b'Reference | SupportCommon Technical Support Questions1.What sample types may be suitable for analysis with 7. What are the differences between our IGH Gene Invivoscribe Gel and Capillary assays?Rearrangement Assays and the IGH Gene Clonality Assays?We recommend high-quality DNA for clonality testing withThe IGH Gene Rearrangement Assay was designed by our assays. This can be extracted from frozen or fresh tissue,Invivoscribe; whereas, the IGH Gene Clonality Assay was peripheral blood, bone marrow, skin biopsies, etc.designed by the EuroClonality/BIOMED-2 Group. Both assays target the conserved IGH framework regions, Framework 1, 2.Framework 2, and Framework 3. The IGH Gene Clonality Assay When should the recommended controls be run with our assays?The no template, positive, and negative controls should bealso targets incomplete D H -J Hrearrangements. The IGH Gene included in every run for each target, per the productClonality Assay includes 33 reactions per master mix and the insert or instructions for use. IGH Gene Rearrangement Assay includes 30 reactions per master mix.8.What do IGH Tubes D and E target do and why are they3.What is the purpose of the Specimen Control Size Ladder and Amplification Control master mix? What is the difference between these master mixes? challenging to interpret?The Specimen Control Size Ladder and Amplification ControlTubes D and E of our IGH Gene Clonality Assays target master mixes are used as troubleshooting tools that allow youincomplete IGHD H- J Hrearrangements. It is common to see to determine if the quality and quantity of your DNA sampleknown amplicons listed in the instructions for use in cases is suitable for use with our assays. The Specimen Control Sizewhere a polyclonal background is absent (this is likely because Ladder amplifies DNA at approximately 100, 200, 300, 400, andthese rearrangements are rare). Some of our customers are 600 base pairs; whereas, the Amplification Control amplifiesconcerned by this, especially because there may be some DNA at 235 bp. samples that have robust germline amplification greater than the valid size range. We do not expect the germline 4.amplification to outcompete true D H- J Hrearrangements.How should the master mix and controls be stored and thawed? PCR amplicons generated from germline templates are much The master mixes should be stored at -65 to -85 C and shouldlarger than true D H- J Hrearrangements. As a result, PCR be thawed at room temperature and vortexed prior to use. Ifproducts of germline amplifications are less robust whenyou intend to use master mixes multiple times, we recommenda specific target is present in samples.aliquoting the master mixes to minimize the number of freeze/ thaw cycles. For the FLT3 CDx Mutation Assay: Opened vials9.Why does the polyclonal control produce a peak around of master mixes stored frozen may incur up to 4 freeze thaw148 bp when amplified with IGK Tube A6FAM?cycles. Opened vials of controls stored frozen may incur up to 8The 148 bp peak is a result of the restricted repertoire of IGK freeze thaw cycles. and this peak commonly appears flanked by several smaller 5.peaks on each side. It is still possible to have a true clonal Where can more information about the primers used in ourrearrangement at this size in samples. If you suspect that this assays be found? peak is clonal in one of your samples, we recommend following Most primer information is proprietary to Invivoscribe andup with heteroduplex analysis. Alternatively, NGS-based cannot be disclosed. We can, however, tell you the target areaLymphoTrack and LymphoTrack Dx Assays provide an easier for the primers in each master mix, if you contact our supportinterpretation for IGK and reduces the number of master mixes team by emailing support@invivoscribe.com or by calling to just one reaction.+1 858-224-6600.10.What T-cell receptor kits would you recommend to detect6.Which targets are recommended for the study of T-cell clonal rearrangements?B-cell malignancies? Ideally, you should perform tests for TRB, TRG, and TRD to The EuroClonality/BIOMED-2 Group has shown that combinedachieve the highest sensitivity. The EuroClonality/BIOMED-2 testing of IGH and IGK achieves a clinical sensitivity of 99%.Group has shown that testing both TRB and TRG offers roughly If purchasing these assays separately is cost prohibitive, ourthe same sensitivity for the detection of T-cell malignancies as IGH + IGK Gene Clonality Assay (does not include IGH Tubes Dtesting all three targets; however, they highly recommend testing and E) may be a feasible alternative option (see Figure 2 andall three assays in parallel to achieve optimal clinical sensitivity. Table 1 in Leukemia (2007) 21, 201-206). We also offer next- TRD is especially useful in cases of suspected immature T-cell generation sequencing LymphoTrack Assays for IGH and IGKproliferations (see Figure 2 and Table 2 in Leukemia (2007) 21, 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.158'