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CAT #: 7100004
LymphoTrack® Flex IGHV Leader Assay
Assay Use
The research use only (RUO) LymphoTrack Flex Assay portfolio is designed to identify rearranged gene sequences in human genomic DNA (gDNA) from peripheral blood, bone marrow and FFPE specimens. These assays provide reagents to generate sequencer-ready IGH FR1, IGHV Leader and/or TRG libraries for the Illumina® NextSeq™1000 System.
Product Details
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Summary and Explanation of the Test
Background
Identifying clonality and determining the frequency of immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements are crucial in understanding lymphoproliferative diseases.1,7 These gene rearrangements serve as markers for clonal lymphocyte populations, aiding in the detection of blood cancers.2During normal lymphocyte development, Ig and TCR genes undergo rearrangement to generate diverse antigen receptors.1 Conversely, in lymphoid malignancies, such as B-cell and T-cell lymphomas, a single clone of lymphocytes proliferates uncontrollably, all sharing the same unique Ig or TCR gene rearrangement.3,4 Detecting these clonal rearrangements is essential for confirming malignancy.5
Traditional methods, such as PCR and Sanger sequencing, have been employed to detect Ig and TCR gene rearrangements for several decades.6 However, the introduction of next-generation sequencing (NGS) has enhanced sensitivity and specificity in clonality assessment, allowing a comprehensive analysis of Ig and TCR gene rearrangements, facilitating the detection of clonal populations even in samples with low tumor cell content. Recent improvements in NGS clonality assays includes providing molecular genetic evidence of clonality in malignant or suspect lymphoproliferative disorders and facilitating minimal residual disease (MRD) assessment.7,9
Summary
The LymphoTrack Flex Assays for hemato-oncology research represent highly sensitive, scalable, NGS library prep reagents that identify gene rearrangement diversity and clonality. Designed to be used with the Illumina NextSeq1000 System to provide flexibility and workflow efficiencies for high-throughput laboratories, LymphoTrack Flex Assay is also optimized for additional applications (such as MRD with DNA input volume flexibility) as well as automated laboratory environments.LymphoTrack Flex Assay kits include PCR master mixes (PCR1 for target amplification and PCR2 to add index primers), positive and negative controls to process a total of 96 samples and controls. The supplementary Linux-based LymphoTrack Enterprise Software analysis generates tabulated output files for sample interpretation that can be easily transferred into a LIMS for automated reporting.
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Principles of the Procedure
LymphoTrack Flex Assays follow a basic NGS workflow in which high-quality genomic DNA (gDNA) is used as the assay input. The gDNA template is amplified via PCR using target-specific primers (PCR1), undergoes purification, then Illumina sequencing adapters and unique dual indices (UDIs) are added to the target amplicons (PCR2). The purified, target-specific, UDI-labelled amplicons are then pooled into Single-Target Libraries, which are then purified and quantified. The Single-Target Libraries are then diluted and combined into a Final Library, which is sequenced on an Illumina NextSeq1000 System. After the sequencing run is complete, LymphoTrack Enterprise Software is used to analyze the resulting data.
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Specimen Requirements
- This assay tests extracted and purified gDNA
- gDNA must be quantified with a method specific for double-stranded DNA (dsDNA) and be free of PCR inhibitors
- The recommended amount of gDNA input for this assay is 200 ng (minimum of 50 ng)
References
1. Tonegawa, S. Somatic generation of antibody diversity. Nature. 1983 Apr;302: 575 -581.
2. Teramo A, Binatti A, Ciabatti E, Schiavoni G, Tarrini G, Barilà G, Calabretto G, Vicenzetto C, Gasparini VR, Facco M, Petrini I, Grossi R, Pisanti N, Bortoluzzi S, Falini B, Tiacci E, Galimberti S, Semenzato G, Zambello R. Defining TCRγδ lymphoproliferative disorders by combined immunophenotypic and molecular evaluation. Nat Commun. 2022 Jun 8;13(1):3298.
3. Logan AC, Vashi N, Faham M, Carlton V, Kong K, Buño I, Zheng J, Moorhead M, Klinger M, Zhang B, Waqar A, Zehnder JL, Miklos DB. Immunoglobulin and T cell receptor gene high-throughput sequencing quantifies minimal residual disease in acute lymphoblastic leukemia and predicts post-transplantation relapse and survival. Biol Blood Marrow Transplant. 2014 Sep;20(9):1307-13.
4. Crane, GM & Loghavi, S (Eds). Precision molecular pathology of aggressive B-cell lymphomas. Springer International Publishing AG, 2024.
5. Rosenquist R, Ghia P, Hadzidimitriou A, Sutton LA, Agathangelidis A, Baliakas P, Darzentas N, Giudicelli V, Lefranc MP, Langerak AW, Belessi C, Davi F, Stamatopoulos K. Immunoglobulin gene sequence analysis in chronic lymphocytic leukemia: updated ERIC recommendations. Leukemia. 2017 Jul;31(7):1477-1481.
6. Langerak, T. (2014 February 11). Complex IG / TCR Clonality Targets: Patterns and Interpretation. 17th Euroclonality Workshop. Nijmegen, Netherlands.
7. Groenen PJTA, van den Brand M, Kroeze LI, Amir AL, Hebeda, K. Read the clonotype: Next-generation sequencing-based lymphocyte clonality analysis and perspectives for application in pathology. Front Oncol. 2023 Feb 07(13) 2234-2243.
8. Montserrat E. Treatment of Chronic Lymphocytic Leukemia: Achieving Minimal Residual Disease -Negative Status As a Goal. J Clin Oncol. 2005 May 1;23(13), pp. 2884 -2885.
9. Faham M, Zheng J, Moorhead M, Carlton VE, Stow P, Coustan-Smith E, Pui CH, Campana D. Deep-sequencing approach for minimal residual disease detection in acute lymphoblastic leukemia. Blood. 2012 Dec 20;120(26):5173-80.
Disclaimer
This product is for RESEARCH USE ONLY; not intended for diagnostic or therapeutic use.
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