150 Reference | Support Invivoscribe 2019 | 151 Reference Support Common Technical Support Questions 1.  What sample types may be suitable for analysis with Invivoscribe Gel and Capillary assays? We recommend high-quality DNA for clonality testing with our assays. This can be extracted from frozen or fresh tissue, peripheral blood, bone marrow, skin biopsies, etc. 2.  When should the recommended controls be run with our assays? The no template, positive, and negative controls should be included in every run for each target, per the product insert or instructions for use. 3.  What is the purpose of the Specimen Control Size Ladder and Amplification Control master mix? What is the difference between these master mixes? The Specimen Control Size Ladder and Amplification Control master mixes are used as troubleshooting tools that allow you to determine if the quality and quantity of your DNA sample is suitable for use with our assays. The Specimen Control Size Ladder amplifies DNA at approximately 100, 200, 300, 400, and 600 base pairs; whereas, the Amplification Control amplifies DNA at 235 bp. 4.  How should the master mix and controls be stored and thawed? The master mixes should be stored at -65 to -85 °C and should be thawed at room temperature and vortexed prior to use. If you intend to use master mixes multiple times, we recommend aliquoting the master mixes to minimize the number of freeze/ thaw cycles. For the FLT3 CDx Mutation Assay: Opened vials of master mixes stored frozen may incur up to 4 freeze thaw cycles. Opened vials of controls stored frozen may incur up to 8 freeze thaw cycles. 5.  Where can more information about the primers used in our assays be found? Most primer information is proprietary to Invivoscribe and cannot be disclosed. We can, however, tell you the target area for the primers in each master mix, if you contact our support team by emailing support@invivoscribe.com or by calling +1 858-224-6600. 6.  Which targets are recommended for the study of B-cell malignancies? The EuroClonality/BIOMED-2 Group has shown that combined testing of IGH and IGK achieves a clinical sensitivity of 99%. If purchasing these assays separately is cost prohibitive, our IGH + IGK Gene Clonality Assay (does not include IGH Tubes D and E) may be a feasible alternative option (see Figure 2 and Table 1 in Leukemia (2007) 21, 201-206). We also offer next-generation sequencing LymphoTrack® Assays for IGH and IGK for use with MiSeq® or Ion PGM™ instruments. In addition, a high percentage of B-ALL patients have TRG rearrangements, which can be detected using our assays to detect TRG gene rearrangements. 7.  What are the differences between our IGH Gene Rearrangement Assays and the IGH Gene Clonality Assays? The IGH Gene Rearrangement Assay was designed by Invivoscribe; whereas, the IGH Gene Clonality Assay was designed by the EuroClonality/BIOMED-2 Group. Both assays target the conserved IGH framework regions, Framework 1, Framework 2, and Framework 3. The IGH Gene Clonality Assay also targets incomplete DH - JH rearrangements. The IGH Gene Clonality Assay includes 33 reactions per master mix and the IGH Gene Rearrangement Assay includes 30 reactions per master mix. 8. What do IGH Tubes D and E target do and why are they challenging to interpret? Tubes D and E of our IGH Gene Clonality Assays target incomplete IGH DH - JH rearrangements. It is common to see known amplicons listed in the instructions for use in cases where a polyclonal background is absent (this is likely because these rearrangements are rare). Some of our customers are concerned by this, especially because there may be some samples that have robust germline amplification greater than the valid size range. We do not expect the germline amplification to outcompete true DH - JH rearrangements. PCR amplicons generated from germline templates are much larger than true DH - JH rearrangements. As a result, PCR products of germline amplifications are less robust when a specific target is present in samples. 9.  Why does the polyclonal control produce a peak around 148 bp when amplified with IGK Tube A – 6FAM? The 148 bp peak is a result of the restricted repertoire of IGK and this peak commonly appears flanked by several smaller peaks on each side. It is still possible to have a true clonal rearrangement at this size in samples. If you suspect that this peak is clonal in one of your samples, we recommend following up with heteroduplex analysis. Alternatively, NGS-based LymphoTrack® and LymphoTrack® Dx Assays provide an easier interpretation for IGK and reduces the number of master mixes to just one reaction. 10.  What T-cell receptor kits would you recommend to detect T-cell clonal rearrangements? Ideally, you should perform tests for TRB, TRG, and TRD to achieve the highest sensitivity. The EuroClonality/BIOMED-2 Group has shown that testing both TRB and TRG offers roughly the same sensitivity for the detection of T-cell malignancies as testing all three targets; however, they highly recommend testing all three assays in parallel to achieve optimal clinical sensitivity. TRD is especially useful in cases of suspected immature T-cell proliferations (see Figure 2 and Table 2 in Leukemia (2007) 21, 201-206). We also offer NGS kits for TRG for use with MiSeq® or Ion PGM™ instruments and for TRB for use with MiSeq® . 11.  What are the differences between the TCRG Gene Clonality Assay and the T-Cell Receptor Gamma Gene Rearrangement Assay 2.0? The TCRG Gene Clonality Assay was designed by the EuroClonality/ BIOMED-2 Group and consists of two master mixes. For polyclonal populations, four Gaussian distributions are Hematologic Malignancy Research and Testing Guide References 1.  Kakizuka, A et al., Cell (1991) 66:675-684. 2. Rimokh, R et al., Blood (1994) 83:1871-1875. 3. Gribben, JG et al., Blood (1994) 83:3800-3807. 4. Radich, JP et al., Leukemia (1994) 8:1688-1695. 5. Gallagher, RE et al., Blood (1995) 86:1540-1547. 6. De Boer, CJ et al., Blood (1995) 86:2715-2723.  7. Mrózek K, Döhner H, and Bloomfield CD. Curr Opin Hematol. (2007) 14:106-114. 8. Rezuke, WN et al., Clinical Chemistry (1997) 43:1814- 1823. 9. Deininger, MW et al., Blood (2000) 96:3343-56. 10. Schlenk RF et al., N Engl J Med. (2008) 358:1909-1918. 11. Gonzalez, D et al., Leukemia (2003) 17:1051-1057. 12. Gonzalez, D et al., Leukemia (2003) 17:1398-1403. 13. Ghia, P et al., Leukemia (2007) 21:1-3. 14. van Krieken, JH et al., Leukemia (2007) 21:201-6. 15. Evans, PAS et al., Leukemia (2007) 21:207-214. 16. Bruggemann, M et al., Leukemia (2007) 21:215-221. 17. Davi, F et al., Leukemia (2008) 22:212-214. 18. Döhner, H et al., Blood (2010) 115:453-474. 19. Huang, W et al., Blood (1993) 82:1264-1269. 20. Hughes, T et al., Blood (2006) 108:28-37. 21. Westbrook, CA et al., Blood (1992) 80:2983-2990. 22. Ralph, QM et al., Blood (1993) 82:202-206. IGH (VH - JH) IGH (DH - JH) IGK IGL IGHV Somatic Hypermutation TRB TRD TRG IGH- CCND1 IGH - BCL2 BCR- ABL1 PML- RARA FLT3 NPM1 Acute Myeloid Leukemia7,10 m m Acute Promyelocytic Leukemia (AML-M3)1,5 m Chronic Myeloid Leukemia9 m Acute Lymphoblastic Leukemia4 m m m u m m m m Chronic Lymphocytic Leukemia13,17 m u m u m u u u Multiple Myeloma11,12 u T-Cell Large Granular Lymphocytic Leukemia14,16 u u m u m T-Cell Prolymphocytic Leukemia14,16 u u u u m u m Anaplastic Large-Cell Lymphoma14,16 m u m Angioimmunoblastic T-Cell Lymphoma14,16 u u u u m u m Diffuse Large B-Cell Lymphoma3,8,14,15 m u m u u u u m Follicular Lymphoma3,8,14,15 m u m u u u u m Mantle Cell Lymphoma2,6,14,15 m u m u u u u m Marginal Zone Lymphoma14,15 m u m u u u u Peripheral T-Cell Lymphoma14,16 u u u m u m Small Lymphocytic Lymphoma13,17 m u m u u u u Suspected B-Cell Proliferations14 m m m u u u u Suspected T-Cell Proliferations14 u u u u m u m Suspected Lymphoid Proliferations of Unknown Origin14 m m m u m u m mRecommended Primary Test uRecommended Secondary Test To receive a complimentary copy of this guide as a wall chart, e-mail marketing@invivoscribe.com.