-IVD or research use only assay whereas; the IGH-BCL2LymphoTrackor LymphoTrackDx Assays? 4.How should the master mix and controls be stored and thawed? amplification to outcompete true D H- J Hrearrangements. t(14;18) Translocation Assay was designed by InvivoscribeThe average size of cfDNA (~170 bps) makes it a suitable sample PCR amplicons generated from germline templates are muchand is only available for research use only. Both of these The master mixes should be stored at -65 to -85 C and shouldlarger than true D H- J Hrearrangements. As a result, PCRassays target MBR and Mcr translocations, but the IGH-BCL2type to run with IGH FR3 master mixes. The use of cfDNA with be thawed at room temperature and vortexed prior to use. Ifproducts of germline amplifications are less robust when Translocation Assay also targets the translocations at the 3TRG master mixes might be possible, but expected amplicon you intend to use master mixes multiple times, we recommenda specific target is present in samples. Mbr. The IGH-BCL2 t(14;18) Translocation Assay was sizes generated with this assay are near the upper limits of the aliquoting the master mixes to minimize the number of freeze/designed as a nested PCR allowing greater sensitivities fragment lengths typically found with this sample type.thaw cycles. For the FLT3 CDx Mutation Assay: Opened vials9.Why does the polyclonal control produce a peak around (1 clonal cell per 10,000 normal cells) to be achieved. The limit 18.Is DNA extracted from FFPE tissue suitable to use with of master mixes stored frozen may incur up to 4 freeze thaw148 bp when amplified with IGK Tube A6FAM? of detection of the IGH-BCL2 Translocation Assay is 1 clonal cycles. Opened vials of controls stored frozen may incur up to 8cell per 100 normal cells. Lastly, the IGH-BCL2 TranslocationInvivoscribe LymphoTrackor LymphoTrackDx Assays? freeze thaw cycles. The 148 bp peak is a result of the restricted repertoire of IGKAssay includes 33 reactions, whereas the IGH-BCL2 t(14;18)To ensure DNA from challenging specimens is of sufficient and this peak commonly appears flanked by several smallerTranslocation Assay includes 30 reactions. quality and quantity to generate a valid result, samples may be 5.Where can more information about the primers used in ourpeaks on each side. It is still possible to have a true clonaltested with the Specimen Control Size Ladder master mix. rearrangement at this size in samples. If you suspect that this assays be found? 13.Do you offer quantitative chromosome translocationpeak is clonal in one of your samples, we recommend following19.On which instruments can I use the LymphoTrack and Most primer information is proprietary to Invivoscribe andup with heteroduplex analysis. Alternatively, NGS-based(e.g., BCR-ABL1) controls? cannot be disclosed. We can, however, tell you the target areaLymphoTrack and LymphoTrack Dx Assays provide an easierOur controls are validated for qualitative use, although ourLymphoTrackDx Assays?for the primers in each master mix, if you contact our supportinterpretation for IGK and reduces the number of master mixescustomers do successfully use them with quantitative assays.We have different versions of our assays for the S5/PGM and team by emailing support@invivoscribe.com or by calling to just one reaction. Unfortunately, we cannot guarantee their performance with anyMiSeq instruments (LymphoTrack TRB is currently available only +1 858-224-6600. assay that was not designed by Invivoscribe. on MiSeq). No other DNA sequencers (e.g. 454) are currently 10.What T-cell receptor kits would you recommend to detect supported. Assays for the Ion S5/PGM and MiSeq platforms6.Which targets are recommended for the study of T-cell clonal rearrangements? 14.Which capillary electrophoresis instruments are currentlydiffer slightly in terms of the total number of indices, etc., butB-cell malignancies? Ideally, you should perform tests for TRB, TRG, and TRD tovalidated for use with our assay kits? both have similar benefits such as a one-step PCR reaction and The EuroClonality/BIOMED-2 Group has shown that combinedachieve the highest sensitivity. The EuroClonality/BIOMED-2Currently the capillary electrophoresis instruments Invivoscribeincluded bioinformatics software.testing of IGH and IGK achieves a clinical sensitivity of 99%.Group has shown that testing both TRB and TRG offers roughlyhas validated include: ABI 3100 and 3130 series for all capillary20.How much DNA is needed for the LymphoTrack and If purchasing these assays separately is cost prohibitive, ourthe same sensitivity for the detection of T-cell malignancies aselectrophoresis detection assays. The ABI 310 and 3500IGH + IGK Gene Clonality Assay (does not include IGH Tubes Dtesting all three targets; however, they highly recommend testinginstrument series have also been validated for the majorityLymphoTrackDx Assays?and E) may be a feasible alternative option (see Figure 2 andall three assays in parallel to achieve optimal clinical sensitivity.of our capillary electrophoresis detection assays. We are not50 ng of high-quality genomic DNA is required for the Ion S5/Table 1 in Leukemia (2007) 21, 201-206). We also offer next- TRD is especially useful in cases of suspected immature T-cellable to support using instruments not listed as validated in thePGM and MiSeq LymphoTrack and LymphoTrack Dx Assays for generation sequencing LymphoTrack Assays for IGH and IGKproliferations (see Figure 2 and Table 2 in Leukemia (2007) 21,instructions for use of our CE-IVD assays.clonality and somatic hypermutation applications.for use with MiSeq or Ion S5/PGM instruments. In addition, a201-206). We also offer NGS kits for TRG for use with MiSeq or high percentage of B-ALL patients have TRG rearrangements,Ion S5/PGM instruments and for TRB for use with MiSeq. which can be detected using our assays to detect TRG gene rearrangements.154Invivoscribe 2021|155"