Table ofContents

2

Introduction

03 Table of Contents
05 Letter to Our Colleagues
06 Board of Directors
08 Executive Leadership
10 Leadership Team
12 Best-in-Class Assays and Reagents
14 LabPMM Clinical Lab Services
16 Streamlined CDx Partnerships
18 Global Distribution Network
20 Global Invivoscribe Locations

Clonality Overview

22 Background
24 Test Algorithm for Suspect Lymphoproliferations

Hematological Test Menu
26 Hematological Test Menu

Next Generation Sequencing (NGS)
28 LymphoTrack® Dx (CE-IVD) Assays
30 LymphoTrack Dx IGHV Leader Somatic Hypermutation Assay
32 LymphoTrack Dx IGH FR1/2/3 Assays
36 LymphoTrack Dx IGK Assays
38 LymphoTrack Dx TRG Assays
40 LymphoTrack Dx TRB Assays
42 LymphoTrack Dx Bioinformatics Software

85 LeukoStrat® CDx Mutation Assays
87 LeukoStrat CDx FLT3 Mutation Assay (IVD) - USA
89 LeukoStrat CDx FLT3 Mutation Assay (CE-marked)
91 LeukoStrat CDx FLT3 Mutation Assay (CE 2797 IVD)
93 LeukoStrat CDx FLT3 Mutation Assay - Japan

Gel and Capillary (RUO) Assays

97 IGH + IGK B-Cell Clonality Assays
99 IGH Gene Rearrangement Assays
101 IGH Gene Clonality Assays
103 IGK Gene Clonality Assays
105 IGL Gene Clonality Assays
107 TCRB Gene Clonality Assays
109 T-Cell Receptor Gamma Gene Rearrangement Assay 2.0
111 T-Cell Receptor Gamma Gene Rearrangement Assays
113 TCRD Gene Clonality Assays
115 BCL1/JH Translocation Assay
117 BCL2/JH t(14;18) Translocation Assay
119 BCL2/JH Translocation Assay
121 BCR/ABL t(9;22) Translocation Assays
123 PML/RARα t(15;17) Translocation Assays
125 IGH Somatic Hypermutation Assays v2.0

FLT3 Mutation (RUO) Assays
127 FLT3 Mutation Assays

Analyte Specific Reagents

129 Analyte Specific Reagents

44 LymphoTrack® (RUO) Assays

Controls, Reagents, and Enzymes

46 LymphoTrack IGHV Leader Somatic Hypermutation Assay
48 LymphoTrack IGH FR1/FR2/FR3 Assays
52 LymphoTrack IGK Assays
54 LymphoTrack TRG Assays
56 LymphoTrack TRB Assays
58 LymphoTrack Bioinformatics Software
60 LymphoTrack Enterprise Software - MiSeqTM

133 DNA Controls
136 LymphoTrack Low Positive Controls
136 LymphoQuant® Internal Controls
137 RNA Controls
140 RNA Sensitivity Panels
141 Master Mix Controls
141 Enzymes

61 MRD Solutions (RUO)
63 LymphoTrack MRD Software
65 FLT3 ITD MRD Assay
67 NPM1 MRD Assay

Capillary (CE-IVD) Assays
69 IdentiClone (CE-IVD) Assays®

71 IdentiClone IGH + IGK B-Cell Clonality Assays
73 IdentiClone IGH Gene Clonality Assays
75 IdentiClone IGK Gene Clonality Assays
77 IdentiClone IGL Gene Clonality Assays
79 IdentiClone TCRB Gene Clonality Assays
81 IdentiClone T-Cell Receptor Gamma Gene Rearrangement Assay 2.0
83 IdentiClone TCRD Gene Clonality Assays

Custom Products
142 Custom Products

Reference

144 Clonality Testing Workflow Methods
146 Illumina® MiSeq™ Workflow Summary
147 Thermo Fisher Scientific® Ion S5™ PGM™ Workflow Summary
148 Next-Generation Sequencing Menu
149 Gel and Capillary Electrophoresis Menu
150 Common Technical Support Questions

Product List

152 Product List by Catalog Number

Table of Contents 3

Improving Lives
with Precision®
Diagnostics
A technology-driven international
company focused on precision
diagnostics with best-in-class products
and services that optimize patientcare and accelerate drug approvals
worldwide.
LEARN MORE:
invivoscribe.com/

4Dear Colleagues,
As we begin 2024, I would like to take this opportunity to extend my heartfelt gratitude to our exceptional team, our valued customers, and our esteemed partners for the accomplishmentsof 2023. It gives me immense pleasure to highlight just some of the many significant milestones we achieved together in 2023, outline the myriad of projects we will advance and complete
this year, and lay out our vision for the future direction of the company. Let’s begin with last year’s accomplishments:
Regulatory Approvals:• We were the second company globally to receive IVDR approval for a companion diagnostic in the EU. Approval of our LeukoStrat® CDx FLT3 Mutation Assay also paved the way for
additional submissions and fueled our team’s drive to complete IVDR validation packages for several other of our molecular diagnostic assays.
• We obtained U.S. Food and Drug Administration (FDA) approval for LeukoStrat® FLT3 CDx Assay to select patients for VANFLYTA treatment in the US, and received updated
reimbursement for newly diagnosed AML patients for the Daiichi Sankyo drug, VANFLYTA, in Japan.
Exciting Collaborations:• Our collaboration with Foundation for the National Institutes of Health (fNIH) Biomarkers Consortium, working with Dr. Chris Hourigan’s lab at the NIH, proved to be groundbreaking
in quantifying and standardizing measurable or minimal residual disease (MRD) as a predictor of relapse for patients with acute myeloid leukemia (AML). The potential clinical value of
our FLT3 ITD MRD assay kits was showcased in an abstract (https://ash.confex.com/ash/2023/webprogram/Paper178839.html) presented at the annual ASH meeting and a forthcoming
publication in JAMA Oncology. We are following this study testing with our internationally standardized NPM1 MRD Assay, confident that this productive partnership will bring further
advancements and benefits to AML patients worldwide.
• Dr. Mark Levis, at Johns Hopkins, also highlighted in his abstract and talk at ASH, the clinical significance of tracking residual disease in patients using the same next-generation
sequencing (NGS) based FLT3 ITD MRD Assay study, demonstrating a tight correlation between log reductions in residual disease and concomitant increase in patient’s overall survival(OS). Notably, there was clean separation and an increase in OS for each log reduction in residual disease as measured with our FLT3 ITD MRD Assay, which is now available both as a
research-use-only (RUO) kit and as a service in our LabPMM centers worldwide.
• Our study collaboration with Dr. Maria Arcila at Memorial Sloan Kettering Cancer Center resulted in a publication highlighting the performance of our LymphoTrack® assays used
to identify clonotypes and track MRD in a range of hematologic malignancies (https://pubmed.ncbi.nlm.nih.gov/38103591/). From the abstract: “Assays performed both at internaland external laboratories showed highly concordant MRD detection (100%) and quantitation (R = 0.97). Overall, this NGS-based MRD assay showed highly reproducible results withquantitation that correlated well with Flow Cytometry MRD assessment, particularly for B-cell neoplasms.”
Launch of New Products/Services:• To fulfill an unmet need for international MRD Assays in AML, we launched two NGS-based MRD (RUO) Assays. Our FLT3 ITD MRD Assay (RUO) with Dockerized FLT3 ITD MRD software(RUO), and NPM1 MRD Assay (RUO) with NPM1 MRD software (RUO). These assays, complemented with Dockerized software, enable high-throughput laboratories to efficiently validate,automate, and streamline their testing and data analyses. Additionally, we introduced a Dockerized version of the LymphoTrack® Enterprise Software (RUO), designed to seamlessly
integrate with our LymphoTrack clonality assays. These products facilitate fully automated testing and data management pipelines, particularly beneficial for large laboratories.
• Our LymphoTrack® RUO assays identify and track the entire spectrum of lymphoid malignancies with sensitivity as low as 10-6 when sufficient cell equivalents are tested. A new
NGS-based service with unprecedented sensitivity, the B-cell MRD Assay, is now available and being used across the LabPMM global network to monitor therapeutic response, and weenvision this test eventually being used to serve as a surrogate endpoint in clinical trials.
• Building on the successful launch of our CAP/CLIA validated multiparametric flow cytometry (MFC) AML MRD Assay on the 12-Color BD FACS Lyric platform, which received great
reviews from our pharma and clinical trial partners, we launched a new CAP/CLIA validated MFC CLL MRD Assay. Our new MFC B-ALL MRD assay will be fully validated in early 2024,
followed by a MFC Multiple Myeloma MRD assay. These highly sensitive and internationally standardized flow assays, using stable dried-down cocktail reagents, will allow us to realize
the promise of performing both flow-based, and NGS-based MRD testing on the same pull of bone marrow and/or peripheral blood specimens for pharma partners conducting clinical
trials anywhere in the world.
Other notable achievements include:• CAP accreditation for our clinical laboratory in Shanghai, contributing to clinical trial testing for key pharmaceutical companies for China studies;• A strategic partnership with Complete Genomics to develop a combination assay for identifying and tracking the most prevalent and clinically actionable MRD markers in AML patients.
Looking ahead, we are focused on further expanding and aligning our services globally. We are actively transferring and validating our service offerings of NGS tests, gene panels,
bioinformatics, multiparametric flow cytometry assays, and MRD applications through our network of fully accredited laboratories in San Diego, Japan, China, and Germany to support
clinical and pharma partners worldwide.
Our commitment to advancing medical research is evident in initiatives like our Biobank Repository. We updated and have made available to partners viably frozen aliquots of some
50,000 AML discarded and anonymized patient specimens, as well as characterized specimens representing other hematologic malignancies. This allows us to assist the clinicalcommunity in more quickly evaluating and validating drugs and diagnostic tests worldwide.
We also initiated work on our MarkerMatch™. This product, immediately connects the subscribing pharma partners with the physician and cancer center location of patients who are
eligible for their clinical trials when identified at any one of 700 participating international clinical testing sites that use our standardized biomarker assays. Our goal is to assist our clinical
trial partners seeking to expand enrollment in their international clinical trials, while maintaining patient confidentiality.
Your support and collaboration have been fundamental to our achievements over the past 30 years. As we embark on another year, we are excited about the prospect of continued
collaboration, innovation, and providing cutting-edge products and services.
We wish you, your colleagues, and your families a safe, joyful, productive, and successful 2024.

Sincerely Yours,

Jeffrey Edward Miller, Ph.D.
Founder, Chief Scientific Officer, Chief Executive Officer & Chairman

CEO Letter 5

Board of Directors

LEARN MORE:
invivoscribe.com/company/board-of-directors/

6

Jeffrey Edward Miller, Ph.D.
Dr. Miller is a scientist, inventor, and entrepreneur focused on Improving Lives with Precision Diagnostics® – coupling drug trials and therapeutic
treatment regimens with optimized clinically-actionable diagnostic methods in order to select the correct patients and then monitor and track their
response throughout the course of their disease. He received his undergraduate degree in Biochemistry from UCLA and a combined Ph.D.
in Biochemistry & Molecular Biology from UCSB. Prior to starting Invivoscribe, Dr. Miller had more than twenty years of combined experience in protein
biochemistry, cellular and molecular immunology, cardiac physiology, virology, and molecular biology, experience he had developed working
in laboratories at the Medical School, Department of Chemistry, Molecular Biology Institute at UCLA, while earning his Ph.D. at UCSB, andas a postdoctoral scientist at Applied Molecular Evolution. He also spent several years at Quest Diagnostics Nichols Institute, setting up the molecular
oncology laboratory and developing and launching PCR-based molecular assays for infectious disease and hematopathology.

James Isaacs, Jr., JD.
James B. Isaacs, Jr. has practiced law since 1983. He currently serves as Licensing and Contracts Counsel at Invivoscribe. Jim attended Stanford
University and Yale Law School, then began his career at the Los Angeles law firm of O’Melveny & Myers. As a trial lawyer and later in-house counsel
with a focus on intellectual property disputes, Jim has successfully defended a myriad of businesses and individuals; in plaintiffs’ actions he has
obtained and collected multi-million dollar judgments in the United States and abroad. As a businessman and co-founder of Invivoscribe, Jim has been
active in the legal and commercial affairs of the company since 1995.

Gary Clouse, JD.
Gary Clouse has practiced as a litigator and business attorney in Southern California for more than three decades. He currently serves as Corporate
Secretary and Legal Counsel for Special Projects at Invivoscribe. Clouse is a graduate of Indiana State University and Northwestern University Schoolof Law. Following law school, he clerked for the federal Seventh Circuit Court of Appeals in Chicago. He began his legal practice at the law firm
O’Melveny & Myers in Los Angeles. Clouse is one of the founders of invivoscribe.

Mitchell Kronenberg, Ph.D.
Dr. Kronenberg received a B.A. from Columbia University, a Ph.D. from the California Institute of Technology, and served on the faculty of the UCLA
School of Medicine from 1986-1997. He joined the La Jolla Institute for Immunology in 1997, and currently is a Professor, the President Emeritus anda member of the institute Board of Directors. Dr. Kronenberg was the President of the institute from 2003 to 2021 and Chief Scientific Officer from 20212023. The Institute has grown in accomplishment and reputation under his leadership. Dr. Kronenberg's research interests include natural killer T cells,
other innate lymphocytes such as MAIT cells and ILC, regulation of mucosal immunology and the pathogenesis of inflammatory bowel disease. He has
co-authored more than 400 publications, and is a fellow of the American Association for the Advancement of Science (AAAS), a Distinguished Fellowof the American Association of Immunologists, recipient of an NIH MERIT award and is an Institute for Scientific Information (ISI) Highly Cited Scientist.
In 2016, he was named the most admired CEO (large nonprofit organization category) by the San Diego Business Journal. He is an advisor to a numberof for-profit and nonprofit organizations, including service as a member of the Board of Scientific Counselors for Basic Science, National Cancer
Institute. He has been involved with Invivoscribe since its founding.

Stephen Wilson, Ph.D.
Dr. Wilson is an immunologist and biotech entrepreneur with more than 30 years of experience in biomedical research, development and executive
management. He earned his Ph.D. from the University of Arizona’s College of Medicine and joined the La Jolla Institute for Immunology (LJI) in 1997
where he was a fellow of the National Multiple Sclerosis Society and National Institutes of Health. Dr. Wilson entered management at LJI, eventuallyserving as LJI’s Chief Operating Officer. During his tenure the Institute grew substantially, becoming a global leader in immunology, conducting nearly$1B in total research operations and ranking as the #1 place to work in the world by The Scientist magazine. His original research has been publishedin high impact journals. Dr. Wilson has served as principal or co-principal investigator on more than $75M in competitive grants and awards, including
the development of the 2021 X-Prize winning rapid CoVID-19 diagnostic test. Currently, Dr. Wilson is serving as the Chief Executive Officer of Foundation
Immune Engineering’s Botnar Institute.

Hardwick Simmons, MBA
Hardwick ‘Wick’ Simmons retired as the Chairman and CEO of The Nasdaq Stock Market, Inc. in May of 2003. Prior to Nasdaq, he served
as President and CEO of Prudential Securities Inc., a major investment management and securities brokerage firm. Simmons is a former chairmanof the Securities Industry Association, a former director of the Chicago Board Options Exchange and a former president of the New York Bond Club.
He is currently a director of Lionsgate Entertainment Corp., president of Stonetex Oil Corp., and a trustee of Woods Hole Oceanographic Institution.
He is a graduate of Harvard College and Harvard Business School and served in the U.S. Marine Corps Reserve from 1960 until 1966.

Board of Directors 7

Executive Leadership

LEARN MORE:
invivoscribe.com/company/leadership-team/8

Jeffrey Edward Miller, Ph.D.
Founder, Chief Scientific Officer, and Chief Executive Officer
Dr. Miller is a scientist, inventor, and entrepreneur focused on Improving Lives with Precision Diagnostics® – coupling drug trials and therapeutic
treatment regimens with optimized clinically-actionable diagnostic methods in order to select the correct patients and then monitor and track their
response throughout the course of their disease. He received his undergraduate degree in Biochemistry from UCLA and a combined Ph.D.
in Biochemistry & Molecular Biology from UCSB. Prior to starting Invivoscribe, Dr. Miller had more than twenty years of combined experiencein protein biochemistry, cellular and molecular immunology, cardiac physiology, virology, and molecular biology, experience he had developed
working in laboratories at the Medical School, Department of Chemistry, Molecular Biology Institute at UCLA, while earning his Ph.D. at UCSB, andas a postdoctoral scientist at Applied Molecular Evolution. He also spent several years at Quest Diagnostics Nichols Institute, setting up the molecular
oncology laboratory and developing and launching PCR-based molecular assays for infectious disease and hematopathology.

Meghna Bhatnagar, MBA
Chief Financial Officer
Meghna Bhatnagar joined Invivoscribe in 2010 as Chief Financial Officer. In this role she is responsible for leading the Invivoscribe global financeorganization, along with human resources and information technology. Since her arrival, Ms. Bhatnagar has played an integral role in directing
all aspects of company strategy, planning and operations. Ms. Bhatnagar has over 20 years of experience building and leading finance and IT teams
in global companies. Prior to joining IVS, she served as COO of Radiant Technologies, a technology company focused on providing business
management solutions to small and medium sized companies where she was responsible for leadership and development of an entire project delivery
team with full P&L responsibility. She played a key role in guiding overall strategy of the company and at the same time provided leadership for
operational improvements.

Dr. Bradley Patay, M.D.
Chief Medical Officer
Dr. Patay is dedicated to improving health by integrating genomic knowledge into medical care. He has authored numerous articles in this field,
presented at multiple conferences and has been featured in Bloomberg Business Week. He has been head of the internal medicine section at Scripps
Torrey Pines, worked as an Assistant Professor at STSI, and has been a founding member of the Board, and Vice President of the College of Genomic
Medicine, which was established in 2010 to educate physicians and other health care professionals about genomic medicine. His diverse clinicalexperience prior to joining Scripps Clinic in 2005 includes four years as an internist and pediatrician at Neighborhood Healthcare, a private, nonprofit
community healthcare practice, and at Palomar Hospital. At these institutions, Brad cared for a wide range of patients, from neonates to the elderly,
in both intensive care and the general wards. Through his service on several committees, he helped improve health care institutions’ systems.

Tania Obranovich, BSc (Hons) PhD LLB
Chief IP and Legal Strategy Officer
Dr. Obranovich has more than 25 years’ experience developing and managing global IP portfolios in the biotechnology sector, in particular in relation to molecular, cellular and genomic technologies as they relate to diagnostics and therapeutics. She has acquired this experience both through private practice,having spent more than a decade as a partner in a top tier IP firm, and as legal counsel for a US based diagnostics company where she ran patent and trade markprosecution and drafting, in addition to overseeing all legal, compliance and corporate governance matters. Tania has practiced throughout her career as botha lawyer and patent attorney. Prior to these roles she completed a PhD and post-doctoral fellowship in immunology. In addition to her legal practice, Tania has
lectured extensively in patent law at The University of Melbourne and Monash University and also speaks widely in relation to IP issues. She was heavily involved withnavigating the Australian and US legal and policy issues that arose as a result of the Myriad gene patent litigation in both these jurisdictions. Tania received
a Federal Government appointment to the Professional Standards Board for Patent and TM Attorneys in 2012 and has held several other Board positions. Tania hasserved as Chief IP and Legal Strategy Officer to Invivoscribe since 2019.

Dr. Meindert Niemeijer, Ph.D., M.Sc.
Chief Information Officer
Dr. Niemeijer came to IVS from Digital Diagnostics where he led the teams that designed, built, documented and put into production the firstautonomous diagnostic Artificial Intelligence (AI), cleared by the FDA. He is an experienced technology leader and deep learning expert passionate
about developing AI based medical devices. During his career, he has built a deep expertise in medical device (software) development processes andthe way in which these interact with quality management and other regulatory requirements. He loves building and developing technology, processes
and teams. Dr. Niemeijer obtained a Ph.D. in Medical Image Analysis and Machine Learning from Utrecht University in the Netherlands. In addition,
he has an M.Sc. in Medical Computer Science, also from Utrecht University.

Andreia de Albuquerque, Ph.D.
Chief Commercial Officer
Dr. de Albuquerque joined Invivoscribe in 2015 and currently serves as the Director of Business Development. In this role, she is responsible forleading Invivoscribe’s global partnerships with key Pharma and Biotech companies, focusing on companion diagnostics development, custom assay
development, clinical trial testing, corporate partnerships and other strategic relationships. Prior to joining Invivoscribe, Dr. de Albuquerque held roles
in precision molecular diagnostics, focusing on development, implementation and validation of PCR and NGS assays for diagnostic and research
purposes. Her work has resulted in several peer-reviewed publications and participations in international conferences. During her career, she has attimes been responsible for overseeing laboratory diagnostic teams and operations. Dr. de Albuquerque's extensive expertise in precision diagnostics
and understanding of assay development and clinical testing, paved the way into Business Development, where she now focuses on accelerating
drug approvals. Her approach is to build successful partnerships, provide technical expertise and work closely with biomarker leaders, lab operations,clinical scientists and regulatory groups.
Dr. de Albuquerque earned her degree in Biology from the University of Coimbra (Portugal) and a combined Ph.D. in Oncology and Molecular Biology
from the University of Dresden (Germany).

Executive Leadership 9

Leadership TeamLEARN MORE:
invivoscribe.com/company/leadership-team/10

Jordan Thornes
V.P., Global Clinical Laboratory Operations
Jordan Thornes joined Invivoscribe in 2004 and currently serves as the Global Director of Clinical Labs. In this role, he is responsible for leading
Invivoscribe’s global network of clinical labs, The Laboratory for Personalized Molecular Medicine®, (LabPMM®). There are currently four such
LabPMM facilities which are strategically located globally in San Diego, USA; Hallbergmoos (Munich), Germany; Shanghai, China and Kawasaki(Tokyo), Japan. LabPMM aids patients and pharmaceutical companies worldwide with globally standardized and clinically-actionable diagnostic
methods. LabPMM utilizes proprietary biomarkers to select the correct patients and monitor and track their critically important response throughout
the course of their disease.

Ying Huang, Ph.D.
V.P., Product Development
Dr. Huang joined Invivoscribe in 2010 and currently serves as the Sr. Director of Product Development. In this role, she is responsible for leading
Invivoscribe’s product development activities to address unmet clinical needs in the fields of molecular diagnostic and precision medicine. Dr. Huang
has over 25 years of research and industry experience with a broad knowledge in technology and assay development. She is passionate aboutdeveloping novel methods using cutting edge technologies to improve lives with precision diagnostics. She has successfully led development teams
through entire product development life cycle, from feasibility to RUO, CLIA and IVD grade product launches and post market support under respective
regulatory standards. Prior to Invivoscribe, Dr. Huang worked at Illumina, ACEA Biosciences, Nanogen and MD Anderson Cancer Center. In thoseroles, she developed a variety of techniques ranging from isolating CTC using microfluidic devices to detecting biowarfare agents using electronic
microarray. Dr. Huang was the PI for research grants from DARPA and NIH. She is the co-author of more than 60 publications and meeting abstracts
and is the co-inventor of 9 patents. Dr. Huang received her Ph.D. in Electrical Engineering from Bangor University, UK and B.E. in Electronic Engineeringfrom Xidian University, China.

Jason Gerhold
V.P., Global Quality, Regulatory, and Clinical Affairs
Jason Gerhold joined Invivoscribe in 2012 and serves as the Global Director of Regulatory, Quality, and Clinical Affairs focusing on developing highquality diagnostics that meet both international pathology needs and support pharma partner’s drug development programs. He directly manages
employees in the US, Japan, and China, and is responsible for worldwide compliance to relevant laws, regulations, and standards. Mr. Gerhold
has over 20 years of experience in the biotech industry with biologic therapeutics and diagnostics, including process development, regulatory,
quality, and clinical roles. Mr. Gerhold has worked in small, medium and large companies; successfully registering IVD and CDx products in several
international markets, developing, improving, and maintaining quality management systems while serving as the management representative
to regulatory authorities, and leading clinical teams managing international registrational studies. He is known for building strong rapport withregulators and working well with research and pharma teams resulting in highly successful partnerships. He has completed trainings and/or
accreditations for ISO 13485 Lead Auditor, Six Sigma Greenbelt, Clinical Trials Administration, Project Management, and CTD writing.

Leadership Team 11

Best-in-Class
Assays andReagents
Invivoscribe® provides a full rangeof standardized CE-marked invitro diagnostic cGMP products forhematology-oncology, as well as RUO
assays, analyte specific reagents (ASRs),
and DNA & RNA controls.
LEARN MORE:
invivoscribe.com/products/

1212Next-Generation
Sequencing (NGS)NGS is a powerful, high-throughput DNA sequencing technology that allows for massively parallel sequencingof millions of DNA fragments in a single sequencing run. Our NGS assays provide comprehensive solutions fromdiagnosis through Measurable Residual Disease tracking.

ABI Fluorescence Detection
and Gel Detection
We exclusively offer a comprehensive selection of PCR-based assays for ABI fluorescence detection and gel detection,
including targeted FLT3 ITD and TKD mutation assays, B- and T-cell clonality assays (based on EuroClonality/BIOMED-2
Concerted Action BMH4-CT98-3936), and translocation assays.

Controls and Reagents
Invivoscribe® offers an extensive range of General Purpose Reagents (GPRs) and Research Use Only (RUO) reagents.
Our high quality, reliable DNA and RNA controls, master mixes, and enzymes, are all manufactured under cGMP
conditions to guarantee reliability.

Companion Diagnostics (CDx)
Streamline drug approvals leveraging our Companion Diagnostic (CDx) assay development, manufacturing andregulatory expertise to accelerate drug approvals. Custom assays are designed and manufactured under an ISO
certified quality management system.

Best-in-Class Assays and Reagents 13

LabPMM
Clinical Lab
Services®

Invivoscribe’s international networkof clinical labs, Laboratory for
Personalized Molecular Medicine®
(LabPMM®), provides globallystandardized testing of novel andproprietary biomarkers that are
critical for patient care.
LEARN MORE:
invivoscribe.com/clinical-lab-services/

14

NGS Cancer Panels
Identification of chromosome abnormalities and actionable driver mutations aid in the stratification of patient risk, and provide critical insight forpatients with leukemia or lymphoma.

NGS MRD Tests
Track disease and trend treatment response by monitoring clonal B- (Ig) and T-cell (TCR) gene rearrangements as well as FLT3 ITD and NPM1
mutations with unprecedented sensitivity.

Clonality Tests
Malignant lymphoproliferative disorders collectively represent a large portion of hematological cancers and are characterized by reduced population
diversity of Ig and TCR rearrangements. Confirm diagnostic suspicions and identify hematological neoplasms with LabPMM’s NGS Clonality services,available for Ig, TCR, and somatic hypermutation (SHM).

Companion Diagnostic (CDx) Tests
CDx enable the identification of biomarkers that indicate the patients most likely to benefit (or alternately not benefit) from specific or combinatorial
targeted therapies. Their utilization enhances patient care and the potential for improved outcomes. The fms related tyrosine kinase 3 (FLT3) is oneof the most commonly mutated genes in acute myeloid leukemia (AML), occurring in approximately 30% of patients at the time of diagnosis.1 Althoughgenerally associated with normal cytogenetics where patients have standard risk of relapse, FLT3 mutations have also been identified in sub-groups ofpatients with chromosomal abnormalities that are associated with high risk of disease relapse.2-3 The LeukoStrat CDx FLT3 Mutation Assay is a globally
standardized test validated for the detection of internal tandem duplications (ITD) and tyrosine kinase domain (TKD) mutations of the FLT3 gene.

Custom Assays
Invivoscribe's experienced regulatory team has over 50 product registrations and multiple companion diagnostic approvals, allowing for optimal
strategy, streamlined development and milestone achievement. With an ever-changing regulatory landscape, why take a chance with anyone else?

Targeted Genes
FLT3 and NPM1 are offered to detect targeted mutations. FLT3 and NPM1 mutations can be used for risk stratification and prognosis of AML.

Multiparametric Flow Cytometry
Complement NGS testing with globally-standardized, 12-color flow cytometry testing. Our comprehensive assay designs allow greater specificity
and sensitivity using smaller sample volumes as compared to 4- or 6-fluorochrome tests. Some of the many MFC applications include leukemia andlymphoma diagnosis and screening and rare event analyses such as MRD.

References
1. Acute Myeloid Leukemia, Clinical Practice Guidelines in Oncology, (v.2.2014)
National Comprehensive Cancer Network.
2. Lowenberg, B. et al. (1999) “Acute myeloid leukemia.” N Engl J Med 341(14):1051-62.
3. Thiede, C. et al. (2002) “Analysis of FLT3-activating mutations in 979 patientswith acute myelogenous leukemia: association with FAB and identificationof subgroups with poor prognosis.” Blood 99(12): 4326-35.

LabPMM® Clinical Lab Services 15

Streamlined
CDx Partnerships
Companion Diagnostics representsa critical milestone in precision
medicine and plays a pivotal rolein the validation of targeted therapies.
Use of a single partner for CDx
development minimizes complexity andrisk which could affect drug approvals.
Our Streamlined CDxTM programsupports all stages of development,
validation, regulatory submission, andcommercialization. Let us help you
accelerate your drug approval.
LEARN MORE:
invivoscribe.com/partnerships/

16

Product Development
Design Controls: All biomarker assays & software developed under design controls.
Bioinformatics Software: Comprehensive solutions to prevent errors, streamline workflow and reduce turn-around times.
Identify and Track: NGS panels and targeted assays to identify clonal architecture and track disease burden.
Custom Development: Custom biomarker assay development.
Complementary MRD Assays: MRD assays may be used as surrogate endpoints for P3 clinical trials.

ManufacturingcGMP Compliant: US FDA/CDRH registered, EN ISO 13485:2016 certified manufacturing facility based in San Diego.
CE-marked IVDs and CDx Manufacturing: CE-Marked, PMDA and FDA approved CDx, and >50 CE-IVDs including gel, capillary electrophoresis
and NGS assays.

Assay Development: IUO & RUO assays, CE-marked IVDs, & CDx.
Controls and Reagents: DNA / RNA controls, ASRs, GPRs, MRD controls & proficiency panels.

Clinical Lab Services
Clinical Lab Experience: More than a dozen years of clinical reference lab experience.
Internationally Standardized: Internationally standardized CDx, flow cytometry and biomarker testing with internationally accredited labs serving
the US, Europe, and Asia.

Comprehensive Panels and Targeted Assays: Comprehensive NGS and MFC-based panels to screen, identify and track clinically actionablebiomarkers.

MOA Profiling of Novel Compounds: Using primary AML patient samples - IC50 determination, mRNA analysis, genetic, and phenotypiccharacterization.

Worldwide Enrollment: Testing services have supported hundreds of enrollment sites worldwide.

Global Regulatory Expertise
Accredited and Proven: EN ISO 13485:2016 accredited. Experienced staff & proven Quality Management System.
Registered Medical Device Establishment: Registered Medical Device Establishment with the US FDA, KFDA, Saudi Arabia, and the MHLW/PMDA.
Multiple CDx Approvals: Multiple companion diagnostics that are CE-marked, FDA and PMDA/MHLW approved.
CE-marked IVDs: More than 50 CE-marked IVDs available in the EU and select ROW markets; >60 tests included in the ARTG in Australia.
Marketing Authorization Holder (MAH): Marketing Authorization Holder (MAH) and national reimbursement for CDx in Japan.

Streamlined CDx Partnerhips 17

GlobalDistribution
Network
A long and proud history of working
with scientists, laboratorians andclinicians from around the world.
We offer product support in over 160
countries. Our distributor partners
are in locations around the globe.
LEARN MORE:
invivoscribe.com/global-distributors/

18

NorthAmerica

EuropeAfricaAsia

Austria

MiddleEast

Algeria

Afghanistan

CanadaBelarusBahrainAngolaArmenia

MexicoBelgium

IranBurkina Faso

Azerbaijan

United States

Bosnia &Iraq

BurundiBangladesh

Herzegovina

IsraelCameroon

China

South
America

Bulgaria

JordanChadHong Kong

Croatia

KuwaitCôte d’Ivoire

India

CyprusLebanon

Egypt

Indonesia

Argentina

Czech Republic

Oman

Equitorial Guinea

Japan

BrazilDenmark

Pakistan

Eswatini

Kyrgyzstan

Chile

Estonia

Palestine

Ethiopia

Malaysia

Colombia

Finland

Qatar

Gabon

Nepal

Peru

FranceSaudi Arabia

Ghana

Philippines

Uruguay

Germany

TurkeyKenya

Singapore

GreeceUnited Arab Emirates

Madagascar

South Korea

Hungary

Yemen

CentralAmerica
Caribbean
Costa Rica
El Salvador
GuatemalaHonduras
Nicaragua
PanamaMali

Sri Lanka

Iceland

MoroccoTaiwanIrelandMozambique

Tajikistan

Italy

Namibia

ThailandKazakhstan

Nigeria

Turkmenistan

LatviaRepublic of the

Uzbekistan

Lithuania

Congo

VietnamLuxembourg

SenegalMacedonia

Sierra Leone

Malta

South Africa

Moldova

Tanzania

MonacoTunisia

Netherlands

UgandaAustralia

NorwayZambiaNew Zealand

PolandZimbabwe

Australia
and New
Zealand

Portugal
Romania
RussiaSerbiaSlovakia
Slovenia
Spain
SwedenSwitzerland
UkraineUnited Kingdom

Global Distribution Network 19

GlobalInvivoscribe
Locations®

A long and proud history of working
with scientists, laboratorians andclinicians from around the world.
We offer product support in over 160
countries. Our distributor partners
are in locations around the globe.
LEARN MORE:
invivoscribe.com/global-distributors/

20

Munich, Germany
San Diego, United States

Shanghai, China

Tokyo, Japan

United States
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Toll Free: +1 866 623 8105 | Phone: +1 858 224 6600
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Global Invivoscribe Locations 21

Background22

PCR and NGS-Based Assessmentof Clonality in Hematologic Malignancies
For nearly 30 years, Invivoscribe® has developed, manufactured, and commercialized the gold-standard molecular hematopathology assays andreagents for gel and capillary electrophoresis detection, and most recently, next-generation sequencing instruments. These standardized, cGMP
manufactured assays and reagents were developed and validated using standardized workflow and optimized primer sets, reagents and controls.
A number of our products were developed in collaboration with studies conducted by the EuroClonality BIOMED-2 concerted action group;
these capillary based products have provided reliable methods for clonality detection that have withstood the test of time.
We have never accepted the status quo, so our comprehensive menu of clonality assays continues to evolve. All of our NGS-based clonality assays
were developed in-house together with accompanying bioinformatics software by our Invivoscribe R&D team. Developed under full ISO 13485 design
control, these assays and bioinformatics software were designed to run on several next-generation sequencing platforms. These NGS-based assays
are several generations ahead of capillary-based products.
Our comprehensive bioinformatics software not only provides critical information on the presence of clonality, but also identifies the sequence
information required to track clones in subsequent samples.
The unique process of gene rearrangement that occurs within the immunoglobulin (Ig) and T-cell receptor (TCR) gene loci during immune cell
development and maturation generates a vast pool of genetically distinct cells. The resulting diverse population of lymphocytes displays
an astonishing number of diverse antigen receptors, each coded in the DNA by a unique sequence, and each displayed on the cell surface,or as antibodies in the blood unique to a given cell.1,2 This diversity allows the adaptive immune system to carry out its role in protecting the humanbody by recognizing the infinite number of pathogens it might encounter during a lifetime.
In sum, lymphoid malignancies are characterized by size- and sequence-specific rearrangements within these loci, which result from thetransformation and subsequent proliferation from a single cell. The associated cellular population typically shares one or more cell-specific or “clonal”
antigen-receptor gene rearrangements. The detection of these clonal cells forms the basis for clonality assessment in leukemia, lymphoma, andhematologic disease. These methods can also be used to assess somatic hypermutation (SHM) and to study measurable residual disease (MRD).
Malignant cells that remain in the bone marrow following treatment are a major cause of disease relapse. MRD testing by NGS offers enhanced
sensitivity and specificity (compared to MRD testing by flow cytometry), and allows residual cells to be identified at very low levels and monitored
throughout the different stages of disease.
Invivoscribe® can provide you with the necessary tools to accommodate your needs. From gel detection to NGS, we can help you accurately identify
and track hematologic biomarkers. For more information on the detection methods available and the biomarkers offered, please refer to therespective product sections of this catalog.

Immunoglobulin and T-Cell Receptor
Gene Rearrangements and the Principle
and Method of Clonality Testing
The adaptive vertebrate immune system produces a repertoire of immunoglobulin and T-cell receptor molecules using a relatively limited numberof heritable germline gene segments. Somatic gene rearrangement is the fundamental mechanism used to generate different immunoglobulin andT-cell receptor molecules, each with unique binding specificity. Lymphocytes undergo gene rearrangements to assemble CDR3 coding regions thatare unique in both size and DNA sequence. Since leukemias and lymphomas arise from the malignant transformation of a single cell, they shareclonal rearrangement(s) of the antigen receptor genes. This is the basis for clonality testing.3

Background 23

Test Algorithm
for Suspect
Lymphoproliferations
Developed in concert with the
EuroClonality/BIOMED-2 group for
PCR-based clonality assessmentof suspected B- and T-cell
lymphoproliferative disorders.

24

If a definitive diagnosis is not possible following:Morphology | Immunohistochemistry | Flow Cytometry

Suspected B-cellproliferations

Mantle Cell
Lymphoma

Follicular
Lymphoma

IGH-CCND1*

IGH-BCL2

Suspected lymphoid proliferationsof unknown origin (B or T)

IGH (VH-JH) preferably with IGKTCRƴ δ+ proliferations
and immature T-cells

Clonal (generally multiple
clonal results)

TRB preferably with TRG

AND

No clonality but still suspected

IGH (DH-JH) preferably with IGL

Suspected T-cellprolifearations

ClonalTRD

No evidence of clonality*Previously known as BCL1/JH. Results should be considered in the context
of all available clinical, histological and immunophenotypic data.

Test Algorithm for Suspect Lymphoproliferations 25

Hematological
Test Menu
References
1. Kakizuka, A et al. (1991) Cell, 66:675-684.

22. Raimondi, S. (2007) Atlas Genet Cytogenet Oncol
Haematol., 11(4):328-339.

2. Rimokh, R et al. (1994) Blood, 83:1871-1875.

23. Vardi, A et al. (2015) Clin Cancer Res., 22(1):167-174.

3. Gribben, JG et al. (1994) Blood, 83:3800-3807.

24. Tan, BT et al. (2008) J Mol Diag., 10(6):502–512.

4. Radich, JP et al. (1994) Leukemia, 8:1688-1695.

25. González, D et al. (2007) Blood, 110:3112-3121.

5. Gallagher, RE et al. (1995) Blood, 86:1540-1547.

26. Langerak AW et al. (2007) Exp Opin Med Diag.,

6. De Boer, CJ et al. (1995) Blood, 86:2715-2723.
7. Deininger, MW et al. (2000) Blood, 96:3343-56.
8. Schlenk RF et al. (2008) N Engl J Med., 358:1909-1918.
9. Gonzalez, D et al. (2003) Leukemia, 17:1051-1057.
10. Gonzalez, D et al. (2003) Leukemia, 17:1398-1403.
11. Ghia, P et al. (2007) Leukemia, 21:1-3.
12. van Krieken, JH et al. (2007) Leukemia, 21:201-6.
13. Evans, PAS et al. (2007) Leukemia, 21:207-214.
14. Bruggemann, M et al. (2007) Leukemia, 21:215-221.
15. Davi, F et al. (2008) Leukemia, 22:212-214.
16. Döhner, H et al. (2010) Blood, 115:453-474.
17. Huang, W et al. (1993) Blood, 82:1264-1269.
18. Hughes, T et al. (2006) Blood, 108:28-37.
19. Westbrook, CA et al. (1992) Blood, 80:2983-2990.
20. Ralph, QM et al. (1993) Blood, 82:202-20.
21. Juárez-Velázquez, MR et al. (2013) Clinical
Epidemiology of Acute Lymphoblastic Leukemia- From the Molecules to the Clinic, 193-235.

26

1(4):451-461.
27. Arcila, ME et al. (2019) J Mol Diag., 2: 330-341.
28. Campo, E et al. (2011) Blood, 117(19): 5019–5032.
29. Smirnova, SY et al. (2016) Acta naturae, 8(4):100–109.
30. Szczepanski, T et al. (2001) Leukemia, 15:1415–1423.
31. Szczepanski, T et al. (1998) Leukemia, 12:1081–1088.
32. Beishuizen, A et al. (1994) Leukemia, 8(12):2228-36.
33. Tümkaya T et al. (2001) Leukemia, 15:121–127.
34. Felix, CA et al. (1990) J Clin Oncol., 8(3):431-42.
35. Rosenquist R et al. (2017) Leukemia, 31:1477–1481.
36. Arons, E et al. (2011) Blood, 117(18): 4844–4851.
37. Lai, R et al. (2006) Modern Pathology, 19:1498–1505.
38. Srour SA et al. (2017) Br J Haematol., 177(2): 331.
39. Ohshima, K et al. (1990) Hematol Oncol., 8(2):111-8.
40. van Dongen, JJM et al. (2003) Leukemia, 17, 2257–2317.

Lymphoid/ Lymphoma

Diseases

Gene Rearrangement
IGH
VH - JH

IGH
DH - JH

IGK

IGL

Marginal Zone Lymphoma(MZL), extranodal12,13,27

88%

58%

84%

Marginal Zone Lymphoma(MZL), nodal13

100%

30%

Mantle Cell Lymphoma(MCL)2,6,7,12,13,27,37

100%

Follicular Lymphoma (FL)3,7,12,13,27,28

IGHV
SHM

Translocation

Mutation

IGHBCL1(CCND1)

FLT3

NPM1

33%

64%

TRB

TRD

TRG

29%

23%

10%

16%

80%

30%

10%

20%

10%

11%

100%

44%

9%

4%

11%

84%

19%

84%

21%

6%

5%

2%

90%

Diffuse Large B-cell Lymphoma(DLBCL)3,12,13,27

80%

30%

80%

28%

21%

14%

15%

30%

Multiple Myeloma (MM) and other
Plasma Cell Neoplasms (PCN)2,9,10,20,25

84%

60%

57%

97%

Chronic Lymphocytic Leukemia(CLL)11,12,13,15,23,27,35

100%

43%

100%

30%

B-cell Acute Lymphoblastic
Leukemia (B-ALL)4,12,14,19,21,22,27,29,30,31,32,33,34

96%

57%

95%

Suspect B-cell Proliferations12,26,27,33

93%

93%

90%

Peripheral T-cell Lymphoma(PTCL)12,13,14,24

35%

4%

T-cell Acute Lymphoblastic Leukemia(T-ALL)12,14,21,22,29,31

24%

25%

4%

Angioimmunoblastic T-cell
Lymphoma (AILT)12,13,14

19%

11%

30%

*

12%

18%

20%

81%

86%

75%

40%

20%

20%

2%

98%

94%

*

92%

68%

95%

99%

35%

92%

Adult T-cell Leukemia/Lymphoma39

97%

Anaplastic Large-Cell Lymphoma(ALCL)12,13,14

74%

12%

74%

T-cell Prolymphocytic Leukemia(T-PLL)12,13,14

3%

T-cell Large Granular Lymphocytic
Leukemia (T-LGL Leukemia)12,13,14
Suspect T-Cell Proliferations12,26,40

10%

3%

PML
RARα
RAR
α*

75%

30%

96%

3%

3%

100%

6%

94%

4%

4%

97%

29%

96%

90%

11%

90%

10%

BCRABL1*

20%
25%

5%

IGHBCL2

Myeloid

Acute Myeloid Leukemia (AML)8,16
Acute Promyelocytic Leukemia(APL)1,5,16,17

90%

Chronic Myeloid Leukemia(CML)7,18,19,21,38

87%

Myeloproliferative Neoplasms(MPNs)38

10%

Note: The percentage of samples within a given disease category were detected using each gene target.
Percentages indicate the highest referenced value.

Recommended
Primary Test

Recommended
Secondary Test

*Some translocation assays, LymphoTrack®, and MRD applications are currently Research Use Only; not for
use in diagnostic procedures. Illumina® and MiSeq™ are registered trademarks of Illumina®, Inc. Ion S5TM and
Ion PGMTM are trademarks of Thermo Fisher Scientific®.

Hematological Test Menu 27

Key Benefits» One-step PCR for amplicon and library generation» Identify, track, and assess mutation status of B- and T-cell gene rearrangements» Sequence amplicons from any LymphoTrack Dx kit together» Included bioinformatics software for easy analysis and interpretation

Next-Generation
Sequencing (NGS)
AssaysKey Benefits
Next-Generation
Squencing(NGS) CE-IVD Assays
	One-step PCR for amplicon and library generation

	Identify, track, and assess mutation status of B- and T-cell gene rearrangements
	Sequence amplicons from any LymphoTrack Dx kit together	Included
software for easy analysis and interpretation
LymphoTrackDx bioinformatics
Assay kits aredesigned for the identification ofgene rearrangements in hematologic
samples utilizing next-generation
sequencing (NGS) technologies.
LEARN MORE:
catalog.invivoscribe.com

Warranty and Liability
Invivoscribe is committed to providing the highest quality products. Invivoscribe warrants that for products which are provided with Instructions for Use, these products meet or exceed theperformance standards described in the Instructions For Use. If a product is covered by product specifications and does not perform as specified, our policy is to replace the product or credit thefull purchase price. No other warranties of any kind, expressed or implied, are provided by Invivoscribe. Invivoscribe liability shall not exceed the purchase price of the product. Invivoscribe shallhave no liability for direct, indirect, consequential, or incidental damages arising from the use, results of use, or inability to use its products; product efficacy under purchaser controlled conditions
in purchaser’s laboratory must be established and continually monitored through purchaser defined and controlled processes including but not limited to testing of positive, negative, and blankcontrols every time a sample is tested. Ordering, acceptance, and use of product constitutes purchaser acceptance of sole responsibility for assuring product efficacy and purchaser agreement to thelimitation of liability set forth in this paragraph.
NoticeThis product is covered by one or more patents owned by or licensed to Invivoscribe, Inc., including US Patent No. 7,785,783, US Patent No. 8,859,748, US Patent No. 10,280,462 European Patent Nos.
1549764 and 2418287 (each validated in 16 countries) European Patent No 2460889, European Patent No. 1633884, Japanese Patent No. 4708029, Japanese Patent No. 6189600, Brazilian Patent No.
PI0410283, Canadian Patent No. 2525122, Indian Patent No. 243620, Mexican Patent No. 286493, Chinese Patent No. 1806051, and Korean Patent No. 10-1215194.
These products use nucleic acid amplification methods such as Polymerase Chain Reaction (PCR). Any necessary license to practice amplification methods or to use reagents, amplification enzymes
or equipment covered by third party patents is the responsibility of the user and no such license is granted by Invivoscribe, Inc., expressly or by implication.

Introduction
These assays take advantage of the wealth and depth of data generated by the Illumina® MiSeq™, Thermo Fisher
Scientific® Ion PGM™ and Ion S5™ platforms.
The Invivoscribe NGS assays offer significant improvements over conventional fragment analysis of B- and T-cell generearrangements by providing detailed information regarding the DNA sequences, sequence frequency, and mutational
status (IGHV Leader and IGH FR1 only) of each clonotype.
LymphoTrack Dx Assay kits are a complete solution. Kits contain ready-to-use indexed amplification master mixes,
necessary controls, and bioinformatics software. As primers are designed with barcoded indices and adapters,
sequencing libraries can be generated with a single PCR, streamlining the overall workflow, eliminating the need fora post-PCR ligation step, and reducing the potential for sample cross contamination.
The per sample testing costs can be reduced by pooling samples from different LymphoTrack Dx Assays into a single
sequencing run. The bioinformatics software will sort the complex NGS data for easy analysis and visualizationof individual samples.
Detailed instructions for use are provided with all kits and the Invivoscribe technical support team is always available
to answer your questions.

Key Benefits» One-step PCR for amplicon and library generation» Identify and assess mutation status of B- and T-cell gene rearrangements» Sequence amplicons from any LymphoTrack kit together» Bioinformatics software is available in desktop or Docker images for analysis and interpretation» Same reagents for clonality, somatic hypermutation (SHM), minimal residual disease (MRD) testing, and tracking/monitoring of immunotherapy constructs

Chapter Contents
NGS Assays
30 LymphoTrack Dx IGHV Leader Somatic Hypermutation Assay
32 LymphoTrack Dx IGH FR1/2/3 Assays
36 LymphoTrack Dx IGK Assays
38 LymphoTrack Dx TRG Assays
40 LymphoTrack Dx TRB Assay
42 LymphoTrack Dx Bioinformatics Software

The LymphoTrack Dx Assays are in vitro diagnostic products and are not available for saleor use within North America. For more information regarding the research use only reagents,
please see the Next-Generation Sequencing - LymphoTrack section.

Next-Generation Sequencing (NGS) | CE-IVD Assays 29

LymphoTrack® Dx IGHV SomaticHypermutation Assay
Assay Description
The LymphoTrack Dx IGHV Leader Somatic Hypermutation Assay for the Illumina® MiSeq™ is an in vitro diagnostic product intended for nextgeneration sequencing (NGS) based determination of the frequency distribution of IGH gene rearrangements, as well as the degree of somatic
hypermutation (SHM) of rearranged genes in patients suspected of having lymphoproliferative disease. This assay aids in the identification oflymphoproliferative disorders as well as providing an aid in determining disease prognosis. If you would like to test for IGHV somatic hypermutation
using the Thermo Fisher® Ion PGM™ or Ion S5™ platform, please refer to the LymphoTrack Dx IGH FR1 Assay (Catalog #: 91210007).

Summary and Explanation of the Test
The NGS LymphoTrack Dx IGHV Leader Somatic Hypermutation Assay for the Illumina® MiSeq™ represents a significant improvement over clonality
assays using fragment analysis as it efficiently detects the majority of IGH gene rearrangements using a single multiplex master mix, identifies the
DNA sequence specific for each clonal gene rearrangement, and assesses the somatic hypermutation rate of clonal samples in the same workflow.
The master mixes target the Leader (VHL) and the joining (JH) gene regions of the IGH locus and are designed with Illumina® adapters and indices(8 included in Kit A and 24 included in the Panel). This allows for a one-step PCR and pooling of amplicons from several different samples and targets
into a single Illumina® MiSeq™ run. No post-PCR ligation step is required.
Positive (clonal positive, SHM negative), negative (polyclonal) and SHM (clonal positive, SHM positive) DNA controls are included in the kits.

Background
The human immunoglobulin heavy chain (IGH) gene locus on chromosome 14 (14q32.3) includes 46-52 functional and 30 nonfunctional variable (VH)
gene segments, 27 functional diversity (DH) gene segments, and 6 functional joining (JH) gene segments spread over 1,250 kilobases.
During B-cell development, genes encoding the IGH protein are assembled from multiple polymorphic gene segments that undergo rearrangements
and selection, generating cell specific VH-DH-JH rearrangements that are unique in both length and sequence.1
Since leukemias and lymphomas originate from the malignant transformation of individual lymphoid cells, all leukemias and lymphomas generally
share one or more cell-specific or "clonal" antigen receptor gene rearrangements. Therefore, tests that detect IGH clonal rearrangements canbe useful in the study of B- cell malignancies. An additional level of diversity is further generated in the antigen receptors by introducing point
mutations in the variable regions, also named SHM. In instances where there is a high degree of SHM, there is the risk that primers located within
the variable region will not be able to bind and clonal products will not amplify. In these cases, the leader primers located upstream of the variable
region can be beneficial for the detection of clonal products, due to the conserved nature of the VHL region. In addition, the SHM rate of the entire
variable gene can be determined using the VHL primers.
Determining the immunoglobulin variable heavy chain gene (IGHV) hypermutation rate is considered a gold standard for determining the prognosisof patients with chronic lymphocytic leukemia (CLL)2 and small lymphocytic lymphoma (SLL). In addition, NGS methods can improve diseasestratification.

Specimen Requirement
50 ng of high-quality genomic DNA.

References
1. Miller et al., Molecular Genetic Pathology (2nd ed.). Springer Science & Business Media. 2013: 302.2.7.13 and 30.2.7.18.
2. Ghia et al., Leukemia 21: 2-3 (2007).

VH L

VH 1

VH 2

VH 3

VH 4

VH 5

VH 6

VH 7

JH

Simplified representation of the immunoglobulin heavy chain (IGH) gene locus on chromosome 14. Depicted are the variable (VH) and downstream consensus joining (JH)
region genes involved in rearrangements. Upstream of the variable gene segments, the leader sequence (VHL) is also depicted. Diversity region genes are not depicted.

30 Next-Generation Sequencing (NGS) | CE-IVD Assays

Reagents - MiSeq™ Detection
Kit A Components

Panel Components (includes all master mixes from Kit A plus the items below)

Master Mix Name

Index #

Master Mix Name

Index #

Master Mix Name

Index #

IGH Leader MiSeq 01

A001

IGH Leader MiSeq 09

A009

IGH Leader MiSeq 18

A018

IGH Leader MiSeq 02

A002

IGH Leader MiSeq 10

A010

IGH Leader MiSeq 19

A019

IGH Leader MiSeq 03

A003

IGH Leader MiSeq 11

A011

IGH Leader MiSeq 20

A020

IGH Leader MiSeq 04

A004

IGH Leader MiSeq 12

A012

IGH Leader MiSeq 21

A021

IGH Leader MiSeq 05

A005

IGH Leader MiSeq 13

A013

IGH Leader MiSeq 22

A022

IGH Leader MiSeq 06

A006

IGH Leader MiSeq 14

A014

IGH Leader MiSeq 23

A023

IGH Leader MiSeq 07

A007

IGH Leader MiSeq 15

A015

IGH Leader MiSeq 25

A025

IGH Leader MiSeq 08

A008

IGH Leader MiSeq 16

A016

IGH Leader MiSeq 27

A027

Controls

Controls

IGH SHM POS (+) Qty. 1

IGH SHM POS (+) Qty. 3

IGH POS (+) Qty. 1

IGH POS (+) Qty. 3

NGS NEG (-) Qty. 1

NGS NEG (-) Qty. 3

Rank

Sequence

Length

Merge
Count

V-geneJ-gene% Totalreads

Cumulative%

Mutation
Rate
PartialV-gene (%)

In-frame(Y/N)

No stop
codon
(Y/N)

V-coverage

1

TTCTCGTGGTG

455

29603

IGHV4-59_08

IGHJ4_02

9.93

9.93

11.26

Y

Y

98.63

2

CTCGCCCTCCT

463

205

IGHV5-51_01

IGHJ4_02

0.07

9.99

0.00

Y

Y

99.66

3

GGTTTTCCTTG

484

201

IGHV3-7_01

IGHJ4_02

0.07

10.06

7.77

Y

Y

100.004

CTCGCCCTCCT

463

185

IGHV5-51_01

IGHJ5_02

0.06

10.12

6.08

Y

Y

99.32

5

CTCGCCCTCCT

469

170

IGHV5-51_01

IGHJ4_02

0.06

10.18

0.00

Y

Y

99.32

6

CTCGCCCTCCT

466

160

IGHV5-51_01

IGHJ4_02

0.05

10.23

0.00

Y

Y

99.66

7

CTGCTGCTGAC

460

159

IGHV2-5_10

IGHJ5_02

0.05

10.29

8.08

Y

Y

97.64

8

GGTTTTCCTTG

493

156

IGHV3-48_02

IGHJ6_02

0.05

10.34

3.72

Y

Y

98.99

9

CTCGCCCTCCT

334

153

IGHV5-51_02

IGHJ2_01

0.05

10.39

3.72

Y

N

27.70

10

CTCGCCCTCCT

334

152

IGHV5-51_02

IGHJ2_01

0.05

10.44

3.38

Y

N

26.01

Example Data. The top 10 sequences from a read summary generated by the LymphoTrack Dx Software - MiSeq™ with the SHM mutation rate and predictions pertaining
to whether a sequence is in-frame or contains a premature stop codon are depicted. To learn more about the LymphoTrack Dx software, please refer to the LymphoTrack
Dx Bioinformatics Software section.

Ordering Information
Catalog #Products

Quantity Components

9-121-0059

LymphoTrack® Dx IGHV Leader Somatic Hypermutation Assay Kit A - MiSeq™

8 indices - 5 sequencing reactions each

9-121-0069

LymphoTrack® Dx IGHV Leader Somatic Hypermutation Assay Panel - MiSeq™

24 indices - 5 sequencing reactions each

9-500-0009

LymphoTrack Dx Software - MiSeq

1 Software Package®

™

These products are CE-IVD assays for in vitro diagnostic use
and available in regions that accept CE-IVD products.

Next-Generation Sequencing (NGS) | CE-IVD Assays 31

LymphoTrack Dx IGH FR1/2/3 Assays®

Assay Description
LymphoTrack Dx IGH FR1 Assays
The LymphoTrack Dx IGH FR1 Assay for the Illumina MiSeqTM or Thermo Fisher Scientific® Ion S5TM and Ion PGMTM is an in vitro diagnostic productintended for next-generation sequencing (NGS) based determination of the frequency distribution of IGH gene rearrangements as well as the degreeof somatic hypermutation of rearranged genes in patients suspected with having lymphoproliferative disease. This assay aids in the identificationof lymphoproliferative disorders as well as providing an aid in determining disease prognosis.
LymphoTrack Dx IGH FR1/2/3 Assays
The LymphoTrack Dx IGH FR1 Assay for the Illumina MiSeqTM or Thermo Fisher Scientific® Ion S5TM and Ion PGMTM is an in vitro diagnostic productintended for next-generation sequencing (NGS) based determination of the frequency distribution of IGH gene rearrangements as well as the degreeof somatic hypermutation of rearranged genes in patients suspected with having lymphoproliferative disease. This assay aids in the identificationof lymphoproliferative disorders as well as providing an aid in determining disease prognosis.
This LymphoTrack Dx IGH FR2 Assay is an in vitro diagnostic product intended for next-generation sequencing (NGS) for the Illumina MiSeqTM
or Thermo Fisher Scientific® Ion S5TM and Ion PGMTM instruments. The assay will determine the frequency distribution of IGH VH-JH generearrangements in patients suspected with having lymphoproliferative disease. This assay aids in the identification of lymphoproliferative disorders.
The LymphoTrack Dx IGH FR3 Assay is an in vitro diagnostic product intended for next-generation sequencing (NGS) for the Illumina MiSeqTM
or Thermo Fisher Scientific® Ion S5TM and Ion PGMTM instruments. The assay will determine the frequency distribution of IGH VH-JH generearrangements in patients suspected with having lymphoproliferative disease. This assay aids in the identification of lymphoproliferative disorders.

Summary and Explanation of the Test
The LymphoTrack Dx IGH Assays represent a significant improvement over conventional clonality assessment methods utilizing fragment analysis
by providing four important and complementary uses in a single workflow:
1. Detection of clonal populations.
2. Identification of sequence information and gene segment utilization.
3. The LymphoTrack Dx IGH framework 1 FR1 master mixes provide the degree of SHM in the immunoglobulin variable heavy chain (IGHV) gene locus.
4. The ability to track sequences in subsequent samples with the Invivoscribe LymphoTrack MRD* Software.
These assays utilize a single multiplex master mix to target each conserved IGH Framework Region (FR1, FR2, and FR3) within the VH and the JH regions
described in lymphoid malignancies. Each master mix targets one of the conserved IGH framework regions (FR1, FR2, or FR3) within the VH and the
JH regions described in lymphoid malignancies. Targeting all three framework regions significantly reduces the risk of not being able to detect thepresence of clonality, as somatic hypermutations in the primer binding sites of the involved VH gene segments can impede DNA amplification.1 The
included primers are designed with Illumina® or Thermo Fisher Scientific adapters and indices (8-24 and 12, respectively). This allows up to 24 samples
on MiSeqTM and 12 samples on Ion PGM™ and Ion S5™ to be sequenced at the same time with any of the individual FRs.
In addition, amplicons generated with different FR master mixes or Invivoscribe LymphoTrack Dx kits (such as IGK or TRG) can be pooled together inthe same sequencing library to reduce testing costs. Positive clonal (SHM negative) and negative polyclonal DNA controls are included in kits. A clonal
SHM positive control can be purchased separately (cat#: 40880008).

Background
The human immunoglobulin heavy chain (IGH) gene locus on chromosome 14 (14q32.3) includes 46-52 functional and 30 non-functional variable(VH), 27 functional diversity (DH), and 6 functional joining (JH) gene segments. The VH gene segments can be further broken down into three conserved
frameworks (FR) and three variable complementarity-determining regions (CDRs).
During development of lymphoid cells, antigen receptor genes undergo somatic gene rearrangements.2 Specifically during B-cell development, IGH
molecules are assembled from multiple polymorphic gene segments that undergo rearrangements generating VH-DH-JH combinations unique in both
length and sequence.3 Since leukemias and lymphomas originate from the malignant transformation of individual lymphoid cells, all leukemias andlymphomas generally share one or more cell-specific or “clonal” antigen receptor gene rearrangements.
In addition, the IGHV hypermutation status obtained with the LymphoTrack Dx IGH FR1 master mixes, provides important prognostic information forpatients with chronic lymphocytic leukemia (CLL) and small lymphocytic lymphoma (SLL). The SHM rate has been shown to have clinical relevance for
CLL, as there is a clear distinction in the median survival of patients with and without SHM.4*MRD Software can be used to track sequences generated by either LymphoTrack Assays MiSeqTM or Ion S5/PGM™. MRD applications are for Research Use Only.

32 Next-Generation Sequencing (NGS) | CE-IVD Assays

Specimen Requirement
50 ng of high-quality genomic DNA.

References
1. Evans, P. A. et al., (2007). Leukemia 21, 207-14.
2. Tonegawa, S. (1983). Nature 302, 575-581.
3. Miller JE. (2013) Molecular Genetic Pathology (2nd Edition.,sections 30.2.7.13 and 30.2.7.18).
4. Ghia et al., Blood 105:1678-1685 (2005).

Simplified Representation of the IGH GeneDH
VL

VH-FR1

VH-FR2

VH-FR3

JH

Simplified depiction of variable (VH) and downstream consensus joining (JH) region genes
involved in gene rearrangements.

JH primers

VH family primers

Reagents - MiSeq™ Detection
The LymphoTrack Dx IGH FR1/2/3 Assays contain components from respective individual FR kit A's or panels.

Master Mix Name

Index #

Master Mix Name

Index#

IGH FR1 MiSeq 01

A001

IGH FR2 MiSeq 01

A001

IGH FR3 MiSeq 01

A001

IGH FR1 MiSeq 02

A002

IGH FR2 MiSeq 02

A002

IGH FR3 MiSeq 02

A002

IGH FR1 MiSeq 03

A003

IGH FR2 MiSeq 03

A003

IGH FR3 MiSeq 03

A003

IGH FR1 MiSeq 04

A004

IGH FR2 MiSeq 04

A004

IGH FR3 MiSeq 04

A004

IGH FR1 MiSeq 05

A005

IGH FR2 MiSeq 05

A005

IGH FR3 MiSeq 05

A005

IGH FR1 MiSeq 06

A006

IGH FR2 MiSeq 06

A006

IGH FR3 MiSeq 06

A006

IGH FR1 MiSeq 07

A007

IGH FR2 MiSeq 07

A007

IGH FR3 MiSeq 07

A007

IGH FR1 MiSeq 08

A008

IGH FR2 MiSeq 08

A008

IGH FR3 MiSeq 08

A008

IGH FR1 MiSeq 09

A009

IGH FR2 MiSeq 09

A009

IGH FR3 MiSeq 09

A009

IGH FR1 MiSeq 10

A010

IGH FR2 MiSeq 10

A010

IGH FR3 MiSeq 10

A010

IGH FR1 MiSeq 11

A011

IGH FR2 MiSeq 11

A011

IGH FR3 MiSeq 11

A011

IGH FR1 MiSeq 12

A012

IGH FR2 MiSeq 12

A012

IGH FR3 MiSeq 12

A012

IGH FR1 MiSeq 13

A013

IGH FR2 MiSeq 13

A013

IGH FR3 MiSeq 13

A013

IGH FR1 MiSeq 14

A014

IGH FR2 MiSeq 14

A014

IGH FR3 MiSeq 14

A014

IGH FR1 MiSeq 15

A015

IGH FR2 MiSeq 15

A015

IGH FR3 MiSeq 15

A015

IGH FR1 MiSeq 16

A016

IGH FR2 MiSeq 16

A016

IGH FR3 MiSeq 16

A016

IGH FR1 MiSeq 18

A018

IGH FR2 MiSeq 18

A018

IGH FR3 MiSeq 18

A018

IGH FR1 MiSeq 19

A019

IGH FR2 MiSeq 19

A019

IGH FR3 MiSeq 19

A019

IGH FR1 MiSeq 20

A020

IGH FR2 MiSeq 20

A020

IGH FR3 MiSeq 20

A020

IGH FR1 MiSeq 21

A021

IGH FR2 MiSeq 21

A021

IGH FR3 MiSeq 21

A021

IGH FR1 MiSeq 22

A022

IGH FR2 MiSeq 22

A022

IGH FR3 MiSeq 22

A022

IGH FR1 MiSeq 23

A023

IGH FR2 MiSeq 23

A023

IGH FR3 MiSeq 23

A023

IGH FR1 MiSeq 25

A025

IGH FR2 MiSeq 25

A025

IGH FR3 MiSeq 25

A025

IGH FR1 MiSeq 27

A027

IGH FR2 MiSeq 27

A027

IGH FR3 MiSeq 27

A027

PANEL

PANEL

PANEL

KIT A

LymphoTrack Dx IGH FR3 Components

Index

KIT A

LymphoTrack Dx IGH FR2 Components

Master Mix NameKIT A

LymphoTrack Dx IGH FR1 Components

Kit A's contain indices IGH FRX A001 to A008. Panels contain all master mixes listed above.

Controls in Individual
FR (1, 2, or 3) Kit A's

Controls in Individual
FR (1, 2, or 3) Panels

Controls in Combo
FR 1/2/3 Kit A

Controls in Combo
FR 1/2/3 Panel

IGH POS (+) Qty. 1

IGH POS (+) Qty 3

IGH POS (+) Qty. 2

IGH POS (+) Qty. 6

NGS NEG (-) Qty. 1

NGS NEG (-) Qty 3

NGS NEG (-) Qty. 2

NGS NEG (-) Qty. 6*MRD Software can be used to track sequences generated by either LymphoTrack Assays MiSeqTM or Ion S5/PGM™. MRD applications are for Research Use Only.

Next-Generation Sequencing (NGS) | CE-IVD Assays 33Reagents - Ion S5™ / PGM™ Detection
The LymphoTrack Dx IGH FR1/2/3 Assays contain components from respective individual FR Assays.

LymphoTrack Dx IGH FR1 Components

LymphoTrack Dx IGH FR2 Components

LymphoTrack Dx IGH FR3 Components

Master Mix Name

Index #

Master Mix Name

Index #

Master Mix Name

Index #

IGH FR1 Ion S5/PGM 01

IonXpress_001

IGH FR2 Ion S5/PGM 01

IonXpress_001

IGH FR3 Ion S5/PGM 01

IonXpress_001

IGH FR1 Ion S5/PGM 02

IonXpress_002

IGH FR2 Ion S5/PGM 02

IonXpress_002

IGH FR3 Ion S5/PGM 02

IonXpress_002

IGH FR1 Ion S5/PGM 03

IonXpress_003

IGH FR2 Ion S5/PGM 03

IonXpress_003

IGH FR3 Ion S5/PGM 03

IonXpress_003

IGH FR1 Ion S5/PGM 04

IonXpress_004

IGH FR2 Ion S5/PGM 04

IonXpress_004

IGH FR3 Ion S5/PGM 04

IonXpress_004

IGH FR1 Ion S5/PGM 07

IonXpress_007

IGH FR2 Ion S5/PGM 07

IonXpress_007

IGH FR3 Ion S5/PGM 07

IonXpress_007

IGH FR1 Ion S5/PGM 08

IonXpress_008

IGH FR2 Ion S5/PGM 08

IonXpress_008

IGH FR3 Ion S5/PGM 08

IonXpress_008

IGH FR1 Ion S5/PGM 09

IonXpress_009

IGH FR2 Ion S5/PGM 09

IonXpress_009

IGH FR3 Ion S5/PGM 09

IonXpress_009

IGH FR1 Ion S5/PGM 10

IonXpress_010

IGH FR2 Ion S5/PGM 10

IonXpress_010

IGH FR3 Ion S5/PGM 10

IonXpress_010

IGH FR1 Ion S5/PGM 11

IonXpress_011

IGH FR2 Ion S5/PGM 11

IonXpress_011

IGH FR3 Ion S5/PGM 11

IonXpress_011

IGH FR1 Ion S5/PGM 12

IonXpress_012

IGH FR2 Ion S5/PGM 12

IonXpress_012

IGH FR3 Ion S5/PGM 12

IonXpress_012

IGH FR1 Ion S5/PGM 13

IonXpress_013

IGH FR2 Ion S5/PGM 13

IonXpress_013

IGH FR3 Ion S5/PGM 13

IonXpress_013

IGH FR1 Ion S5/PGM 14

IonXpress_014

IGH FR2 Ion S5/PGM 14

IonXpress_014

IGH FR3 Ion S5/PGM 14

IonXpress_014

Controls in Individual FR (1,2, or 3) Kits

Controls in FR 1/2/3 Kit

IGH POS (+) Qty. 2

IGH POS (+) Qty. 4

NGS NEG (-) Qty. 2

NGS NEG (-) Qty. 4

LymphoTrack Dx IGH Assay - V -J Sequence Frequencies

LymphoTrack Dx FR1

LT Dx FR1

V-J Sequence Frequencies. The LymphoTrack Dx Software provides a stacked bar graphdepicting the relative frequencies of the 200 most prevalent VH-JH rearrangements identified
in a sample. To learn more about the LymphoTrack Dx software, please refer to the
LymphoTrack Dx Bioinformatics Software section.

34 Next-Generation Sequencing (NGS) | CE-IVD Assays

Comparison of SHM Analysis Methods. The SHM rate of 51 CLL samples wasdetermined by the LymphoTrack Dx IGH FR1 Assay – MiSeqTM and analyzed
with both the LymphoTrack Dx Software - MiSeqTM and IMGT analysis.

Ordering
Information for Reagents - Miseq™ Detection
Ordering Information
Catalog #Products

9-121-0129

LymphoTrack® Dx IGH FR1/2/3 Assay Kit A - MiSeqTM

Quantity Components
8 + 8 + 8 indices - 5 sequencing reactions each

9-121-0139

LymphoTrack Dx IGH FR1/2/3 Assay Panel - MiSeq

24 + 24 + 24 indices - 5 sequencing reactions each

9-121-0009

LymphoTrack® Dx IGH FR1 Assay Kit A - MiSeqTM

8 indices - 5 sequencing reactions each

9-121-0039

LymphoTrack Dx IGH FR1 Assay Panel - MiSeq

24 indices - 5 sequencing reactions each

9-121-0089

LymphoTrack Dx IGH FR2 Assay Kit A - MiSeq

8 indices - 5 sequencing reactions each

9-121-0099

LymphoTrack® Dx IGH FR2 Assay Panel - MiSeqTM

24 indices - 5 sequencing reactions each

9-121-0109

LymphoTrack Dx IGH FR3 Assay Kit A - MiSeq

8 indices - 5 sequencing reactions each

9-121-0119

LymphoTrack® Dx IGH FR3 Assay Panel - MiSeqTM

24 indices - 5 sequencing reactions each

9-500-0009

LymphoTrack Dx Software - MiSeq

1 Software Package

TM

®

TM

®

TM

®

TM

®

®

TM

Ordering Information for Reagents - Ion S5™ / PGM™ Detection
Catalog #Products

Quantity Components

9-121-0057

LymphoTrack® Dx IIGH FR1/2/3 Assay - S5/PGMTM

12 + 12 + 12 indices - 5 sequencing reactions each

9-121-0007

LymphoTrack Dx IGH FR1 Assay - S5/PGM

12 indices - 5 sequencing reactions eachTM®

9-121-0037

TM

LymphoTrack Dx IGH FR2 Assay - S5/PGM

12 indices - 5 sequencing reactions each

9-121-0047

LymphoTrack® Dx IGH FR3 Assay - S5/PGMTM

12 indices - 5 sequencing reactions each

9-500-0007

LymphoTrack Dx Software - S5/PGM

1 Software Package

®

®

TM

These products are CE-IVD assays for in vitro diagnostic use
and available in regions that accept CE-IVD products.

Next-Generation Sequencing (NGS) | CE-IVD Assays 35

LymphoTrack Dx IGK Assay®

Assay Description
The LymphoTrack Dx IGK Assays for the Illumina® MiSeq™, or Thermo Fisher Scientific® Ion PGM™ and Ion S5™ instruments are in vitro diagnostic products
intended for next-generation sequencing (NGS) based determination of the frequency distribution of IGK gene rearrangements in patients suspectedof having lymphoproliferative disease. These assays aid in the identification of lymphoproliferative disorders.

Summary and Explanation of the Test
In contrast to the IdentiClone® fragment analysis assays for clonality that utilize two master mixes, these NGS assays contain a single multiplex
master mix to target conserved regions of the IGK gene locus described in lymphoid malignancies, thereby reducing sample DNA requirements andsimplifying the testing workflow. The LymphoTrack Dx IGK master mix primers are also designed with Illumina® or Thermo Fisher Scientific adaptersand up to 24 different indices. This allows amplicons generated from different indexed IGK master mixes to be pooled into a single library for loading
onto one MiSeq™ flow cell, Ion PGM™ or Ion S5™ chips.
Positive clonal and negative polyclonal DNA controls are included in kits.
LymphoTrack Dx Software is capable of sorting complex NGS data generated by LymphoTrack Dx Assays by gene target, providing users the ability
to reduce per sample testing costs by sequencing amplicons from any LymphoTrack Dx Assay (e.g. IGH, IGK, TRB, TRG) at the same time. In addition,
the LymphoTrack Dx Software provides an easy and streamlined method for visualization of data and guidelines provided in the instructions for use
allow samples to be interpreted for evidence or no evidence of clonality.

Background
The LymphoTrack Dx IGK Assays represent a significant improvement over existing fragment analysis clonality assays by providing two important andcomplementary uses:
1. Detection of clonal populations.
2. Identification of sequence information and gene segment utilization.
3. Ability to track sequences in subsequent samples with the use of the Invivoscribe LymphoTrack MRD Software.
The human immunoglobulin kappa (IGK) gene locus on chromosome 2 (2p11.2) includes 76 V (variable) region genes spanning 7 subgroups and5 J (joining) region gene segments upstream of the (Cƙ) region.
During lymphoid cell development, antigen receptor genes undergo somatic gene rearrangements.1 Specifically, during B-cell development, genes
encoding IGK molecules are assembled from multiple polymorphic gene segments that generate V-J combinations unique in both length andsequence.2
In addition, the kappa deleting element (Kde), approximately 24 kb downstream of the Jƙ-Cƙ region can also rearrange with Vƙ gene segments and theisolated recombination signal sequence in the Jƙ-Cƙ intron (Jƙ-Cƙ INTR).
Since leukemias and lymphomas originate from the malignant transformation of individual lymphoid cells, all leukemias and lymphomas generally
share one or more cell-specific or “clonal” antigen receptor gene rearrangements. Therefore, tests that detect IGK clonal rearrangements canbe useful in the study of B- cell malignancies and complement IGH testing, as the IGK receptor is less susceptible to somatic mutations.

Specimen Requirement
50 ng of high-quality genomic DNA.

References
1. Tonegawa, S. (1983). Nature 302, 575-581.
2. Miller JE. (2013) Molecular Genetic Pathology (2nd Edition.,sections 30.2.7.13 and 30.2.7.18).

Simplified Representation of the IGK GeneKde
Kde

36 Next-Generation Sequencing (NGS) | CE-IVD Assays

Depicted are the variable region (Vƙ) or variable intragenic
Jƙ-Cƙ intron (Jƙ-Cƙ INTR) genes involved in IGK generearrangements in addition to the downstream consensus
joining region genes (Jƙ) or kappa deleting element (Kde).

Reagents - MiSeq™ Detection
Kit A Components

Panel Components (includes all master mixes from Kit A plus the items below)

Master Mix Name

Index #

Master Mix Name

Index #

Master Mix Name

Index #IGK MiSeq 01

A001

IGK MiSeq 09

A009

IGK MiSeq 18

A018

IGK MiSeq 02

A002

IGK MiSeq 10

A010

IGK MiSeq 19

A019

IGK MiSeq 03

A003

IGK MiSeq 11

A011

IGK MiSeq 20

A020

IGK MiSeq 04

A004

IGK MiSeq 12

A012

IGK MiSeq 21

A021

IGK MiSeq 05

A005

IGK MiSeq 13

A013

IGK MiSeq 22

A022

IGK MiSeq 06

A006

IGK MiSeq 14

A014

IGK MiSeq 23

A023

IGK MiSeq 07

A007

IGK MiSeq 15

A015

IGK MiSeq 25

A025

IGK MiSeq 08

A008

IGK MiSeq 16

A016

IGK MiSeq 27

A027

Controls

Controls

IGK POS (+) Qty. 1

IGK POS (+) Qty. 3

NGS NEG (-) Qty. 1

NGS NEG (-) Qty. 3

Reagents - Ion S5™/PGM™ Detection
Assay Components
Master Mix Name

Index #

Master Mix Name

Index #IGK S5/PGM 01

IonXpress_001

IGK S5/PGM 11

IonXpress_011

IGK S5/PGM 02

IonXpress_002

IGK S5/PGM 12

IonXpress_012

IGK S5/PGM 04

IonXpress_004

IGK S5/PGM 13

IonXpress_013

IGK S5/PGM 08

IonXpress_008

IGK S5/PGM 14

IonXpress_014

IGK S5/PGM 09

IonXpress_009

IGK S5/PGM 16

IonXpress_016

IGK S5/PGM 010

IonXpress_010

IGK S5/PGM 17

IonXpress_017

Controls
IGK POS (+) Qty. 2

NGS NEG (-) Qty. 2

LymphoTrack Dx IGK Assay - V -J Sequence Frequencies

V-J Sequence Frequencies. The LymphoTrack Dx Software provides a stacked bar graph depicting the relative frequencies forthe most prevalent rearrangements identified in a sample. To learn more about the LymphoTrack Dx software, please refer tothe LymphoTrack Dx Bioinformatics Software section.

Ordering Information
Catalog #Products

Quantity Components

9-122-0009

LymphoTrack Dx IGK Assay Kit A - MiSeq

8 indices - 5 sequencing reactions each

9-122-0019

LymphoTrack® Dx IGK Assay Panel - MiSeq™

24 indices - 5 sequencing reactions each

9-500-0009

LymphoTrack® Dx Software - MiSeq™

1 Software Package

9-122-0007

LymphoTrack Dx IGK Assay - S5/PGM

12 indices - 5 sequencing reactions each

9-500-0007

LymphoTrack® Dx Software - S5/PGM™

1 Software Package

®

®

™

™

These products are CE-IVD assays for in vitro diagnostic use
and available in regions that accept CE-IVD products.

Next-Generation Sequencing (NGS) | CE-IVD Assays 37

LymphoTrack® Dx TRG Assay
Assay Description
The LymphoTrack Dx TRG Assays for the Illumina® MiSeq™, Thermo Fisher Scientific® Ion PGM™ or Ion S5™ instruments are in vitro diagnostic products
intended for next-generation sequencing (NGS) based determination of the frequency distribution of TRG gene rearrangements in patients suspected withhaving lymphoproliferative disease. These assays aid in the identification of lymphoproliferative disorders.

Summary and Explanation of the Test
This assay utilizes a single multiplex master mix to target conserved V and J regions of the human TRG gene locus described in lymphoidmalignancies, thereby reducing sample DNA requirements and simplifying the testing workflow. Primers are designed with Illumina® or Thermo Fisher
Scientific adapters and up to 24 different indices; thereby allowing amplicons generated from different TRG master mixes to be pooled together forsequencing on a single MiSeq™ flow cell, or Ion PGM™ or Ion S5™ chip.
Positive clonal and negative polyclonal DNA controls are included in kits.
LymphoTrack Dx Software sorts complex NGS data generated by LymphoTrack Dx Assays by gene target,, providing users the ability to reduce persample testing costs by sequencing amplicons generated with any LymphoTrack Dx Assay (e.g. IGH, IGK, TRB, TRG) at the same time. In addition, theLymphoTrack Dx Software provides an easy and streamlined method for data visualization and guidelines provided in the instructions for use allow
samples to be interpreted for evidence or no evidence of clonality.

Background
The LymphoTrack Dx TRG Assays represent a significant improvement over existing fragment analysis clonality assays by providing two importantand complementary uses:
1. Detection of initial clonal populations.
2. Identification of sequence information required to track clonal rearrangements in subsequent samples.
The TRG gene locus on chromosome 7 (7q14) includes 14 V (variable region) genes (Group I, II, III, and IV), 5 J (joining region) gene segments, and2 C (constant region) genes spread over 200 kilobases.
During development of lymphoid cells, antigen receptor genes undergo somatic gene rearrangements.1 Specifically during T-cell development,
genes encoding TRG molecules are assembled from multiple polymorphic gene segments that undergo rearrangement generating V-J combinations
unique in both length and sequence.2 Since leukemias and lymphomas originate from the malignant transformation of individual lymphoid cells, allleukemias and lymphomas generally share one or more cell-specific or “clonal” antigen receptor gene rearrangement. Therefore, tests that detect
TRG clonal rearrangements can be useful in the study of B- and T-cell malignancies.
Note: For a more thorough explanation of the locus and the targeted deep sequencing strategy, please refer to Principle of Immunoglobulin and
T-Cell Receptor Gene Rearrangement.2

Specimen Requirement
50 ng of high-quality genomic DNA.

References

1. Tonegawa, S. (1983). Nature 302, 575-581.
2. Miller JE. (2013) Molecular Genetic Pathology (2nd Edition.,sections 30.2.7.13 and 30.2.7.18).

Simplified Representation of the TRG Gene
Depicted are the variable region (V) genes and downstream consensus joining region genes (J) that are involved in TRG gene rearrangements.

38 Next-Generation Sequencing (NGS) | CE-IVD Assays

Reagents - MiSeq™ Detection
Kit A Components

Panel Components (includes all master mixes from Kit A plus the items below)

Master Mix Name

Index #

Master Mix Name

Index #

Master Mix Name

Index #TRG MiSeq 01

A001

TRG MiSeq 09

A009

TRG MiSeq 18

A018

TRG MiSeq 02

A002

TRG MiSeq 10

A010

TRG MiSeq 19

A019

TRG MiSeq 03

A003

TRG MiSeq 11

A011

TRG MiSeq 20

A020

TRG MiSeq04

A004

TRG MiSeq 12

A012

TRG MiSeq 21

A021

TRG MiSeq 05

A005

TRG MiSeq 13

A013

TRG MiSeq 22

A022

TRG MiSeq 06

A006

TRG MiSeq 14

A014

TRG MiSeq 23

A023

TRG MiSeq 07

A007

TRG MiSeq 15

A015

TRG MiSeq 25

A025

TRG MiSeq 08

A008

TRG MiSeq 16

A016

TRG MiSeq 27

A027

Controls

Controls
TRG POS (+) Qty. 1

NGS NEG (-) Qty. 1

TRG POS (+) Qty. 3

NGS NEG (-) Qty. 3

Reagents - Ion S5™/PGM™ Detection
Assay components
Master Mix Name

Index #

Master Mix Name

Index #

Master Mix Name

Index #TRG S5/PGM 01

IonXpress_001

TRG S5/PGM 07

IonXpress_007

TRG S5/PGM 11

IonXpress_011

TRG S5/PGM 02

IonXpress_002

TRG S5/PGM 08

IonXpress_008

TRG S5/PGM 12

IonXpress_012

TRG S5/PGM 03

IonXpress_003

TRG S5/PGM 09

IonXpress_009

TRG S5/PGM 13

IonXpress_013

TRG S5/PGM 04

IonXpress_004

TRG S5/PGM 10

IonXpress_010

TRG S5/PGM 14

IonXpress_014

Controls
TRG POS (+) Qty. 2

NGS NEG (-) Qty. 2

LymphoTrack Dx TRG Assay - V -J Sequence Frequencies

V-J Sequence Frequencies. The LymphoTrack Dx bioinformatics software provides PDF
reports which include Top 10 Merged Read Summary as well as a stacked bar graphdepicting the relative frequencies of the V-J rearrangements identified in the sample.
To learn more about the LymphoTrack Dx software, please refer to the LymphoTrack Dx
Bioinformatics Software section.

Ordering Information
Catalog #Products

Quantity Components

9-227-0019

LymphoTrack Dx TRG Assay Kit A - MiSeq

8 indices - 5 sequencing reactions each

9-227-0009

LymphoTrack® Dx TRG Assay Panel - MiSeq™

24 indices - 5 sequencing reactions each

9-500-0009

LymphoTrack® Dx Software - MiSeq™

1 Software Package

9-227-0007

LymphoTrack Dx TRG Assay - S5/PGM

12 indices - 5 sequencing reactions each

9-500-0007

LymphoTrack® Dx Software - S5/PGM™

1 Software Package

®

®

™

™

These products are CE-IVD assays for in vitro diagnostic use
and available in regions that accept CE-IVD products.

Next-Generation Sequencing (NGS) | CE-IVD Assays 39

LymphoTrack Dx TRB Assay®

Assay Description
The LymphoTrack® Dx TRB Assay for the Illumina MiSeq™ is an in vitro diagnostic product intended for next-generation sequencing (NGS) based
determination of the frequency distribution of TRB gene rearrangements in patients suspected of having lymphoproliferative disease. This assay aidsin the identification of lymphoproliferative disorders.

Summary and Explanation of the Test
This assay utilizes a single multiplex master mix to target conserved V and J regions of the human TRB gene locus described in lymphoid malignancies,
thereby reducing sample DNA requirements and simplifying the testing workflow. The LymphoTrack Dx TRB master mix primers are designed with
Illumina® adapters and 8 (Kit A) or 24 (Panel) different indices. This allows amplicons generated from different indexed TRB master mixes to be pooled
into a single library for loading onto one MiSeq™ flow cell.
Positive clonal and negative polyclonal DNA controls are included in kits.
LymphoTrack Dx Software is capable of sorting complex NGS data generated by LymphoTrack Dx Assays - MiSeq™ by gene target, offering a second
layer of multiplexing. This provides users the ability to reduce per sample testing costs by sequencing amplicons from any LymphoTrack Dx Assay from
the MiSeq instrument (e.g. TRB, TRG, IGH, IGK) at the same time. In addition, the LymphoTrack Dx Software provides an easy and streamlined method
for visualization of data. Guidelines to interpret samples for evidence or no evidence of clonality are included in the instructions for use provided witheach kit.

Background
The LymphoTrack Dx TRB Assays represent a significant improvement over fragment analysis methods for clonality testing by providing two importantand complementary uses:
1. Detection of initial clonal populations.
2. Identification of sequence information required to track clonal rearrangements in subsequent samples.
Analysis of the rearranged TRB locus increases the probability of identifying T cell receptor gene rearrangements, as compared to testing for TRG generearrangements only. As a result, combining the analysis of TRB and TRG loci increases the sensitivity of clonality detection.
The human T-cell receptor beta (TRB) gene locus on chromosome 7 (7q34) includes 65 Vβ (variable) gene segments, followed by two separate clustersof genes each containing a Dβ (diversity) gene, several Jβ (joining) genes, and a Cβ (constant) region spread over 685 kilobases. The 2 Cβ genes, TRBC1
and TRBC2, encode highly homologous products with no functional difference.
During lymphoid cell development, antigen receptor genes undergo somatic gene rearrangements.1 Specifically, during T-cell development genes
encoding TRB molecules are assembled from multiple polymorphic gene segments that generate Vβ - Dβ - Jβ combinations unique in both lengthand sequence.2
Since leukemias and lymphomas originate from the malignant transformation of individual lymphoid cells, all leukemias and lymphomas generally
share one or more cell-specific or “clonal” antigen receptor gene rearrangements. Therefore, tests that detect TRB clonal rearrangements canbe useful in the study of B- and T-cell malignancies.
Note: For a more thorough explanation of the locus and the targeted deep sequencing strategy, please refer to Principle of Immunoglobulin and T-Cell
Receptor Gene Rearrangement.2

Specimen Requirement
50 ng of high-quality genomic DNA.

References
1. Tonegawa, S. (1983). Nature 302, 575-581.
2. Miller JE. (2013) Molecular Genetic Pathology (2nd Edition.,sections 30.2.7.13 and 30.2.7.18).
3. JE Miller et al., Molecular Genetic Pathology (2nd ed.). Springer Science & Business Media. 2013: 30.2.7.13.

40 Next-Generation Sequencing (NGS) | CE-IVD Assays

Simplified Representation of the TRB Gene
Vβ

Dβ1

Jβ1

Cβ1

Dβ2

Jβ2

Cβ2

Depicted are the variable (Vβ), diversity (Dβ), and joining (Jβ) gene regions involved in TRB gene rearrangements, in addition to the downstream consensus (Cβ) gene regions.

Reagents - MiSeq™ Detection
Kit A Components

Panel Components (includes all master mixes from Kit A plus the items below)

Master Mix Name

Index #

Master Mix Name

Index #

Master Mix Name

Index #TRB MiSeq 01

A001

TRB MiSeq 09

A009

TRB MiSeq 18

A018

TRB MiSeq 02

A002

TRB MiSeq 10

A010

TRB MiSeq 19

A019

TRB MiSeq 03

A003

TRB MiSeq 11

A011

TRB MiSeq 20

A020

TRB MiSeq 04

A004

TRB MiSeq 12

A012

TRB MiSeq 21

A021

TRB MiSeq 05

A005

TRB MiSeq 13

A013

TRB MiSeq 22

A022

TRB MiSeq 06

A006

TRB MiSeq 14

A014

TRB MiSeq 23

A023

TRB MiSeq 07

A007

TRB MiSeq 15

A015

TRB MiSeq 25

A025

TRB MiSeq 08

A008

TRB MiSeq 16

A016

TRB MiSeq 27

A027

Controls

Controls

TRB POS (+) Qty. 1

TRB POS (+) Qty. 3

NGS NEG (-) Qty. 1

NGS NEG (-) Qty. 3

LymphoTrack Dx TRB Assay - V -J Sequence Frequencies
V-J Sequence Frequencies. The LymphoTrack Dx bioinformatics
software provides a stacked bar graph depicting the relative
frequencies for the 200 most prevalent rearrangements
sequenced and identified in the sample. To learn more aboutthe LymphoTrack Dx software, please refer to the LymphoTrack Dx
Bioinformatics
Software section.

Ordering Information
Catalog #Products

9-225-0009

LymphoTrack® Dx TRB Assay Kit A - MiSeqTM

Quantity Components

9-225-0019

LymphoTrack Dx TRB Assay Panel - MiSeq

24 indices - 5 sequencing reactions each

9-500-0009

LymphoTrack® Dx Software - MiSeqTM

1 Software Package®

8 indices - 5 sequencing reactions each
TM

These products are CE-IVD assays for in vitro diagnostic use
and available in regions that accept CE-IVD products.

Next-Generation Sequencing (NGS) | CE-IVD Assays 41

LymphoTrack Dx
Bioinformatics Software®

Software Use
The LymphoTrack Dx Bioinformatics Software package is designed to analyze raw FASTQ files generated by LymphoTrack Dx Assays for clonality
analysis of single or multiple target data sets (IGHV Leader, IGH FR1, IGH FR2, IGH FR3, IGK, TRB, or TRG). For data generated with LymphoTrack Dx
IGHV Leader or IGH FR1 Assays, the software provides additional information, including the rate of somatic hypermutation and whether a clone would
be functional based upon the presence of a premature stop codon. The software can also predict whether an open reading frame would be in- or
out-of-frame.
The LymphoTrack Dx software is composed of two distinct parts:
1. A bioinformatics Data Analysis Application
2. Microsoft Excel® Data Visualization spreadsheets and automated Sample-to-PDF Reports for streamlined data analysis for VJ usage and VJ
sequence frequency graphs.

Sequence Frequency Graph
LymphoTrack
IGH Assay-AssayVAssay- J- VSequence
Frequencies
LymphoTrack
Dx IGHIGH-JVSequence- J Sequence
Frequencies
LymphoTrack–
Frequencies
6

Unique sequence
5% Total Reads

4

CATCTGGATACACCTTCACCAGCTACTATATGCACTGGGTGCGACAGGCCCCTGGACAAGG
GCTTGAGTGGATGGGAATAATCAACCCTAGTGGTGGTAGCACAAGCTACGCACAGAAGTTCC
AGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAG
CCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCTAGAGATCTCACAGGTTGTATTAGTACCAGCTGCTATCCTCCGAACTACTTTGACTACTGGGGCCAGGGAACCCT

3

2

Gene rearrangement family
IGHV1-46_03-

IGHJ4_02

10

The stacked bar graph depicts the top 200 sequencing reads for a sample. Each individual colored bar represents a unique population of cells. Different colors stacked at the same point on the x-axis
represent unique sequences that utilize the same V and J gene families. The amplicons of these products vary in sequence and may also vary in product size.

42 Next-Generation Sequencing (NGS) | CE-IVD Assays

Sequencing Summary
Using the merged read summary, along with the easy to follow flow charts in the LymphoTrack Dx Assay instructions for use (IFU), interpretationis quick and easy.
Sample Name

1
Total reads = 32,458

3

2

4

5

Rank

Sequence

Length

Merge
count

V-geneJ-gene% Totalreads

Cumulative%

Mutation ratepartial V-gene(%)

1

TTCTCGTGGTG

455

29603

IGHV4-59_08

IGHJ4_02

9.93

9.93

11.26

In-frame(Y/N)

No stop
codon
(Y/N)

V-coverage

Y

Y

98.63

2

CTCGCCCTCCT

463

205

IGHV5-51_01

IGHJ4_02

0.07

9.99

0.00

Y

Y

99.66

3

GGTTTTCCTTG

484

201

IGHV3-7_01

IGHJ4_02

0.07

10.06

7.77

Y

Y

100.004

CTCGCCCTCCT

463

185

IGHV5-51_01

IGHJ5_02

0.06

10.12

6.08

Y

Y

99.32

5

CTCGCCCTCCT

469

170

IGHV5-51_01

IGHJ4_02

0.06

10.18

0.00

Y

Y

99.32

6

CTCGCCCTCCT

466

160

IGHV5-51_01

IGHJ4_02

0.05

10.23

0.00

Y

Y

99.66

7

CTGCTGCTGAC

460

159

IGHV2-5_10

IGHJ5_02

0.05

10.29

8.08

Y

Y

97.64

8

GGTTTTCCTTG

493

156

IGHV3-48_02

IGHJ6_02

0.05

10.34

3.72

Y

Y

98.99

9

CTCGCCCTCCT

334

153

IGHV5-51_02

IGHJ2_01

0.05

10.39

3.72

Y

N

27.70

10

CTCGCCCTCCT

334

152

IGHV5-51_02

IGHJ2_01

0.05

10.44

3.38

Y

N

26.01

1.1 T
 he sample name is clearly identified and the total number of reads (= Read Depth) generated for the sample is provided. Following the IFU,
it is easy to determine whether the data generated for a sample can be assessed for the presence or absence of clonality.
2 The sequence of clonal populations is provided and populations are ranked from most abundant to least prevalent. Sequences that differ by 1-2
2.
bp are automatically merged to account for possible sequencing errors and to improve the accuracy and ease of sample interpretation.
33.
Sequences are aligned with reference genes to allow for easy identification of specific types of gene rearrangements such as IGHV3-21, which
is characteristic of some CLL cases and correlates with a poor prognosis.
4 The percentage that a unique sequence contributes to the total number of reads for a sample is calculated. Following the guidelines in the IFU,
4.
samples can be interpreted for the evidence indicating the presence or absence of clonality.
5 The software for LymphoTrack Dx IGHV Leader and IGH FR1 Assays automatically calculates the somatic hypermutation status of a sequence5.
by comparing the identified sequence with a germline reference. In addition, predictions on whether the sequence would be functional can be
drawn by the provided information regarding the presence of a premature stop codon or an open reading frame that is out-of-frame.

Ordering Information
Catalog #Products

Quantity Components

9-500-0009

LymphoTrack® Dx Software - MiSeq™

1 Software Package

9-500-0007

LymphoTrack® Dx Software - S5/PGM™

1 Software Package

These products are CE-IVD assays for in vitro diagnostic use
and available in regions that accept CE-IVD products.

Next-Generation Sequencing (NGS) | CE-IVD Assays 43

Next-Generation Squencing (NGS)
Research Use Only (RUO) Assays
LymphoTrack Assay kits are designed
for the identification of generearrangements in hematologic
samples utilizing next-generation
sequencing (NGS) technologies.
LEARN MORE:
catalog.invivoscribe.com

Warranty and Liability
Invivoscribe is committed to providing the highest quality products. Invivoscribe warrants that for products which are provided with Instructions for Use, these products meet or exceed theperformance standards described in the Instructions For Use. If a product is covered by product specifications and does not perform as specified, our policy is to replace the product or credit thefull purchase price. No other warranties of any kind, expressed or implied, are provided by Invivoscribe. Invivoscribe liability shall not exceed the purchase price of the product. Invivoscribe shallhave no liability for direct, indirect, consequential or incidental damages arising from the use, results of use, or inability to use its products; product efficacy under purchaser controlled conditions
in purchaser’s laboratory must be established and continually monitored through purchaser defined and controlled processes including but not limited to testing of positive, negative, and blankcontrols every time a sample is tested. Ordering, acceptance and use of product constitutes purchaser acceptance of sole responsibility for assuring product efficacy and purchaser agreement
to the limitation of liability set forth in this paragraph.
NoticeThis product is covered by one or more patents owned by or licensed to Invivoscribe, Inc., including US Patent No. 7,785,783, US Patent No. 8,859,748, US Patent No. 10,280,462 European Patent No.
1549764 and 2418287 (each validated in 16 countries), European Patent No 2460889, European Patent No. 1633884, Japanese Patent No. 4708029, Japanese Patent No. 6189600, Brazilian Patent No.
PI0410283, Canadian Patent No. 2525122, Indian Patent No. 243620, Mexican Patent No. 286493, Chinese Patent No. 1806051, and Korean Patent No. 10-1215194.
These products use nucleic acid amplification methods such as Polymerase Chain Reaction (PCR). Any necessary license to practice amplification methods or to use reagents, amplification enzymes
or equipment covered by third party patents is the responsibility of the user and no such license is granted by Invivoscribe, Inc., expressly or by implication.

Introduction
These assays take advantage of the wealth and depth of NGS data generated with either the Illumina® MiSeq™
or the Thermo Fisher Scientific® Ion PGM™ and Ion S5™ platforms. They offer significant improvements overconventional clonality testing methods, by providing the distribution of gene rearrangements, DNA sequences, themutational status (IGH only), and the ability to track specific gene rearrangements all with the same workflow.
Primers included in the master mixes are designed with Illumina® adapters and indices (8 or 24 indices configurations
for a total possible 24 or *48 unique indices [*FR1 only] per framework kits) or Thermo Fisher adapters and indices(12 indices per framework kits). By offering multiple kit configurations (8-or-24 indices for MiSeq™, 12 for Ion S5/
PGM™), Invivoscribe provides laboratories the ability to choose the optimal kit for their sample throughput andread-depth requirements. Testing costs can be reduced by sequencing in a single run, with the possibility to combine:a) samples with up to 48 different indices and b) amplicons from other LymphoTrack Assays.

Key Benefits» One-step PCR for amplicon and library generation» Identify and assess mutation status of B- and T-cell gene rearrangements» Sequence amplicons from any LymphoTrack kit together» Bioinformatics software for analysis and interpretation» Same reagents for clonality, somatic hypermutation (SHM), measurable residual disease (MRD) testing, and tracking/monitoring of immunotherapy constructs

Chapter Contents
NGS Assays
46 LymphoTrack IGHV Leader Somatic Hypermutation Assay
48 LymphoTrack IGH FR1/2/3 Assays
52 LymphoTrack IGK Assays
54 LymphoTrack TRG Assays
56 LymphoTrack TRB Assay
58 LymphoTrack Bioinformatics Software
60 LymphoTrack Enterprise Software - MiSeqTM

Research Use Only (RUO) assays are not for sale in Europe and other global markets where
equivalent CE-IVD assays are available and registered with the appropriate regulatory
agencies. For more information regarding the CE-marked LymphoTrack® Dx Assays,
please refer to the preceding pages.

Next-Generation Sequencing (NGS) | Research Use Only (RUO) 45

LymphoTrack® IGHV Leader SomaticHypermutation Assays
Assay Uses
This research use only (RUO) assay for next-generation sequencing (NGS), identifies clonal IGH VH-JH rearrangements, the associated VH-JH
DNA sequences, and the frequency distribution of VH region and JH region segment utilization. The assay also uses the Illumina® MiSeq™ platform
to define the extent of somatic hypermutation (SHM) present in the IGHV gene of analyzed samples. If you would like to test for IGHV somatic
hypermutation using the Thermo Fisher® Ion PGM™ or Ion S5™ platform please refer to the LymphoTrack IGH FR1 Assay (71210007).

Summary and Explanation of the Test
The LymphoTrack IGHV Leader Somatic Hypermutation Assay for NGS provides significant improvements over clonality assays using fragment
analysis and Sanger sequencing. The assay efficiently detects the majority of IGH gene rearrangements using a single multiplex master mix,
identifies the DNA sequence specific for each clonal gene rearrangement, and calculates the degree of SHM for each sample.
The master mixes included in this assay target the Leader (VHL) and the joining (JH) gene regions of IGH and are designed with Illumina® adaptersand indices (8 included in Kit A and 24 included in Panels). Including adapters and indices in the primer design allows for a one-step PCR approachto generate sequence-ready amplicons, followed by direct pooling of samples for sequencing on a Illumina® MiSeq™ flow cell.
Positive (clonal positive, SHM negative), negative (polyclonal), and SHM positive (clonal positive, SHM positive) controls are included in the kit.
LymphoTrack Software is designed to sort complex NGS data generated from LymphoTrack Assays by gene target, providing easy bioinformatics
analyses and data visualization. Once the clonal sequence(s) are identified, they can be monitored/tracked using the LymphoTrack MRD
Software in subsequent samples. Please see the LymphoTrack MRD software section to learn how the LymphoTrack Assays can be applied
to MRD studies, or email marketing@invivoscribe.com.

Background
The human immunoglobulin heavy chain (IGH) gene locus on chromosome 14 (14q32.3) includes 46 - 52 functional and 30 nonfunctionalvariable (VH) gene segments, 27 functional diversity (DH) gene segments, and 6 functional joining (JH) gene segments spread over 1250 kilobases.
During B-cell development, genes encoding the IGH molecules are assembled from multiple polymorphic gene segments that undergo
rearrangements and selection, generating VH-DH-JH combinations that are unique in both length and sequence.1
Since leukemias and lymphomas originate from the malignant transformation of individual lymphoid cells, all leukemias and lymphomas
generally share one or more cell-specific or "clonal" antigen receptor gene rearrangements. Therefore, tests that detect IGH clonalrearrangements can be useful in the study of B-cell malignancies. An additional level of diversity is generated in the antigen receptors bysomatic point mutations in the variable regions and this mutation status provides important prognostic information for chronic lymphocytic
leukemia (CLL) 2 and small lymphocytic lymphoma (SLL). In addition, NGS methods can improve disease stratification and elucidate subclone
gene profiles.

Specimen Requirement
50 ng of high-quality genomic DNA.

References
1. Miller et al., Molecular Genetic Pathology (2nd ed.). Springer Science & Business Media. 2013: 302.2.7.13 and 30.2.7.18.
2. Ghia et al., Leukemia 21: 2-3 (2007).

VH L

VH 1

VH 2

VH 3

VH 4

VH 5

VH 6

VH 7

JH

Simple representation of the organization of the immunoglobulin heavy chain (IGH) gene on chromosome 14. Depicted are the variable region (VH) genes anddownstream consensus joining region genes (JH) that are involved in rearrangements. Upstream of the variable gene segments, the leader sequence (VHL) is
also depicted. Diversity region genes are not depicted.

46 Next-Generation Sequencing (NGS) | Research Use Only (RUO)

Reagents - MiSeq™ Detection
Kit A Components

Panel Components (includes all master mixes from Kit A plus the items below)

Master Mix Name

Index #

Master Mix Name

Index #

Master Mix Name

Index #

IGH Leader MiSeq 01

A001

IGH Leader MiSeq 09

A009

IGH Leader MiSeq 18

A018

IGH Leader MiSeq 02

A002

IGH Leader MiSeq 10

A010

IGH Leader MiSeq 19

A019

IGH Leader MiSeq 03

A003

IGH Leader MiSeq 11

A011

IGH Leader MiSeq 20

A020

IGH Leader MiSeq 04

A004

IGH Leader MiSeq 12

A012

IGH Leader MiSeq 21

A021

IGH Leader MiSeq 05

A005

IGH Leader MiSeq 13

A013

IGH Leader MiSeq 22

A022

IGH Leader MiSeq 06

A006

IGH Leader MiSeq 14

A014

IGH Leader MiSeq 23

A023

IGH Leader MiSeq 07

A007

IGH Leader MiSeq 15

A015

IGH Leader MiSeq 25

A025

IGH Leader MiSeq 08

A008

IGH Leader MiSeq 16

A016

IGH Leader MiSeq 27

A027

Controls

Controls

IGH SHM POS (+) Qty. 1

IGH SHM POS (+) Qty. 3

IGH POS (+) Qty. 1

IGH POS (+) Qty. 3

NGS NEG (-) Qty. 1

NGS NEG (-) Qty. 3

Rank

Sequence

Length

Merge
count

V-geneJ-gene% Totalreads

Cumulative%

Mutation ratepartial V-gene(%)

In-frame(Y/N)

No stop
codon
(Y/N)

V-coverage

1

TTCTCGTGGTG

455

29603

IGHV4-59_08

IGHJ4_02

9.93

9.93

11.26

Y

Y

98.63

2

CTCGCCCTCCT

463

205

IGHV5-51_01

IGHJ4_02

0.07

9.99

0.00

Y

Y

99.66

3

GGTTTTCCTTG

484

201

IGHV3-7_01

IGHJ4_02

0.07

10.06

7.77

Y

Y

100.004

CTCGCCCTCCT

463

185

IGHV5-51_01

IGHJ5_02

0.06

10.12

6.08

Y

Y

99.32

5

CTCGCCCTCCT

469

170

IGHV5-51_01

IGHJ4_02

0.06

10.18

0.00

Y

Y

99.32

6

CTCGCCCTCCT

466

160

IGHV5-51_01

IGHJ4_02

0.05

10.23

0.00

Y

Y

99.66

7

CTGCTGCTGAC

460

159

IGHV2-5_10

IGHJ5_02

0.05

10.29

8.08

Y

Y

97.64

8

GGTTTTCCTTG

493

156

IGHV3-48_02

IGHJ6_02

0.05

10.34

3.72

Y

Y

98.99

9

CTCGCCCTCCT

334

153

IGHV5-51_02

IGHJ2_01

0.05

10.39

3.72

Y

N

27.70

10

CTCGCCCTCCT

334

152

IGHV5-51_02

IGHJ2_01

0.05

10.44

3.38

Y

N

26.01

Example Data. Depicted are the top 10 sequences from a read summary generated by the LymphoTrack Software - MiSeqTM. Highlighted columns represent fields that are unique to SHM analysis andinclude the SHM mutation rate and predictions pertaining to whether a sequence is in-frame or contains a premature stop codon. To learn more about the LymphoTrack software, please refer to the
LymphoTrack Bioinformatics Software section.

Ordering Information
Catalog #Products

7-121-0059

LymphoTrack® IGHV Leader Somatic Hypermutation Assay Kit A - MiSeq™

Quantity Components

7-121-0069

LymphoTrack IGHV Leader Somatic Hypermutation Assay Panel - MiSeq

24 indices - 5 sequencing reactions each

7-500-0009

LymphoTrack® Software - MiSeq™

1 Software Package®

8 indices - 5 sequencing reactions each™

These products are for Research Use Only (RUO). Not intended for diagnostic purposes.

Next-Generation Sequencing (NGS) | Research Use Only (RUO) 47

LymphoTrack® IGH FR1/FR2/FR3 Assays
Assay Uses
These research use only (RUO) assays for next-generation sequencing (NGS), identify clonal IGH VH-JH rearrangements, the associated VH-JH region
DNA sequences, the frequency distribution of VH region and JH region segment utilization. The LymphoTrack FR1 can also identify the degree of somatic
hypermutation (SHM) of rearranged genes using the Illumina® MiSeq™, Thermo Fisher Scientific® Ion PGM™ or Ion S5™. The LymphoTrack IGH FR1, IGH FR2,and IGH FR3 Assays contain primers that target the conserved framework 1 (FR1), framework 2 (FR2), and framework 3 (FR3) regions, respectively.
The LymphoTrack IGH FR1/2/3 Assay kits contain the master mixes of all three frameworks.

Summary and Explanation of the Test
The LymphoTrack IGH Assays represent a significant improvement over clonality assays that utilize fragment analysis by providing four importantand complementary uses:
1. Detection of clonal populations.
2. Identification of sequence information and VH-JH segment utilization.
3. The LymphoTrack IGH FR1 Assays provide the degree of SHM of the immunoglobulin variable heavy chain (IGHV).
4. The ability to track sequences in subsequent samples with the Invivoscribe LymphoTrack MRD Software.
Each single multiplex master mix targets one of the conserved IGH framework regions (FR1, FR2, or FR3) within the VH and the JH regions describedin lymphoid malignancies. Targeting all three framework regions significantly reduces the risk of not being able to detect the presence of clonality,
as somatic hypermutations in the primer binding sites of the involved VH gene segments can impede DNA amplification.1
Primers included in the master mixes are designed with Illumina® adapters and indices (8 included in Kit A, 24 included in the Panel, and an
independent 24 included in the Panel B) or Thermo Fisher adaptors and indices (12 indices per framework kits). This allows for a one-step PCR
reaction to generate sequence-ready amplicons and pooling of several different samples on the same Illumina® MiSeq™ cell, Ion S5 or Ion PGM chip.
Positive clonal (SHM negative) and negative polyclonal DNA controls are included in kits. A clonal SHM positive control can be purchased separately(Catalog # 40880008).
LymphoTrack Software is designed to sort complex NGS data generated from LymphoTrack Assays by gene target, providing easy bioinformatics
analyses and data visualization. Once the clonal sequence(s) are identified, they can be monitored/tracked using the LymphoTrack MRD Softwarein subsequent samples.. Please see the LymphoTrack MRD software section to learn how the LymphoTrack Assays can be applied to MRD studies, oremail marketing@invivoscribe.com.

Background
The human immunoglobulin heavy chain (IGH) gene locus on chromosome 14 (14q32.3) includes 46-52 functional and 30 nonfunctional variable(VH) gene segments, 27 functional diversity (DH) gene segments, and 6 functional joining (JH) gene segments spread over 1250 kilobases.
During development of lymphoid cells, the antigen receptor genes go through somatic gene rearrangements.1 For example, during B-celldevelopment, genes encoding the IGH molecules are assembled from multiple polymorphic gene segments that undergo rearrangements andselection, generating VH-DH-JH combinations. Since leukemias and lymphomas originate from the malignant transformation of individual lymphoidcells, all leukemias and lymphomas generally share one or more cell-specific or “clonal” antigen receptor gene rearrangements. Therefore, tests that
detect IGH clonal populations can be useful in the study of B- and T-cell malignancies.

Specimen Requirement
50 ng of high-quality genomic DNA.

Reference
1. S Tonegawa. Nature 302: 575-581 (1983).

DH
VL

VH-FR1

VH-FR2JH

VH-FR3

JH primers

VH family primers

Simple representation of the organization of the immunoglobulin heavy chain (IGH) gene locus on chromosome 14. Depicted are the variable region (VH) genes and downstream consensus joining region
genes segments (JH) that are involved in rearrangements.

48 Next-Generation Sequencing (NGS) | Research Use Only (RUO)

V-J Sequence Frequencies. The LymphoTrack Software provides a stacked bar graph depicting the relative frequencies for the 200 most prevalent
VH-JH rearrangements identified in a sample. To learn more about the LymphoTrack software, please refer to the LymphoTrack Bioinformatics
Software section.

Ordering Information
Catalog #Products

7-121-0129

LymphoTrack® IGH FR1/2/3 Assay Kit A - MiSeq™

Quantity Components

7-121-0139

LymphoTrack IGH FR1/2/3 Assay Panel - MiSeq

Indices 1-24 (5 sequencing reactions each)

7-121-0009

LymphoTrack® IGH FR1 Assay Kit A - MiSeq™

Indices 1-8 (5 sequencing reactions each)

7-121-0039

LymphoTrack IGH FR1 Assay Panel - MiSeq

Indices 1-24 (5 sequencing reactions each)

7-121-0149

LymphoTrack® IGH FR1 Assay Panel B - MiSeq™

Indices 25-48 (5 sequencing reaction each)

7-121-0089

LymphoTrack® IGH FR2 Assay Kit A - MiSeq™

Indices 1-8 (5 sequencing reactions each)

7-121-0099

LymphoTrack IGH FR2 Assay Panel - MiSeq

7-121-0109

LymphoTrack® IGH FR3 Assay Kit A - MiSeq™

7-121-0119

LymphoTrack IGH FR3 Assay Panel - MiSeq

Indices 1-24 (5 sequencing reactions each)

7-500-0009

LymphoTrack® Software - MiSeq™

1 Software Package

7-121-0057

LymphoTrack IGH FR1/2/3 Assay - S5/PGM

12 + 12 + 12 indices - 5 sequencing reactions each

7-121-0007

LymphoTrack® IGH FR1 Assay - S5/PGM™

12 indices - 5 sequencing reactions each

7-121-0037

LymphoTrack® IGH FR2 Assay - S5/PGM™

12 indices - 5 sequencing reactions each

7-121-0047

LymphoTrack IGH FR3 Assay - S5/PGM

12 indices - 5 sequencing reactions each

7-500-0007

LymphoTrack® Software - S5/PGM™

1 Software Package

7-500-0008

LymphoTrack MRD Software

1 Software Package

®

®

™

®

™

™

®

™

™

®

Indices 1-24 (5 sequencing reactions each)
Indices 1-8 (5 sequencing reactions each)®

®

Indices 1-8 (5 sequencing reactions each)™

These products are for Research Use Only (RUO). Not intended for diagnostic purposes.

Next-Generation Sequencing (NGS) | Research Use Only (RUO) 49

Reagents - MiSeqTM Detection
The LymphoTrack IGH FR1/2/3 Assays contain components from respective individual FR Kit A's, Panel's or Panel B.

LymphoTrack IGH FR2 Components

LymphoTrack IGH FR3 Components

Index #

Master Mix Name

Index #

IGH FR2 MiSeq 01

A001

IGH FR3 MiSeq 01

A001

IGH FR1 MiSeq 02

A002

IGH FR2 MiSeq 02

A002

IGH FR3 MiSeq 02

A002

IGH FR1 MiSeq 03

A003

IGH FR2 MiSeq 03

A003

IGH FR3 MiSeq 03

A003

IGH FR1 MiSeq 04

A004

IGH FR2 MiSeq 04

A004

IGH FR3 MiSeq 04

A004

IGH FR1 MiSeq 05

A005

IGH FR2 MiSeq 05

A005

IGH FR3 MiSeq 05

A005

IGH FR1 MiSeq 06

A006

IGH FR2 MiSeq 06

A006

IGH FR3 MiSeq 06

A006

IGH FR1 MiSeq 07

A007

IGH FR2 MiSeq 07

A007

IGH FR3 MiSeq 07

A007

IGH FR1 MiSeq 08

A008

IGH FR2 MiSeq 08

A008

IGH FR3 MiSeq 08

A008

IGH FR1 MiSeq 09

A009

IGH FR2 MiSeq 09

A009

IGH FR3 MiSeq 09

A009

IGH FR1 MiSeq 10

A010

IGH FR2 MiSeq 10

A010

IGH FR3 MiSeq 10

A010

IGH FR1 MiSeq 11

A011

IGH FR2 MiSeq 11

A011

IGH FR3 MiSeq 11

A011

IGH FR1 MiSeq 12

A012

IGH FR2 MiSeq 12

A012

IGH FR3 MiSeq 12

A012

IGH FR1 MiSeq 13

A013

IGH FR2 MiSeq 13

A013

IGH FR3 MiSeq 13

A013

IGH FR1 MiSeq 14

A014

IGH FR2 MiSeq 14

A014

IGH FR3 MiSeq 14

A014

IGH FR1 MiSeq 15

A015

IGH FR2 MiSeq 15

A015

IGH FR3 MiSeq 15

A015

IGH FR1 MiSeq 16

A016

IGH FR2 MiSeq 16

A016

IGH FR3 MiSeq 16

A016

IGH FR1 MiSeq 18

A018

IGH FR2 MiSeq 18

A018

IGH FR3 MiSeq 18

A018

IGH FR1 MiSeq 19

A019

IGH FR2 MiSeq 19

A019

IGH FR3 MiSeq 19

A019

IGH FR1 MiSeq 20

A020

IGH FR2 MiSeq 20

A020

IGH FR3 MiSeq 20

A020

IGH FR1 MiSeq 21

A021

IGH FR2 MiSeq 21

A021

IGH FR3 MiSeq 21

A021

IGH FR1 MiSeq 22

A022

IGH FR2 MiSeq 22

A022

IGH FR3 MiSeq 22

A022

IGH FR1 MiSeq 23

A023

IGH FR2 MiSeq 23

A023

IGH FR3 MiSeq 23

A023

IGH FR1 MiSeq 25

A025

IGH FR2 MiSeq 25

A025

IGH FR3 MiSeq 25

A025

IGH FR1 MiSeq 27

A027

IGH FR2 MiSeq 27

A027

IGH FR3 MiSeq 27

A027

IGH FR1 MiSeq 17

A017

IGH FR1 MiSeq 24

A024

IGH FR1 MiSeq 26

A026

IGH FR1 MiSeq 28

A028

IGH FR1 MiSeq 29

A029

IGH FR1 MiSeq 30

A030

IGH FR1 MiSeq 31

A031

IGH FR1 MiSeq 32

A032

IGH FR1 MiSeq 33

A033

IGH FR1 MiSeq 34

A034

IGH FR1 MiSeq 35

A035

IGH FR1 MiSeq 36

A036

IGH FR1 MiSeq 37

A037

PANEL

PANEL
PANEL B

50 Next-Generation Sequencing (NGS) | Research Use Only (RUO)

KIT A

Master Mix Name

A001

KIT A

Index #

IGH FR1 MiSeq 01

KIT A

Master Mix Name

PANEL

LymphoTrack IGH FR1 ComponentsReagents - MiSeq™ Detection Continued
The LymphoTrack IGH FR1/2/3 Assays contain components from respective individual FR Kit A's, Panel's or Panel B.

LymphoTrack IGH FR1 Components

LymphoTrack IGH FR2 Components

LymphoTrack IGH FR3 Components

Master Mix Name

Index #

Master Mix Name

Master Mix Name

IGH FR1 MiSeq 38

A038

IGH FR1 MiSeq 39

A039

IGH FR1 MiSeq 40

A040

IGH FR1 MiSeq 41

A041

IGH FR1 MiSeq 42

A042

IGH FR1 MiSeq 43

A043

IGH FR1 MiSeq 44

A044

IGH FR1 MiSeq 45

A045

IGH FR1 MiSeq 46

A046

IGH FR1 MiSeq 47

A047

IGH FR1 MiSeq 48

A048

IGH FR1 MiSeq 49

A049

Controls in Individual
FR (1, 2, or 3) Kit A's

Controls in Individual
FR (1,2, or 3) Panels

Controls in Combo
FR 1/2/3 Kit A

Controls in Combo
FR 1/2/3 Panel

IGH POS (+) Qty. 1

IGH POS (+) Qty. 3

IGH POS (+) Qty. 2

IGH POS (+) Qty. 6

NGS NEG (-) Qty. 1

NGS NEG (-) Qty. 3

NGS NEG (-) Qty. 2

NGS NEG (-) Qty. 6

Index #PANEL B

Index #

Reagents - Ion S5™/PGM™ Detection
The LymphoTrack IGH FR1/2/3 Assays contain components from respective individual FR Assays.

LymphoTrack IGH FR1 Components

LymphoTrack IGH FR2 Components

LymphoTrack IGH FR3 Components

Master Mix Name

Index #

Master Mix Name

Index #

Master Mix Name

Index #

IGH FR1 S5/PGM 01

IonXpress_001

IGH FR2 S5/PGM 01

IonXpress_001

IGH FR3 S5/PGM 01

IonXpress_001

IGH FR1 S5/PGM 02

IonXpress_002

IGH FR2 S5/PGM 02

IonXpress_002

IGH FR3 S5/PGM 02

IonXpress_002

IGH FR1 S5/PGM 03

IonXpress_003

IGH FR2 S5/PGM 03

IonXpress_003

IGH FR3 S5/PGM 03

IonXpress_003

IGH FR1 S5/PGM 04

IonXpress_004

IGH FR2 S5/PGM 04

IonXpress_004

IGH FR3 S5/PGM 04

IonXpress_004

IGH FR1 S5/PGM 07

IonXpress_007

IGH FR2 S5/PGM 07

IonXpress_007

IGH FR3 S5/PGM 07

IonXpress_007

IGH FR1 S5/PGM 08

IonXpress_008

IGH FR2 S5/PGM 08

IonXpress_008

IGH FR3 S5/PGM 08

IonXpress_008

IGH FR1 S5/PGM 09

IonXpress_009

IGH FR2 S5/PGM 09

IonXpress_009

IGH FR3 S5/PGM 09

IonXpress_009

IGH FR1 S5/PGM 10

IonXpress_010

IGH FR2 S5/PGM 10

IonXpress_010

IGH FR3 S5/PGM 10

IonXpress_010

IGH FR1 S5/PGM 11

IonXpress_011

IGH FR2 S5/PGM 11

IonXpress_011

IGH FR3 S5/PGM 11

IonXpress_011

IGH FR1 S5/PGM 12

IonXpress_012

IGH FR2 S5/PGM 12

IonXpress_012

IGH FR3 S5/PGM 12

IonXpress_012

IGH FR1 S5/PGM 13

IonXpress_013

IGH FR2 S5/PGM 13

IonXpress_013

IGH FR3 S5/PGM 13

IonXpress_013

IGH FR1 S5/PGM 14

IonXpress_014

IGH FR2 S5/PGM 14

IonXpress_014

IGH FR3 S5/PGM 14

IonXpress_014

Controls in Individual FR (1,2, or 3) Kits

Controls in FR 1/2/3 Kit

IGH POS (+) Qty. 2

IGH POS (+) Qty. 4

NGS NEG (-) Qty. 2

NGS NEG (-) Qty. 4

These products are for Research Use Only (RUO). Not intended for diagnostic purposes.

Next-Generation Sequencing (NGS) | Research Use Only (RUO) 51

LymphoTrack® IGK Assay
Assay Uses
This research use only (RUO) assay for next-generation sequencing (NGS), identifies clonal IGK Vƙ-Jƙ, Vƙ-Kde, and intron-Kde (INTR-Kde)rearrangements, the corresponding DNA sequences, and provides the distribution frequency of Vƙ-Jƙ, Vƙ-Kde, and INTR-Kde segment utilization
using the Illumina® MiSeq™ or Thermo Fisher Scientific® Ion PGM™ and Ion S5™ platforms.

Summary and Explanation of the Test
The LymphoTrack IGK Assay represents a significant improvement over clonality assays that utilize fragment analysis by providing three importantand complementary uses:
1. Detection of clonal populations.
2. Identification of sequence information and gene segment utilization.
3. Ability to track sequences in subsequent samples with the use of the LymphoTrack MRD Software.
Unlike conventional fragment analysis assays, this NGS method utilizes a single multiplex master mix to target conserved regions of IGK that aredescribed in lymphoid malignancies. Primers are designed with Illumina® adapters and indices (8-24) or Thermo Fisher Scientific adaptors andindices (12), thereby allowing for a one-step PCR reaction to generate sequence-ready amplicons. To reduce per sample testing costs, amplicons
from different samples (amplified with different indexed master mixes) or LymphoTrack kits can be sequenced together on a single Illumina®
MiSeq™ flow cell, Ion S5™ or PGM™ chips.
Positive (clonal) and negative (polyclonal) DNA controls are included in kits.
LymphoTrack Software is designed to sort complex NGS data generated from LymphoTrack Assays by gene target, providing easy bioinformatics
analyses and data visualization. Once the clonal sequence(s) are identified, they can be monitored/tracked using the LymphoTrack MRD
Software in subsequent samples. Please see the LymphoTrack MRD software section to learn how the LymphoTrack Assays can be applied
to MRD studies, or email marketing@invivoscribe.com.

Background
The human immunoglobulin kappa (IGK) gene locus on chromosome 2 (2p11.2) includes 76 V (variable) region genes spanning 7 subgroups and 5 J(joining) region gene segments upstream of the Cƙ region. The Kde, approximately 24 kb downstream of the Jƙ-Cƙ region, can also rearrange with Vƙgene segments and the isolated recombination signal sequence in the Jƙ-Cƙ intronic region.
During development of lymphoid cells, antigen receptor genes undergo somatic gene rearrangements.1 Specifically during B-cell development, genes
encoding IGK molecules are assembled from multiple polymorphic gene segments that undergo rearrangements generating V-J combinations uniquein both length and sequence.2 Since leukemias and lymphomas originate from the malignant transformation of individual lymphoid cells, all leukemias
and lymphomas generally share one or more cell-specific or “clonal” antigen receptor gene rearrangements. Therefore, tests that detect IGK clonal
populations can be useful in the study of B-cell malignancies and complement IGH testing, as the IGK receptor is less susceptible to somatic mutations.

Specimen Requirement
50 ng of high-quality genomic DNA.

References
1. S Tonegawa et al., Nature 302: 575-581 (1983).
2. JE Miller et al., Molecular Genetic Pathology (2nd ed.). Springer Science & Business Media. 2013: 30.2.7.13 and 30.2.7.18.

Simplified Representation of the IGK Gene
Depicted are the variable region (Vƙ) genes or variable intragenic Jƙ-Cƙ intron (Jƙ-Cƙ INTR) and downstream consensus joining region genes (Jƙ) or kappa deleting
element (Kde) that are involved in IGK gene rearrangements.

Kde
Kde

52 Next-Generation Sequencing (NGS) | Research Use Only (RUO)

Reagents - MiSeq™ Detection
Kit A Components

Panel Components (includes all master mixes from Kit A plus the items below)

Master Mix Name

Index #

Master Mix Name

Index #

Master Mix Name

Index #IGK MiSeq 01

A001

IGK MiSeq 09

A009

IGK MiSeq 18

A018

IGK MiSeq 02

A002

IGK MiSeq 10

A010

IGK MiSeq 19

A019

IGK MiSeq 03

A003

IGK MiSeq 11

A011

IGK MiSeq 20

A020

IGK MiSeq 04

A004

IGK MiSeq 12

A012

IGK MiSeq 21

A021

IGK MiSeq 05

A005

IGK MiSeq 13

A013

IGK MiSeq 22

A022

IGK MiSeq 06

A006

IGK MiSeq 14

A014

IGK MiSeq 23

A023

IGK MiSeq 07

A007

IGK MiSeq 15

A015

IGK MiSeq 25

A025

IGK MiSeq 08

A008

IGK MiSeq 16

A016

IGK MiSeq 27

A027

Controls
IGK POS (+) Qty. 1

Controls
NGS NEG (-) Qty. 1

IGK POS (+) Qty. 3

NGS NEG (-) Qty. 3

Reagents - Ion S5/PGM™ Detection
Assay Components
Master Mix Name

Index #

Master Mix Name

Index #IGK S5/PGM 01

IonXpress_001

IGK S5/PGM 11

IonXpress_011

IGK S5/PGM 02

IonXpress_002

IGK S5/PGM 12

IonXpress_012

IGK S5/PGM 04

IonXpress_004

IGK S5/PGM 13

IonXpress_013

IGK S5/PGM 08

IonXpress_008

IGK S5/PGM 14

IonXpress_014

IGK S5/PGM 09

IonXpress_009

IGK S5/PGM 16

IonXpress_016

IGK S5/PGM 010

IonXpress_010

IGK S5/PGM 17

IonXpress_017

Controls
IGK POS (+) Qty. 2

NGS NEG (-) Qty. 2

V-J Sequence Frequencies.
The LymphoTrack bioinformatics software provides a stacked bar graph depicting
the relative frequencies for the 200 most prevalent rearrangements sequenced
and identified in the sample. To learn more about the LymphoTrack software,please refer to the LymphoTrack Bioinformatics Software section.

Ordering Information
Catalog #Products

7-122-0009

LymphoTrack® IGK Assay Kit A - MiSeq™

Quantity Components

7-122-0019

LymphoTrack IGK Assay Panel - MiSeq

24 indices - 5 sequencing reactions each

7-500-0009

LymphoTrack® Software - MiSeq™

1 Software Package

7-122-0007

LymphoTrack IGK Assay - S5/PGM

12 indices - 5 sequencing reactions each

7-500-0007

LymphoTrack® Software - S5/PGM™

1 Software Package

7-500-0008

LymphoTrack MRD Software*

1 Software Package

®

®

®

8 indices - 5 sequencing reactions each™

™

*MRD Software can be used to track sequences generated by either
LymphoTrack Assays - MiSeq™ or Ion S5/PGM™.
These products are for Research Use Only (RUO). Not intended for
diagnostic purposes.

Next-Generation Sequencing (NGS) | Research Use Only (RUO) 53

LymphoTrack® TRG Assay
Assay Uses
This research use only (RUO) assay for next-generation sequencing (NGS) identifies clonal TRG V-J rearrangements and the associated V-J region
DNA sequences. It also provides the frequency distribution of V-J segment utilization using the Illumina® MiSeq™ or Thermo Fisher Scientific® Ion S5™
and PGM™ instruments.

Summary and Explanation of the Test
The LymphoTrack TRG Assay represents a significant improvement over existing clonality assays that utilize fragment analysis by providing threeimportant and complementary uses:
1. Detection of clonal populations.
2. Identification of sequence information and gene segment utilization.
3. Ability to track sequences in subsequent samples with the use of the Invivoscribe MRD Software.
This assay utilizes a single multiplex master mix to target conserved V and J regions of the human TRG gene locus described in lymphoidmalignancies, thereby reducing sample DNA requirements and simplifying the testing workflow. Primers are designed with Illumina® adapters andindices (8-24) or Thermo Fisher Scientific adaptors and indices (12), thereby allowing for a one-step PCR reaction to generate sequence-ready
amplicons. In addition, amplicons from different samples (amplified with different indexed master mixes) or LymphoTrack kits can be sequenced
together on a single Illumina® MiSeq™ flow cell Ion S5™ or PGM™ chip to reduce per sample testing costs.
Positive (clonal) and negative (polyclonal) DNA controls are included in kits.
LymphoTrack Software is designed to sort complex NGS data generated from LymphoTrack Assays by gene target, providing easy bioinformatics
analyses and data visualization. Once the clonal sequence(s) are identified, they can be monitored/tracked using the LymphoTrack MRD Softwarein subsequent samples. Please see the LymphoTrack MRD software section to learn how the LymphoTrack Assays can be applied to MRD studies,or email marketing@invivoscribe.com.

Background
The human T-cell receptor gamma (TRG) gene locus on chromosome 7 (7q14) includes 14 variable region (Group I, II, III, and IV), 5 joining region(J) gene segments, and 2 constant (C) genes spread over 200 kilobases.
During development of lymphoid cells, the antigen receptor genes (including gene segments within the TRG locus), undergo somatic generearrangement.1 These developmentally regulated gene rearrangements generate V-J combinations that are unique for each cell.2 Since leukemias
and lymphomas originate from the malignant transformation of individual lymphoid cells, all leukemias and lymphomas generally share one or morecell-specific or “clonal” antigen receptor gene rearrangements. Therefore, tests that detect TRG clonal populations can be useful in the study of T-celland certain B-cell malignancies. Since the TRG locus rearranges before the TRB locus, and all unsuccessful or unproductive rearrangements are
retained in the cells, examination of the TRG locus can identify both clonal Δ/ƴ as well as clonal α/β T-cells. Clonal α/β T-cells would be expected
to have biallelic TRG gene rearrangements.
Note | For a more thorough explanation of the locus and the targeted deep sequencing strategy, please refer to Principle of Immunoglobulin and
T-Cell Receptor Gene Rearrangement.2

Specimen Requirement
50 ng of high-quality genomic DNA.

References
1. Tonegawa, S. (1983). Nature 302, 575-581.
2. Miller JE. (2013) Molecular Genetic Pathology (2nd Edition.,sections 30.2.7.13 and 30.2.7.18).

Simplified Representation of the TRG Gene
Simple representation of the organization of the T-cell receptor gamma gene on chromosome 7. Depicted are the variable region genes (V ƴ 2–V ƴ 11) anddownstream joining region genes (J ƴ 1/2–J ƴ P1/2) that are involved in rearrangements in T-cell lymphomas.

54 Next-Generation Sequencing (NGS) | Research Use Only (RUO)

Reagents - MiSeq™ Detection
Kit A Components

Panel Components (includes all master mixes from Kit A plus the items below)

Master Mix Name

Index #

Master Mix Name

Index #

Master Mix Name

Index #TRG MiSeq 01

A001

TRG MiSeq 09

A009

TRG MiSeq 18

A018

TRG MiSeq 02

A002

TRG MiSeq 10

A010

TRG MiSeq 19

A019

TRG MiSeq 03

A003

TRG MiSeq 11

A011

TRG MiSeq 20

A020

TRG MiSeq 04

A004

TRG MiSeq 12

A012

TRG MiSeq 21

A021

TRG MiSeq 05

A005

TRG MiSeq 13

A013

TRG MiSeq 22

A022

TRG MiSeq 06

A006

TRG MiSeq 14

A014

TRG MiSeq 23

A023

TRG MiSeq 07

A007

TRG MiSeq 15

A015

TRG MiSeq 25

A025

TRG MiSeq 08

A008

TRG MiSeq 16

A016

TRG MiSeq 27

A027

Controls
TRG POS (+) Qty. 1

Controls
TRG POS (+) Qty. 3

NGS NEG (-) Qty. 1

NGS NEG (-) Qty. 3

Reagents - Ion S5/PGM™ Detection
Assay Components
Master Mix Name

Index #

Master Mix Name

Index #

Master Mix Name

Index #TRG S5/PGM 01

IonXpress_001

TRG S5/PGM 07

IonXpress_007

TRG S5/PGM 11

IonXpress_011

TRG S5/PGM 02

IonXpress_002

TRG S5/PGM 08

IonXpress_008

TRG S5/PGM 12

IonXpress_012

TRG S5/PGM 03

IonXpress_003

TRG S5/PGM 09

IonXpress_009

TRG S5/PGM 13

IonXpress_013

TRG S5/PGM 04

IonXpress_004

TRG S5/PGM 10

IonXpress_010

TRG S5/PGM 14

IonXpress_014

Controls
TRG POS (+) Qty. 2

NGS NEG (-) Qty. 2

V - J Sequence Frequencies.
The LymphoTrack bioinformatics software provides a stacked bar graphdepicting the relative frequencies for the V-J rearrangements identified andsequenced in a sample. To learn more about the LymphoTrack software,please refer to the LymphoTrack Bioinformatics Software section.

Ordering Information
Catalog #Products

7-227-0019

LymphoTrack® TRG Assay Kit A - MiSeq™

Quantity Components

7-227-0009

LymphoTrack TRG Assay Panel - MiSeq

24 indices - 5 sequencing reactions each

7-500-0009

LymphoTrack® Software - MiSeq™

1 Software Package

7-227-0007

LymphoTrack TRG Assay - S5/PGM

12 indices - 5 sequencing reactions each

7-500-0007

LymphoTrack® Software - S5/PGM™

1 Software Package

7-500-0008

LymphoTrack® MRD Software*

1 Software Package®

®

8 indices - 5 sequencing reactions each™

™

*MRD Software can be used to track sequences generated by either LymphoTrack Assays - MiSeq™ or Ion S5/PGM™.
These products are for Research Use Only (RUO). Not intended for diagnostic purposes.

Next-Generation Sequencing (NGS) | Research Use Only (RUO) 55

LymphoTrack® TRB Assay
Assay Uses
This research use only (RUO) assay for next-generation sequencing (NGS) identifies clonal TRB Vβ-(Dβ-)Jβ rearrangements, the associated Vβ-(Dβ-)Jβregion DNA sequences, and provides the frequency distribution of Vβ, Dβ, and Jβ region segment utilization using the Illumina® MiSeq™ platform.
Analysis of the rearranged TRB locus increases the probability of identifying T-cell receptor gene rearrangements, as compared to testing for TRG
gene rearrangements only. As a result, combining the analysis of TRB and TRG loci increases the sensitivity of clonality detection.

Summary and Explanation of the Test
This assay utilizes a single multiplex master mix to target conserved V, D and J regions of the human TRB gene locus described in lymphoidmalignancies, thereby reducing sample DNA requirements and simplifying the testing workflow.
The LymphoTrack TRB master mix primers are also designed with Illumina® adapters and 8 indices (Kit A) or 24 indices (Panel). This allows amplicons
generated from different indexed TRB master mixes to be pooled into a single sequencing library.
Positive (clonal) and negative (polyclonal) DNA controls are included in kits.
LymphoTrack Software is designed to sort complex NGS data generated from LymphoTrack Assays by gene target, providing easy bioinformatics
analyses and data visualization. Once the clonal sequence(s) are identified, they can be monitored/tracked using the LymphoTrack MRD Softwarein subsequent samples. Please see the LymphoTrack MRD software section to learn how the LymphoTrack Assays can be applied to MRD studies,or email marketing@invivoscribe.com.

Background
The LymphoTrack TRB Assay represent a significant improvement over clonality assessment by fragment analysis by providing two importantand complementary uses:
1. Detection of clonal populations.
2. Identification of sequence information and gene segment utilization.
3. Ability to track sequences in subsequent samples with the use of the Invivoscribe MRD Software.
The human T-cell receptor beta (TRB) gene locus on chromosome 7 (7q34) includes 65 Vβ (variable) gene segments, followed by two separate clustersof genes each containing a Dβ (diversity) gene, several Jβ (joining) genes, and a Cβ (constant) region spread over 685 kilobases. The two Cβ genes,
TRBC1 and TRBC2, encode highly homologous products with no functional difference.
During lymphoid cell development, antigen receptor genes undergo somatic gene rearrangements.1 Specifically, during T-cell development, genes
encoding TRB molecules are assembled from multiple polymorphic gene segments that generate Vβ-Dβ-Jβ combinations unique in both length andsequence.2
Since leukemias and lymphomas originate from the malignant transformation of individual lymphoid cells, all leukemias and lymphomas generally
share one or more cell-specific or “clonal” antigen receptor gene rearrangements. Therefore, tests that detect TRB clonal rearrangements canbe useful in the study of B- and T-cell malignancies.
Note: For a more thorough explanation of the locus and the targeted deep sequencing strategy, please refer to Principle of Immunoglobulin
and T-Cell Receptor Gene Rearrangement.2

Specimen Requirement
50 ng of high-quality genomic DNA.

References
1. Tonegawa, S. (1983). Nature 302, 575-581.
2. Miller JE. (2013) Molecular Genetic Pathology (2nd Edition.,sections 30.2.7.13 and 30.2.7.18).

56 Next-Generation Sequencing (NGS) | Research Use Only (RUO)

Simplified Representation of the TRB Gene
Vβ

Dβ1

Jβ1

Cβ1

Dβ2

Jβ2

Cβ2

Depicted are the variable (Vβ), diversity (Dβ), and joining (Jβ) gene regions involved in TRB gene rearrangements, in addition to the downstream
consensus (Cβ) gene regions.

Reagents - MiSeq™ Detection
Kit A Components

Panel Components (includes all master mixes from Kit A plus the items below)

Master Mix Name

Index #

Master Mix Name

Index #

Master Mix Name

Index #TRB MiSeq 01

A001

TRB MiSeq 09

A009

TRB MiSeq 18

A018

TRB MiSeq 02

A002

TRB MiSeq 10

A010

TRB MiSeq 19

A019

TRB MiSeq 03

A003

TRB MiSeq 11

A011

TRB MiSeq 20

A020

TRB MiSeq 04

A004

TRB MiSeq 12

A012

TRB MiSeq 21

A021

TRB MiSeq 05

A005

TRB MiSeq 13

A013

TRB MiSeq 22

A022

TRB MiSeq 06

A006

TRB MiSeq 14

A014

TRB MiSeq 23

A023

TRB MiSeq 07

A007

TRB MiSeq 15

A015

TRB MiSeq 25

A025

TRB MiSeq 08

A008

TRB MiSeq 16

A016

TRB MiSeq 27

A027

Controls
TRB POS (+) Qty. 1

Controls
TRB POS (+) Qty. 3

NGS NEG (-) Qty. 1

NGS NEG (-) Qty. 3

V-J Sequence Frequencies.
The LymphoTrack bioinformatics software provides
PDF reports which include Top 10 Merged Read
Summary as well as a stacked bar graph depicting
the relative frequencies of the 200 most prevalent
rearrangements sequenced and identified in thesample. To learn more about the LymphoTracksoftware, please refer to the LymphoTrack
Bioinformatics Software section.

Ordering Information
Catalog #Products

Quantity Components

7-225-0009

LymphoTrack® TRB Assay Kit A - MiSeqTM

8 indices - 5 sequencing reactions each

7-225-0019

LymphoTrack® TRB Assay Panel - MiSeqTM

24 indices - 5 sequencing reactions each

7-500-0009

LymphoTrack Software - MiSeq

1 Software Package

7-500-0008

LymphoTrack® MRD Software*

1 Software Package®

TM

These products are for Research Use Only (RUO). Not intended for diagnostic purposes.

Next-Generation Sequencing (NGS) | Research Use Only (RUO) 57

LymphoTrack® Bioinformatics Software
Software Use
The LymphoTrack Bioinformatics Software package is used with LymphoTrack Assays to analyze raw FASTQ files for clonality analysis of single
or multiple target data sets (IGHV Leader), IGH FR1, IGH FR2, IGH FR3, IGK, TRG, TRB). For data generated with LymphoTrack IGHV Leader
or IGH FR1 Assays the software provides additional information, including the rate of somatic hypermutation (SHM) and whether a clone will
be functional based upon the presence of a premature stop codon. The software can also predict whether an open reading frame would
be in- or out-of-frame, so no external data analysis is required for sample interpretation.
The provided software is composed of two distinct parts:
1. A bioinformatics Data Analysis Application
2. Microsoft Excel® Data Visualization spreadsheets for each gene target and automated Sample-to-PDF Reports for streamlined data analysis.

Sequence Frequency Graph
LymphoTrack LymphoTrack
IGH Assay- V - J Sequence Frequencies
IGH Assay - V – J Sequence Frequencies
6

5% Total Reads

4

Unique sequence
CATCTGGATACACCTTCACCAGCTACTATATGCACTGGGTGCGACAGGCCCCTGGACAAGG
GCTTGAGTGGATGGGAATAATCAACCCTAGTGGTGGTAGCACAAGCTACGCACAGAAGTTCC
AGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAG
CCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCTAGAGATCTCACAGGTTGTATTAGTACCAGCTGCTATCCTCCGAACTACTTTGACTACTGGGGCCAGGGAACCCT

3

2

Gene rearrangement family
IGHV1-46_03-

IGHJ4_02

10

The stacked bar graph depicts the top 200 sequencing reads for a sample. Each individual colored bar represents a unique population of cells. Different colors stacked at the same point on the x-axis
represent unique sequences that utilize the same V and J gene families. The amplicons of these products vary in sequence and may also vary in product size.

58 Next-Generation Sequencing (NGS) | Research Use Only (RUO)

Sequencing Summary
Using the merged read summary interpretation is quick and easy.

1Easy identification of specific types of gene rearrangements such as IGHV3-21.

Rank

Sequence

Length

Merge
count

V-geneJ-gene% Totalreads

Cumulative%

Mutation ratepartial V-gene (%)

In-frame(Y/N)

No stop
codon
(Y/N)

V-coverage

1

TTCTCGTGGTG

455

29603

IGHV4-59_08

IGHJ4_02

9.93

9.93

11.26

Y

Y

98.63

2

CTCGCCCTCCT

463

205

IGHV5-51_01

IGHJ4_02

0.07

9.99

0.00

Y

Y

99.66

3

GGTTTTCCTTG

484

201

IGHV3-7_01

IGHJ4_02

0.07

10.06

7.77

Y

Y

100.004

CTCGCCCTCCT

463

185

IGHV5-51_01

IGHJ5_02

0.06

10.12

6.08

Y

Y

99.32

5

CTCGCCCTCCT

469

170

IGHV5-51_01

IGHJ4_02

0.06

10.18

0.00

Y

Y

99.32

6

CTCGCCCTCCT

466

160

IGHV5-51_01

IGHJ4_02

0.05

10.23

0.00

Y

Y

99.66

7

CTGCTGCTGAC

460

159

IGHV2-5_10

IGHJ5_02

0.05

10.29

8.08

Y

Y

97.64

8

GGTTTTCCTTG

493

156

IGHV3-48_02

IGHJ6_02

0.05

10.34

3.72

Y

Y

98.99

9

CTCGCCCTCCT

334

153

IGHV5-51_02

IGHJ2_01

0.05

10.39

3.72

Y

N

27.70

10

CTCGCCCTCCT

334

152

IGHV5-51_02

IGHJ2_01

0.05

10.44

3.38

Y

N

26.01

2

3

Identification of clonal sequences forfollow up tracking with LymphoTrack
MRD Software.

SHM rate and indicators to determine
whether a clone is productive. Only
provided for IGHV Leader and IGH FR1.

The read summary provides sequences from a sample ranked from most abundant to least prevalent. The total read count for individual sequences
is provided and no independent analysis is required to determine V and J gene families and predictions for SHM when analyzing data fromLymphoTrack IGHV Leader or IGH FR1 Assays. Additionally, the software provides raw and merged data in which reads that differ by 1-2 bp areautomatically merged to account for possible sequencing errors and to improve the accuracy and ease of sample interpretation.

Ordering Information
Catalog #Products

Quantity Components

7-500-0009

LymphoTrack® Software- MiSeq™

1 Software Package

7-500-0007

LymphoTrack® Software - S5/PGM™

1 Software Package

These products are for Research Use Only (RUO). Not intended for diagnostic purposes.

Next-Generation Sequencing (NGS) | Research Use Only (RUO) 59

LymphoTrack® Enterprise
Software - MiSeqTM
Software Use
When used with the LymphoTrack® Assays, the LymphoTrack® Enterprise
Software enables fully automated pipeline bioinformatics data analysis forclonality using the raw FASTQ files from the Illumina® MiSeqTM. The DockerizedLinux-based software is built for portability, flexibility and can easily be integrated
into downstream reporting systems and/or LIMS.

Product Details
The LymphoTrack® Enterprise Software – MiSeqTM package was designed to enable automated pipelines for laboratories that generate highvolumes of sequencing data using the LymphoTrack Assays. This Docker image enables clonal analysis of single or multiple targets (IGHV Leader,
IGH FR1, IGH FR2, IGH FR3, IGK, TRG and/or TRB) and additionally provides the somatic hypermutation (SHM) status of samples run with theLeader or IGH FR1 assays. The FASTQ files that are output from a MiSeqTM sequencing run of samples prepared with LymphoTrack Assays, areautomatically processed and output into a tab-separated value (.tsv) file using the LymphoTrack Enterprise Software. The analysis is initiatedby either REST-API or command line interface. The data output can then be visualized in Microsoft Excel or imported into custom reports.

Ordering Information
Catalog #Products

Quantity Components

INQUIRE

LymphoTrack® Enterprise Software- MiSeq™

1 Software Package

This product is for Research Use Only (RUO). Not intended for diagnostic purposes.

60 Next-Generation Sequencing (NGS) | Research Use Only (RUO)

Measurable Residual
Disease (MRD) Solutions
Research Use Only (RUO)
MRD testing with Next-Generation
Sequencing is a proven tool that may
be used to develop hematologic
malignancy management strategies.
LEARN MORE:
catalog.invivoscribe.com

Warranty and Liability
Invivoscribe is committed to providing the highest quality products. Invivoscribe warrants that for products which are provided with Instructions for Use, these products meet or exceed theperformance standards described in the Instructions For Use. If a product is covered by product specifications and does not perform as specified, our policy is to replace the product or credit
the full purchase price. No other warranties of any kind, expressed or implied, are provided by Invivoscribe. Invivoscribe liability shall not exceed the purchase price of the product. Invivoscribe
shall have no liability for direct, indirect, consequential or incidental damages arising from the use, results of use, or inability to use its products; product efficacy under purchaser controlled
conditions in purchaser’s laboratory must be established and continually monitored through purchaser defined and controlled processes including but not limited to testing of positive, negative,
and blank controls every time a sample is tested. Ordering, acceptance and use of product constitutes purchaser acceptance of sole responsibility for assuring product efficacy and purchaser
agreement to the limitation of liability set forth in this paragraph.
NoticeSome of these products are covered by one or more patents owned by or licensed to Invivoscribe, Inc., including US Patent No. 7,785,783, US Patent No. 8,859,748, US Patent No. 10,280,462
European Patent No. 1549764 and 2418287 (each validated in 16 countries) European Patent No 2460889, European Patent No. 1633884, Japanese Patent No. 4708029, Japanese Patent No.
6189600, Brazilian Patent No. PI0410283, Canadian Patent No. 2525122, Indian Patent No. 243620, Mexican Patent No. 286493, Chinese Patent No. 1806051, and Korean Patent No. 10-1215194.
These products use nucleic acid amplification methods such as Polymerase Chain Reaction (PCR). Any necessary license to practice amplification methods or to use reagents, amplification
enzymes or equipment covered by third party patents is the responsibility of the user and no such license is granted by Invivoscribe, Inc., expressly or by implication.
Research Use Only (RUO) assays are not for sale in Europe and other global markets where equivalent CE-IVD assays are available and registered with the appropriate regulatory agencies.

61 Measurable Residual Disease (MRD) Solution | Research Use Only (RUO)

Introduction
A number of investigators have described NGS-based approaches that have demonstrated success in detecting
and monitoring MRD in Chronic Lymphocytic Leukemia (CLL), Acute Lymphoblastic Leukemia (ALL) and otherlymphoid malignancies.1,2 LymphoTrack Assays are NGS-based deep sequencing assays that detect virtually all clonalrearrangements within targeted T-cell receptor (TCR) or immunoglobulin (Ig) antigen receptor loci. Once a specificrearrangement (the clonotype) has been identified, LymphoTrack assays can be used to track these clonotype
populations to a sensitivity as low as 10-6. The LymphoTrack MRD solution includes assays, controls and software
to facilitate objective MRD assessments over time.
B- or T-cell bundles may be purchased that include the LymphoTrack Assay, LymphoTrack MRD Bioinformatics
Software, a LymphoTrack Low Positive Control and a LymphoQuant Internal Control. Each bundle facilitates thestandardization of MRD testing by providing controls suitable for longitudinal MRD tracking with test sensitivity
assurance. LymphoTrack MRD Software further simplifies clonal tracking due to rich sequence specific data analyses.
This software enables objective monitoring of clonal populations by providing multiple functionalities to the user
including project planning features and automated bioinformatics applications.
When monitoring MRD, a highly sensitive detection method such as NGS-based LymphoTrack may aid in the early
detection of lymphoproliferative disease relapse. However, MRD test results are dependent on DNA amounts
interrogated, as well as the confidence level of the test. Controls tracking MRD test sensitivity are thus necessary
when reporting MRD test results. Designed for MRD testing, the LymphoTrack Low Positive Control confirms thesensitivity of respective LymphoTrack MRD runs match or exceed a 10-4 (or 1 in 10,000) level. Detection of theLymphoTrack Low Positive Control thus lessens false negative reporting concerns at 10-4, and is further necessary
to report MRD negative results with confidence at 10-4.
Consistent use of a spike-in internal control enables objective monitoring of clonality over time and test
standardization. Invivoscribe now offers both a LymphoQuant B-cell and T-cell Internal Control. Addition of a spikein LymphoQuant B-cell or T-cell Internal Control to samples allows the software to convert percent clonal reads into
estimated clonal cell equivalents.

Key Benefits» Complete solution for MRD» Ensures test sensitivity to enable confidence in reporting» Facilitates standardization of clonotype tracking» Longitudinal assessment of mutation status of FLT3 ITD, NPM1, B- and T-cell gene rearrangements and somatic
hypermutation (SHM)» LymphoTrack Assays formatted for both Illumina® and Thermo Fisher® NGS Platforms

Chapter Contents
RUO MRD Assays
63 LymphoTrack MRD Software
65 FLT3 ITD MRD Assay
67 NPM1 MRD Assay

References
1. Leukemia. 27:1659-1665, 2013.
2. Blood. 120:5173-5180, 2012.

Measurable Residual Disease (MRD) Solution | Research Use Only (RUO) 62

LymphoTrack® MRD Software
Software Use
The LymphoTrack MRD Bioinformatics Software package may be purchased to aid in the evaluation of treatment response in many lymphoidmalignancies such as Acute Lymphoblastic Leukemia and Multiple Myeloma to minimize the risk of patients relapsing. The exceptional sensitivity
and precision of NGS-based MRD tracking can accelerate clinical trials and drug development. This MRD software is intended to detect thepresence of clonotype sequences within the output files generated using the Invivoscribe LymphoTrack Assays and accompanying LymphoTrackbioinformatics software; it is not intended to define the significance of these findings. Once a specific rearrangement sequence (the clonotype)has been identified in a primary sample, the MRD software enables streamlined tracking of clonal populations at a sensitivity of 10-4, or evenlower limits provided sufficient DNA is tested. The MRD software can be used to Create, Save and Load projects to objectively track up to 5 clonalsequences to monitor for disease relapse and for use in drug development studies.
The provided software is composed of four distinct parts:
1. A Project tool that can be used to plan experiments with sufficient confidence based on read depth, replicate count, and DNA input.
Projects can also be Saved and Loaded for use in subsequent time points.
2. A bioinformatics data analysis application.
3. A PDF Sample Report identifying the clonal sequence(s) if present, a summary of the degree of mismatches, calculations of the read frequency
and the degree of confidence if clonal sequence(s) are not detected at various sensitivities.
4. A PDF Summary Report that will automatically generate longitudinal graphs of up to 5 clonal frequencies for a Subject and summary tables
with estimates of clonal cell equivalents and clonal frequencies for all queried sequences if LymphoQuant Internal Controls is used.

MRD Project Planner
The Project Planner can be used to calculate the confidence of a true negative by adding replicate counts, resequencing counts, sequencing
depth, and DNA input amount. The software assumes that the same sequencing depth and DNA input is used for each replicate.

Research Use Only (RUO) assays are not for sale in Europe and other global
markets where equivalent CE-IVD assays are available and registered with theappropriate regulatory agencies.

63 Measurable Residual Disease (MRD) Solution | Research Use Only (RUO)

LymphoTrack MRD Sample Report
This report summarizes the overall call, i.e. if a clonotype was detected or not detected, the number of checked replicates, the total DNA input,
total reads analyzed and the location of all output files. The values in the PDF report are also found in the generated text files.

MRD Summary Graph

Note: The MRD Report may slightly differ from what is shown.

Ordering Information
Catalog #Products

Quantity Components

7-500-0008

LymphoTrack® MRD Software**

1 Software Package** MRD Software can be used to track sequences generated using either LymphoTrack®
Assays formatted for either the Illumina® or Thermo Fisher® NGS platforms.
MRD applications are for Research Use Only (RUO). Not intended for diagnostic purposes.

Measurable Residual Disease (MRD) Solution | Research Use Only (RUO) 64

FLT3 ITD MRD Assay
Assay Use
This Research Use Only (RUO) assay identifies internal tandem duplications (ITD) of the FLT3 gene.

Summary and Explanation of the Test
FLT3 ITD MRD is an NGS-based assay for use on the Illumina® MiSeq™. FLT3 ITD MRD Assay tracks internal tandem duplication mutations of the FLT3
gene to identify measurable residual disease in subjects following acute myeloid leukemia diagnosis (AML) and treatment. The assay is an ampliconbased approach which uses polymerase chain reaction to amplify the region of interest and next-generation sequencing to detect the regionof interest.
The assay kit includes 24 master mixes, along with a ready to use positive and negative controls. Primers included in the master mix are designedwith Illumina® adapters containing unique dual-indexes allowing for multiplexing at two levels. First, the multiplexing of up to 21 unique individuals
in parallel in a single sequencing run, and second, the multiplexing of multiple targets in a single sequencing run.
The FLT3 ITD MRD Assay is for use with the Illumina® MiSeq™ platform. It was designed to produce sequencing data that can be analyzed using the
FLT3 ITD MRD Software package. The FLT3 ITD MRD Software is a Dockerized Linux-based package built for portability, flexibility, and can easily
be integrated into downstream reporting system and/or LIMS. It provides automated bioinformatic analyses of raw FASTQ files generated by the
Illumina® MiSeq™, when used with the FLT3 ITD MRD Assay. Individual sample and aggregate sample data output is generated in under an hourin the form of .tsv (tab-separated value) files.
This deep-sequencing assay can detect internal tandem duplications up to 252 bp at an RUO validated allelic sensitivity of 5x10-5.

Background
FLT3 mutations are one of the most common mutations in AML, occurring in roughly ~30% of adult AML subjects.1 The internal tandem duplicationis the most common FLT3 mutation and is present in about 25% of AML subjects.1,2 FLT3 ITD is considered a driver mutation at diagnosis andin relapsed AML,1,3 conferring a higher relapse rate and poor prognosis.1 Since FLT3 ITD mutations are present in 75% of relapsed subjects
which were FLT3 ITD-mutated at diagnosis,3 ITDs can serve as a biomarker to monitor for hematological disease progression and can guidetherapeutic decisions.

Specimen Requirements
700 ng DNA isolated from whole blood or bone marrow.

References
1. Levis, M. Hematology Am Soc Hematol Educ Program (2013) 2013:220-6.
2. Daver, N. et al., Leukemia (2019) 33:299-312.
3. Krönke, J. et al., Blood (2013) 122(1):100-8.
Figure 1. FLT3 ITD MRD Assay Workflow

06

SOFTWARE

65 Measurable Residual Disease (MRD) Solution | Research Use Only (RUO)

Reagents
Dual-indexed Master Mix

Index # (I7 Index ID)

Index # (I5 Index ID)

FLT3 ITD IA13

N701

N502

FLT3 ITD IA14

N702

N504

FLT3 ITD IA15

N703

N506

FLT3 ITD IA16

N704

N508

FLT3 ITD IA17

N705

N501

FLT3 ITD IA18

N706

N503

FLT3 ITD IA19

N707

N505

FLT3 ITD IA20

N708

N507

FLT3 ITD IA21

N709

N502

FLT3 ITD IA22

N710

N504

FLT3 ITD IA23

N711

N506

FLT3 ITD IA24

N712

N508

FLT3 ITD IB01

N702

N501

FLT3 ITD IB02

N703

N503

FLT3 ITD IB03

N704

N505

FLT3 ITD IB04

N705

N507

FLT3 ITD IB05

N706

N502

FLT3 ITD IB06

N707

N504

FLT3 ITD IB07

N708

N506

FLT3 ITD IB08

N709

N508

FLT3 ITD IB09

N710

N501

FLT3 ITD IB10

N711

N503

FLT3 ITD IB11

N712

N505

FLT3 ITD IB12

N701

N507

Controls

Concentration

Units in 96 Reaction Assay

FLT3 ITD MRD POS (+)

20 ng/µL

2 x 500 µL tube

FLT3 ITD MRD NEG (-)

20 ng/µL

2 x 500 µL tube

Catalog #Products

Quantity Components

14120019

FLT3 ITD Assay

24 indices - 4 sequencing reactions each

S100005

FLT3 ITD MRD Software

1 Software Package

Ordering Information

These products are for Research Use Only (RUO). Not intended for diagnostic purposes.

Measurable Residual Disease (MRD) Solution | Research Use Only (RUO) 66

NPM1 MRD Assay
Assay Use
This Research Use Only (RUO) assay identifies four base pair insertions of the NPM1 gene.

Summary and Explanation of the Test
NPM1 MRD is an NGS-based assay for use on the Illumina® MiSeq™. NPM1 MRD Assay tracks four base pair insertions in the NPM1 gene to identify
measurable residual disease in subjects following acute myeloid leukemia diagnosis (AML) and treatment. The assay is an amplicon-based approach
which uses polymerase chain reaction to amplify the region of interest and next-generation sequencing to detect the region of interest.
The assay kit includes 24 master mixes, along with a ready to use positive and negative controls. Primers included in the master mix are designedwith Illumina® adapters containing unique dual-indexes allowing for multiplexing at two levels. First, the multiplexing of up to 21 unique individuals
in parallel in a single sequencing run, and second, the multiplexing of multiple targets in a single sequencing run.
The NPM1 MRD Assay is for use with the Illumina® MiSeq™ platform was designed to produce sequencing data that can be analyzed using the NPM1MRD Software package. The NPM1 MRD Software is a Dockerized Linux-based package built for portability, flexibility, and can easily be integrated
into downstream reporting system and/or LIMS. It provides automated bioinformatic analyses of raw FASTQ files generated by the Illumina® MiSeq™,
when used with the NPM1 MRD Assay. Individual sample and aggregate sample data output is generated in under an hour in the form of .tsv
(tab-separated value) files.
This deep-sequencing assay can detect four base mutation insertions characterized as ‘A’, ‘B’, ‘D’, or ‘Other’ at an RUO validated allelic sensitivityof 5x10-5.

Background
One of the most commonly mutated genes in AML, NPM1 gene mutations occur in about one-third of the cases of primary AML in adults.1 While risk
stratification of NPM1-mutated AML is ever evolving,2 NPM1 gene mutations are common,1 AML specific,3 and its mutant transcripts are expressedat high levels,2 making the mutation an ideal target for monitoring measurable residual disease2 to guide therapeutic decisions.

Specimen Requirements
700 ng DNA isolated from whole blood or bone marrow.

References
1. Falini, B. et al., N Engl J Med. (2005) 352:254-266.
2. Falini, B. et al., Leukemia (2021) 35:3113-3126.
3. Liso, A. et al., Leukemia (2008) 22:1285–1289.

Figure 1. NPM1 MRD Assay Workflow

06

SOFTWARE

67 Measurable Residual Disease (MRD) Solution | Research Use Only (RUO)

Reagents
Dual-indexed Master Mix

Index # (I7 Index ID)

Index # (I5 Index ID)

NPM1 MRD IA01

N701

N501

NPM1 MRD IA02

N702

N503

NPM1 MRD IA03

N703

N505

NPM1 MRD IA04

N704

N507

NPM1 MRD IA06

N706

N504

NPM1 MRD IA07

N707

N506

NPM1 MRD IA08

N708

N508

NPM1 MRD IA09

N709

N501

NPM1 MRD IA10

N710

N503

NPM1 MRD IA11

N711

N505

NPM1 MRD IA12

N712

N507

NPM1 MRD IA25

N705

N502

NPM1 MRD IB13

N702

N502

NPM1 MRD IB14

N703

N504

NPM1 MRD IB15

N704

N506

NPM1 MRD IB16

N705

N508

NPM1 MRD IB17

N706

N501

NPM1 MRD IB18

N707

N503

NPM1 MRD IB19

N708

N505

NPM1 MRD IB21

N710

N502

NPM1 MRD IB22

N711

N504

NPM1 MRD IB23

N712

N506

NPM1 MRD IB24

N701

N508

NPM1 MRD IB26

N709

N507

Controls

Concentration

Units in 96 Reaction Assay

NPM1 MRD POS (+)

20 ng/µL

2 x 500 µL tube

NPM1 MRD NEG (-)

20 ng/µL

2 x 500 µL tube

Catalog #Products

Quantity Components

14160019

NPM1 MRD Assay

24 indices - 4 sequencing reactions each

S100004

NPM1 MRD Software

1 Software Package

Ordering Information

For information regarding pricing and availability, please contact Invivoscribe at inquiry@invivoscribe.com

These products are for Research Use Only (RUO). Not intended for diagnostic purposes.

Measurable Residual Disease (MRD) Solution | Research Use Only (RUO) 68

Capillary - CE-IVD Assays

IdentiClone Assay kits are CE-marked
in vitro diagnostic products.*

LEARN MORE:
catalog.invivoscribe.com

Warranty and Liability
Invivoscribe is committed to providing the highest quality products. Invivoscribe warrants that for products which are provided with Instructions for Use, these products meet or exceed theperformance standards described in the Instructions For Use. If a product is covered by product specifications and does not perform as specified, our policy is to replace the product or credit thefull purchase price. No other warranties of any kind, expressed or implied, are provided by Invivoscribe. Invivoscribe liability shall not exceed the purchase price of the product. Invivoscribe shallhave no liability for direct, indirect, consequential or incidental damages arising from the use, results of use, or inability to use its products; product efficacy under purchaser controlled conditions
in purchaser’s laboratory must be established and continually monitored through purchaser defined and controlled processes including but not limited to testing of positive, negative, and blankcontrols every time a sample is tested. Ordering, acceptance and use of product constitutes purchaser acceptance of sole responsibility for assuring product efficacy and purchaser agreement
to the limitation of liability set forth in this paragraph.
NoticeThis product is covered by one or more patents licensed to Invivoscribe Inc, including European Patent No. 1549764 and 2418287 (each validated in 16 countries), European Patent No. 2460889,
Japanese Patent No. 4708029, United States Patent No. 8859748 and United States Patent No. 10280462.
These products require nucleic acid amplification methods such as Polymerase Chain Reaction (PCR). No license under these patents to use amplification processes or enzymes is conveyed expressly
or by implication to the purchaser by the purchase of these products.
Identiclone® is a registered trademark of Invivoscribe®.

Introduction
These kits are intended for PCR-based detection of clonal gene rearrangements and translocations in patients withsuspected lymphoproliferations, using capillary electrophoresis methods.
The Invivoscribe CE-marked IdentiClone Assays represent a simple, cost-effective approach to PCR-based clonality
testing. These standardized assays were carefully optimized testing positive and negative control samples using
multiplex master mixes. Assay development was followed by extensive validation including the testing of more than 400
clinical samples using Revised European/American Lymphoma (REAL) Classification. Testing was performed at morethan thirty prominent independent testing centers throughout Europe in a collaborative study known as the BIOMED-2
Concerted Action.1 These PCR-based tests include standardized Instructions For Use (IFUs) with interpretation guidelines
describing the use of master mixes and controls. The same thermal cycler program and similar detection methods areused for all IdentiClone kits to improve consistency, reduce human error, and facilitate cross-training.

Chapter Contents
B-cell Assays

T-cell Assays

71 IdentiClone® IGH + IGK B-Cell Clonality Assays
73 IdentiClone® IGH Gene Clonality Assays
75 IdentiClone® IGK Gene Clonality Assays
77 IdentiClone® IGL B-Cell Clonality Assays

79 IdentiClone® TCRB Gene Clonality Assays
81 IdentiClone® T-Cell Receptor Gama Gene Rearrangement Assay 2.0
83 IdentiClone® TCRD Gene Clonality Assays

Reference:
1. JJM van Dongen et al., (2003) Leukemia 17:2257-2317.

IdentiClone Assays are in vitro diagnostic products and are not available for sale or use
within North America. For more information regarding the research use only reagents,
please see the Gel & Capillary Research Use Only Assays section.

®
Capillary | CE-IVD Assays 70

IdentiClone® IGH + IGK B-Cell
Clonality Assays
Assay Description
The IdentiClone IGH + IGK B-Cell Clonality Assay is an in vitro diagnostic product intended for PCR-based detection of clonal immunoglobulin
heavy chain and kappa light chain gene rearrangements in patients with suspect lymphoproliferations. Specifically, the IGH + IGK B-Cell
Clonality Assay can be used to:• Identify clonality in atypical lymphoproliferative disorders• Support a differential diagnosis between reactive lesions and hematologic malignancies4• Assign presumptive lineage in mature monoclonal lymphoproliferative disorders• Identify tumor-specific markers (IGH and IGK gene rearrangements) for post-treatment monitoring• Monitor and evaluate disease recurrence

Summary and Explanation of the Test
These kits include six master mixes to test for rearrangements of both IGH and IGK. The IGH Tube A, B, and C master mixes target the framework
1, 2, 3 regions (respectively) within the variable (VH) region, and the joining (JH) region of the immunoglobulin heavy chain locus. The IGK Tube
A master mix targets the variable (Vƙ) and the joining (Jƙ) region. IGK Tube B master mix targets kappa deleting element (Kde) rearrangementswith the variable (Vƙ) region and the intragenic Jƙ-Cƙ region. The resulting Vƙ-Kde and Jƙ-Cƙ intron-Kde rearrangements are a result of unsuccessful
rearrangements retained by the B cell. For best sensitivity, it is recommended to test suspect B-cell malignancies for both IGH and IGK.3 The
included 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.

Performance Characteristics
Data from two independent studies that tested more than 300 patient samples of varying types suggests the diagnostic accuracy of selected
IdentiClone tests to be 96%. In both peer-reviewed studies, there were no clear false-positive results generated using the IdentiClone tests,
and there was a high level of precision.2 The clinico-histopathological diagnosis correlated well with PCR results in a higher numberof patients when compared with Southern Blot (SB) results, as seen below:
PCR/SB concordance:1
IGH:
93% sensitivity / 92% specificity
IGK:
90% sensitivity / 90% specificity
PCR vs. SB analysis relative to histopathology and final diagnosis:
PCR/SB concordance:2
IGH + IGK: 85%

PCR sensitivity:98%

SB sensitivity:
39%

References1 . JJM van Dongen et al., Leukemia 17:2257-2317 (2003).
2. Y Sandberg et al., J. Mol. Diag. 7(4):495-503 (2005).
3. Van Krieken, JHJM et al., Leukemia 21:201 - 206 (2007).

This assay is based on the EuroClonality/BIOMED-2 Concerted Action BMH4-CT98-3936.

®
71 Capillary | CE-IVD Assays

κ

Figure Legend: Simple representation of the organization of a rearranged immunoglobulin heavy chain (IGH) gene on chromosome 14q32.33 and the immunoglobulin
kappa light chain gene on chromosome 2p11.2. Black arrows represent the relative positions of primers that target the conserved framework regions (FR1-3) and thedownstream consensus JH gene segments for IGH and the Vƙ, Jƙ, INTR and Kde primers which are included in the IGK master mix tubes.

κReagents
Controls

Concentration

Units in Assay

Units in Assay MegaKit

IVS-0030 Clonal Control DNA

200 μg/mL

1 x 100 μL tube

5 x 100 μL tubes

IVS-0019 Clonal Control DNA

200 μg/mL

1 x 100 μL tube

5 x 100 μL tubes

IVS-0007 Clonal Control DNA

200 μg/mL

1 x 100 μL tube

5 x 100 μL tubes

IVS-0000 Polyclonal Control DNA

200 μg/mL

1 x 100 μL tube

5 x 100 μL tubes

Master Mixes

TargetUnits in Assay

Units in Assay MegaKit

IGH Tube A

Framework 1 + JH

1 x 1500 μL tube

10 x 1500 μL tubes

IGH Tube B

Framework 2 + JH

1 x 1500 μL tube

10 x 1500 μL tubes

IGH Tube C

Framework 3 + JH

1 x 1500 μL tube

10 x 1500 μL tubes

IGK Tube AVƙ-Jƙ

1 x 1500 μL tube

10 x 1500 μL tubes

IGK Tube B

Vƙ-Kde, Intron-Kde

1 x 1500 μL tube

10 x 1500 μL tubes

Specimen Control Size LadderMultiple Genes

1 x 1500 μL tube

10 x 1500 μL tubes

Ordering Information
Catalog #Products

Quantity

9-100-0031

IdentiClone IGH + IGK B-Cell Clonality Assay - ABI Fluorescence Detection

33 reactions

9-100-0041

IdentiClone IGH + IGK B-Cell Clonality Assay MegaKit - ABI Fluorescence Detection

330 reactions®

®

These are in vitro diagnostic products and are available in regions that accept CE-IVD products.

®
Capillary | CE-IVD Assays 72

IdentiClone® IGH Gene Clonality Assays
Assay Description
The IdentiClone IGH Gene Clonality Assay is an in vitro diagnostic product intended for PCR-based detection of clonal immunoglobulin heavy chain
gene rearrangements in patients with suspect lymphoproliferations.
Specifically, the IGH Gene Clonality Assay can be used to:• Identify clonality in atypical lymphoproliferative disorders• Support a differential diagnosis between reactive lesions and hematologic malignancies4• Assign presumptive lineage in mature monoclonal lymphoproliferative disorders• Identify tumor-specific markers (IGH gene rearrangements) for post-treatment monitoring• Monitor and evaluate disease recurrence

Summary and Explanation of the Test
These test kits include six master mixes. The IGH Tube A, B, and C master mixes target the framework 1, 2, and 3 regions (respectively) within thevariable (VH) region and the joining (JH) region of the immunoglobulin heavy chain locus. The IGH Tube D and E master mixes target the diversity andjoining regions. 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.

Performance Characteristics
Data from two independent studies that tested more than 300 patient samples of varying types suggests the diagnostic accuracy of selected
IdentiClone tests to be 96%. In both peer-reviewed studies, there were no clear false-positive results generated using the IdentiClone tests, andthere was a high level of precision.2 The clinico-histopathological diagnosis correlated well with PCR results in a higher number of patients whencompared with Southern Blot (SB) results, as seen below:
PCR/SB concordance:1		
IGH:
93% sensitivity / 92% specificity
PCR vs. SB analysis relative to histopathology and final diagnosis:
PCR/SB concordance:2
IGH + IGK: 85%

PCR sensitivity:98%

SB sensitivity:
39%

References
1. JJM van Dongen et al., Leukemia 17:2257-2317 (2003).
2. Y Sandberg et al., J. Mol. Diag. 7(4):495-503 (2005).

This assay is based on the EuroClonality/BIOMED-2 Concerted Action BMH4-CT98-3936.

®
73 Capillary | CE-IVD Assays

Figure Legend: Simple representation of the organization of a rearranged immunoglobulin heavy chain gene on chromosome 14q32.33. Black arrows represent therelative positions of primers that target the conserved framework (FR1-3) and diversity (DH1-7) regions, and the downstream consensus JH gene segments. The amplicon
products generated from each of these regions can be differentially detected when fluorescent primer sets are used with capillary electrophoresis instruments that employ
differential fluorescence detection.

Reagents
Controls

Concentration

Units in Assay

Units in Assay MegaKit

IVS-0030 Clonal Control DNA

200 μg/mL

1 x 100 μL tube

5 x 100 μL tubes

IVS-0019 Clonal Control DNA

200 μg/mL

1 x 100 μL tube

5 x 100 μL tubes

IVS-0024 Clonal Control DNA

200 μg/mL

1 x 100 μL tube

5 x 100 μL tubes

IVS-0008 Clonal Control DNA

200 μg/mL

1 x 100 μL tube

5 x 100 μL tubes

IVS-0000 Polyclonal Control DNA

200 μg/mL

1 x 100 μL tube

5 x 100 μL tubes

Master Mixes

TargetUnits in Assay

Units in Assay MegaKit

IGH Tube A

Framework 1 + JH

1 x 1500 μL tube

10 x 1500 μL tubes

IGH Tube B

Framework 2 + JH

1 x 1500 μL tube

10 x 1500 μL tubes

IGH Tube C

Framework 3 + JH

1 x 1500 μL tube

10 x 1500 μL tubes

IGH Tube D

DH1-6 + JH

1 x 1500 μL tube

10 x 1500 μL tubes

IGH Tube E

DH7 + JH

1 x 1500 μL tube

10 x 1500 μL tubes

Specimen Control Size LadderMultiple Genes

1 x 1500 μL tube

10 x 1500 μL tubes

Ordering Information
Catalog #Products

Quantity

9-101-0061

IdentiClone IGH Gene Clonality Assay - ABI Fluorescence Detection

33 reactions

9-101-0081

IdentiClone® IGH Gene Clonality Assay MegaKit - ABI Fluorescence Detection

330 reactions®

These are in vitro diagnostic products and are available in regions that accept CE-IVD products.

®
Capillary | CE-IVD Assays 74

IdentiClone® IGK Gene Clonality Assays
Assay Description
The IdentiClone IGK Gene Clonality Assay is an in vitro diagnostic product intended for PCR-based detection of clonal immunoglobulin kappa lightchain gene rearrangements in patients with suspect lymphoproliferations.
Specifically, the IGK Gene Clonality Assay can be used to:• Identify clonality in atypical lymphoproliferative disorders• Support a differential diagnosis between reactive lesions andhematologic malignancies• Assign presumptive lineage in mature monoclonal lymphoproliferative disorders• Identify tumor-specific markers (IGK and IGK-Kde rearrangements) for post-treatment monitoring• Monitor and evaluate disease recurrence

Summary and Explanation of the Test
These test kits include three master mixes. The IGK Tube A master mix targets the variable (Vƙ) and the joining (Jƙ) regions of the immunoglobulin kappalight chain locus, whereas the IGK Tube B master mix targets kappa deleting element (Kde) rearrangements with the Vƙ regions and the intragenic Jƙ-Cƙ
regions. The Vƙ-Kde and Jƙ-Cƙ intron-Kde rearrangements are a result of unsuccessful rearrangements retained by the B cell. The third master mix, theSpecimen Control Size Ladder, 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.

Performance Characteristics
Data from two independent studies that tested more than 300 patient sample of varying types suggests the diagnostic accuracy of selected
IdentiClone tests to be 96%. In both peer-reviewed studies, there were no clear false-positive results generated using the IdentiClone tests, andthere was a high level of precision.2 The clinico-histopathological diagnosis correlated well with PCR results in a higher number of patients whencompared with Southern Blot (SB) results, as seen below:
PCR/SB concordance:1		
IGK:
90% sensitivity / 90% specificity
PCR vs. SB analysis relative to histopathology and final diagnosis:
PCR/SB concordance:2
IGH + IGK: 85%

PCR sensitivity:98%

SB sensitivity:
39%

References
1. JJM van Dongen et al., Leukemia 17:2257-2317 (2003).
2. Y Sandberg et al., J. Mol. Diag. 7(4):495-503 (2005).

This assay is based on the EuroClonality/BIOMED-2 Concerted Action BMH4-CT98-3936.

®
75 Capillary | CE-IVD Assays

Figure Legend: Schematic diagram of the immunoglobulin kappa light chain gene complex on chromosome 2p11.2. Shown are the relative positions and orientations for
the Vƙ-Jƙ, and Kde primers, which are included in the IGK master mix tubes.

Reagents
Controls

Concentration

Units in Assay

Units in Assay MegaKit

IVS-0007 Clonal Control DNA

200 μg/mL

1 x 100 μL tube

5 x 100 μL tubes

IVS-0000 Polyclonal Control DNA

200 μg/mL

1 x 100 μL tube

5 x 100 μL tubes

Master Mixes

TargetUnits in Assay

Units in Assay MegaKit

IGK Tube AVƙ - Jƙ

1 x 1500 μL tube

10 x 1500 μL tubes

IGK Tube B

Vƙ-Kde, Intron-Kde

1 x 1500 μL tube

10 x 1500 μL tubes

Specimen Control Size LadderMultiple Genes

1 x 1500 μL tube

10 x 1500 μL tubes

Ordering Information
Catalog #Products

Quantity

9-102-0021

IdentiClone® IGK Gene Clonality Assay - ABI Fluorescence Detection

33 reactions

9-102-0031

IdentiClone IGK Gene Clonality Assay MegaKit - ABI Fluorescence Detection

330 reactions®

These are in vitro diagnostic products and are available in regions that accept CE-IVD products.

®
Capillary | CE-IVD Assays 76

IdentiClone® IGL Gene Clonality Assays
Assay Description
The IdentiClone IGL Gene Clonality Assay is an in vitro diagnostic product intended for PCR-based detection of clonal immunoglobulin lambda lightchain gene rearrangements in patients with suspect lymphoproliferations and can be used to:Specifically, the IdentiClone IGL Gene Clonality Assays can be used to:• Identify clonality in atypical lymphoproliferative disorders• Support a differential diagnosis between reactive lesions and hematologic malignancies• Assign presumptive lineage in mature monoclonal lymphoproliferative disorders• Identify tumor-specific markers (IGL gene rearrangements) for post-treatment monitoring• Monitor and evaluate disease recurrence

Summary and Explanation of the Test
These test kits include two master mixes. The IGL Tube master mix targets the variable (Vλ) region and the joining (Jλ) region of the immunoglobulin
lambda light chain gene locus (IGL). The Specimen Control Size Ladder 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.

Performance Characteristics
Data from two independent studies that tested more than 300 patient samples of varying types suggests the diagnostic accuracy of selected
IdentiClone tests to be 96%. In both peer-reviewed studies, there were no clear false-positive results generated using the IdentiClone tests, andthere was a high level of precision.2The clinico-histopathological diagnosis correlated well with PCR results in a higher number of patients whencompared with Southern Blot (SB) results, as seen below:
IGL:

PCR/SB concordance:1		
86% sensitivity / 92% specificity

References
1. JJM van Dongen et al., Leukemia 17:2257-2317 (2003).
2. Y Sandberg et al., J. Mol. Diag. 7(4):495-503 (2005).

This assay is based on the EuroClonality/BIOMED-2 Concerted Action BMH4-CT98-3936.

®
77 Capillary | CE-IVD Assays

Figure Legend: Schematic diagram of the immunoglobulin lambda light chain gene complex on chromosome 22q11.2. Shown are the relative positions and orientations for
the Vλ and Jλ primers, which are included in the IGL master mix tube. The two Vλ primers only target Vλ1, 2, and 3 because these three V families cover approximately 70%
of rearrangeable Vλ gene segments, and approximately 90% of all IGL gene rearrangements involve these three families. Similarly, the single Jλ primer only targets Jλ1, 2,
and 3 because these three J segments are involved in 98% of all IGL gene rearrangements.

Reagents
Controls

Concentration

Units in Assay

Units in Assay MegaKit

IVS-0010 Clonal Control DNA

200 μg/mL

1 x 100 μL tube

5 x 100 μL tubes

IVS-0029 Clonal Control DNA

200 μg/mL

1 x 100 μL tube

5 x 100 μL tubes

IVS-0000 Polyclonal Control DNA

200 μg/mL

1 x 100 μL tube

5 x 100 μL tubes

Master Mixes

TargetUnits in Assay

Units in Assay MegaKit

IGL TubeVλ -Jλ1 x 1500 μL tube

10 x 1500 μL tubes

Specimen Control Size LadderMultiple Genes

1 x 1500 μL tube

10 x 1500 μL tubes

Ordering Information
Catalog #Products

Quantity

9-103-0011

IdentiClone® IGL Gene Clonality Assay - ABI Fluorescence Detection

33 reactions

These are in vitro diagnostic products and are available in regions that accept CE-IVD products.

®
Capillary | CE-IVD Assays 78

IdentiClone® TCRB Gene Clonality Assays
Assay Description
The IdentiClone TCRB Gene Clonality Assay is an in vitro diagnostic product intended for PCR-based detection of clonal T-cell receptor beta chain
gene rearrangements in patients with suspect lymphoproliferations.
Specifically, the TCRB Gene Clonality Assay can be used to:• Identify clonality in suspect lymphoproliferations• Support a differential diagnosis between reactive lesions and T-cell and some immature B-cell malignancies• Determine lineage involvement in mature lymphoproliferative disorders• Monitor and evaluate disease recurrence

Summary and Explanation of the Test
These kits include four master mixes. TCRB Tubes A and B target framework regions within the variable region, and the joining region (Vβ) of the TCR
beta chain locus. TCRB Tube C targets the diversity and joining (Jβ) regions of the TCR beta chain locus. The Specimen Control Size Ladder mastermix included targets multiple genes and generates a series of amplicons of approximately 100, 200, 300, 400, and 600 base pairs to ensure that thequality and quantity of input DNA is adequate to yield a valid result.

Performance Characteristics
Data from two independent studies that tested more than 300 patient samples of varying types suggests the diagnostic accuracy of selected
IdentiClone tests to be 96%. In both peer-reviewed studies, there were no clear false-positive results generated using the IdentiClone tests, andthere was a high level of precision.2 The clinico-histopathological diagnosis correlated well with PCR results in a higher numberof patients when compared with Southern Blot (SB) results, as seen below:
TRB:

PCR/SB concordance:1		
86% sensitivity / 98% specificity

PCR vs. SB analysis relative to histopathology and final diagnosis:
TRB:

PCR/SB concordance:2
85% 96%

PCR sensitivity:
35%

SB sensitivity:

References
1. JJM van Dongen et al., Leukemia 17:2257-2317 (2003).
2. Y Sandberg et al., J. Mol. Diag. 7(4):495-503 (2005).

This assay is based on the EuroClonality/BIOMED-2 Concerted Action BMH4-CT98-3936.

®
79 Capillary | CE-IVD Assays

Figure Legend: Simplified diagram of a representative rearranged T-cell receptor beta gene showing the approximate placement of the upstream and downstream DNA
primers. The numbers of primers and their specificity are listed for Master Mix Tubes A, B, and C.

Reagents
Controls

Concentration

Units in Assay

Units in Assay MegaKit

IVS-0009 Clonal Control DNA

200 μg/mL

1 x 100 μL tube

5 x 100 μL tubes

IVS-0004 Clonal Control DNA

200 μg/mL

1 x 100 μL tube

5 x 100 μL tubes

IVS-0000 Polyclonal Control DNA

200 μg/mL

1 x 100 μL tube

5 x 100 μL tubes

IVS-0021 Clonal Control DNA

200 μg/mL

1 x 100 μL tube

5 x 100 μL tubes

Master Mixes

TargetUnits in Assay

Units in Assay MegaKit

TCRB Tube A

Multiple Vβ + Jβ1/2

1 x 1500 μL tube

10 x 1500 μL tubes

TCRB Tube B

Multiple Vβ + Jβ2

1 x 1500 μL tube

10 x 1500 μL tubes

TCRB Tube C

Multiple Dβ + Jβ1/2

1 x 1500 μL tube

10 x 1500 μL tubes

Specimen Control Size LadderMultiple Genes

1 x 1500 μL tube

10 x 1500 μL tubes

Ordering Information
Catalog #Products

Quantity

9-205-0011

IdentiClone TCRB Clonality Assay - ABI Fluorescence Detection

33 reactions

9-205-0021

IdentiClone TCRB Clonality Assay MegaKit - ABI Fluorescence Detection

330 reactions®
®

These are in vitro diagnostic products and are available in regions that accept CE-IVD products.

®
Capillary | CE-IVD Assays 80

IdentiClone® T-Cell Receptor Gamma
Gene Rearrangement Assay 2.0
Assay Description
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 thatare 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.1
This assay allows for amplification of the TRG region with fluorescent labeled primers, yielding products that can be grouped under a single Gaussian
distribution when separated by size using capillary electrophoresis. In addition, the product size facilitates increased success when testing FFPE
samples. The included analysis algorithm aids in the interpretation of data and identification of significant clonal peaks. Presence or absenceof molecular clonality can support the differential diagnosis of reactive lesions and certain B- and T-cell malignancies, provided that the results areinterpreted in the context of all available clinical, histological, and immunophenotypic data.

Performance Characteristics
To assess the performance of the TCRG 2.0 Assay, testing was performed on cell lines with known clonal rearrangements followed by testing
on previously sequenced clinical samples.
When used in combination with the provided TCRG Algorithm worksheet, the assay was capable of detecting DNA from 6 control cell lines (200 ng/µL) diluted into polyclonal tonsil DNA (200 ng/µL) at 5% (v/v).
Furthermore, the performance of the TCRG 2.0 Assay was evaluated on clinical samples for which the T-cell receptor gamma gene rearrangement
status had been identified by Roche 454 sequencing. For the 7 samples that had been identified as clonal by sequencing, the TCRG 2.0 assay had
100% concordance. For the 12 samples that were either negative for a clonal event or were oligoclonal, concordance of the TCRG 2.0 assay was 75%.
Sample types included peripheral blood, bone marrow, and formalin-fixed, paraffin embedded (FFPE) tissue.
Always interpret the results of molecular clonality tests in the context of clinical, histological and immunophenotypic data.

References
1. Miller JE, Wilson SS, Jaye DJ, and Kronenberg M. Mol. Diag. 1999, 4(2):101-117.
2. Armand, Marine et al. HemaSphere, 2019;3:3.

This assay is based on the EuroClonality/BIOMED-2 Concerted Action BMH4-CT98-3936.
The performance of this assay was reviewed and validated by the EuroClonality/BIOMED-2 Group.2®
81 Capillary | CE-IVD Assays

Figure Legend: Simple representation of the organization of the T-cell receptor gamma gene on chromosome 7p14. Black arrows represent the relative positions of primersthat target the variable region genes and the downstream joining region gene segments that are involved in rearrangements in T-cell lymphomas. The downstream
primers are fluorescently labeled through the incorporation of a 6FAM fluorophore. The amplicon products generated from these rearrangements are detected bycapillary electrophoresis.

Reagents
Controls

Concentration

Units in Assay

Units in Assay MegaKit

5% TCRG Positive Control DNA

50 μg/mL

1 x 50 μL tube

5 x 50 μL tube

TCRG Negative Control DNA

50 μg/mL

1 x 50 μL tube

5 x 50 μL tube

Master Mixes

TargetUnits in Assay

Units in Assay MegaKit

TCRG - 6FAM

Vƴ 1-Vƴ 11 + Jƴ 1/Jƴ 2, Jƴ P, Jƴ P1/Jƴ P2

1 x 1500 μL tube

10 x 1500 μL tubes

Specimen Control Size LadderMultiple Genes

1 x 1500 μL tube

10 x 1500 μL tubes

Ordering Information
Catalog #Products

Quantity

9-207-0101

IdentiClone T-Cell Receptor Gamma Gene Rearrangement Assay 2.0 - ABI Fluorescence Detection

33 reactions

9-207-0111

IdentiClone® T-Cell Receptor Gamma Gene Rearrangement Assay MegaKit 2.0 - ABI Fluorescence Detection

330 reactions®

These are in vitro diagnostic products and are available in regions that accept CE-IVD products.

®
Capillary | CE-IVD Assays 82

IdentiClone® TCRD Gene Clonality Assays
Assay Description
The IdentiClone TCRD Gene Clonality Assay is an in vitro diagnostic product intended for PCR-based detection of clonal T-cell receptor delta chain
gene rearrangements in patients with suspect lymphoproliferations and can be used to:• Identify clonality in suspect lymphoproliferations• Support a differential diagnosis between reactive lesions and T-cell and some immature B-cell malignancies• Determine lineage involvement in mature lymphoproliferative disorders• Monitor and evaluate disease recurrence

Summary and Explanation of the Test
These test kits include two master mixes. The TCRD tube targets the framework regions within the variable region, the diversity region, and the joining
region of the T-cell receptor delta chain locus (TRD, formerly known as TCRD). 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 isadequate to yield a valid result.

Performance Characteristics
Data from an independent, peer-reviewed study suggests the diagnostic accuracy of selected IdentiClone tests to be 96%. There were no clear
false-positive results generated using the IdentiClone tests, and there was a high level of precision. The clinico- histopathological diagnosis correlates
well with PCR results in a higher number of patients when compared with Southern Blot (SB) results, as seen below:
TRD:

PCR/SB concordance:1
83% sensitivity / 95% specificity

Reference
1. JJM van Dongen et al., Leukemia 17:2257-2317 (2003).

This assay is based on the EuroClonality/BIOMED-2 Concerted Action BMH4-CT98-3936.

®
83 Capillary | CE-IVD Assays

Figure Legend: Simplified diagram of a representative rearranged T-cell receptor delta gene showing the approximate placement of the upstream and downstream DNA
primers. The numbers of primers and their specificity are listed for the TRD Tube Master Mix tube.

Reagents
Controls

Concentration

Units in Assay

Units in Assay MegaKit

IVS-0021 Clonal Control DNA

200 μg/mL

1 x 100 μL tube

5 x 100 μL tubes

IVS-0000 Polyclonal Control DNA

200 μg/mL

1 x 100 μL tube

5 x 100 μL tubes

Master Mixes

TargetUnits in Assay

Units in Assay MegaKit

TCRD Tube

Multiple Vδ + Dδ +Jδ

1 x 1500 μL tube

10 x 1500 μL tubes

Specimen Control Size LadderMultiple Genes

1 x 1500 μL tube

10 x 1500 μL tubes

Ordering Information
Catalog #Products

Quantity

9-206-0011

IdentiClone® TCRD Gene Clonality Assay - ABI Fluorescence Detection

33 reactions

9-206-0021

IdentiClone® TCRD Gene Clonality Assay MegaKit - ABI Fluorescence Detection

330 reactions

These are in vitro diagnostic products and are available in regions that accept CE-IVD products.

®
Capillary | CE-IVD Assays 84

Capillary Detection - CE-IVD Assays and
IVD Companion Diagnostic Assays
LeukoStrat Assay Kits are in vitro diagnostic
products intended for PCR-based detectionof FLT3 activating mutations (ITD, TKD) in patientswith acute myelogenous leukemia (AML) using
capillary electrophoresis methods, and include
companion diagnostic assays.

LEARN MORE:
catalog.invivoscribe.com

Warranty and Liability
Invivoscribe is committed to providing the highest quality products. Invivoscribe warrants that for products which are provided with Instructions for Use, these products meet or exceed theperformance standards described in the Instructions For Use. If a product is covered by product specifications and does not perform as specified, our policy is to replace the product or credit thefull purchase price. No other warranties of any kind, expressed or implied, are provided by Invivoscribe. Invivoscribe liability shall not exceed the purchase price of the product. Invivoscribe shallhave no liability for direct, indirect, consequential or incidental damages arising from the use, results of use, or inability to use its products; product efficacy under purchaser controlled conditions inpurchaser’s laboratory must be established and continually monitored through purchaser defined and controlled processes including but not limited to testing of positive, negative, and blank controls
every time a sample is tested. Ordering, acceptance and use of product constitutes purchaser acceptance of sole responsibility for assuring product efficacy and purchaser agreement
to the limitation of liability set forth in this paragraph.
Use of this product may require nucleic acid amplification methods such as Polymerase Chain Reaction (PCR). Any necessary license to practice amplification methods or to use reagents,
amplification enzymes or equipment covered by third party patents is the responsibility of the user and no such license is granted by Invivoscribe, Inc., expressly or by implication.
Disclaimer
The products in the section that follows are in vitro diagnostic products. CE-IVD products are not available for sale or use within North America.

Introduction
All tests include PCR master mixes and controls for ITD and TKD detection, along with Instructions For Use (IFUs).
Master mixes are composed of a buffered magnesium chloride solution, deoxynucleotides, and primers targeting
the gene segments of interest. These assay master mixes are complete other than Taq DNA polymerase.

Companion Diagnostics
The LeukoStrat CDx FLT3 Mutation Assays are companion diagnostics, and provide a complete solution with
software automated PDF reports including mutant identification, mutant to wild type signal ratios, and clinicalinterpretation. Additional reagents of Taq DNA Polymerase, EcoRV Enzyme, and NEBuffer r3.1 are included in IVD kits
except the CE-marked kit.

Chapter Contents
CDx Assays
87 LeukoStrat CDx FLT3 Mutation Assay (IVD) - United States
89 LeukoStrat CDx FLT3 Mutation Assay (CE-marked)
91 LeukoStrat CDx FLT3 Mutation Assay (CE 2797 IVD)
93 LeukoStrat CDx FLT3 Mutation Assay - Japan

LeukoStrat Assays are in vitro diagnostic products. CE-marked and CE 2797 IVD products
are not available for sale or use within North America. IVD products are available for saleor use where registered. Refer to product page for respective detail. For more information
regarding the research use only reagents, please see the Gel and Capillary Research Use
Only Assays section.

Capillary Detection | CE-IVD Assays and IVD Companion Diagnostic Assays 86

LeukoStrat® CDx FLT3 Mutation Assay
FDA Approved Companion Diagnostic
Available in the USA - In Vitro Diagnostic Kit

FDA approved assay for assessment of acute myeloid leukemia (AML) patients eligible for treatment with RYDAPT® (midostaurin), XOSPATA®
(gilteritinib), or VANFLYTA® (quizartinib), now available as US distributed kit. This FLT3 companion diagnostic includes reagents along with software that
identiﬁes ITD and TKD mutations, generates mutant/wildtype signal ratios, and predicts response to gilteritinib, midostaurin, and quizartinib.
Clinical Signiﬁcance of FLT3 Mutation Status: Each year approximately 21,000 patients in the United States are diagnosed with AML. Of thosediagnosed with AML, ~1 out of 3 are expected to have presence of FLT3 mutations, (FLT3mut+). Since FLT3mut+ AML is clinically actionable,
stratiﬁcation of AML patients by testing for FLT3 mutation status has become a standard of care.

Intended Use
The LeukoStrat CDx FLT3 Mutation Assay is a PCR-based in vitro diagnostic test designed to detect internal tandem duplication (ITD) and tyrosine
kinase domain (TKD) mutations D835 and I836 in the FLT3 gene in genomic DNA extracted from mononuclear cells obtained from peripheral blood orbone marrow aspirates of patients diagnosed with acute myelogenous leukemia (AML).
The LeukoStrat CDx FLT3 Mutation Assay is used as an aid in the assessment of patients with AML for whom RYDAPT® (midostaurin) treatment is being
considered.
The LeukoStrat CDx FLT3 Mutation Assay is used as an aid in the assessment of patients with AML for whom XOSPATA® (gilteritinib) treatment is being
considered.
The LeukoStrat CDx FLT3 Mutation Assay is used as an aid in the assessment of patients with FLT3-ITD+ AML for whom VANFLYTA® (quizartinib)
treatment is being considered.
The test is for use on the 3500xL Dx Genetic Analyzer.

Summary and Explanation of the Test
Acute myelogenous leukemia (AML) in general has a poor prognosis. Many studies in AML have shown that the presence of FLT3 activating mutations
portends a poor prognosis making it an attractive target for treatment.1,2
The LeukoStrat CDx FLT3 Mutation Assay targets regions of the FLT3 gene to identify internal tandem duplication (ITD) mutations and tyrosine kinasedomain (TKD) mutations, such as the D835 and I836 mutations.
The LeukoStrat CDx FLT3 Mutation Assay includes reagents, software and procedures for isolating mononuclear cells and extracting DNA from patient
peripheral blood or bone marrow specimens to determine if FLT3 mutations are present.
DNA is ampliﬁed via PCR and the amplicons are detected via capillary electrophoresis. FLT3 mutation status is determined by the LeukoStrat CDx FLT3
Software. A FLT3 ITD and/or TKD mutation is reported as Positive if the mutant:wild-type signal ratio meets or exceeds the clinical cutoff of 0.05.

Method Description
ITD Mutations of FLT3
The LeukoStrat CDx FLT3 Mutation Assay uses ﬂuorescently labeled primers that are in and around the JM region. Wild-type FLT3 alleles will amplify
and produce a product at 327±1 bp as measured by this assay, while alleles that contain ITD mutations will produce a product that exceeds 327±1 bp(see Figure, right).
TKD Mutations of FLT3
The LeukoStrat CDx FLT3 Mutation Assay uses primers that lie on either side of the TKD region. The FLT3 target region is ampliﬁed using PCR andthen an EcoRV restriction digest is performed. Wild-type alleles of the FLT3 gene yield digestion products of 79±1 bp whereas mutant alleles yieldproducts of 125±1 bp or 127±1 bp from the original undigested amplicon product of 145±1 bp or 147±1 bp, as measured by this assay (see Figure, right).

87 Capillary Detection | CE-IVD Assays and IVD Companion Diagnostic Assays

Method Schematic: FLT3 ITD and TKD Mutant Detection

Depicted is a representation of the FLT3 juxtamembrane (JM) region (TM = transmembrane) and the activating loop of the tyrosine kinase (TK) domain. Black arrows represent the relative
positions of primers that target in and around the JM region for ITD or the activating loop of the kinase domain for TKD. Colored dots represent fluorophores on labeled primers. The yellow box hasvertical black lines that represent the position of the EcoRV restriction digest sites.

Reagents
Reagent Name

Units in Assay

FLT3 Extraction Control

1 x 1800 μL tube

FLT3 ITD Master Mix

1 x 1500 μL tube

FLT3 TKD Master Mix

1 x 1500 μL tube

FLT3 ITD Positive Control

1 x 100 μL tube

FLT3 TKD Positive Control

1 x 100 μL tube

FLT3 No Template Control

1 x 200 μL tube

Taq DNA Polymerase

1 x 200 μL tube

EcoRV Enzyme

1 x 200 μL tube

NEBuffer r3.1

1 x 1250 μL tube

Ordering Information
Catalog #Products

Quantity

K-412-0361

LeukoStrat CDx FLT3 Mutation Assay

33 reactions

K-412-0371

LeukoStrat CDx FLT3 Software

CD complimentary with purchaseK-412-0401

LeukoStrat® CDx Assay Installer

USB complimentary with purchase®
®

References
1. Murphy KM et al., A Clinical PCR/Capillary Electrophoresis Assay for the Detection of Internal Tandem Duplication and Point Mutation of the FLT3
Gene. J. Mol. Diag. 5:96-102 (2003).
2. Yamamoto, Y., et al., Activating mutation of D835 within the activation loop of FLT3 in human hematologic malignancies. Blood, 97(8):2434-9 (2001).

These are in vitro diagnostic (IVD) products, and are available for sale or use in the United States only

Capillary Detection | CE-IVD Assays and IVD Companion Diagnostic Assays 88

LeukoStrat® CDx FLT3 Mutation Assay
CE-marked Companion Diagnostic
The only internationally standardized CE-IVD assay for FLT3 Signal Ratio mutation analysis for assessment of acute myeloid leukemia (AML) patients
eligible for treatment with RYDAPT® (midostaurin) or XOSPATA® (gilteritinib fumarate).

Intended Use
The LeukoStrat CDx FLT3 Mutation Assay is a PCR-based in vitro diagnostic test designed to detect internal tandem duplications (ITD) and tyrosine
kinase domain (TKD) mutations D835 and I836 in the FLT3 gene in genomic DNA extracted from mononuclear cells obtained from peripheral bloodor bone marrow aspirates of patients diagnosed with acute myelogenous leukemia (AML).
In regions where midostaurin is available, the LeukoStrat CDx FLT3 Mutation Assay is used as an aid in the assessment of patients with AML for whom
RYDAPT® (midostaurin) treatment is being considered.
In regions where gilteritinib fumarate is available, the LeukoStrat CDx FLT3 Mutation Assay is used as an aid in the assessment of patients with AML
for whom XOSPATA® (gilteritinib fumarate) treatment is being considered.

Summary and Explanation of the Test
AML in general has a poor prognosis. Assessment of the mutation status of the FLT3 (fms related tyrosine kinase 3) receptor gene in karyotype normal
AML is the most important prognostic indicator of disease outcome, which is often substantial, as many studies in AML have shown that the presenceof FLT3 activating mutations portends a poor prognosis.1,2
The LeukoStrat CDx FLT3 Mutation Assay targets regions of the FLT3 gene to identify ITD mutations and TKD mutations, such as the D835 and I836
mutations, and has been validated in an international clinical trial.
The LeukoStrat CDx FLT3 Mutation Assay includes reagents, equipment, software and procedures for isolating mononuclear cells and extracting DNA frompatient specimens to determine if FLT3 mutations are present. DNA is amplified via PCR and the amplicons are detected via capillary electrophoresis.
FLT3 mutation status is determined by the LeukoStrat CDx FLT3 Software. A FLT3 ITD and/or TKD mutation is reported as Positive if the mutant:wild-typesignal ratio meets or exceeds the clinical cutoff of 0.05.

Method Description
ITD Mutations of FLT3
The LeukoStrat CDx FLT3 Mutation Assay uses fluorescently labeled primers that are in and around the JM region. Wild-type FLT3 alleles will amplify
and produce a product at 327±1 bp as measured by this assay, while alleles that contain ITD mutations will produce a product that exceeds 327±1 bp(see Figure, right).

TKD Mutations of FLT3
The LeukoStrat CDx FLT3 Mutation Assay uses primers that lie on either side of the TKD region. The FLT3 target region is amplified using PCR and thenan EcoRV restriction digest is performed. Wild-type alleles of the FLT3 gene yield digestion products of 79±1 bp whereas mutant alleles yield productsof 125±1 bp or 127±1 bp from the original undigested amplicon product of 145±1 bp or 147±1 bp, as measured by this assay (please see Figure, right).

References
1. Murphy KM et al., A Clinical PCR/Capillary Electrophoresis Assay for the Detection of Internal Tandem Duplication and Point Mutation of the FLT3
Gene. J. Mol. Diag. 5:96-102 (2003).
2. Yamamoto, Y., et al., Activating mutation of D835 within the activation loop of FLT3 in human hematologic malignancies. Blood, 97(8):2434-9 (2001).

89 Capillary Detection | CE-IVD Assays and IVD Companion Diagnostic Assays

Depicted is a representation of the FLT3 juxtamembrane (JM) region (TM = transmembrane) and the activating loop of the tyrosine kinase (TK)domain. Black arrows represent the relative positions of primers that target in and around the JM region for ITD or the activating loop of the kinasedomain for TKD. Colored dots represent fluorophores on labeled primers. The yellow box has vertical black lines that represent the position of the
EcoRV restriction digest sites.

Reagents
Controls

Units in Assay

FLT3 Extraction Control

1 x 1800 μL tube

FLT3 ITD Master Mix

1 x 1500 μL tube

FLT3 TKD Master Mix

1 x 1500 μL tube

FLT3 ITD Positive Control

1 x 100 μL tube

FLT3 TKD Positive Control

1 x 100 μL tube

FLT3 No Template Control

1 x 200 μL tube

Ordering Information
Catalog #Products

Quantity

K-412-0291

LeukoStrat® CDx FLT3 Mutation Assay

33 reactions

K-412-0281

LeukoStrat CDx FLT3 Software

CD complimentary with purchase®

These are in vitro diagnostic products and are available in regions that accept CE-IVD products.

Capillary Detection | CE-IVD Assays and IVD Companion Diagnostic Assays 90

LeukoStrat® CDx FLT3 Mutation Assay
CE 2797 IVD Companion Diagnostic
The only internationally standardized CE 2797 IVD assay for FLT3 Signal Ratio mutation analysis for assessment of acute myeloid leukemia (AML)
patients eligible for treatment with XOSPATA® (gilteritinib fumarate).

Intended Use
The LeukoStrat CDx FLT3 Mutation Assay is a PCR-based in vitro diagnostic test designed to detect internal tandem duplication (ITD) and tyrosine
kinase domain (TKD) mutations D835 and I836 in the FLT3 gene in genomic DNA extracted from mononuclear cells obtained from peripheral blood orbone marrow aspirates of patients diagnosed with acute myelogenous leukemia (AML). The LeukoStrat CDx FLT3 Mutation Assay may be used as acompanion diagnostic for the following therapeutic:
In regions where XOSPATA® (gilteritinib fumarate) is available, the LeukoStrat CDx FLT3 Mutation Assay is used as an aid in the assessment of patientswith AML for whom XOSPATA (gilteritinib fumarate) treatment is being considered.
The qualitative, non-automated test is for use on the 3500xL or 3500xL Dx Genetic Analyzers.

Summary and Explanation of the Test
AML in general has a poor prognosis. Assessment of the mutation status of the FLT3 (fms related tyrosine kinase 3) receptor gene in karyotype normal
AML is the most important prognostic indicator of disease outcome, which is often substantial, as many studies in AML have shown that the presenceof FLT3 activating mutations portends a poor prognosis.1,2
The LeukoStrat CDx FLT3 Mutation Assay targets regions of the FLT3 gene to identify ITD mutations and TKD mutations, such as the D835 and I836
mutations, and has been validated in an international clinical trial.
The LeukoStrat CDx FLT3 Mutation Assay includes reagents, equipment, software and procedures for isolating mononuclear cells and extracting DNA frompatient specimens to determine if FLT3 mutations are present. DNA is amplified via PCR and the amplicons are detected via capillary electrophoresis.
FLT3 mutation status is determined by the LeukoStrat CDx FLT3 Software. A FLT3 ITD and/or TKD mutation is reported as Positive if the mutant:wild-typesignal ratio meets or exceeds the clinical cutoff of 0.05.

Method Description
ITD Mutations of FLT3
The LeukoStrat CDx FLT3 Mutation Assay uses fluorescently labeled primers that are in and around the JM region. Wild-type FLT3 alleles will amplify
and produce a product at 327±1 bp as measured by this assay, while alleles that contain ITD mutations will produce a product that exceeds 327±1 bp(see Figure, right).

TKD Mutations of FLT3
The LeukoStrat CDx FLT3 Mutation Assay uses primers that lie on either side of the TKD region. The FLT3 target region is amplified using PCR and thenan EcoRV restriction digest is performed. Wild-type alleles of the FLT3 gene yield digestion products of 79±1 bp whereas mutant alleles yield productsof 125±1 bp or 127±1 bp from the original undigested amplicon product of 145±1 bp or 147±1 bp, as measured by this assay (please see Figure, right).

References
1. Murphy KM et al., A Clinical PCR/Capillary Electrophoresis Assay for the Detection of Internal Tandem Duplication and Point Mutation of the FLT3
Gene. J. Mol. Diag. 5:96-102 (2003).
2. Yamamoto, Y., et al., Activating mutation of D835 within the activation loop of FLT3 in human hematologic malignancies. Blood, 97(8):2434-9 (2001).

91 Capillary Detection | CE-IVD Assays and IVD Companion Diagnostic Assays

Depicted is a representation of the FLT3 juxtamembrane (JM) region (TM = transmembrane) and the activating loop of the tyrosine kinase (TK)domain. Black arrows represent the relative positions of primers that target in and around the JM region for ITD or the activating loop of the kinasedomain for TKD. Colored dots represent fluorophores on labeled primers. The yellow box has vertical black lines that represent the position of the
EcoRV restriction digest sites.

Reagents
Controls

Units in Assay

FLT3 Extraction Control

1 x 1800 μL tube

FLT3 ITD Master Mix

1 x 1500 μL tube

FLT3 TKD Master Mix

1 x 1500 μL tube

FLT3 ITD Positive Control

1 x 100 μL tube

FLT3 TKD Positive Control

1 x 100 μL tube

FLT3 No Template Control

1 x 200 μL tube

Taq DNA Polymerase

1 x 200 μL tube

EcoRV Enzyme

1 x 200 μL tube

NEBuffer r3.1

1 x 1250 μL tube

Ordering Information
Catalog #Products

Quantity

K-412-0431

LeukoStrat CDx FLT3 Mutation Assay

33 reactions

K-412-0441

LeukoStrat® CDx FLT3 Software

CD complimentary with purchase®

These are in vitro diagnostic products and are available in regions that accept CE-IVD products.

Capillary Detection | CE-IVD Assays and IVD Companion Diagnostic Assays 92

LeukoStrat® CDx FLT3 Mutation Assay
PMDA/MHLW Approved Companion Diagnostic
Available in the JapanThe only internationally standardized assay for FLT3 Signal Ratio mutation analysis for assessment of acute myeloid leukemia (AML) patients eligible
for treatment with Gilteritinib Fumarate or Quizartinib Hydrochloride.

Intended Use
The LeukoStrat CDx FLT3 Mutation Assay is a PCR-based, in vitro diagnostic test designed to detect internal tandem duplication (ITD) mutations andtyrosine kinase domain (TKD) mutations D835 and I836 in the FLT3 gene in genomic DNA extracted from mononuclear cells obtained from peripheral
blood or bone marrow aspirates of patients diagnosed with acute myelogenous leukemia.
The LeukoStrat CDx FLT3 Mutation Assay is used as an aid in the assessment of patients with AML for whom Gilteritinib Fumarate treatment
is being considered.
The LeukoStrat CDx FLT3 Mutation Assay is used as an aid in the assessment of patients with AML for whom Quizartinib Hydrochloride treatment
is being considered.

Summary and Explanation of the Test
AML in general has a poor prognosis. Assessment of the mutation status of the FLT3 (fms related tyrosine kinase 3) receptor gene in karyotype normal
AML is the most important prognostic indicator of disease outcome, which is often substantial, as many studies in AML have shown that the presenceof FLT3 activating mutations portends a poor prognosis.1,2 The LeukoStrat CDx FLT3 Mutation Assay targets regions of the FLT3 gene to identify ITD
mutations and TKD mutations, such as the D835 and I836 mutations, and has been validated in an international clinical trial.
The LeukoStrat CDx FLT3 Mutation Assay includes reagents, equipment, software and procedures for isolating mononuclear cells and extracting
DNA from patient specimens to determine if FLT3 mutations are present. DNA is amplified via PCR with fluorescently labeled primers, amplicon is
enzymatically digested (TKD), and the amplicons are detected via capillary electrophoresis. FLT3 mutation status is determined by the LeukoStrat CDx
FLT3 Software. A FLT3 ITD and/or TKD mutation is reported as Positive if the mutant:wild-type signal ratio meets or exceeds the clinical cutoff of 0.05.

Method Description
ITD Mutations of FLT3
The LeukoStrat CDx FLT3 Mutation Assay uses fluorescently labeled primers that are in and around the JM region. Wild-type FLT3 alleles will amplify
and produce a product at 327±1 bp as measured by this assay, while alleles that contain ITD mutations will produce a product that exceeds 327±1 bp(see Figure, right).

TKD Mutations of FLT3
The LeukoStrat CDx FLT3 Mutation Assay uses primers that lie on either side of the TKD region. The FLT3 target region is amplified using PCR andthen an EcoRV restriction digest is performed. Wild-type alleles of the FLT3 gene yield digestion products of 79±1 bp whereas mutant alleles yieldproducts of 125±1 bp or 127±1 bp from the original undigested amplicon product of 145±1 bp or 147±1 bp, as measured by this assay (see Figure, right).

References
1. Murphy KM et al., A Clinical PCR/Capillary Electrophoresis Assay for the Detection of Internal Tandem Duplication and Point Mutation of the FLT3
Gene. J. Mol. Diag. 5:96-102 (2003).
2. Yamamoto, Y., et al., Activating mutation of D835 within the activation loop of FLT3 in human hematologic malignancies. Blood, 97(8):2434-9 (2001).

93 Capillary Detection | CE-IVD Assays and IVD Companion Diagnostic Assays

Depicted is a representation of the FLT3 juxtamembrane (JM) region (TM = transmembrane) and the activating loop of the tyrosine kinase(TK) domain. Black arrows represent the relative positions of primers that target in and around the JM region for ITD or the activating loopof the kinase domain for TKD. Colored dots represent fluorophores on labeled primers. The yellow box has vertical black lines that represent
the position of the EcoRV restriction digest sites.

Reagents
Reagent Name

Units in Assay

FLT3 Extraction Control

1 x 1800 μL tube

FLT3 ITD Master Mix

1 x 1500 μL tube

FLT3 TKD Master Mix

1 x 1500 μL tube

FLT3 ITD Positive Control

1 x 100 μL tube

FLT3 TKD Positive Control

1 x 100 μL tube

FLT3 No Template Control

1 x 200 μL tube

Taq DNA Polymerase Enzyme

1 x 200 μL tube

EcoRV Enzyme

1 x 200 μL tube

NEBuffer r3.1

1 x 1250 μL tube

Ordering Information
Catalog #Products

Quantity

K-412-0331

LeukoStrat CDx FLT3 Mutation Assay (Japan)

33 reactions

K-412-0341

LeukoStrat® CDx FLT3 Software (Japan)

CD complimentary with purchase®

These are in vitro diagnostic products, and are available for sale or use within Japan.

Capillary Detection | CE-IVD Assays and IVD Companion Diagnostic Assays 94

Gel and Capillary
Research Use Only (RUO) Assays

Invivoscribe offers an array of assays for
B- and T-cell gene clonality/rearrangements,
mutations, and chromosome translocations
for the study of hematologic malignancies.

LEARN MORE:
catalog.invivoscribe.com

Warranty and Liability
Invivoscribe is committed to providing the highest quality products. Invivoscribe warrants that for products which are provided with Instructions for Use, these products meet or exceed theperformance standards described in the Instructions For Use. If a product is covered by product specifications and does not perform as specified, our policy is to replace the product or credit thefull purchase price. No other warranties of any kind, expressed or implied, are provided by Invivoscribe. Invivoscribe liability shall not exceed the purchase price of the product. Invivoscribe shallhave no liability for direct, indirect, consequential or incidental damages arising from the use, results of use, or inability to use its products; product efficacy under purchaser controlled conditions
in purchaser’s laboratory must be established and continually monitored through purchaser defined and controlled processes including but not limited to testing of positive, negative, and blankcontrols every time a sample is tested. Ordering, acceptance and use of product constitutes purchaser acceptance of sole responsibility for assuring product efficacy and purchaser agreement to thelimitation of liability set forth in this paragraph.
NoticeSome of these products are covered by one or more patents licenses to Invivoscribe Inc, including European Patent No. 1549764 and 2418287 (each validated in 16 countries), European Patent No.
2460889, Japanese Patent No. 4708029, United States Patent No. 8859748 and United States Patent No. 10280462.
These products require nucleic acid amplification methods such as Polymerase Chain Reaction (PCR). No license under these patents to use amplification processes or enzymes is conveyed expressly
or by implication to the purchaser by the purchase of these products.

Introduction
These (RUO) assays are available for either ABI capillary electrophoresis fluorescence, or PAGE/agarose gel detection,and contain the PCR master mixes, recommended controls, and Instructions For Use.
On the following pages, you will find detailed information on each RUO assay, including: assay use, backgroundinformation, typical output data, kit contents, and ordering information. These assays are available in regular sizes(30 or 33 reactions). Select assays are also available in high-volume MegaKit formats (300 or 330 reactions).
These pages contain Research Use Only products which are not for use in diagnostic procedures. Research
Use Only (RUO) assays are not for sale in Europe and other global markets where equivalent CE-IVD assays areavailable and registered with the appropriate regulatory agencies. Refer to the preceding pages for information
regarding our IdentiClone® and LeukoStrat® CE-IVD Assays.
For more information, please visit www.invivoscribe.com

Chapter Contents
B-cell Assays

T-cell Assays

97 IGH + IGK B-cell Clonality Assays
99 IGH Gene Rearrangement Assays
101 IGH Gene Clonality Assays
103 IGK Gene Clonality Assays
105 IGL Gene Clonality Assays

107 TCRB Gene Clonality Assays
109 T-Cell Receptor Gama Gene Rearrangement Assays 2.0
111 T-Cell Receptor Gama Gene Rearrangement Assays
113 TCRD Gene Clonality Assays

Translocation Assays

SHM Assays

115 BCL1/JH Translocation Assay
117 BCL2/JH t(14;18) Translocation Assay
119 BCL2/JH Translocation Assay
121 BCR/ABL t(9;22) Translocation Assays
123 PML/RARα t(15;17) Translocation Assays

125 IGH Somatic Hypermutation Assays v2.0

FLT3 Mutation Assays
127 FLT3 Mutation Assays

Gel and Capillary | Research Use Only (RUO) Assays 96

IGH + IGK B-Cell Clonality Assays
Assay Use
Immunoglobulin heavy chain (IGH) and Kappa light chain (IGK) gene clonality assays are useful for identification of B-cell clonality, studying clonal
B-cell populations, and evaluation of new research and methods in malignancy studies.

Summary and Explanation of the Test
Five PCR master mixes are included in these test kits to test for rearrangements of both IGH and IGK. IGH Tubes A, B, and C target the conserved
framework 1, 2, and 3 regions (respectively) within the variable (VH) region and the joining (JH) region of the IGH locus. IGK tubes A and B target thevariable (Vƙ), intragenic and joining (Jƙ), and kappa deleting element (Kde) regions of the IGK locus.
Positive and negative controls, as well as Specimen Control Size Ladder Master Mix are included. PCR products can be analyzed by capillaryelectrophoresis or heteroduplex analysis. Clonality is indicated if any one of the master mixes generates clonal products.

Background
The immunoglobulin heavy chain (IGH) gene locus on chromosome 14 (14q32.33, formerly 14q32.3) includes 46-52 functional and 30 nonfunctionalvariable (VH), 27 functional diversity (DH), and 6 functional joining (JH) gene segments spread over 1250 kilobases.1,2 The most frequently used VH genesegments in normal and malignant B cells belong to VH3, VH4, and VH1 families, which together cover 75–95% of VH usage. The VH gene segments
contain three framework regions (FR) and two complementarity determining regions (CDR). The FRs are characterized by their similarity among
the various VH segments, whereas the CDRs are highly different even within the same VH family. The CDRs represent the preferred target sequences
for somatic hypermutations; however, somatic mutations can also occur in the FRs. Therefore, family-specific primers in the three different FRs
were designed to increase the detection rate of clonal IGH B-cell populations and decrease the occurrence of false-negative results due to somatic
hypermutation in primer binding sites.1
The human immunoglobulin kappa (IGK) light chain locus on the short arm of chromosome 2 (2p11.2) spans 1820 kb. It is made up of 76 variable (Vƙ)
gene segments belonging to seven subgroups, five joining (Jƙ) gene segments, and one constant (Cƙ) gene segment. Productive assembly of the
kappa gene is successful in about 60% of human B lymphocytes2; however, even when unsuccessful, clonal B cells generally retain the rearranged
kappa genes. The Vƙ segments encode the first 95 N-terminal amino acids. Positions 96-108 are encoded by one of five joining (Jƙ) gene segments.
The constant (Cƙ) portion of the kappa light chain (amino acids 109-214) is encoded by a single constant (Cƙ) region separated from the Jƙ region
by an intron.

Specimen Requirements
This assay tests extracted and purified genomic DNA (gDNA)

References
1. M Hummel et al., Leukemia 17: 2266-2272 (2003).
2. AW Langerak et al., Leukemia 17: 2272-2275 (2003).
3. EP Rock, PR Sibbald, MM Davis, and YH Chien. J. Exp. Med. 179(1): 323-328 (1994).
4. JJM van Dongen et al., Leukemia 17: 2257-2317 (2003).

This assay is based on the EuroClonality/BIOMED-2 Concerted Action BMH4-CT98-3936.

97 Gel and Capillary | Research Use Only (RUO) Assays

κ

Figure Legend: Simple representation of the organization of a rearranged immunoglobulin heavy chain (IGH) gene on chromosome 14 and the immunoglobulin kappalight chain gene on chromosome 2p11.2. Black arrows represent the relative positions of primers that target the conserved framework regions (FR1-3) and the downstream
consensus JH gene segments for IGH and the Vƙ, κ
Jƙ, INTR and Kde primers which are included in the IGK master mix tubes.

Reagents
Controls

Concentration

Units in Assay

Units in Assay MegaKit

IVS-0030 Clonal Control DNA

200 μg/mL

1 x 100 μL tube

5 x 100 μL tubes

IVS-0019 Clonal Control DNA

200 μg/mL

1 x 100 μL tube

5 x 100 μL tubes

IVS-0007 Clonal Control DNA

200 μg/mL

1 x 100 μL tube

5 x 100 μL tubes

IVS-0000 Polyclonal Control DNA

200 μg/mL

1 x 100 μL tube

5 x 100 μL tubes

Master Mixes

TargetUnits in Assay

Units in Assay MegaKit

IGH Tube A

Framework 1 + JH

1 x 1500 μL tube

10 x 1500 μL tubes

IGH Tube B

Framework 2 + JH

1 x 1500 μL tube

10 x 1500 μL tubes

IGH Tube C

Framework 3 + JH

1 x 1500 μL tube

10 x 1500 μL tubes

IGK Tube AVƙ-Jƙ

1 x 1500 μL tube

10 x 1500 μL tubes

IGK Tube B

Vƙ-Kde1 x 1500 μL tube

10 x 1500 μL tubes

Specimen Control Size LadderMultiple Genes

1 x 1500 μL tube

10 x 1500 μL tubes

Ordering Information
Catalog #Products

Quantity

1-100-0010

IGH + IGK B-Cell Clonality Assay - Gel Detection

33 reactions

1-100-0031

IGH + IGK B-Cell Clonality Assay - ABI Fluorescence Detection

33 reactions

1-100-0041

IGH + IGK B-Cell Clonality Assay MegaKit - ABI Fluorescence Detection

330 reactions

These products are for Research Use Only (RUO).
Not intended for diagnostic purposes.

Gel and Capillary | Research Use Only (RUO) Assays 98

IGH Gene Rearrangement Assays
Assay Use
Immunoglobulin heavy chain (IGH) gene clonality assays are useful for the study of identifying clonal B-cell populations and evaluation of new
research and methods in malignancy studies.

Summary and Explanation of the Test
Genomic DNA is amplified using three PCR master mixes that target the three conserved framework regions (FR1, FR2, and FR3) of the IGH gene andthe joining (JH) region. These regions flank the unique, hypervariable, antigen-binding, complementarity determining region 3 (CDR3). All positive
and negative DNA controls, as well as an Amplification Control master mix, are included. PCR products can be analyzed by capillary electrophoresis.
Clonality is indicated if one or more of the three framework master mixes generates clonal products.

Background
Genes encoding immunoglobulin heavy chain (IGH) molecules are assembled from multiple polymorphic gene segments that undergo
rearrangement and selection during B-cell development.2 Rearrangement of these variable (VH), diversity (DH), and joining (JH) genetic segments
result in VDJ products of unique length and sequence.1,2 Clonal IGH rearrangements can be rapidly identified through analyses of the size distributionsof DNA products amplified from conserved sequences that flank this region.2 For example, DNA isolated from a normal polyclonal population of B cells
produces a Gaussian distribution (bell-shaped size curve) of amplified products; whereas, DNA amplified from a clonal B-cell population generates
one or two product(s) of unique size that reflect proliferation of a single rearranged clone.1

Specimen Requirements
This assay tests extracted and purified genomic DNA (gDNA).

References
1. JE Miller, SS Wilson, DL Jaye, and M Kronenberg. J. Mol. Diag. 4: 101-117 (1999).
2. S Tonegawa. Nature 302: 575-581 (1983).

99 Gel and Capillary | Research Use Only (RUO) Assays

Figure Legend: Genomic organization of a rearranged immunoglobulin heavy chain gene on chromosome 14. The blue and green arrows represent primers targeting theconserved framework regions within the variable region gene. The relative location, size range of valid products, and colors correspond to the products generated fromeach of these regions when differential fluorescence detection methods are used.

Reagents
Controls

Concentration

Units in Assay

Units in Assay MegaKit

IVS-0030 Clonal Control DNA

200 μg/mL

1 x 100 μL tube

5 x 100 μL tubes

IVS-0029 Clonal Control DNA

200 μg/mL

1 x 100 μL tube

5 x 100 μL tubes

IVS-0000 Polyclonal Control DNA

200 μg/mL

1 x 100 μL tube

5 x 100 μL tubes

Master Mixes

TargetUnits in Assay

Units in Assay MegaKit

IGH Framework 1

Framework 1 + JH

1 x 1500 μL tube

10 x 1500 μL tubes

IGH Framework 2

Framework 2 + JH

1 x 1500 μL tube

10 x 1500 μL tubes

IGH Framework 3

Framework 3 + JH

1 x 1500 μL tube

10 x 1500 μL tubes

Amplification Control

HLA-DQa1 x 1500 μL tube

10 x 1500 μL tubes

Ordering Information
Catalog #Products

Quantity

1-101-0051

IGH Gene Rearrangement Assay - ABI Fluorescence Detection

30 reactions

1-101-0071

IGH Gene Rearrangement Assay MegaKit - ABI Fluorescence Detection

300 reactions

These products are for Research Use Only (RUO).
Not intended for diagnostic purposes.

Gel and Capillary | Research Use Only (RUO) Assays 100

IGH Gene Clonality Assays
Assay Use
Immunoglobulin heavy chain (IGH) clonality assays are useful for identification of B-cell clonality, studying clonal B-cell populations, and evaluationof new research and methods in malignancy studies.

Summary and Explanation of the Test
Five master mixes target conserved regions within the variable (VH), diversity (DH), and the joining (JH) regions that flank the unique hypervariable,
antigen-binding, complementarity determining region 3 (CDR3). Tube A contains six framework region 1 (FR1) primers and a consensus JH region
primer. Tube B contains seven framework region 2 (FR2) primers and a consensus JH primer. Tube C contains seven framework region 3 (FR1)
primers and a consensus JH primer. Tube D contains six DH region primers and a consensus JH region primer. Tube E contains a DH7 region primer
and a consensus JH primer. Positive and negative controls, as well as the Specimen Control Size Ladder Master Mix are included. PCR products canbe analyzed by capillary electrophoresis or heteroduplex analysis. Clonality is indicated if any one of the master mixes generates a clonal product.

Background
The immunoglobulin heavy chain (IGH) gene locus on chromosome 14 (14q32.33, formerly 14q32.3) includes 46-52 functional and 30 nonfunctionalvariable (VH), 27 functional diversity (DH), and 6 functional joining (JH) gene segments spread over 1250 kilobases.1,2 The most frequently used VH genesegments in normal and malignant B cells belong to the VH3, VH4, and VH1 family, together covering 75–95% of VH usage. The VH gene segments contain
three framework regions (FR) and two complementarity determining regions (CDR).
The FRs are characterized by their similarity among the various VH segments, whereas the CDRs are highly different even within the same VH family.
The CDRs represent the preferred target sequences for somatic hypermutations; however, somatic mutations can also occur in the FRs. Therefore,family-specific primers in the three different FRs were designed to increase the detection rate of clonal IGH B-cell populations and decrease theoccurrence of false-negative results due to somatic hypermutation in primer binding sites.1 In addition to VH-JH rearrangements, incomplete DH-JH
rearrangements have been found in mature and immature B-cell malignancies. Therefore, DH-JH PCR analysis may be of added value for clonality
assessment.2

Specimen Requirements
This assay tests extracted and purified genomic DNA (gDNA).

References
1. M Hummel et al., Leukemia 17:2266-2272 (2003).
2. AW Langerak et al., Leukemia 17:2272-2275 (2003).
3. JJM van Dongen et al., Leukemia 17:2257-2317 (2003).

This assay is based on the EuroClonality/BIOMED-2 Concerted Action BMH4-CT98-3936.

101 Gel and Capillary | Research Use Only (RUO) Assays

Figure Legend: Simple representation of the organization of a rearranged immunoglobulin heavy chain gene on chromosome 14. Black arrows represent the relative
positions of primers that target the conserved framework (FR1-3) and diversity (DH1-7) regions, and the downstream consensus JH gene segments. The amplicon products
generated from each of these regions can be differentially detected when fluorescent primer sets are used with capillary electrophoresis instruments that employ
differential fluorescence detection.

Reagents
Controls

Concentration

Units in Assay

Units in Assay MegaKit

IVS-0030 Clonal Control DNA

200 μg/mL

1 x 100 μL tube

5 x 100 μL tubes

IVS-0019 Clonal Control DNA

200 μg/mL

1 x 100 μL tube

5 x 100 μL tubes

IVS-0024 Clonal Control DNA

200 μg/mL

1 x 100 μL tube

5 x 100 μL tubes

IVS-0008 Clonal Control DNA

200 μg/mL

1 x 100 μL tube

5 x 100 μL tubes

IVS-0000 Polyclonal Control DNA

200 μg/mL

1 x 100 μL tube

5 x 100 μL tubes

Master Mixes

TargetUnits in Assay

Units in Assay MegaKit

IGH Tube A

Framework 1 + JH

1 x 1500 μL tube

10 x 1500 μL tubes

IGH Tube B

Framework 2 + JH

1 x 1500 μL tube

10 x 1500 μL tubes

IGH Tube C

Framework 3 + JH

1 x 1500 μL tube

10 x 1500 μL tubes

IGH Tube D

DH1-6 + JH

1 x 1500 μL tube

10 x 1500 μL tubes

IGH Tube E

DH7 + JH

1 x 1500 μL tube

10 x 1500 μL tubes

Specimen Control Size LadderMultiple Genes

1 x 1500 μL tube

10 x 1500 μL tubes

Ordering Information
Catalog #Products

Quantity

1-101-0020

IGH Gene Clonality Assay - Gel Detection

33 reactions

1-101-0061

IGH Gene Clonality Assay - ABI Fluorescence Detection

33 reactions

1-101-0081

IGH Gene Clonality Assay MegaKit - ABI Fluorescence Detection

330 reactions

These products are for Research Use Only (RUO).
Not intended for diagnostic purposes.

Gel and Capillary | Research Use Only (RUO) Assays 102

IGK Gene Clonality Assays
Assay Use
Immunoglobulin kappa light chain (IGK) gene clonality assays are useful for the identification of B-cell clonality, studying clonal B-cell populations,and evaluation of new research and methods in malignancy studies.

Summary and Explanation of the Test
Two master mixes target conserved regions within the variable (Vƙ1-7) and the joining (Jƙ1-5) regions that flank the unique hypervariable,
antigen-binding, complementarity determining region 3 (CDR3). Other primers target the Kde and intragenic regions.
Tube A contains six upstream primers and two Jƙ region primers. Tube B contains six upstream Vƙ region primers, an upstream intragenic
primer and a downstream Kde primer. Positive and negative controls, as well as a Specimen Control Size Ladder Master Mix, are included.
PCR products can be analyzed by capillary electrophoresis or heteroduplex analysis. Clonality is indicated if any one of the master mixes
generates clonal products.

Background
The human immunoglobulin kappa (IGK) light chain locus on the short arm of chromosome 2 (2p12, formerly 2p11.2) spans 1820 kb. It is madeup of 76 variable (Vƙ) gene segments belonging to 7 subgroups, 5 joining (Jƙ) gene segments, and one constant (Cƙ) gene segment.
Productive assembly of the kappa gene is successful in about 60% of human B lymphocytes.1 However, even when unsuccessful, clonal B cells
generally retain the rearranged kappa genes. The Vƙ segments encode the first 95 N-terminal amino acids. Positions 96-108 are encoded by oneof five joining (Jƙ) gene segments. The constant (Cƙ) portion of the kappa light chain (amino acids 109-214) is encoded by a single constant (Cƙ) region
separated from the Jƙ region by an intron. The length of the hypervariable complementarity determining region 3 (CDR3) in kappa light chain genes
is limited and rearrangements in this region display significant skewing (platykurtosis).2
Therefore, clonal CDR3 products generated from this region are most easily and reliably identified by heteroduplex analysis using standard
polyacrylamide gels. Alternatively, capillary electrophoresis or gene sequencing instruments coupled with differential fluorescence detection canbe used for analysis.

Specimen Requirements
This assay tests extracted and purified genomic DNA (gDNA).

References
1. AW Langerak et al., Leukemia 17: 2275-2280 (2003).
2. EP Rock, PR Sibbald, MM Davis, and YH Chien. J. Exp. Med. 179(1): 323-328 (1994).
3. JJM van Dongen et al., Leukemia 17: 2257-2317 (2003).

This assay is based on the EuroClonality/BIOMED-2 Concerted Action BMH4-CT98-3936.

103 Gel and Capillary | Research Use Only (RUO) Assays

Figure Legend: Schematic diagram of the immunoglobulin kappa light chain gene complex on chromosome 2p11.2. Shown are the relative positions and orientations
for the Vƙ-Jƙ, and Kde primers, which are included in the IGK master mix tubes.

Reagents
Controls

Concentration

Units in Assay

Units in Assay MegaKit

IVS-0007 Clonal Control DNA

200 μg/mL

1 x 100 μL tube

5 x 100 μL tubes

IVS-0000 Polyclonal Control DNA

200 μg/mL

1 x 100 μL tube

5 x 100 μL tubes

Master Mixes

TargetUnits in Assay

Units in Assay MegaKit

IGK Tube AVƙ-Jƙ

1 x 1500 μL tube

10 x 1500 μL tubes

IGK Tube B

Vƙ-Kde1 x 1500 μL tube

10 x 1500 μL tubes

Specimen Control Size LadderMultiple Genes

1 x 1500 μL tube

10 x 1500 μL tubes

Ordering Information
Catalog #Products

Quantity

1-102-0020

IGK Gene Clonality Assay - Gel Detection

33 reactions

1-102-0021

IGK Gene Clonality Assay - ABI Fluorescence Detection

33 reactions

1-102-0031

IGK Gene Clonality Assay MegaKit - ABI Fluorescence Detection

330 reactions

These products are for Research Use Only (RUO).
Not intended for diagnostic purposes.

Gel and Capillary | Research Use Only (RUO) Assays 104

IGL Gene Clonality Assays
Assay Use
Immunoglobulin lambda light chain (IGL) gene clonality assays are useful for the identification of B-cell clonality, studying clonal B-cell populations,and evaluation of new research and methods in malignancy studies.

Summary and Explanation of the Test
The IGL Tube master mix targets conserved regions within the variable (Vλ1-3) and the joining (Jλ1-3) regions that flank the unique, hypervariable,
antigen-binding, complementarity determining region 3 (CDR3). Positive and negative controls, as well as a Specimen Control Size Ladder Master
Mix, are included. PCR products can be analyzed by capillary electrophoresis or heteroduplex analysis. Clonality is indicated if the master mix
generates clonal products.

Background
The human immunoglobulin lambda (IGL) light chain locus is located on the long arm of chromosome 22 (22q11.2) and spans 1050 kilobases.
It is made up of 73-74 variable (Vλ) gene segments (spread over 900 kilobases), 7-11 joining (Jλ) gene segments and 7-11 constant (Cλ) genesegments depending on the haplotypes. Of the 73-74 Vλ region genes, only 30-33 are functional and can be grouped into 11 families and 3 clans.1
The Jλ and Cλ region genes are organized in tandem with a Jλ segment preceding a Cλ gene. Typically there are 7 Jλ-Cλ segments of which four are
functional and encode the four Ig lambda isotypes.
IGL gene rearrangements (Vλ-Jλ) rearrangements potentially represent an attractive PCR target for clonality studies to compensate for false-negative
IGH VH-JH PCR results mainly caused by somatic hypermutations. The limited size of the junctional region may create a challenge to distinguishpolyclonal from monoclonal rearrangements when running a simple agarose or polyacrylamide gel.1 Therefore, clonal Vλ-Jλ PCR products are mosteasily and reliably identified by heteroduplex analysis using standard polyacrylamide gels. Alternatively, capillary electrophoresis or gene sequencing
instruments coupled with differential fluorescence detection can also be used for analysis.1

Specimen Requirements
This assay tests extracted and purified genomic DNA (gDNA).

References
1. F Davi et al., Leukemia 17:2280-2283 (2003).
2. JJM van Dongen et al., Leukemia 17:2257-2317 (2003).

This assay is based on the EuroClonality/BIOMED-2 Concerted Action BMH4-CT98-3936.

105 Gel and Capillary | Research Use Only (RUO) Assays

Figure Legend: Schematic diagram of the immunoglobulin lambda light chain gene complex on chromosome 22q11.2. Shown are the relative positions and orientations for
the Vλ and Jλ primers, which are included in the IGL master mix tube. The two Vλ primers only target Vλ1, 2, and 3 because these three V families cover approximately 70% of
rearrangeable Vλ gene segments, and approximately 90% of all IGL gene rearrangements involve these three families. Similarly, the single Jλ primer only targets Jλ1, 2, and3 because these three J segments are involved in 98% of all IGL gene rearrangements.

Reagents
Controls

Concentration

Units in Assay

Units in Assay MegaKit

IVS-0010 Clonal Control DNA

200 μg/mL

1 x 100 μL tube

5 x 100 μL tubes

IVS-0029 Clonal Control DNA

200 μg/mL

1 x 100 μL tube

5 x 100 μL tubes

IVS-0000 Polyclonal Control DNA

200 μg/mL

1 x 100 μL tube

5 x 100 μL tubes

Master Mixes

TargetUnits in Assay

Units in Assay MegaKit

IGL TubeVλ-Jλ

1 x 1500 μL tube

10 x 1500 μL tubes

Specimen Control Size LadderMultiple Genes

1 x 1500 μL tube

10 x 1500 μL tubes

Ordering Information
Catalog #Products

Quantity

1-103-0010

IGL Gene Clonality Assay - Gel Detection

33 reactions

1-103-0011

IGL Gene Clonality Assay - ABI Fluorescence Detection

33 reactions

1-103-0021

IGL Gene Clonality Assay MegaKit - ABI Fluorescence Detection

330 reactions

These products are for Research Use Only (RUO).
Not intended for diagnostic purposes.

Gel and Capillary | Research Use Only (RUO) Assays 106

TCRB Gene Clonality Assays
Assay Use
T-Cell receptor beta (TCRB) gene clonality assays are useful for the identification of T-cell clonality, studying clonal T-cell populations, and evaluationof new research and methods in malignancy studies.

Summary and Explanation of the Test
Three multiplex master mixes target conserved regions within the variable (Vβ), diversity (Dβ), and the joining (Jβ) regions that flank the unique
hypervariable, antigen-binding, complementarity determining region 3 (CDR3) of the T-cell receptor beta locus. Tube A contains 23 Vβ primers, six
Jβ1 primers, and three Jβ2 primers. Tube B contains 23 Vβ and four Jβ2 primers. Tube C contains two Dβ and 13 Jβ primers. Positive and negative DNA
controls, as well as a Specimen Control Size Ladder Master Mix, are included. PCR products can be analyzed by capillary electrophoresisor heteroduplex analysis. Clonality is indicated, if any one of the master mixes generates clonal products.

Background
The human T-cell receptor beta (TRB, formerly known as TCRB) gene locus on chromosome 7 (7q34, formerly 7q35) includes 64-67 variable (Vβ) genesegments (belonging to 30 subgroups), two diversity (Dβ) gene segments, and 13 joining (Jβ) gene segments, spread over 685 kilobases. The diversityof this locus has complicated PCR-based testing and extended dependence on Southern blot analysis in many testing centers. However, thisstandardized multiplex PCR assay detects the vast majority of clonal TRB gene rearrangements using only three multiplex master mixes.1

Specimen Requirements
This assay tests extracted and purified genomic DNA (gDNA).

References
1. M Brüggemann et al., Leukemia 17: 2283-2289 (2003).
2. JJM van Dongen et al., Leukemia 17: 2257-2317 (2003).

This assay is based on the EuroClonality/BIOMED-2 Concerted Action BMH4-CT98-3936.

107 Gel and Capillary | Research Use Only (RUO) Assays

Figure Legend: Simplified diagram of a representative rearranged T-cell receptor beta gene showing the approximate placement of the upstream and downstream DNA
primers. The numbers of primers and their specificity are listed for Master Mix Tubes A, B, and C.

Reagents
Controls

Concentration

Units in Assay

Units in Assay MegaKit

IVS-0009 Clonal Control DNA

200 μg/mL

1 x 100 μL tube

5 x 100 μL tubes

IVS-0004 Clonal Control DNA

200 μg/mL

1 x 100 μL tube

5 x 100 μL tubes

IVS-0021 Clonal Control DNA

200 μg/mL

1 x 100 μL tube

5 x 100 μL tubes

IVS-0000 Polyclonal Control DNA

200 μg/mL

1 x 100 μL tube

5 x 100 μL tubes

Master Mixes

TargetUnits in Assay

Units in Assay MegaKit

TCRB Tube A

Multiple Vβ + Jβ1/2

1 x 1500 μL tube

10 x 1500 μL tubes

TCRB Tube B

Multiple Vβ + Jβ2

1 x 1500 μL tube

10 x 1500 μL tubes

TCRB Tube C

Multiple Dβ + Jβ1/2

1 x 1500 μL tube

10 x 1500 μL tubes

Specimen Control Size LadderMultiple Genes

1 x 1500 μL tube

10 x 1500 μL tubes

Ordering Information
Catalog #Products

Quantity

1-205-0010

TCRB Gene Clonality Assay - Gel Detection

33 reactions

1-205-0020

TCRB Gene Clonality Assay MegaKit - Gel Detection

330 reactions

1-205-0011

TCRB Gene Clonality Assay - ABI Fluorescence Detection

33 reactions

1-205-0021

TCRB Gene Clonality Assay MegaKit - ABI Fluorescence Detection

330 reactions

These products are for Research Use Only (RUO).
Not intended for diagnostic purposes.

Gel and Capillary | Research Use Only (RUO) Assays 108

T-Cell Receptor Gamma
Gene Rearrangement Assay 2.0
Assay Use
This Research Use Only assay identifies T-cell receptor gamma (TCRG) chain gene rearrangements and is useful for the identification of clonal T-cell
populations and evaluation of new research and methods in malignancy studies.

Summary and Explanation of the Test
This T-Cell Receptor Gamma Gene Rearrangement Assay 2.0 contains a single multiplex master mix which includes primers for all known groups ofTCR gamma variable (Vƴ) region genes and joining (Jƴ) region genes involved in rearrangements. In addition, positive and negative controls, as well
as a Specimen Control Size Ladder Master Mix are included. PCR products can be analyzed by polyacrylamide or capillary electrophoresis detection.

Background
The human T-cell receptor gamma (TRG, formerly known as TCRG) gene locus on chromosome 7 (7q14) includes 14 Vƴ genes belonging to four
subgroups, five Jƴ segments, and two Cƴ genes spread over 200 kilobases. The diversity of this locus has historically complicated PCR-based testing.
This multiplex PCR assay represents an improvement over other assays as it can detect the vast majority of TCR gamma gene rearrangements witha single multiplex master mix. This master mix targets all conserved regions within the variable (Vƴ) and joining (Jƴ) region genes, providing a morecomprehensive analysis to include Vƴ and Jƴ regions that would not be identified with a single Vƴ (1-8) and Jƴ 1/Jƴ 2 primer set.
In addition, competitive amplification of all TRG gene rearrangements allows for identification of a quantitative threshold for a positive result andhelps to avoid false positive results. The average size of the TRG gene rearrangement PCR amplicons is 190 nucleotides, with a normal distributionof product sizes between 159 and 207 nucleotides. This protocol leads to improved product formation from formalin-fixed, paraffin-embedded (FFPE)
samples compared to other protocols that yield products of 260 nucleotides or larger.

Specimen Requirements
This assay tests extracted and purified genomic DNA (gDNA).

References
1. TC Greiner et al., JMD 4: 137-143 (2002).
2. LC Lawnickie et al., JMD 5: 82-87 (2003).
3. Y Sandberg et al., Leukemia 21: 21 (2007).
4. Armand, Marine et al. HemaSphere, 2019;3:3.

This assay was developed by Invivoscribe.
The performance of this assay was reviewed and validated by the EuroClonality/BIOMED-2 Group.4

109 Gel and Capillary | Research Use Only (RUO) Assays

Figure Legend: Simple representation of the organization of the T-cell receptor gamma gene on chromosome 7. Black arrows represent the relative positions of primersthat target the variable region genes and the downstream joining region gene segments that are involved in rearrangements in T-cell lymphomas. The downstream
primers are fluorescently labeled through the incorporation of a 6FAM fluorophore. The amplicon products generated from these rearrangements are detected bycapillary electrophoresis.

Reagents
Controls

Concentration

Units in Assay

Units in Assay MegaKit

5% TCRG Positive Control DNA

50 μg/mL

1 x 50 μL tube

5 x 50 μL tube

TCRG Negative Control DNA

50 μg/mL

1 x 50 μL tube

5 x 50 μL tube

Master Mixes

TargetUnits in Assay

Units in Assay MegaKit

TCRG - 6FAM

Vƴ 1-Vƴ 11 + Jƴ 1/Jƴ 2, Jƴ P, Jƴ P1/Jƴ P2

1 x 1500 μL tube

10 x 1500 μL tubes

Specimen Control Size LadderMultiple Genes

1 x 1500 μL tube

10 x 1500 μL tubes

Ordering Information
Catalog #Products

Quantity

1-207-0101

T-Cell Receptor Gamma Gene Rearrangement Assay 2.0 - ABI Fluorescence Detection

33 reactions

1-207-0111

T-Cell Receptor Gamma Gene Rearrangement Assay MegaKit 2.0 - ABI Fluorescence Detection

330 reactions

These products are for Research Use Only (RUO).
Not intended for diagnostic purposes.

Gel and Capillary | Research Use Only (RUO) Assays 110

T-Cell Receptor Gamma
Gene Rearrangement Assays
Assay Use
T-Cell receptor gamma (TCRG) gene clonality assays are useful for the identification of T-cell clonality, studying clonal T-cell populations, andevaluation of new research and methods in malignancy studies.

Summary and Explanation of the Test
Sample genomic DNA is amplified using two master mixes that independently target conserved regions within the variable (Vƴ) and joining (Jƴ)
regions that flank the unique, hypervariable, antigen-binding, complementarity determining region 3 (CDR3). This assay targets Vƴ 1-9 and Jƴ genesegments. Positive and negative DNA controls, as well as an internal Amplification Control Master Mix, are included. PCR products can be analyzed
by capillary electrophoresis.

Background
The T-cell receptor gamma (TRG, formerly known as TCRG) chain locus spans 160 kilobases on chromosome 7 (7p14). The locus consists of 14 variable(Vƴ) gene segments in six subgroups, and five joining (Jƴ) gene segments interspersed between two constant (Cƴ) gene segments. However, therepertoire of functional TRG molecules is limited to 4-6 functional Vƴ gene segments that belong to two subgroups.2
Rearrangement of the Vƴ and Jƴ gene segments of the TRG locus results in Vƴ-Jƴ products of unique length and sequence. Clonal TRG rearrangements
can be most rapidly identified by analyzing the size distribution of DNA products amplified from conserved sequences that flank this Vƴ-Jƴ region.1
DNA isolated from a normal heterogeneous population of polyclonal T-cells produces a Gaussian distribution (bell-shaped size curve) of amplified
products. DNA amplified from a clonal T-cell population generates one or two product(s) of unique size that reflects proliferation of a single
rearranged clone.1,2Specimen Requirements
This assay tests extracted and purified genomic DNA (gDNA).

References
1. JE Miller, SS Wilson, DL Jaye, and M Kronenberg. J. Mol. Diag. 4: 101-117 (1999).
2. K Beldjord et al., Leukemia 17: 2289-2292 (2003).

111 Gel and Capillary | Research Use Only (RUO) Assays

Figure Legend: Simplified figure representing the organization of a rearranged T-cell receptor gamma chain gene on chromosome 7. Colored arrows represent conserved
regions within the variable region gene segments targeted by primers. Primers are represented by arrows with the size range of valid products generated with each of themaster mixes indicated below the figure. Colors correspond to the peak colors assigned to products when differential fluorescence detection methods are used.

Reagents
Controls

Concentration

Units in Assay

Units in Assay MegaKit

IVS-0009 Clonal Control DNA

200 μg/mL

1 x 100 μL tube

5 x 100 μL tubes

IVS-0000 Polyclonal Control DNA

200 μg/mL

1 x 100 μL tube

5 x 100 μL tubes

Master Mixes

TargetUnits in Assay

Units in Assay MegaKit

T-Cell Receptor Gamma Mix 1

Vƴ1-8,9 + Jƴ1/2

1 x 1500 μL tube

10 x 1500 μL tubes

T-Cell Receptor Gamma Mix 2

Alt Vƴ + Jƴ1/2

1 x 1500 μL tube

10 x 1500 μL tubes

Amplification Control

HLA-DQa1 x 1500 μL tube

10 x 1500 μL tubes

Ordering Information
Catalog #Products

Quantity

1-207-0051

T-Cell Receptor Gene Rearrangement Assay - ABI Fluorescence Detection

30 reactions

This product is for Research Use Only (RUO).
Not intended for diagnostic purposes.

Gel and Capillary | Research Use Only (RUO) Assays 112

TCRD Gene Clonality Assays
Assay Use
T-Cell receptor delta (TCRD) gene clonality assays are useful for the identification of T-cell clonality, studying clonal T-cell populations, and evaluationof new research and methods in malignancy studies.

Summary and Explanation of the Test
The TCRD Tube Master Mix targets conserved regions within the variable (Vδ1-6), the diversity (Dδ2-3) and the joining (Jδ1-4) regions that flank theunique, hypervariable, antigen-binding, complementarity determining region 3 (CDR3) of the T-cell receptor delta (TRD, formerly known as TCRD).
Positive and negative controls, as well as a Specimen Control Size Ladder Master Mix, are included. PCR products can be analyzed by capillaryelectrophoresis or heteroduplex analysis. Clonality is indicated if the master mix generates clonal products.

Background
The human T-cell receptor delta (TRD, formerly known as TCRD) gene locus is comprised of a cluster of 10 genes located on chromosome 14 (14q11.2)
spread over 60 kilobases, localized between the T-cell receptor alpha (TRA, formerly known as TCRA) variable (Vα) and joining (Jα) gene segments.
It is made up of eight variable (Vδ), three diversity (Dδ), and four joining (Jδ) gene segments.1 At least five of the eight Vδ gene segments can also
rearrange to Jδ gene segments and other Vδ gene segments may also be utilized in TRD gene rearrangements in rare cases. Although the small
number of Vδ, Dδ, and Jδ gene segments available for recombination limits the potential combinatorial diversity, the complementarity determining
region 3 (CDR3) or junctional diversity is extensive due to the addition of N regions, P regions, and random deletion of nucleotides by recombinases.
This diversity is also extended by the recombination of up to three Dδ segments and therefore up to four N regions within the rearranged TRD locus.
This limited germline diversity encoded at the TRD locus in conjunction with extensive junctional diversity results in a useful target for PCR analysis.
TRD recombination events have been used most extensively as clonal markers in both T- and B-cell ALL. This standardized multiplex PCR assay
detects the vast majority of clonal TRD gene rearrangements using a single multiplex master mix1.

Specimen Requirements
This assay tests extracted and purified genomic DNA (gDNA).

References
1. FL Lavender et al., Leukemia 17: 2292-2296 (2003).
2. JJM van Dongen et al., Leukemia 17: 2257-2317 (2003).

This assay is based on the EuroClonality/BIOMED-2 Concerted Action BMH4-CT98-3936.

113 Gel and Capillary | Research Use Only (RUO) Assays

Figure Legend: Simplified diagram of a representative rearranged T-cell receptor delta gene showing the approximate placement of the upstream and downstream DNA
primers. The numbers of primers and their specificity are listed for the TRD Tube Master Mix tube.

Reagents
Controls

Concentration

Units in Assay

Units in Assay MegaKit

IVS-0021 Clonal Control DNA

200 μg/mL

1 x 100 μL tube

5 x 100 μL tubes

IVS-0000 Polyclonal Control DNA

200 μg/mL

1 x 100 μL tube

5 x 100 μL tubes

Master Mixes

TargetUnits in Assay

Units in Assay MegaKit

TCRD Tube

Multiple Vδ + Dδ + Jδ

1 x 1500 μL tube

10 x 1500 μL tubes

Specimen Control Size LadderMultiple Genes

1 x 1500 μL tube

10 x 1500 μL tubes

Ordering Information
Catalog #Products

Quantity

1-206-0010

TCRD Gene Clonality Assay - Gel Detection

33 reactions

1-206-0011

TCRD Gene Clonality Assay - ABI Fluorescence Detection

33 reactions

1-206-0021

TCRD Gene Clonality Assay MegaKit - ABI Fluorescence Detection

330 reactions

These products are for Research Use Only (RUO).
Not intended for diagnostic purposes.

Gel and Capillary | Research Use Only (RUO) Assays 114

BCL1/JH Translocation Assay
Assay Use
These assays identify BCL1/JH t(11;14) translocations and are useful for the evaluation of new research and methods in malignancy studies.

Summary and Explanation of the Test
Two master mixes are included in this assay kit. The BCL1/JH Master Mix targets the major translocation cluster (MTC) of the CCND1 locus (formerly
known as BCL1) and the joining region (JH) of the immunoglobulin heavy chain locus (IGH). 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 andquantity of input DNA is adequate to yield a valid result. Positive and negative controls are included. PCR products can be analyzed using
standard gel electrophoresis with ethidium bromide staining. A CCND1 translocation is indicated if the master mix generates product(s) within thevalid size range.

Background
This aberrant BCL1/JH t(11;14) translocation juxtaposes genes of the immunoglobulin heavy chain (IGH) gene on chromosome 14q32 with the cyclin
D1 gene on chromosome 11q13. The juxtaposition of IGH-sequences results in the transcriptional activation of cyclin D1.2,3 Cyclin D1 is involved in theregulation of the G1 progression and G1/S transition of the cell cycle.3 Translocation does not lead to expression of a fusion protein. In fact, oncogenesis
is due to a promoter/enhancer exchange, wherein the immunoglobulin gene enhancer stimulates the expression of cyclin D1. Overexpression of cyclin
D1, in turn, accelerates passage of transformed cells through the G1 phase.

Specimen Requirements
This assay tests extracted and purified genomic DNA (gDNA).

References
1. P Wijers et al., Leukemia 17: 2296-2298 (2003).
2. JJM van Dongen et al., Leukemia 17: 2257-2317 (2003).
3. Shimazaki C, et. al., (1997). International Journal of Hematology. 66(1):111-5.

This assay is based on the EuroClonality/BIOMED-2 Concerted Action BMH4-CT98-3936.

115 Gel and Capillary | Research Use Only (RUO) Assays

Figure Legend: Schematic diagram of the IGH-CCND1 t(11;14) translocation showing the cyclin D1 (CCND1) gene on the left and the Ig heavy chain (IGH) gene on the right.
Shown are the relative positions and orientations for the BCL1/MTC primer and the JH primer, which are included in the BCL1/JH Master Mix tube.

Reagents
Controls

Concentration

Units in Assay

Units in Assay MegaKit

IVS-0010 Clonal Control DNA

200 μg/mL

1 x 100 μL tube

5 x 100 μL tubes

IVS-0000 Polyclonal Control DNA

200 μg/mL

1 x 100 μL tube

5 x 100 μL tubes

Master Mixes

TargetUnits in Assay

Units in Assay MegaKit

BCL1/JH Tube

MTC of CCND1 + IGH JH

1 x 1500 μL tube

10 x 1500 μL tubes

Specimen Control Size LadderMultiple Genes

1 x 1500 μL tube

10 x 1500 μL tubes

Ordering Information
Catalog #Products

Quantity

1-308-0010

BCL1/JH Translocation Assay - Gel Detection

33 reactions

1-308-0020

BCL1/JH Translocation Assay MegaKit - Gel Detection

330 reactions

These products are for Research Use Only (RUO).
Not intended for diagnostic purposes.

Gel and Capillary | Research Use Only (RUO) Assays 116

BCL2/JH t(14;18) Translocation Assays
Assay Use
This Research Use Only assay identifies BCL2/JH t(14;18) translocations.

Summary and Explanation of the Test
Five master mixes are included in this assay kit. Two master mixes target BCL2 major break point (MBR) translocations and two target BCL2 minor
cluster region (mcr) translocations. An Amplification Control Master Mix is also included to ensure the quality and quantity of sample DNA. Positive
and negative controls are also included. This assay can be run either in a standard or nested assay format. PCR products can be analyzed
by standard gel electrophoresis with ethidium bromide staining. A BCL2 translocation is indicated if just one of the 2nd round master mixes (mixes
ending in b) generates product(s) within the valid size range.

Background
BCL2 translocations are reciprocal chromosome exchanges that place the bcl-2 proto-oncogene, located on chromosome 18, under aberrant
transcriptional control of the immunoglobulin heavy chain gene, located on chromosome 14. The bcl-2 protein is an antagonist to apoptosis(programmed cell death), a normal process designed to eliminate unneeded and damaged cells during hematopoiesis. Increased expressionof the bcl-2 protein leads to an increase in the levels of B-cells in the body.

Specimen Requirements
This assay tests extracted and purified genomic DNA (gDNA).

References
1. MS Lee et al., Science 237: 175-178 (1987).
2. M Crescenzi et al., Proc. Natl. Acad. Sci. USA 85: 4869-4873 (1988).

BCL2 Gene

Chromosome 18q21

Heavy Chain Gene
Chromosome 14q32

BCL2/IgH Fusion Gene
MBR JH Breakpoints

BCL2/IgH Fusion Gene
mcr JH Breakpoints

Figure Legend: Simplified view of the genomic organization of the BCL2 and IGH genes on chromosomes 18 and 14, respectively. Yellow boxes represent the exon regionsof the BCL2 gene. Exons of the immunoglobulin heavy chain gene are represented in other colors. The solid black lines represents intron regions, which have been left
incompletely spliced to assist in demarcation of the exon segments. MBR and mcr type t(14;18) translocations are shown in the lower portions of the figure with the relative
positions of primers and the size of the amplicons generated from the positive control DNAs indicated.

117 Gel and Capillary | Research Use Only (RUO) Assays

Reagents
Controls

Concentration

Units in Assay

Units in Assay MegaKit

IVS-0030 Clonal Control DNA

200 μg/mL

1 x 100 μL tube

5 x 100 μL tubes

IVS-0031 Clonal Control DNA

200 μg/mL

1 x 100 μL tube

5 x 100 μL tubes

IVS-0009 Clonal Control DNA

200 μg/mL

1 x 100 μL tube

5 x 100 μL tubes

Master Mixes

TargetUnits in Assay

Units in Assay MegaKit

BCL2/JH t(14;18) (MBR) Mix 1b

Inside BCL2 MBR

1 x 1500 μL tube

10 x 1500 μL tubes

BCL2/JH t(14;18) (mcr) Mix 2b

Inside BCL2 mcr

1 x 1500 μL tube

10 x 1500 μL tubes

BCL2/JH t(14;18) (MBR) Mix 1a

Outside BCL2 MBR

1 x 1500 μL tube

10 x 1500 μL tubes

BCL2/JH t(14;18) (mcr) Mix 2a

Outside BCL2 mcr

1 x 1500 μL tube

10 x 1500 μL tubes

Amplification Control

HLA-DQa1 x 1500 μL tube

10 x 1500 μL tubes

Ordering Information
Catalog #Products

Quantity

1-309-0010

BCL2/JH t(14;18) Translocation Assay - Gel Detection

30 reactions

This product is for Research Use Only (RUO).
Not intended for diagnostic purposes.

Gel and Capillary | Research Use Only (RUO) Assays 118

BCL2/JH Translocation Assay
Assay Use
This Research Use Only assay identifies BCL2/JH translocations.

Summary and Explanation of the Test
Four master mixes are included in this assay. Three are used to identify translocations in the major breakpoint region (MBR) and minor cluster region(mcr) of BCL2. 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. This assay includes negative
control DNA and positive control DNAs for both the MBR and mcr. PCR products can be analyzed using standard gel electrophoresis with ethidium
bromide staining. A BCL2 translocation is indicated if any one of the master mixes generates product(s) within the valid size range.

Background
BCL2 translocations are reciprocal chromosome exchanges that place the bcl-2 proto-oncogene, located on chromosome 18, under aberrant
transcriptional control of the immunoglobulin heavy chain gene, located on chromosome 14. The bcl-2 protein is an antagonist to apoptosis(programmed cell death), a normal process designed to eliminate unneeded and damaged cells during hematopoiesis. Increased expressionof the bcl-2 protein leads to an increase in the levels of B-cells in the body.

Specimen Requirements
This assay tests extracted and purified genomic DNA (gDNA).

References
1. PAS Evans et al., Leukemia 17: 2298-2301 (2003).
2. JJM van Dongen et al., Leukemia 17: 2257-2317 (2003).

H

H

H

Figure Legend: Schematic diagram of the IGH-BCL2 t(14;18) translocation showing the BCL2 gene on the left and the Ig heavy chain (IGH) gene on the right. Shown are
the relative positions and orientations for the major breakpoint region (MBR) primers, the minor cluster region (mcr) primers, and the JH primer, which are included in the
3 BCL2/JH master mix tubes.

This assay is based on the EuroClonality/BIOMED-2 Concerted Action BMH4-CT98-3936.

119 Gel and Capillary | Research Use Only (RUO) Assays

Reagents
Controls

Concentration

Units in Assay

Units in Assay MegaKit

IVS-0030 Clonal Control DNA

200 μg/mL

1 x 100 μL tube

5 x 100 μL tubes

IVS-P002 Clonal Control DNA

1600 pg/mL

1 x 100 μL tube

5 x 100 μL tubes

IVS-0031 Clonal Control DNA

200 μg/mL

1 x 100 μL tube

5 x 100 μL tubes

IVS-0000 Polyclonal Control DNA

200 μg/mL

1 x 100 μL tube

5 x 100 μL tubes

Master Mixes

TargetUnits in Assay

Units in Assay MegaKit

BCL2/JH Tube A

BCL2 MBR + IGH JH

1 x 1500 μL tube

10 x 1500 μL tubes

BCL2/JH Tube B

BCL2 3’ MBR + IGH JH

1 x 1500 μL tube

10 x 1500 μL tubes

BCL2/JH Tube C

BCL2 mcr + IGH JH

1 x 1500 μL tube

10 x 1500 μL tubes

Specimen Control Size LadderMultiple Genes

1 x 1500 μL tube

10 x 1500 μL tubes

Ordering Information
Catalog #Products

Quantity

1-309-0020

BCL2/JH Translocation Assay - Gel Detection

33 reactions

1-309-0040

BCL2/JH Translocation Assay MegaKit - Gel Detection

330 reactions

These products are for Research Use Only (RUO).
Not intended for diagnostic purposes.

Gel and Capillary | Research Use Only (RUO) Assays 120

BCR/ABL t(9;22) Translocation Assays
Assay Use
This Research Use Only assay identifies BCR/ABL t(9;22) translocations.

Summary and Explanation of the Test
The master mixes are included in these assay kits used to amplify complementary DNA (cDNA) produced from specimen(s), and positive and negative
RNA controls (included). Primers target an internal control transcript (Abl) and p190-, p210-, and p230-type transcripts expressed from BCR-ABL1
translocations. Amplicon products can be analyzed by capillary electrophoresis or standard gel electrophoresis with ethidium bromide staining. A
BCR-ABL1 translocation is indicated if just one of the 2nd round master mixes (Mix 2b, Mix 2c, Mix 3b, Mix 3c, or Mix 3d) generates product(s) of thevalid size. Reagents for RNA extraction and reverse transcription are not included. This assay is compatible with all standard RNA extraction and cDNA
synthesis methods. This is a qualitative assay and has not been validated for quantitative use.

Background
BCR/ABL translocations are associated with a variety of hematologic malignancies. The Philadelphia chromosome (Ph1) is a specific chromosomal
abnormality that results from reciprocal t(9;22)(q34;ql1) chromosomal rearrangements fuse coding regions of the BCR gene, located on chromosome
22, with the ABL receptor-independent tyrosine kinase gene on chromosome 9. This assay detects and identifies the variety of p190-, p210- and p230type transcripts produced from all known BCR/ABL translocations.

Specimen Requirements
This assay tests complementary DNA (cDNA) template.

References
1. R Kurzrock et al., Ann. Intern. Med. 138: 819-30 (2003).
2. JV Melo. Blood 88: 2375-2384 (1996).
3. JP Radich et al., Blood 85: 2632-2638 (1995).

BCR

Chromosome 22

ABL

Chromosome 9

BCR/ABL t(9;22) Translocation
p210 Type, b3a2 junction

ABL cDNA Control

Figure Legend: This figure shows the genomic organization of the BCR and ABL genes on chromosomes 22 and 9, respectively. Boxes represent exon regions of the ABL (red boxes) and BCR encodingexons (other colors). The solid black line represents intron regions, which have been left incompletely spliced to assist in demarcation of the exon segments. The location of exon regions targeted bylabeled and unlabeled primers are indicated by arrows. A p210-type BCR-ABL1 translocation (b3a2 junction) is depicted in the lower portion of the figure along with the control ABL transcript control.

121 Gel and Capillary | Research Use Only (RUO) Assays

Reagents
Controls

Concentration

Units in Assay

Units in Assay MegaKit

IVS-0032 Clonal Control RNA

400 μg/mL

1 x 100 μL tube

5 x 100 μL tubes

IVS-0011 Clonal Control RNA

400 μg/mL

1 x 100 μL tube

5 x 100 μL tubes

IVS-0035 Clonal Control RNA

400 μg/mL

1 x 100 μL tube

5 x 100 μL tubes

Master Mixes

TargetUnits in Assay

Units in Assay MegaKit

BCR/ABL t(9;22) Mix 1a

Abl

1 x 1500 μL tube

10 x 1500 μL tubes

BCR/ABL t(9;22) Mix 2ap190

1 x 1500 μL tube

10 x 1500 μL tubes

BCR/ABL t(9;22) Mix 3ap210+230

1 x 1500 μL tube

10 x 1500 μL tubes

BCR/ABL t(9;22) Mix 1b

Abl

1 x 1500 μL tube

10 x 1500 μL tubes

BCR/ABL t(9;22) Mix 2bp190

1 x 1500 μL tube

10 x 1500 μL tubes

BCR/ABL t(9;22) Mix 2c

p190

1 x 1500 μL tube

10 x 1500 μL tubes

BCR/ABL t(9;22) Mix 3bp210+230

1 x 1500 μL tube

10 x 1500 μL tubes

BCR/ABL t(9;22) Mix 3c

p210+230

1 x 1500 μL tube

10 x 1500 μL tubes

BCR/ABL t(9;22) Mix 3d

p210+230

1 x 1500 μL tube

10 x 1500 μL tubes

Ordering Information
Catalog #Products

Quantity

1-310-0010

BCR/ABL t(9;22) Translocation Assay - Gel Detection

30 reactions

1-310-0031

BCR/ABL t(9;22) Translocation Assay - ABI Fluorescence Detection

30 reactions

These products are for Research Use Only (RUO).
Not intended for diagnostic purposes.

Gel and Capillary | Research Use Only (RUO) Assays 122

PML/RARα t(9;22) Translocation Assays
Assay Use
This Research Use Only assay identifies PML/RARα t(15;17) translocations.

Summary and Explanation of the Test
Four master mixes are included in these assay kits. Master mixes are used to amplify complementary DNA (cDNA) produced from specimen(s), as wellas positive and negative RNA controls (included). Primers target an internal control transcript and the variety of Bcr1, Bcr2, and Bcr3 type transcripts
expressed from PML-RARα translocations. Amplicon products can be analyzed by differential fluorescence detection using capillary electrophoresis.
A PML-RARα translocation is indicated if just one of the 2nd round master mixes (Mix 2b or Mix 2c) generates product(s) of the valid size. Reagents for
RNA extraction and reverse transcription are not included. This assay is compatible with all standard RNA extraction and cDNA synthesis methods.
This is a qualitative assay and has not been validated for quantitative use.

Background
Three PML/RARα translocation patterns have been identified in samples with acute myelogenous leukemia (AML): type A is the short (S-form); thebreakpoint occurs within breakpoint cluster region 3 (Bcr-3). Type B is the long (L-form); the breakpoint occurs within Bcr-1. There is a third typeB variant or variable (V-form); the breakpoint is within Bcr-2. Identification of the PML/RARα t(15;17) rearrangements are commonly used in the studyof APL because it is correlated with responsiveness to treatment. This RT-PCR method directly identifies the chimeric PML/RARα transcripts expressedfrom all three forms of PML/RARα translocations.

Specimen Requirements
This assay tests complementary DNA (cDNA) template.

References
1. H De Thé et al., Nature 347: 558-561 (1990).
2. H De Thé et al., Cell 66: 675-684 (1991).
3. A Kakizuka et al., Cell 66: 663-674 (1991).
4. WH Miller et al., Proc. Natl. Acad. Sci. 89: 2694-2698 (1992).

PML Gene

Chromosome 15

α GeneRARA
Chromosome 17ααFigure Legend: This figure shows the genomic organization of the PML and RARα genes on chromosomes 15 and 17, respectively. Boxes represent exon regions of the PML (red boxes) and RARα(orange) encoding exons. The solid black line represents intron regions, which were left incompletely spliced to assist in demarcation of the exon segments. Primers are indicated by arrows, and thesize of several of the products are indicated below the translocated gene segments. S-form (Bcr3) and L-form (Bcr1) PML-RARα translocations are depicted in the lower portion of the figure.

123 Gel and Capillary | Research Use Only (RUO) Assays

Reagents
Controls

Concentration

Units in Assay

Units in Assay MegaKit

IVS-0020 Clonal Control RNA

400 μg/mL

1 x 100 μL tube

5 x 100 μL tubes

IVS-0035 Clonal Control RNA

400 μg/mL

1 x 100 μL tube

5 x 100 μL tubes

Master Mixes

TargetUnits in Assay

Units in Assay MegaKit

PML/RARα t(15;17) Mix 1

RARA

1 x 1500 μL tube

10 x 1500 μL tubes

PML/RARα t(15;17) Mix 2a

PML-RARA

1 x 1500 μL tube

10 x 1500 μL tubes

PML/RARα t(15;17) Mix 2b

S- and L-Forms

1 x 1500 μL tube

10 x 1500 μL tubes

PML/RARα t(15;17) Mix 2c

L-Form1 x 1500 μL tube

10 x 1500 μL tubes

Ordering Information
Catalog #Products

Quantity

1-311-0011

PML/RARα t(15;17) Translocation Assay - ABI Fluorescence Detection

30 reactions

This product is for Research Use Only (RUO).
Not intended for diagnostic purposes.

Gel and Capillary | Research Use Only (RUO) Assays 124

IGH Somatic HyperMutation Assays v2.0
Assay Use
The Research Use Only IGH Somatic Hypermutation Assay v2.0 is used to identify clonal rearrangements of the immunoglobulin heavy (IGH) chain
gene and determine the somatic mutation status of the variable (V) gene sequence and is useful for the study of:
1. Identifying clonal rearrangements of the IGH chain gene
2. Assessing the extent of somatic hypermutation in the variable region of the immunoglobulin heavy chain gene
3. Evaluating new research and methods in malignancy studies

Summary and Explanation of the Test
These assays amplify either genomic DNA or complementary DNA (cDNA) that lies between the upstream leader (VHL) or framework 1 (FR1) regions
and the downstream joining (JH) region of the IGH gene. The assays employ two different master mixes: Hypermutation Mix 1 and Hypermutation Mix
2. The Hypermutation Mix 1 targets sequences between the leader (VHL) and joining (JH) regions. Therefore the amplicon product(s) span the entire
variable (VH) region, which contains all framework (FR) and complementarity-determining regions (CDR). The Hypermutation Mix 2 targets sequences
between the framework 1 (FR1) and joining (JH) regions. The resulting amplicons include a portion of the FR1 region to the downstream JH region.
The primers that target the VHL and FR1 regions have been redesigned to include a universal sequencing tag at the 5'end. This design allows forbi-directional sequencing of clonal PCR products with just one sequencing-tag specific forward primer and one JH reverse primer, thus ensuringa more reliable and complete coverage of clonal products. Positive and negative DNA, positive RNA, as well as an amplification control are includedin the assay. Clonality is indicated if any one of the master mixes generates clonal products.

BackgroundRearrangements of the antigen receptor genes occur during ontogeny in B and T lymphocytes. These gene rearrangements are unique in lengthand sequence for each cell. Therefore, polymerase chain reaction (PCR) assays can be used to identify lymphocyte populations derived froma single cell by detecting the unique V-J gene rearrangements present within these antigen receptor loci.1 This PCR-based assay employs multiple
consensus DNA primers that target conserved genetic regions within the immunoglobulin heavy chain (IGH) gene. This test is used to detect andsequence the majority of clonal IGH rearrangements from either genomic DNA (gDNA) or complementary DNA (cDNA). Clonal products canbe detected using a variety of methods, including gel and capillary electrophoresis. The presence of IGH somatic hypermutation (SHM) is definedas greater or equal to 2% difference from the germline variable (V) gene sequence, whereas less than 2% difference is considered evidenceof no somatic hypermutation.3

Specimen Requirements
This assay tests extracted and purified genomic DNA (gDNA).

References
1. P Ghia et al., Leukemia 21: 1-3 (2007).
2. P Ghia et al., Blood 105: 1678-1685 (2005).
3. F Davi et al., Leukemia 22: 212-214 (2008).

Figure Legend: Simple representation of the organization of a rearranged immunoglobulin heavy chain gene on chromosome 14. Black arrows represent the relative positions of primers that target theconserved Leader (L) and Framework 1 (FR1) regions, and the downstream consensus JH gene segments.

125 Gel and Capillary | Research Use Only (RUO) Assays

Reagents
Controls

Concentration

Units in Assay

Units in Assay MegaKit

IVS-0013 Clonal Control DNA

200 μg/mL

1 x 100 μL tube

5 x 100 μL tubes

IVS-0013 Clonal Control RNA

400 μg/mL

1 x 100 μL tube

5 x 100 μL tubes

IVS-0000 Polyclonal Control DNA

200 μg/mL

1 x 100 μL tube

5 x 100 μL tubes

Master Mixes

TargetUnits in Assay

Units in Assay MegaKit

Hypermutation Mix 1 v2.0

Leader + JH

1 x 1500 μL tube

10 x 1500 μL tubes

Hypermutation Mix 2 v2.0

Framework 1 + JH

1 x 1500 μL tube

10 x 1500 μL tubes

Specimen Control Size LadderMultiple Genes

1 x 1500 μL tube

10 x 1500 μL tubes

Primers

TargetUnits in Assay

Units in Assay MegaKit

Primer - Hypermutation

Leader + Framework 1

1 x 10 μL tube at 100 μM

5 x 10 μL tube

JH

1 x 10 μL tube at 100 μM

5 x 10 μL tube

IGH JH Primer

Ordering Information
Catalog #Products

Quantity

5-101-0030

IGH Somatic Hypermutation Assay v2.0 - Gel Detection

33 reactions

5-101-0040

IGH Somatic Hypermutation Assay v2.0 MegaKit - Gel Detection

330 reactions

5-101-0031

IGH Somatic Hypermutation Assay v2.0 - ABI Fluorescence Detection

33 reactions

5-101-0041

IGH Somatic Hypermutation Assay v2.0 MegaKit - ABI Fluorescence Detection

330 reactions

These products are for Research Use Only (RUO).
Not intended for diagnostic purposes.

Gel and Capillary | Research Use Only (RUO) Assays 126

FLT3 Mutation Assays
Assay Use
These Research Use Only assays identify FLT3 mutations.

Summary and Explanation of the Test
FLT3 Mutation Assays target regions of the FLT3 gene to identify internal tandem duplication (ITD) mutations and tyrosine kinase domain (TKD) mutations,such as the D835 and I836 mutations. DNA is amplified by PCR, TKD amplicon is enzymatically digested, and FLT3 mutations are detected via agarosegel (catalog #14120010) or capillary (catalog #14120031) electrophoresis.
Assay kits include three PCR master mixes, along with positive and negative controls. FLT3 ITD master mix tests for internal tandem duplication mutations.
FLT3 D835 master mix tests for TKD region mutations. The Specimen Control Size Ladder master mix targets multiple genes and generates a series ofamplicons 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.
Master mixes contain fluorophore-labeled (capillary) or unlabeled (gel) primer sets as appropriate to kit detection method.

Background
FLT3 is a receptor tyrosine kinase that is normally expressed on many cell types including hematologic stem cells. Mutation of the FLT3 receptor,
by either internal tandem duplication (ITD) of the juxtamembrane domain or point mutation in the activation loop of the tyrosine kinase domain (TKD),
causes constitutive activation of the FLT3 receptor.
Such gain-of-function mutations in the FMS related tyrosine kinase 3 (FLT3) gene are the subject of research studies and multiple clinical trials
targeting Acute Myeloid Leukemia (AML) subjects. The most prevalent type of FLT3 mutation is an internal tandem duplication in and around thejuxtamembrane domain. The second most common mutation type in the FLT3 gene is a TKD point mutation in aspartate (D835) or isoleucine (I836).

Specimen Requirements
High quality genomic DNA

References
https://clinicaltrials.gov
1. Acute Myeloid Leukemia, Clinical Practice Guidelines in Oncology, National Comprehensive Cancer Network (v.2.2014).
2. Lowenberg, B. et al. “Acute myeloid leukemia.” N Engl J Med 341(14):1051-62 (1999).
3. Thiede, C. et al. “Analysis of FLT3-activating mutations in 979 patients with acute myelogenous leukemia: association with FAB and identification ofsubgroups with poor prognosis.” Blood 99(12): 4326-35 (2002).
4. Nakao, M. et al. “Internal tandem duplication of the FLT3 gene found in acute myeloid leukemia.” Leukemia 10(12):1911-18 (1996).
5. Yamamoto, Y et al. Activating mutation of D835 within the activation loop of FLT3 in human hematologic malignancies. Blood, 97(8):2434-9 (2001).
6. Gilliland, DG et al. The roles of FLT3 in hematopoiesis and leukemia. Blood 100(5):1532-154 (2002).

These products are not available for sale or use in the United States.

127 Gel and Capillary | Research Use Only (RUO) Assays

Reagents
Controls

Concentration

Units in 33 Reaction Assay

IVS-0017 Clonal Control DNA

200 μg/mL

1 x 100 μL tube

IVS-P001 Clonal Control DNA

200 μg/mL

1 x 100 μL tube

IVS-0000 Polyclonal Control DNA

200 μg/mL

1 x 100 μL tube

Master Mixes

TargetUnits in 33 Reaction Assay

FLT3 ITD Master Mix

FLT3 ITD

1 x 1500 μL tube

FLT3 D835 Master Mix

FLT3 TKD

1 x 1500 μL tube

Specimen Control Size LadderMultiple Genes

1 x 1500 μL tube

Gel Electrophoresis Detection
Data was generated using the FLT3 ITD Master Mix, and amplified products were run on a 2% TBE agarose gel alongside a 100bp DNA size ladder.
Lane 1 is a FLT3 ITD control*; lane 2 is a 10% dilution of a FLT3 ITD control; and lane 3 is IVS-0000, which is representative of a wild type product.
*IVS-0050 performs comparable to IVS-0017 clonal control DNA, which is included in the kit as the positive control.

Capillary Electrophoresis Detection (ABI)
Data was generated using the FLT3 ITD Master Mix and amplified products were run on an ABI 3100 instrument. Templates analyzed follow: Panel 1 isthe FLT3 ITD positive control; panel 2 is a 10% dilution of the positive control; and Panel 3 is IVS-0000, which is representative of a wild type product.

Ordering Information
Catalog #Products

Quantity

1-412-0010

FLT3 Mutation Assay - Gel Detection

33 reactions

1-412-0031

FLT3 Mutation Assay - ABI Fluorescence Detection

33 reactions

These products are for Research Use Only (RUO). Not intended for diagnostic purposes., and additionally notavailable for sale or use in regions where CE-IVD products are registered.
These products are not available for sale or use in the United States. Please see the LeukoStrat CDx FLT3
Mutation Assay product pages for IVD product availability in the United States and other global regions.

Gel and Capillary | Research Use Only (RUO) Assays 128

Analyte Specific Reagents

Invivoscribe® Analyte Specific Reagents (ASRs)
target B- and T-cell antigen receptor loci, FLT3
ITD and TKD, or chromosome translocations(IGH - BCL2, BCR-ABL1, PML-RARA).

LEARN MORE:
invivoscribe.com/products/reagents

Warranty and Liability
Invivoscribe is committed to providing the highest quality products. Invivoscribe warrants that for products which are provided with Instructions for Use, these products meet or exceed theperformance standards described in the Instructions For Use. If a product is covered by product specifications and does not perform as specified, our policy is to replace the product or credit
the full purchase price. No other warranties of any kind, expressed or implied, are provided by Invivoscribe. Invivoscribe liability shall not exceed the purchase price of the product. Invivoscribe
shall have no liability for direct, indirect, consequential or incidental damages arising from the use, results of use, or inability to use its products; product efficacy under purchaser controlled
conditions in purchaser’s laboratory must be established and continually monitored through purchaser defined and controlled processes including but not limited to testing of positive, negative,
and blank controls every time a sample is tested. Ordering, acceptance and use of product constitutes purchaser acceptance of sole responsibility for assuring product efficacy and purchaser
agreement to the limitation of liability set forth in this paragraph.
NoticeThe products in this section are Analyte Specific Reagents. The analytical and performance characteristics are not established. Some of these products are covered by one or more patents
licensed to Invivoscribe Inc, including European Patent No. 1549764 and 2418287 (each validated in 16 countries), European Patent No. 2460889, Japanese Patent No. 4708029, United States
Patent No. 8859748 and United States Patent No. 10280462.
These products require nucleic acid amplification methods such as Polymerase Chain Reaction (PCR). No license under these patents to use amplification processes or enzymes is conveyed
expressly or by implication to the purchaser by the purchase of these products.

Analyte Specific Reagents
Invivoscribe Analyte Specific Reagents (ASRs) target B- and T-cell antigen receptor loci.
The ASRs are available as a single tube containing a volume of 1500 μL. To ensure the highest quality and reliability of reagents ASRs are manufactured
under cGMP and ISO 13485 standards.
Per the current US FDA regulations, ASRs may only be sold to in vitro diagnostic manufacturers, CLIA accredited high complexity laboratories, VHA regulated
clinical laboratories, and laboratories not intending to use ASRs as a part of a diagnostic test.
Analytical and performance characteristics are not established. ASRs are not available for sale or use outside of the USA.

IGH (Immunoglobulin Heavy Chain
Gene Locus)

TRB (T-Cell Receptor Beta Chain
Gene Locus)

Description

Catalog #Description

Catalog #IGH Framework 1 - 6FAM

A-101-0061

TCRB V - J1 + 2 - 6FAM & HEX

A-205-0011

IGH Framework 2 - 6FAM

A-101-0091

TCRB V - J2 - 6FAM

A-205-0021

IGH Framework 3 - HEX

A-101-0081

TCRB D - J1 + 2 - 6FAM & HEX

A-205-0031

IGH FR1 - 6FAM

A-101-0011

IGH FR2 - 6FAM

A-101-0101

IGH FR3 - HEX

A-101-0031

IGH DH1 - 6 - HEX

A-101-0041

IGH DH7 - 6FAM

A-101-0051

IGK (Immunoglobulin Kappa Light
Chain Gene Locus)

TRG (T-Cell Receptor Gamma Chain
Gene Locus)

Description

Catalog #Description

Catalog #IGK V - J - 6FAM

A-102-0011

TCRG V(2-5,8-11) J 1 + 2+P - 6FAM

A-207-0091

IGK V - Kde - 6FAM

A-102-0021

TCRG V(1-8,9) J - 6FAM

A-207-0071

TCRG V(1-8) J - HEX

A-207-0021

Analyte Specific Reagents 130

Controls, Reagents, and Enzymes

Invivoscribe® offers an extensive rangeof General Purpose Reagents (GPRs)and Research Use Only (RUO) nucleic
acid controls.

LEARN MORE:
invivoscribe.com/products/controls

Warranty and Liability
Invivoscribe is committed to providing the highest quality products. Invivoscribe warrants that for products which are provided with Instructions for Use, these products meet or exceed theperformance standards described in the Instructions For Use. If a product is covered by product specifications and does not perform as specified, our policy is to replace the product or credit thefull purchase price. No other warranties of any kind, expressed or implied, are provided by Invivoscribe. Invivoscribe liability shall not exceed the purchase price of the product. Invivoscribe shallhave no liability for direct, indirect, consequential or incidental damages arising from the use, results of use, or inability to use its products; product efficacy under purchaser controlled conditions
in purchaser’s laboratory must be established and continually monitored through purchaser defined and controlled processes including but not limited to testing of positive, negative, and blankcontrols every time a sample is tested. Ordering, acceptance and use of product constitutes purchaser acceptance of sole responsibility for assuring product efficacy and purchaser agreement
to the limitation of liability set forth in this paragraph.

Introduction
Controls are available in DNA extracted from tissue or cell lines, or RNA extracted from cell lines. These controls canbe purchased in various dilutions or as complete dilution sets and panels for several purposes, such as to help withassay validation, sensitivity or proficiency testing, or troubleshooting.
The following pages will provide an overview of available controls, along with a number of tables and reference guides,
to help you decide which Invivoscribe control(s) will be suitable for your application.

Chapter Contents
DNA Controls

RNA Controls

133 Quick Reference for DNA Controls
136 LymphoTrack Low Positive Controls
136 LymphoQuant Internal Controls

137 Quick Reference for RNA Controls
139 BCR/ABL1 RNA Dilution Sets

Control Panels

Reagents

140 RNA Sensitivity Panels

141 ABI Detection Reagents

Mastermix Controls

Enzyme141 Specimen Control Size Ladder

141 FalconTaq DNA Polymerase

Controls, Reagents, and Enzymes 132

DNA Controls
Every laboratory needs suitable controls (positive and negative) for sensitivity and proficiency testing, as well as for troubleshooting. Since patient samples
cannot serve as true controls (due to a lack of characterization and inter-sample variability), Invivoscribe offers a multitude of high quality, reliable DNA
controls manufactured under cGMP conditions.
These controls can be used for most assays targeting B- and T-cell antigen receptor loci, or IGH-BCL2, BCR-ABL1, and PML-RARa chromosome translocations.

Quick Reference for DNA Controls
The vast majority of our high-quality DNA controls, including sensitivity controls and panels, are supplied in aliquots of 100 μL and are adjusted
to a final concentration of 200 μg/mL in 1/10 TE (1 mM Tris- HCl (pH 8.0), 0.1 mM EDTA).

Immunoglobulin
Rearrangements

Positive for

IGH

IGK

IGL

Mutations
IGHV SHM

T-CellReceptor Gene
Rearrangements

Translocations
IGH-CCND1 **IVS-0001

IGH-BCL2

TRG

u

u

u

u

u

u

u

u

★

★

★

★

TRD

u

IVS-0004
IVS-0007

u

IVS-0008€

m

u

u

u

IVS-0009
IVS-0010uu

IVS-0013uu

IVS-0019uu

IVS-0021uu

IVS-0024uu

IVS-0029uu

IVS-0030 ‡uu

IVS-0031uuLymphoQuant B-cell Internal Control

★

★

★

★

u

u

LymphoQuant T-cell Internal Control
LymphoTrack B-cell Low Positive Control

TRB

LymphoTrack T-cell Low Positive Control

These controls can be used as SHM positive controls with ≥2% mutational rates compared to the germline sequence.
€
This control does not contain a complete IGH VH-JH rearrangement and may only be suitable for IGH DH-JH rearrangements.
**IGH-CCND1 was previously referred to as BCL1/JH.

‡u Gene rearrangementm Partial IGH DH-JH rearrangement★ Recommended for NGS

Tissue DNA
Standard Concentrations

IVS-0000 Polyclonal Control DNA

Our high-quality DNA controls are supplied in aliquots of 100 μL andare adjusted to a final concentration of 200 μg/mL in 1/10 TE (1 mM
Tris- HCl (pH 8.0), 0.1 mM EDTA). This diluent provides sufficientbuffering capacity and EDTA to protect the DNA without interferingwith the Mg2+ concentrations required for robust amplification
reactions.

Tissue DNA controls are extracted from normal, disease-free tissue
and are tested extensively to ensure quality and reproducibility of yourtest results. IVS-0000 Polyclonal Control DNA consists of genomic DNA
isolated from the tissue of normal human tonsils. This control represents
an excellent negative control for gene rearrangements, chromosometranslocations, and mutation tests and is included in all of our PCR
DNA-based assay kits. This DNA is supplied at a volume of 100 μL andat a concentration of 200 μg/mL.

Catalog #Description

4-092-0010

IVS-0000 Polyclonal Control DNA*These controls are general purpose reagents (GPRs).

133 Controls, Reagents, and Enzymes

Cell Line DNA
Reliable Positive Controls
Cell Line DNA controls are extracted from established cell lines grown under cell culture conditions recommended by the supplier.
Our controls are tested extensively to ensure quality and reproducibility of your test results. Please note, these controls are for
qualitative use only.
Note: n/c is used to indicate that the control has not been fully characterized; there may be additional rearrangements, translocations
or mutations associated with the control.

Standard Concentrations
Our high-quality DNA controls are supplied in aliquots of 100 μL and are provided at a final concentration of 200 μg/mL in 1/10 TE (1 mM
Tris-HCl (pH 8.0), 0.1 mM EDTA). This diluent provides sufficient buffering capacity and EDTA to protect the DNA controls without interferingwith the Mg2+ concentrations required for robust amplification reactions.

IVS-0001 Clonal Control DNA

IVS-0008 Clonal Control DNA‡

IVS-0001 Clonal Control DNA can be used as a positive control for:

IVS-0008 Clonal Control DNA can be used as a positive control for:

Gene Rearrangements:
Chromosome Translocations:
Mutations:Gene Rearrangements:
Chromosome Translocations:
Mutations:n/c
IGH-BCL2 t(14;18) mcr
n/c

Catalog #Description

4-088-0010

IVS-0001 Clonal Control DNA

IGH DH-JH‡, TRB, TRG
n/cn/cCatalog #Description

4-088-0430

IVS-0008 Clonal Control DNA

IVS-0004 Clonal Control DNA

IVS-0009 Clonal Control DNA

IVS-0004 Clonal Control DNA can be used as a positive control for:

IVS-0009 Clonal Control DNA can be used as a positive control for:

Gene Rearrangements:
Chromosome Translocations:
Mutations:Gene Rearrangements:
Chromosome Translocations:
Mutations:

TRB, TRG
n/cn/c

TRB, TRGn/cn/c

Catalog #Description

Catalog #Description

4-088-0190

IVS-0004 Clonal Control DNA

4-088-0490

IVS-0009 Clonal Control DNA

IVS-0007 Clonal Control DNA
IVS-0007 Clonal Control DNA can be used as a positive control for:
Gene Rearrangements:
Chromosome Translocations:
Mutations:

IGH, IGK, IGL
IGH-BCL2 t(14;18) Mbrn/cCatalog #Description

4-088-0370

IVS-0007 Clonal Control DNA hese controls are general purpose reagents (GPRs).
T‡
This control does not contain a complete IGH VH-JH rearrangement and may only
be suitable for IGH DH-JH rearrangements.

Controls, Reagents, and Enzymes 134

Cell Line DNA
IVS-0010 Clonal Control DNA

IVS-0029 Clonal Control DNA

IVS-0010 Clonal Control DNA can be used as a positive control for:

IVS-0029 Clonal Control DNA can be used as a positive control for:

Gene Rearrangements:
Chromosome Translocations:
Mutations:Gene Rearrangements:
Chromosome Translocations:
Mutations:

IGH, IGK, IGL
IGH-BCL1 t(11;14)n/cCatalog #Description

4-088-0550

IVS-0010 Clonal Control DNA

IGH, IGK, IGLn/cn/cCatalog #Description

4-088-1690

IVS-0029 Clonal Control DNA

IVS-0013 Clonal Control DNA

IGH SHM Positive Control DNA

IVS-0013 Clonal Control DNA can be used as a positive control for:IGH SHM Postive Control can be used as a positive control for:

Gene Rearrangements:
Chromosome Translocations:
Mutations:Gene Rearrangements:
Chromosome Translocations:
Mutations:

IGH, IGK, IGL
n/c
n/c

IGH
n/c
IGH SHM

Catalog #Description

Catalog #Description

4-088-0730

IVS-0013 Clonal Control DNA

4-088-0008

IGH SHM Positive Control DNA

IVS-0019 Clonal Control DNA

IVS-0030 Clonal Control DNA

IVS-0019 Clonal Control DNA can be used as a positive control for:

IVS-0030 Clonal Control DNA can be used as a positive control for:

Gene Rearrangements:
Chromosome Translocations:
Mutations:Gene Rearrangements:
Chromosome Translocations:
Mutations:

IGH, IGKn/cn/c

Catalog #Description

4-088-1090

IVS-0019 Clonal Control DNA

IVS-0021 Clonal Control DNA
IVS-0021 Clonal Control DNA can be used as a positive control for:
Gene Rearrangements:
Chromosome Translocations:
Mutations:

TRB, TRD, TRG
n/cn/cCatalog #Description

4-088-1210

IVS-0021 Clonal Control DNA

IGH, IGK
IGH-BCL2 t(14;18) Mbrn/cCatalog #Description

4-088-1750

IVS-0030 Clonal Control DNA

IVS-0031 Clonal Control DNA
IVS-0031 Clonal Control DNA can be used as a positive control for:
Gene Rearrangements:
Chromosome Translocations:
Mutations:

IGH, IGK
IGH-BCL2 t(14;18) mcr
n/c

Catalog #Description

4-088-1810

IVS-0031 Clonal Control DNA

IVS-0024 Clonal Control DNA
IVS-0024 Clonal Control DNA can be used as a positive control for:
Gene Rearrangements:
Chromosome Translocations:
Mutations:

IGH, IGKn/cn/c

Catalog #Description

4-088-1390

IVS-0024 Clonal Control DNA
These controls are general purpose reagents (GPRs).

135 Controls, Reagents, and Enzymes

LymphoTrack® Low Positive Controls
Measurable Residual Disease (MRD) testing is a valuable tool that allows investigators to study and monitor multiple myeloma (MM) , chronic
lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML) and other hematologic diseases.
Recent treatment advances have led to significantly increased clinical response and overall survival, but ultimately most subjects will relapse, driving
the need for sensitive MRD monitoring. Sensitive and standardized testing such as NGS-based MRD may one day enable identification of those casesthat will eventually relapse versus those who are potentially cured. In addition to the need for more sensitive tracking, it is clear that standardized
methods are needed. Currently, MRD methods are highly subjective and recommendations are often based on consensus expert-shared knowledge
and experience, not on a validated, objective method. Once specific rearrangements have been identified, LymphoTrack assays can be used with
LymphoQuant and LymphoTrack Low Positive Controls to track these clonotype populations to a sensitivity as low as 10-4.

LymphoTrack® B-cell Low Positive Control

LymphoTrack® T-cell Low Positive Control

LymphoTrack B-cell Low Positive Control can be used as a control for:LymphoTrack T-cell Low Positive Control can be used as a control for:

Gene Rearrangements:
Chromosome Translocations:
Mutations:Gene Rearrangements:
Chromosome Translocations:
Mutations:

IGH
n/c
n/c

TRB, TRGn/cn/c

Catalog #Description

Catalog #Description

4-088-0098

LymphoTrack® B-cell Low Positive Control*

4-088-0108

LymphoTrack® T-cell Low Positive Control*

LymphoQuant® Internal Controls
LymphoQuant T-cell or B-cell Internal Controls may be spiked into specimens to estimate the respective number of clonotype T-cell or IGH equivalents
present. Addition of the LymphoQuant Internal Control to the specimen PCR facilitates clonotype tracking over time without any additional sequencing
cost. Consistent use of a LymphoQuant Internal Control enables investigators to objectively monitor the disease over time with a highly standardized,
sensitive method. The LymphoTrack MRD software helps researchers that use the LymphoQuant Internal Control, calculate and report an estimatednumber of clonotype cell equivalents and the percent clonotype in the sample, enabling researchers and pharmaceutical companies to accurately
monitor hematologic disease in longitudinal studies.

LymphoQuant® B-cell Internal Control

LymphoQuant® T-cell Internal Control

LymphoQuant B-cell Internal Control can be used to objectively track
Ig clonotypes.

LymphoQuant T-cell Internal Control can be used to objectively track
TCR clonotypes.

Gene Rearrangements:
Chromosome Translocations:
Mutations:Gene Rearrangements:
Chromosome Translocations:
Mutations:

IGH
n/c
n/c

TRB, TRGn/cn/c

Catalog #Description

Catalog #Description

4-088-0118

LymphoQuant® B-cell Internal Control*

4-088-0128

LymphoQuant® T-cell Internal Control**LymphoTrack® Low Positive Controls and LymphoQuant® Internal Controls areresearch use only (RUO), not for diagnostic procedures.

Controls, Reagents, and Enzymes 136

RNA Controls
Quick Reference for RNA Controls
Reliable Assay Controls
Our RNA controls are extracted from well characterized cell lines grown under standard and carefully controlled culture conditions. The general
purpose reagent (GPR) controls are tested to ensure linearity and reproducible results. Since this RNA is extracted from cell lines, these controls
can be used with any of the standard housekeeping genes.

Standard Concentrations
Each RNA single control tube (as separate control tube, RNA sensitivity panel and proficiency panel) is supplied in aliquots of 100 μL at a final
concentration of 400 μg/mL in water. Each BCR/ABL RNA dilution set member is supplied in aliquots of 50 μL at a final concentration of 400 μg/
mL in water. To ensure maximum stability, store the dilutions at -85 °C to -65 °C and minimize the number of freeze-thaw cycles .

RNAs positive for chromosome translocations
Chromosome Translocation

Clonal Control RNA

Chromosome Translocation

Clonal Control RNA

BCR-ABL1 t(9;22) p210 e13a2 (b2a2)

IVS-0003

CBFB-MYH11 inv(16)IVS-0015

BCR-ABL1 t(9;22) p210 e14a2 (b3a2)

IVS-0011

E2A-PBX1 t(1;19)(q23;p13)

IVS-0002

BCR-ABL1 t(9;22) p190 e1a2

IVS-0032

PML-RARA t(15;17)(q22;q11)

IVS-0020

RNAs negative for chromosome translocations
IVS-0035 Clonal Control RNA is negative for BCR-ABL t(9;22) and PML-RARa t(15;17) chromosome translocations.
IVS-0035 may be used as a negative control for other chromosome translocations or diluents for other chromosome translocation positive
controls. Please do not hesitate to contact us at support@invivoscribe.com so we can evaluate whether this control may work for your testing needs.

These controls are for Research Use Only (RUO).
Not intended for diagnostic purposes.

137 Controls, Reagents, and Enzymes

Cell Line RNA
Reliable Positive and Negative Controls

IVS-0015 Clonal Control RNA

Cell Line RNA controls are extracted from established cell lines grownunder cell culture conditions recommended by the supplier. Our GPR
controls are tested extensively to ensure quality and reproducibility ofyour test results. Please note, these controls are for qualitative use only.

IVS-0015 Clonal Control RNA can be used as a positive control
for the chromosome translocation: CBFB-MYH11 inv(16)Catalog #Description

Standard Concentrations

4-089-1270

IVS-0015 Clonal Control RNA*Our GMP-manufactured high-quality RNA controls, including sensitivity
controls and proficiency panel samples, are supplied in aliquotsof 100 μL and are adjusted to a final concentration of 400 μg/mL
in RNase-free glass-distilled water. The pH of distilled water is slightly
acidic; this protects the RNA from hydrolysis. RNA dilutions are diluted
volume to volume in our negative control RNA, IVS-0035 Clonal
Control RNA.

IVS-0020 Clonal Control RNA
IVS-0020 Clonal Control RNA can be used as a positive control
for the chromosome translocation: PML-RARA t(15;17) L-Form.

Catalog #Description

4-089-1720

IVS-0020 Clonal Control RNA*

IVS-0002 Clonal Control RNA
IVS-0002 Clonal Control RNA can be used as a positive control forthe chromosome translocation: E2A-PBX1 t(1;19) (q23;p13).

Catalog #Description

4-089-0100

IVS-0002 Clonal Control RNA*

IVS-0032 Clonal Control RNA
IVS-0032 Clonal Control RNA can be used as a positive control
for the chromosome translocation: BCR-ABL1 t(9;22) p190 e1a2.
This control is also available as several ready-to-use dilutions
into a standard negative control as listed in the table below.

Catalog #Description

4-089-2800

IVS-0032 Clonal Control RNA*

IVS-0003 Clonal Control RNA can be used as a positive control forthe chromosome translocation: B
 CR-ABL1 t(9;22) p210 e13a2 (b2a2).

4-089-2810

10-1 IVS-0032 Clonal Control RNA

4-089-2820

10-2 IVS-0032 Clonal Control RNA

This control is also available as several ready-to-use dilutions intoa standard negative control as listed in the table below.

4-089-2830

10-3 IVS-0032 Clonal Control RNA

4-089-2840

10-4 IVS-0032 Clonal Control RNA

Catalog #Description

4-089-2850

10-5 IVS-0032 Clonal Control RNA

4-089-0190

IVS-0003 Clonal Control RNA*4-089-0200

10-1 IVS-0003 Clonal Control RNA

4-089-0210

10-2 IVS-0003 Clonal Control RNA

4-089-0220

10-3 IVS-0003 Clonal Control RNA

4-089-0230

10-4 IVS-0003 Clonal Control RNA

4-089-0240

10-5 IVS-0003 Clonal Control RNA

IVS-0003 Clonal Control RNA

IVS-0011 Clonal Control RNA

IVS-0035 Clonal Control RNA
IVS-0035 Clonal Control RNA can be used as a negative control for
BCR-ABL1 t(9;22) and PML-RARa t(15; 17) chromosome translocations.

Catalog #Description

4-089-3070

IVS-0035 Clonal Control RNA*

IVS-0011 Clonal Control RNA can be used as a positive control forthe chromosome translocation: B
 CR-ABL1 t(9;22) p210 e14a2 (b3a2).
This control is also available as several ready-to-use dilutions intoa standard negative control as listed in the table below.

Catalog #Description

4-089-0910

IVS-0011 Clonal Control RNA*4-089-0920

10-1 IVS-0011 Clonal Control RNA

4-089-0930

10-2 IVS-0011 Clonal Control RNA

4-089-0940

10-3 IVS-0011 Clonal Control RNA

4-089-0950

10-4 IVS-0011 Clonal Control RNA

4-089-0960

10-5 IVS-0011 Clonal Control RNA*These controls are general purpose reagents (GPRs). All others areresearch use only (RUO).

Controls, Reagents, and Enzymes 138

BCR/ABL RNA Dilution Sets
Our BCR/ABL b2a2, b3a2, and e1a2 RNA Dilution Sets consist of RNA that has been extracted from BCR-ABL1 expressing and BCR-ABL1 negative celllines. Each set is composed of several dilutions (10-1, 10-2, 10-3, 10-4, 10-5) of the BCR-ABL1 positive RNA diluted (v/v) into RNA purified from a cell linethat does not contain a BCR-ABL1 translocation. Also included in these sets is a 100% BCR-ABL1 negative RNA.
The individual BCR/ABL b2a2, b3a2, and e1a2 RNA Dilution Sets can be used as reference and validation materials with assays that target the maintranscripts of BCR-ABL1 t(9;22) translocations: p210 (e13a2 (b2a2), e14a2 (b3a2), and p190 (e1a2). These products may be used as the following:
1. Routine testing controls for cDNA synthesis, amplification and detection
2. Controls to establish a standard reference curve
3. Proficiency controls
4. Sensitivity controls for specific target assays

Data
Plot of Ct values (5 replicates) for the 10-1, 10-2, 10-3, 10-4, and 10-5 dilutions.

Ordering Information - e14a2 (b3a2)
Catalog #Description

4-085-0210

BCR/ABL b3a2 RNA Dilution Set(10-1, 10-2, 10-3, 10-4, 10-5 dilutions and negative)

Ordering Information - e13a2 (b2a2)
Catalog #Description

4-085-0310

BCR/ABL b2a2 RNA Dilution Set(10-1, 10-2, 10-3, 10-4, 10-5 dilutions and negative)

Ordering Information - e1a2
Catalog #Description

4-085-0110

BCR/ABL e1a2 RNA Dilution Set(10-1, 10-2, 10-3, 10-4, 10-5 dilutions and negative)

These products are for Research Use Only (RUO).
Not intended for diagnostic purposes.

139 Controls, Reagents, and EnzymesRNA Control Panels
RNA Sensitivity Panels
RNA sensitivity panels are 7 member panels that consist of 100% clonal RNA extracted from a positive control cell line and 10-1, 10-2, 10-3, 10-4, 10- 5,
and 10-6 (1:10 - 1:1 000 000) dilutions of the positive clonal RNA diluted (v/v) into our standard negative control RNA, IVS-0035 Clonal Control RNA.
Each tube contains 100 μL of RNA at 400 μg/mL in RNase-free glass-distilled water. The pH of distilled water is slightly acidic thereby protecting
the RNA from hydrolysis. Please note, these controls are for qualitative use only.

RNA Sensitivity Panels
Catalog #Description

Can be used as a positive control for:4-087-0030

Sensitivity Panel for IVS-0003 Clonal Control RNA

BCR-ABL1 t(9;22) p210 e13a2 (b2a2)

4-087-0110

Sensitivity Panel for IVS-0011 Clonal Control RNA

BCR-ABL1 t(9;22) p210 e14a2 (b3a2)

4-087-0150

Sensitivity Panel for IVS-0015 Clonal Control RNA

CBFB/MYH11 inv16

4-087-0200

Sensitivity Panel for IVS-0020 Clonal Control RNA

PML-RARA t(15;17) L-form

4-087-0320

Sensitivity Panel for IVS-0032 Clonal Control RNA

BCR-ABL1 t(9;22) p190 e1a2

These products are for Research Use Only (RUO).
Not intended for diagnostic purposes.

Controls, Reagents, and Enzymes 140

Master Mix Controls
These master mixes serve as control for many of our DNA assays to ensure that sample DNA is of sufficient quality and integrity to generate
a valid result.

Specimen Control Size Ladder
Our Specimen Control Size Ladder master mix targets four different housekeeping genes producing products of approximately 100, 200,
300, 400, and 600 base pair in size to ensure that the quality and quantity of the sample DNA is adequate to yield a valid result with thespecific assay(s). This master mix is based on the BIOMED-2 Concerted Action BMH4-CT98-3936 from the EuroClonality Group andis available for Gel Detection (unlabeled) or ABI detection (labeled with 6FAM).

Catalog #Description

2-096-0020

Specimen Control Size Ladder - Unlabeled

2-096-0021

Specimen Control Size Ladder - 6FAM

These master mixes are general purpose reagents (GPRs).

Reagents
ABI Detection Reagents
Invivoscribe also offers highly deionized (Hi-Di) Formamide with ROX size standards for ABI fluorescence detection with the ABI 310 or 3100 series.
Hi-Di Formamide is used to stabilize single strands of denatured PCR amplicons. The ROX size standards are fluorescent labeled DNA standards
which cover the 50 to 400 base pair size range. Sizes of the individual standards are: 50, 60, 90, 100, 120, 150, 160, 180, 190, 200, 220, 240, 260, 280, 290,
300, 320, 340, 360, 380, and 400 base pair.
For samples tested on an ABI 3100 series, we recommend using 10 μL of the Hi-Deionized Formamide with ROX Size Standards mixture for eachmicroliter of PCR product. Please note that the ABI 3100 series require different concentrations of ROX size standards and the different Hi-Deionized
Formamide with ROX Size Standards cannot be used interchangeably.
For samples tested on an ABI 3500 series, GeneScan™ 600® LIZ dye Size Standard v2.0 can be purchased from Thermo Fisher Scientific.

Catalog #Description

6-098-0061

Hi-Deionized Formamide with ROX Size | Standard (ABI 3100), 1 mL

Available through Thermo Fisher Scientific : 4408399®

GeneScan™ 600 LIZ® dye v2.0 | Standard (ABI 3500), 800 reactions

EnzymeFalconTaqTM DNA Polymerase
FalconTaq™ DNA Polymerase can be used for amplification using PCR to obtain high specificity, sensitivity, and yield. This enzyme has been provento minimize extension of non-specifically bound primers. Generate reliable results by using FalconTaq™ DNA Polymerase for robust performance.

Catalog #Description

6-097-0130

FalconTaq™ DNA Polymerase 250 U, 5 U/μL

FalconTaq™ DNA Polymerase is replacing EagleTaq DNA Polymerase, please contact
support@invivoscribe.com if you have questions.

These products are for Research Use Only (RUO).
Not intended for diagnostic purposes.

141 Controls, Reagents, and Enzymes

Custom Products

The Invivoscribe® team of experts can help
develop your ideas into customized products.
Allow us to partner with you to take a basic
concept through design, development,
validation, regulatory approval, and release.

LEARN MORE:
invivoscribe.com/clinical-lab-services

Warranty and Liability
Invivoscribe, Inc. (Invivoscribe®) is committed to providing the highest quality products. Invivoscribe® warrants that the products meet or exceed the performance standards described in theInstructions For Use, as to products with such an insert. If a product is covered by product specifications and does not perform as specified, our policy is to replace the product or credit thefull purchase price. No other warranties of any kind, expressed or implied, are provided by Invivoscribe®. Invivoscribe® liability shall not exceed the purchase price of the product. Invivoscribe
shall have no liability for direct, indirect, consequential, or incidental damages arising from the use, results of use, or inability to use its products; product efficacy under purchaser controlled
conditions in purchaser’s laboratory must be established and continually monitored through purchaser defined and controlled processes including but not limited to testing of positive, negative,
and blank controls every time a sample is tested. Ordering, acceptance, and use of product constitutes purchaser acceptance of sole responsibility for assuring product efficacy and purchaser
agreement to the limitation of liability set forth in this paragraph.

Customized Products to Meet Your Needs
The Invivoscribe team of experts can help develop your ideas into customized products. Allow us to partner with you to take a basic concept
through design, development, validation, regulatory approval (if applicable), and release. For more information, please call our San Diego officeat +1 858.224.6600 or send an email to inquiry@invivoscribe.com.

Custom Designed Assays
In response to the FDA announcing its intention to dramatically expand its regulatory oversight of laboratory developed tests (LDTs), Invivoscribe
is partnering with laboratories worldwide to help facilitate the conversion of LDTs into FDA-cleared assays, as we know the barriers to bringing new
assays online are often the availability of resources and the cost of validation.
By leveraging the power of our regulatory expertise, provided through each milestone, we can help ensure safety, efficacy and quality.
Our customizable reagent manufacturing capabilities can reduce your LDT costs and lead to higher-quality testing.
To date, we have partnered with more than 40 laboratories around the world to develop, validate, and launch a variety of molecular products.
A number of these partnerships have also led to the release of US and CE-marked in vitro diagnostic products and services. Learn how Invivoscribe
can help you develop assays for new products, services, and novel applications.

Custom Controls and Validation Panels
We offer a large selection of well-characterized DNA and RNA controls that are used to define the performance characteristics of a wide variety
of molecular reagents. To address your specific requirements, we can partner with you to design, validate, and provide custom controls and validation
panels. If necessary, we are willing to acquire, characterize, and engineer custom controls for your specific application. We can produce DNA, RNA,or cDNA at any specified concentration, dilution, or volume. Please contact us with your requirements and we will be happy to provide controls to suit
your needs.

Invivoscribe is a Comprehensive Partner for Companion Diagnostic Development
From biomarker identification through commercialization, Invivoscribe has expertise at every stage of companion diagnostics development.
• Discovery and Patient Stratification: We offer comprehensive gene panels to identify biomarkers and define patient populations, thus reducing
development costs and improving the success of clinical trials.
• Clinical Trials: Our network of global laboratories accelerates sample acquisition and harmonizes testing to ensure accurate results.
• Regulatory Approval: Our in-house experts have experience seeking approval with global agencies.
• Commercialization: Our cGMP manufacturing expertise and distribution channels allow approved CDx to reach all global markets.
Invivoscribe is an ISO 13485-accredited and FDA/CDRH registered medical device manufacturer with a long record of successful partnerships.
We are the industry-leading assay and software development company, providing full QSR design control and a complete range of cGMP
manufactured assays, controls, reagents, and services to CLIA-accredited clinical laboratory and pharmaceutical communities.
Please contact us at +1 858.224.6600 or inquiry@invivoscribe.com for more details about partnering with Invivoscribe for the development andmanufacturing of companion diagnostics, in vitro diagnostics, molecular reagents, and/or nucleic acid controls.

Custom Products 143

B- or T-cell Clonality
Testing Workflow

The Invivoscribe® European Conformity marked
in vitro diagnostics (CE-IVD) and Research
Use Only (RUO) clonality assays detect clonal
populations in just a few easy steps.

LEARN MORE:
invivoscribe.com/products/controls

Warranty and Liability
Invivoscribe, Inc. (Invivoscribe®) is committed to providing the highest quality products. Invivoscribe® warrants that the products meet or exceed the performance standards described in theInstructions For Use, as to products with such an insert. If a product is covered by product specifications and does not perform as specified, our policy is to replace the product or credit the fullpurchase price. No other warranties of any kind, expressed or implied, are provided by Invivoscribe®. Invivoscribe® liability shall not exceed the purchase price of the product. Invivoscribe® shallhave no liability for direct, indirect, consequential, or incidental damages arising from the use, results of use, or inability to use its products; product efficacy under purchaser controlled conditions
in purchaser’s laboratory must be established and continually monitored through purchaser defined and controlled processes including but not limited to testing of positive, negative, and blankcontrols every time a sample is tested. Ordering, acceptance, and use of product constitutes purchaser acceptance of sole responsibility for assuring product efficacy and purchaser agreement
to the limitation of liability set forth in this paragraph.

Peripheral blood, Tissue, or FFPE sample
DNA Extraction
PCR Reaction Setup(Master Mix + Taq* + DNA sample or control)

PCR Amplification

Gel Electrophoresis
with Heteroduplex
Analysis
STEP 1Heat denature
94 °C / 5 minutes

STEP 2

Snap chill on ice
1 hour

STEP 3

Load and run
non-denaturing
polyacrylamide gel

Capillary
Electrophoresis

STEP 1

Mix with Hi-Di
Formamide andsize standards

STEP 3

Enrich** andprepare library

STEP 4

Load and run
instrument

Load and run
capillaryelectrophoresis

STEP 5

UV illumination

STEP 2

Quantify andnormalize library

Snap chill on ice
5 minutesl

Staining

STEP 1

Purify PCR
amplicons

STEP 2Heat denature
95 °C / 3 minutes

STEP 4Next-Genereation
Sequencing

STEP 5

Analyze withfragment analysis
software

STEP 3

STEP 4

STEP 5

Process withbioinformatics
softwareData Interpretation

The Invivoscribe European Conformity marked in vitro diagnostics (CE-IVD) and Research Use Only (RUO) clonality assays detectclonal populations in just a few easy steps. These steps include PCR amplification of the immunoglobulin or T-cell receptor genes
of interest, followed by detection with non-denaturing polyacrylamide gels, capillary electrophoresis, or next-generation sequencing
using an Illumina® MiSeq™, Thermo Fisher Scientific® Ion S5™ or Ion PGM™ instrument. A flowchart illustrating this workflow is shown
below.

*Or equivalent DNA Polymerase.
**For LymphoTrack and LymphoTrack Dx Assays run on the Ion S5 and PGM only.
CE-marked in vitro diagnostic products are not available for sale or use within North America.

Clonality Testing Workflow 145

LymphoTrack® Dx and LymphoTrack®
Workflow Summary
Illumina MiSeq®

TM

STEP 1 Prepare DNA samples
STEP 2 Run PCR with a LymphoTrack® Assay
STEP 3 Purify amplicons
STEP 4 Quantify and prepare library
STEP 5 Load onto MiSeqTM flow cell and run
STEP 6 Process raw data with bioinformatics software
STEP 7 Analyze results

Notice: The LymphoTrack Dx Assays are in vitro diagnostic products and are available in regions that accept CE-IVD products.
*Image courtesy of Illumina, Inc.

146 LymphoTrack Dx and LymphoTrack Workflow Summary

LymphoTrack® Dx and LymphoTrack®
Workflow Summary
Thermo Fisher Scientific® Ion S5/PGMTM

STEP 1 Prepare DNA samples
STEP 2 Run PCR with a LymphoTrack® Assay
STEP 3 Purify amplicons
STEP 4 Quantify, prepare, and enrich library
STEP 5 Load onto Ion S5 /PGM™ chip and run
STEP 6 Process raw data with bioinformatics software
STEP 7 Analyze results

Notice: The LymphoTrack Dx Assays are in vitro diagnostic products and are available in regions that accept CE-IVD products.
*Image courtesy of Thermo Fisher Scientific

LymphoTrack Dx and LymphoTrack Workflow Summary 147

Next-Generation Sequencing Menu
Invivoscribe offers LymphoTrack and LymphoTrack Dx Assays for the analysis of B- and T-cell clonality, somatic hypermutation, and measurable
residual disease studies**. Assays are designed for use on both industry standard next-generation sequencing (NGS) platforms: the Illumina® MiSeq™
and Thermo Fisher Scientific® Ion PGM™ and Ion S5™ instruments.
Invivoscribe assays for the Illumina® MiSeq™ platform offer the ability to analyze up to twenty two samples and two controls per gene target and themultiplexing capabilities to generate a sequencing library that combines amplicons from different Invivoscribe LymphoTrack and LymphoTrack Dx Assays
onto the same flow cell. Our software then sorts and assigns the correct sequences to their corresponding sample.
Invivoscribe assays for the Ion PGM™ and Ion S5™ platform offer the ability to analyze up to ten samples and two controls per gene target and themultiplexing capability to generate a sequencing library that combines amplicons from different Invivoscribe LymphoTrack and LymphoTrack Dx
Assays onto the same sequencing chip, reducing per sample testing costs.
All LymphoTrack and LymphoTrack Dx Assays allow for fast and easy analysis and data visualization using the LymphoTrack bioinformatics software.
The LymphoTrack software sorts and assigns the sequences to their corresponding sample and provides information such as the prevalence, genesegment usage, and the mutation rate (IGH Leader and IGH FR1 only). In addition, the LymphoTrack Measurable Residual Disease (MRD) Software
allows for clonotype sequences to be tracked in subsequent samples for research applications.
The table below indicates which LymphoTrack (Research Use Only) and LymphoTrack Dx (CE-IVD Marked) Assays are currently available.

CE-Marked IVD Assays

MiSeq™

Ion S5/PGM™

LymphoTrack® Dx IGHV Leader Somatic Hypermutation Assays

AVAILABLENOT AVAILABLE

LymphoTrack® Dx IGH FR1 Assays

AVAILABLEAVAILABLE

LymphoTrack Dx IGH FR2 Assays

AVAILABLEAVAILABLE

LymphoTrack® Dx IGH FR3 Assays

AVAILABLEAVAILABLE®

LymphoTrack Dx IGH FR1/2/3 Assays

AVAILABLEAVAILABLE

LymphoTrack® Dx IGK Assays

AVAILABLEAVAILABLE

LymphoTrack® Dx TRG Assays

AVAILABLEAVAILABLE

LymphoTrack Dx TRB Assays

AVAILABLENOT AVAILABLE

Research Use Only (RUO) Assays

MiSeq™

Ion S5/PGM™

LymphoTrack® IGHV Somatic Hypermutation Assays

AVAILABLENOT AVAILABLE

LymphoTrack® IGH FR1 Assays

AVAILABLEAVAILABLE

LymphoTrack IGH FR2 Assays

AVAILABLEAVAILABLE

LymphoTrack® IGH FR3 Assays

AVAILABLEAVAILABLE®

LymphoTrack IGH FR1/2/3 Assays

AVAILABLEAVAILABLE®

LymphoTrack IGK Assays

AVAILABLEAVAILABLE

LymphoTrack® TRG Assays

AVAILABLEAVAILABLE

LymphoTrack TRB Assay

AVAILABLENOT AVAILABLE

®

®

®

®

CE-marked assays are in vitro diagnostic products and are not available for sale or use within North America.
**Measurable residual disease (MRD) applications are currently for research use only.

148 Next-Generation Sequencing Menu

Gel and Capillary Electrophoresis Menu
Invivoscribe offers assays that can be analyzed using two conventional methods of fragment analysis: gel electrophoresis or capillaryelectrophoresis. Gel electrophoresis kits offer a comparatively easy and inexpensive solution for clonality, translocation, and mutational testing andare often the method of choice for laboratories new to using these methods and techniques. PCR products are analyzed using non-denaturing
polyacrylamide gels (PAGE) and often require a heteroduplex step for resolution of generated amplicons.
Capillary electrophoresis kits are supplied with fluorescently labeled primers, allowing the resulting PCR products to be analyzed on Applied
Biosystems (ABI) platforms e.g. 3130, 3500, 3500xL, 3500xL Dx. Fragment analysis by capillary electrophoresis offers the ability to detect fragmentswith a high level of accuracy and analytical sensitivity and allows for greater sample throughput compared to gel detection methods. In addition,
capillary electrophoresis detection often facilitates a more objective interpretation of results than gel-based detection.
The table below summarizes which detection methods are available for our clonality, translocation and FLT3 mutation assays either as RUO,
CE-IVD, or IVD.

CE-Marked IVD Assays

Gel

ABI

IdentiClone® IGH + IGK B-Cell Clonality Assay

NOT AVAILABLEAVAILABLE

IdentiClone IGH Gene Clonality Assay

NOT AVAILABLEAVAILABLE

IdentiClone IGK Gene Clonality Assay

NOT AVAILABLEAVAILABLE

IdentiClone® IGL Gene Clonality Assay

NOT AVAILABLEAVAILABLE

IdentiClone TCRB Gene Clonality Assay

NOT AVAILABLEAVAILABLE

IdentiClone® T-Cell Receptor Gamma Gene Rearrangement Assay 2.0

NOT AVAILABLEAVAILABLE

IdentiClone® TCRD Gene Clonality Assay

NOT AVAILABLEAVAILABLE

LeukoStrat® CDx FLT3 Mutation Assay (CE-marked)

NOT AVAILABLEAVAILABLE

LeukoStrat CDx FLT3 Mutation Assay (CE 2797 IVD)NOT AVAILABLEAVAILABLEIVD Companion Diagnostic Assays

Gel

ABI

LeukoStrat CDx FLT3 Mutation Assay IVD (USA)NOT AVAILABLEAVAILABLE

LeukoStrat CDx FLT3 Mutation Assay (JP)NOT AVAILABLEAVAILABLE

LeukoStrat® CDx FLT3 Mutation Assay (CE-marked)

NOT AVAILABLEAVAILABLE

LeukoStrat CDx FLT3 Mutation Assay (CE 2797 IVD)NOT AVAILABLEAVAILABLE

Research Use Only (RUO) Assays

Gel

ABI

FLT3 Mutation Assay

AVAILABLEAVAILABLE

IGH + IGK B-Cell Clonality Assay

AVAILABLEAVAILABLE

IGH Gene Rearrangement Assay

NOT AVAILABLEAVAILABLE

IGH Gene Clonality Assay

AVAILABLEAVAILABLE

IGH Somatic Hypermutation Assay v2.0

AVAILABLEAVAILABLE

IGL Gene Clonality Assay

AVAILABLEAVAILABLETCRB Gene Clonality Assay

AVAILABLEAVAILABLE

T-Cell Receptor Gamma Gene Rearrangement Assay

NOT AVAILABLEAVAILABLE

T-Cell Receptor Gamma Gene Rearrangement Assay 2.0

AVAILABLEAVAILABLE

TCRD Gene Clonality Assay

AVAILABLEAVAILABLE

BCL1/JH Translocation Assay

AVAILABLENOT AVAILABLE

BCL2/JH Translocation Assay

AVAILABLENOT AVAILABLE

BCL2/JH t(14;18) Translocation Assay

AVAILABLENOT AVAILABLE

BCR/ABL t(9;22) Translocation Assay

AVAILABLEAVAILABLE

PML/RARα t(15;17) Translocation Assay

NOT AVAILABLEAVAILABLE

®
®

®

®
®

CE-marked assays are in vitro diagnostic products and are available in regions that accept CE-IVD products.
IVD assays are in vitro diagnostic products, and are available for sale or use in regions indicated.

Gel and Capillary Electrophoresis Menu 149

Common Technical Support Questions
1.

 hat sample types may be suitable for analysis withW
Invivoscribe Gel and Capillary assays?

9.

We recommend high-quality DNA for clonality testing with our assays. This can beextracted from frozen or fresh tissue, peripheral blood, bone marrow, skin biopsies, etc.

2.

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 trueclonal rearrangement at this size in samples. If you suspect that this peak is clonal in oneof 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.

When should the recommended controls be run with our assays?
The no template, positive, and negative controls should be included in every run for eachtarget, per the product insert or instructions for use.

3.

 hat is the purpose of the Specimen Control Size LadderW
and Amplification Control master mix? What is the difference
between these master mixes?

10.

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.

11.

 here can more information about the primers used in ourWassays be found?

 hich targets are recommended for the study of B-cellWmalignancies?

12.

 hat are the differences between our IGH Gene Rearrangement
W
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.

 hat do IGH Tubes D and E target do and why are theyWchallenging 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 foruse in cases where a polyclonal background is absent (this is likely because theserearrangements are rare). Some of our customers are concerned by this, especiallybecause there may be some samples that have robust germline amplification greaterthan the valid size range. We do not expect the germline amplification to outcompetetrue DH - JH rearrangements. PCR amplicons generated from germline templates aremuch 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.

150 Common Technical Support Questions

What are the differences between the IGH-BCL2 Translocation
Assay and the IGH-BCL2 t(14;18) Translocation Assay?
The IGH-BCL2 Translocation Assay was designed by the EuroClonality/BIOMED-2
Group and is available as either a CE-IVD or research use only assay whereas; the IGHBCL2 t(14;18) Translocation Assay was designed by Invivoscribe and is only available
for research use only. Both of these assays target MBR and Mcr translocations, but
the IGH-BCL2 Translocation Assay also targets the translocations at the 3’ Mbr. The
IGH-BCL2 t(14;18) Translocation Assay was designed as a nested PCR allowing greater
sensitivities (1 clonal cell per 10,000 normal cells) to be achieved. The limit of detectionof the IGH-BCL2 Translocation Assay is 1 clonal cell per 100 normal cells. Lastly, theIGH-BCL2 Translocation Assay includes 33 reactions, whereas the IGH-BCL2 t(14;18)
Translocation Assay includes 30 reactions.

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 afeasible 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 S5/PGM™ instruments. In addition, a high percentage of B-ALL patients
have TRG rearrangements, which can be detected using our assays to detect TRG generearrangements.

7.

 hat are the differences between the TCRG GeneW
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 andconsists of two master mixes. For polyclonal populations, four Gaussian distributions are
generated. The T-Cell Receptor Gamma Gene Rearrangement Assay 2.0 was designed
by Invivoscribe and it's performance was subsequently reviewed and validated by the
EuroClonality/BIOMED-2 Group. It targets all functional VH - JH rearrangements in a single
master mix and produces smaller amplicons grouped under a single Gaussian distribution.
This allows for easier interpretation and makes the assay more suitable for DNA extracted
from FFPE tissue, which may consist of partially degraded DNA that would not amplify wellwith the larger valid size range of the TCRG Gene Clonality Assay.

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.

 hat T-cell receptor kits would you recommend to detect
W
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 threetargets; 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-cellproliferations (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 S5/PGM™ instruments and for TRB for use with
MiSeq™.

The Specimen Control Size Ladder and Amplification Control master mixes are used astroubleshooting 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.

 hy does the polyclonal control produce a peak aroundW
148 bp when amplified with IGK Tube A – 6FAM?

13.

 o you offer quantitative chromosome translocation
D(e.g., BCR-ABL1) controls?
Our controls are validated for qualitative use, although our customers do successfully use
them with quantitative assays. Unfortunately, we cannot guarantee their performancewith any assay that was not designed by Invivoscribe.

14. W
 hich capillary electrophoresis instruments are currently
validated for use with our assay kits?
Currently the capillary electrophoresis instruments Invivoscribe has validated include:
ABI 3100 and 3130 series for all capillary electrophoresis detection assays. The ABI 310
and 3500 instrument series have also been validated for the majority of our capillaryelectrophoresis detection assays. We are not able to support using instruments not listed
as validated in the instructions for use of our CE-IVD assays.

15.

What are the recommended settings for my ABI instrument?
Instruments should be calibrated with the DS-30 matrix standards (Dye set D) for the ABI
310, 3100, or 3130 instrument series. For the ABI 3500 sequencer series, we advise that you
calibrate the instrument with DS-33 matrix standards. We also recommend using either
POP-4 or POP-7 depending on which ABI instrument you are using. If your equipment
supports POP-7, we recommend using this polymer as it can be utilized for both fragment
analysis and sequencing; whereas, POP-4 can only be utilized for fragment analysis.

16. H ow should peaks outside the valid size range be interpreted
when using assay kits?
You should not interpret peaks outside of the valid size range; although, in theory,
it is possible to have a true rearrangement fall outside this region. If you are concerned
about a suspect peak, you may sequence your product for confirmation. Please notethat samples should always be interpreted within the context of all available clinicalinformation.

17.

I s cell-free DNA (cfDNA) a suitable sample type for Invivoscribe
LymphoTrack® or LymphoTrack® Dx Assays?
The average size of cfDNA (~170 bps) makes it a suitable sample type to run with IGH FR3
master mixes. The use of cfDNA with TRG master mixes might be possible, but expected
amplicon sizes generated with this assay are near the upper limits of the fragment
lengths typically found with this sample type.

26. Why am I getting a low percent passing filter and Q30 score?
Low Q30 and percent passing filter (%PF) scores could be an indication that the flow
cell is overloaded. If this is suspected, verify your amplicon and library calculations andquantifications are correct. Low run metrics can also be attributed to many additional
factors including poor quality DNA, contamination, flow cell or instrument issues, etc.
Please refer to your Illumina MiSeq™ user guides and contact Illumina® Support.

27. W
 hy is the same VH-JH rearrangement combination andsequence shared by two groups of reads, one of which is several
bases shorter than the other when looking at the Read Summary
tab of the excel document created by the LymphoTrack®
Visualization Tool?
Our software was designed to list every unique sequence separately in order for thecustomer to see all of the data and make their own determination on how to interpret
it. The several base pair difference can be due to a number of factors including
amplification errors and sequencing errors. It could also be a result of similarities
between some of the primer sequences that were designed to ensure maximum
coverage. We also include a Merged Read Summary report for your reference thatcombines sequences that only differ by 1 or 2 basepairs.

18. I s DNA extracted from FFPE tissue suitable to use with
Invivoscribe LymphoTrack® or LymphoTrack® Dx Assays?
To ensure DNA from challenging specimens is of sufficient quality and quantity
to generate a valid result, samples may be tested with the Specimen Control Size
Ladder master mix.

28. D
 o I need to perform an adapter ligation prior to sequencing
my products?

19. O
 n which instruments can I use the LymphoTrack® andLymphoTrack® Dx Assays?
We have different versions of our assays for the S5/PGM™ and MiSeq™ instruments(LymphoTrack TRB is currently available only on MiSeq™). No other DNA sequencers(e.g. 454) are currently supported. Assays for the Ion S5/PGM™ and MiSeq™ platforms
differ slightly in terms of the total number of indices, etc., but both have similar benefits
such as a one-step PCR reaction and available bioinformatics software.

Performing an adapter ligation is not needed. The primers included in our LymphoTrackand LymphoTrack Dx master mixes already include the appropriate index barcodes andadapter sequences. After PCR amplification, you will be able to proceed with ampliconpurification, amplicon quantification, library pooling, and sequencing.

29. I f the LymphoTrack® or LymphoTrack® Dx software generated anerror, what information should I submit to Technical Support?
Please submit the *.txt Log file that should have been created by the software in the
output folder, a screenshot of the sample directory, and the Lot Number of the software
you are using to support@invivoscribe.com.

20. H
 ow much DNA is needed for the LymphoTrack® andLymphoTrack® Dx Assays?
50 ng of high-quality genomic DNA is required for the Ion S5/PGM™ and MiSeq™
LymphoTrack and LymphoTrack Dx Assays for clonality and somatic hypermutation
applications.

21.

30. Are
controls provided with the kits? Can you purchaseadditional controls? How are they supplied?
Each kit contains the necessary positive and negative controls required to perform theassay; additional controls may also be purchased separately. Single-tube DNA controls
are provided as 100 μL aliquots of 200 μg/mL in 1/10 TE Buffer, 50 μL aliquots of 50 μg/
mL in 1/10 TE Buffer, and 45 μL aliquots of 15 μg/mL in 1/10 TE Buffer. Single-tube RNA
controls are provided as 100 μL aliquots of 400 μg/ml in RNAse free in glass distilledwater.

Can I use a different library quantification method or kit?
We recommend using the KAPA™ kit for MiSeq™ assays and either the 2100 Bioanalyzer®
or the LabChip® GX for the Ion S5/PGM™ assays.

22. W
 ill the LymphoTrack® or LymphoTrack® Dx analysis software
work on my computer?
The software requires Microsoft Windows 7 and Windows 10 (64-bit) and Excel 2007, 2010,or 2013 and will work with most desktop or laptop PCs. For specific requirements pleaserefer to the software instructions for use.

23. C
 an I use the LymphoTrack or LymphoTrack Dx bioinformatics
software with a different assay?
®

®

No, the software will only work with datasets obtained by our LymphoTrack andLymphoTrack Dx Assays.

24. Whatcharacters can I use when naming my samples and the filepathways? What types of files are accepted by the LymphoTrack®
and LymphoTrack® Dx Software - MiSeq™ ?
Our software only recognizes file names and pathways that contain the following
characters (A-Z, a-z, 0-9, . (dot), _ (underscore), - (hyphen)). In addition, spaces in thepathname for the data files or software (pathnames include file folders and file names)
should be avoided. If the software encounters a character that is not listed above or
extra spaces, an error message may be generated. Furthermore, the software is only
compatible with adaptor-trimmed fastq.gz files that are generated by the MiSeq™
Reporter Software when the MiSeq™ instrument is used. An example of the naming
format that the MiSeq™ Reporter uses: SampleName_S1_L001_R1_001.fastq.gz andSampleName_S1_L001_R2_001.fastq.gz.

25. D
 o the Invivoscribe MiSeq™ indices correspond to theIllumina® indices?
The indices included in our MiSeq™ master mixes follow Illumina®’s TruSeq LT
nomenclature. For instance, IGH FR1 MiSeq™ 01 corresponds to A001. Information for theother indices can be found in the instructions for use on how to set up the MiSeq™ Sample
Sheet to detect the appropriate indices.

31.

 hat are the differences between dilution sets, sensitivity
Wpanels, and proficiency panels?
31a. RNA Dilution Sets
BCR/ABL b3a2 (Cat# 4-085-0210), BCR/ABL b2a2 (Cat# 4-085-0310), and BCR/ABL e1a2 (Cat# 4-085-0110). These sets contain six tubes: 100% negative control
RNA and volume to volume (v/v) dilutions (10-1, 10-2, 10-3, 10-4, and 10-5) of thepositive control RNA into the negative control RNA (IVS-0048). The RNA Dilution
Sets are supplied at a concentration of 400 μg/mL, and each tube contains 50
μL. These dilution sets may be used to establish a standard reference curve,
as proficiency controls, as sensitivity controls for specific target assays, and as
routine testing controls for cDNA synthesis, amplification and detection.
31b. RNA Sensitivity Panels
These panels consist of seven tubes: 100% positive control RNA and v/v dilutions(10-1, 10-2, 10-3, 10-4, 10-5, and 10-6) of the positive control RNA into the negative
control RNA (IVS-0035). The RNA Sensitivity Panels are supplied at a concentrationof 400 μg/mL, and each tube contains 100 μL. The RNA Sensitivity Panels may be
used as sensitivity controls for specific target assays, and as routine testing controls
for cDNA synthesis, amplification and detection.
31c. DNA Sensitivity Panels
Consist of six tubes: 100% clonal DNA and v/v dilutions of the clonal DNA into
negative polyclonal DNA (IVS-0000) to make 30%, 20%, 10%, 5%, and 1% dilutions.
The DNA Sensitivity Panels are supplied at a concentration of 200 μg/mL and eachtube contains 100 μL. The DNA Sensitivity Panels may be used as sensitivity controls
for specific target assays.
31d. RNA Proficiency Panel
The proficiency panel for BCR-ABL1 t(9;22) can be used as a sensitivity control
for specific target assays, and as routine testing controls for cDNA synthesis,
amplification and detection. It consists of ten tubes: 100% positive control RNA andv/v dilutions (10-2 and 10-4) of IVS-0003, IVS-0011 and IVS-0032. It also includes
BCR-ABL1 Negative Clonal Control RNA (IVS-0035).

Common Technical Support Questions 151

Product List by Catalog Number
Companion Diagnostic Assays
Capillary Analysis
K-412-0291
K-412-0281
K-412-0331
K-412-0341
K-412-0361
K-412-0371
K-412-0431
K-412-0441

LeukoStrat® CDx FLT3 Mutation Assay (CE-marked)
LeukoStrat® CDx FLT3 Software (CE-marked)
LeukoStrat® CDx FLT3 Mutation Assay (JP)LeukoStrat® CDx FLT3 Software (JP)LeukoStrat® CDx FLT3 Mutation Assay (USA)
LeukoStrat® CDx FLT3 Software (USA)
LeukoStrat® CDx FLT3 Mutation Assay (CE 2797 IVD)
LeukoStrat® CDx FLT3 Software (CE 2797 IVD)

Research Use Only Assays
Capillary & Gel Fragment Analysis
1-100-0010
1-100-0031
1-100-0041
1-101-0020
1-101-0051
1-101-0061
1-101-0071
1-101-0081
1-102-0020
1-102-0021
1-102-0031
1-103-0010
1-103-0011
1-103-0021
1-205-0010
1-205-0011
1-205-0020
1-205-0021
1-206-0010
1-206-0011
1-206-0021
1-207-0051
1-207-0101

IGH + IGK B-Cell Clonality Assay – Gel Detection
IGH + IGK B-Cell Clonality Assay – ABI Fluorescence Detection
IGH + IGK B-Cell Clonality Assay MegaKit – ABI Fluorescence Detection
IGH Gene Clonality Assay – Gel Detection
IGH Gene Rearrangement Assay – ABI Fluorescence Detection
IGH Gene Clonality Assay – ABI Fluorescence Detection
IGH Gene Rearrangement Assay MegaKit – ABI Fluorescence Detection
IGH Gene Clonality Assay MegaKit – ABI Fluorescence Detection
IGK Gene Clonality Assay – Gel Detection
IGK Gene Clonality Assay – ABI Fluorescence Detection
IGK Gene Clonality Assay MegaKit – ABI Fluorescence Detection
IGL Gene Clonality Assay – Gel Detection
IGL Gene Clonality Assay – ABI Fluorescence Detection
IGL Gene Clonality Assay MegaKit – ABI Fluorescence Detection
TCRB Gene Clonality Assay – Gel Detection
TCRB Gene Clonality Assay – ABI Fluorescence Detection
TCRB Gene Clonality Assay MegaKit – Gel Detection
TCRB Gene Clonality Assay MegaKit – ABI Fluorescence Detection
TCRD Gene Clonality Assay – Gel Detection
TCRD Gene Clonality Assay – ABI Fluorescence Detection
TCRD Gene Clonality Assay MegaKit – ABI Fluorescence Detection
T-Cell Receptor Gamma Gene Rearrangement Assay – ABI Fluorescence Detection
T-Cell Receptor Gamma Gene Rearrangement Assay 2.0 –ABI Fluorescence Detection
1-207-0111
T-Cell Receptor Gamma Gene Rearrangement Assay 2.0 MegaKit –ABI Fluorescence Detection
1-308-0010 BCL1/JH Translocation Assay – Gel Detection
1-308-0020 BCL1/JH Translocation Assay MegaKit – Gel Detection
1-309-0010 BCL2/JH t(14;18) Translocation Assay – Gel Detection
1-309-0020 BCL2/JH Translocation Assay – Gel Detection
1-309-0040 BCL2/JH Translocation Assay MegaKit – Gel Detection
1-310-0010 BCR/ABL t(9;22) Translocation Assay – Gel Detection
1-310-0031 BCR/ABL t(9;22) Translocation Assay – ABI Fluorescence Detection
1-311-0011
PML/RARa t(15;17) Translocation Assay – ABI Fluorescence Detection
1-412-0010 FLT3 Mutation Assay - Gel Detection
1-412-0031 FLT3 Mutation Assay - ABI Fluorescence Detection

Master Mixes
2-096-0020
2-096-0021
2-101-0011
2-101-0031
2-101-0041
2-101-0051
2-101-0061
2-101-0081
2-101-0091
2-101-0101
2-101-0180
2-102-0011
2-102-0021
2-103-0011
2-205-0011
2-205-0021
2-205-0031
2-206-0011
2-207-0010
2-207-0020
2-207-0091
2-308-0010
2-309-0010

Specimen Control Size Ladder – Unlabeled
Specimen Control Size Ladder – 6FAM
IGH Tube A – 6FAM
IGH Tube C – HEX
IGH Tube D – HEX
IGH Tube E – 6FAM
IGH Framework 1 (FR1) – 6FAM
IGH Framework 3 (FR3) – HEX
IGH Framework 2 (FR2) – 6FAM
IGH Tube B – 6FAM
Hypermutation Mix 2 v2.0 – Unlabeled
IGK Tube A – 6FAM
IGK Tube B – 6FAM
IGL Tube – 6FAM
TCRB Tube A – 6FAM & HEX
TCRB Tube B – 6FAM
TCRB Tube C – 6FAM & HEX
TCRD Tube – 6FAM & HEX
T-Cell Receptor Gamma Mix 1 – Unlabeled
T-Cell Receptor Gamma Mix 2 – Unlabeled
TCRG – 6FAM
BCL1/JH Tube – Unlabeled
BCL2/JH t(14;18) (Mbr) Mix 1b – Unlabeled

152 Product List by Catalog Number

2-309-0020
2-309-0030
2-309-0040
2-309-0050
2-309-0060
2-309-0070
2-310-0040
2-310-0050
2-310-0060
2-310-0070
2-310-0080
2-310-0090
2-311-0011
2-311-0031
2-311-0041

BCL2/JH t(14;18) (mcr) Mix 2b – Unlabeled
BCL2/JH t(14;18) (Mbr) Mix 1a – Unlabeled
BCL2/JH t(14;18) (mcr) Mix 2a – Unlabeled
BCL2/JH Tube A – Unlabeled
BCL2/JH Tube B – Unlabeled
BCL2/JH Tube C – Unlabeled
BCR/ABL t(9;22) Mix 1b – Unlabeled
BCR/ABL t(9;22) Mix 2b – Unlabeled
BCR/ABL t(9;22) Mix 2c – Unlabeled
BCR/ABL t(9;22) Mix 3b – Unlabeled
BCR/ABL t(9;22) Mix 3c – Unlabeled
BCR/ABL t(9;22) Mix 3d – Unlabeled
PML/RARa t(15;17) Mix 1 – HEX
PML/RARa t(15;17) Mix 2b – HEX
PML/RARa t(15;17) Mix 2c – HEX

BCR/ABL RNA Dilution Sets
4-085-0110
4-085-0210
4-085-0310

BCR/ABL e1a2 RNA Dilution Set
BCR/ABL b3a2 RNA Dilution Set
BCR/ABL b2a2 RNA Dilution Set

Next-Generation Sequencing
RUO LymphoTrack Assays
7-121-0059
7-121-0069
7-121-0129
7-121-0139
7-121-0009
7-121-0039
7-121-0089
7-121-0099
7-121-0109
7-121-0119
7-121-0057
7-121-0007
7-121-0037
7-121-0047
7-122-0009
7-122-0019
7-122-0007
7-225-0009
7-225-0019
7-227-0019
7-227-0009
7-227-0007
7-500-0007
7-500-0008
7-500-0009

LymphoTrack® IGHV Somatic Hypermutation Assay Kit A – MiSeq™
LymphoTrack® IGHV Somatic Hypermutation Assay Panel – MiSeq™
LymphoTrack® IGH FR1/2/3 Assay Kit A – MiSeq™
LymphoTrack® IGH FR1/2/3 Assay Panel – MiSeq™
LymphoTrack® IGH FR1 Assay Kit A – MiSeq™
LymphoTrack® IGH FR1 Assay Panel – MiSeq™
LymphoTrack® IGH FR2 Assay Kit A – MiSeq™
LymphoTrack® IGH FR2 Assay Panel – MiSeq™
LymphoTrack® IGH FR3 Assay Kit A – MiSeq™
LymphoTrack® IGH FR3 Assay Panel – MiSeq™
LymphoTrack® IGH FR1/2/3 Assay – S5/PGM™
LymphoTrack® IGH FR1 Assay – S5/PGM™
LymphoTrack® IGH FR2 Assay – S5/PGM™
LymphoTrack® IGH FR3 Assay – S5/PGM™
LymphoTrack® IGK Assay Kit A – MiSeq™
LymphoTrack® IGK Assay Panel – MiSeq™
LymphoTrack® IGK Assay – S5/PGM™
LymphoTrack® TRB Assay Kit A - MiSeq™
LymphoTrack® TRB Assay Panel - MiSeq™
LymphoTrack® TRG Assay Kit A – MiSeq™
LymphoTrack® TRG Assay Panel – MiSeq™
LymphoTrack® TRG Assay – S5/PGM™
LymphoTrack® Software – S5/PGM™
LymphoTrack® MRD Software
LymphoTrack® Software – MiSeq™

Capillary Fragment Analysis CE-IVD IdentiClone® Assays
9-100-0031
9-100-0041
9-101-0061
9-101-0081
9-102-0021
9-102-0031
9-103-0011
9-205-0011
9-205-0021
9-206-0011
9-206-0021
9-207-0101
9-207-0111

IdentiClone® IGH + IGK B-Cell Clonality Assay – ABI Fluorescence Detection
IdentiClone® IGH + IGK B-Cell Clonality Assay MegaKit –ABI Fluorescence Detection
IdentiClone® IGH Gene Clonality Assay – ABI Fluorescence Detection
IdentiClone® IGH Gene Clonality Assay MegaKit –ABI Fluorescence Detection
IdentiClone® IGK Gene Clonality Assay – ABI Fluorescence Detection
IdentiClone® IGK Gene Clonality Assay MegaKit –ABI Fluorescence Detection
IdentiClone® IGL Gene Clonality Assay – ABI Fluorescence Detection
IdentiClone® TCRB Gene Clonality Assay – ABI Fluorescence Detection
IdentiClone® TCRB Gene Clonality Assay MegaKit – ABI Fluorescence Detection
IdentiClone® TCRD Gene Clonality Assay – ABI Fluorescence Detection
IdentiClone® TCRD Gene Clonality Assay MegaKit –ABI Fluorescence Detection
IdentiClone® T-Cell Receptor Gamma Gene Rearrangement Assay 2.0 –ABI Fluorescence Detection
IdentiClone® T-Cell Receptor Gamma Gene Rearrangement Assay 2.0
MegaKit – ABI Fluorescence Detection

Capillary Fragment Analysis CE-IVD LeukoStrat® Assays
K-412-0291
K-412-0281

LeukoStrat® CDx FLT3 Mutation Assay 33 reactions – ABI Fluorescence Detection
LeukoStrat® CDx FLT3 Mutation Assay SoftwareAnalyte Specific Reagents
A-412-0071
A-412-0081
A-101-0011
A-101-0020
A-101-0030
A-101-0031
A-101-0041
A-101-0051
A-101-0061
A-101-0070
A-101-0080
A-101-0081
A-101-0091
A-101-0101
A-102-0010
A-102-0011
A-102-0020
A-102-0021
A-103-0011
A-205-0010
A-205-0011
A-205-0020
A-205-0021
A-205-0031
A-207-0021
A-207-0071
A-207-0091
A-309-0050
A-309-0060
A-309-0070

FLT3 ITD MM - 6FAM & HEX - ASR
FLT3 TKD MM - 6FAM - ASR
IGH FR1 – 6FAM
IGH FR2 – Unlabeled
IGH FR3 – Unlabeled
IGH FR3 – HEX
IGH DH1 - 6 – HEX
IGH DH7 – 6FAM
IGH Framework 1 – 6FAM
IGH Framework 2 – Unlabeled
IGH Framework 3 – Unlabeled
IGH Framework 3 – HEX
IGH Framework 2 – 6FAM
IGH FR2 – 6FAM
IGK V - J – Unlabeled
IGK V - J – 6FAM
IGK V - Kde – Unlabeled
IGK V - Kde – 6FAM
IGL V - J – 6FAM
TCRB V - J1+2 – Unlabeled
TCRB V - J1+2 – 6FAM & HEX
TCRB V - J2 – Unlabeled
TCRB V - J2 – 6FAM
TCRB D - J1+ 2 – 6FAM & HEX
TCRG V(1-8)J – HEXTCRG V(1-8,9)J – 6FAM
TCRG V(2-5,8-11)J1+2+P – 6FAM
BCL2/JH Mbr – Unlabeled
BCL2/JH 3’Mbr – Unlabeled
BCL2/JH mcr – Unlabeled

BCR/ABL RNA Dilution Sets
4-085-0110
4-085-0210
4-085-0310

BCR/ABL e1a2 RNA Dilution Set
BCR/ABL b3a2 RNA Dilution Set
BCR/ABL b2a2 RNA Dilution Set

RNA Sensitivity Panels
4-087-0030
4-087-0110
4-087-0150
4-087-0200
4-087-0320

Sensitivity Panel – IVS-0003 Clonal Control RNA
Sensitivity Panel – IVS-0011 Clonal Control RNA
Sensitivity Panel – IVS-0015 Clonal Control RNA
Sensitivity Panel – IVS-0020 Clonal Control RNA
Sensitivity Panel – IVS-0032 Clonal Control RNA

Cell Line DNA Controls
4-088-0008
4-088-0010
4-088-0098
4-088-0108
4-088-0118
4-088-0128
4-088-0190
4-088-0370
4-088-0430
4-088-0490
4-088-0550
4-088-0730
4-088-1090
4-088-1210
4-088-1390
4-088-1690
4-088-1750
4-088-1810

IGH SHM Positive Control DNA
IVS-0001 Clonal Control DNA
LymphoTrack® B-cell Low Positive Control
LymphoTrack® T-cell Low Positive Control
LymphoQuant® B-cell Internal Control
LymphoQuant® T-cell Internal Control
IVS-0004 Clonal Control DNA
IVS-0007 Clonal Control DNA
IVS-0008 Clonal Control DNA
IVS-0009 Clonal Control DNA
IVS-0010 Clonal Control DNA
IVS-0013 Clonal Control DNA
IVS-0019 Clonal Control DNA
IVS-0021 Clonal Control DNA
IVS-0024 Clonal Control DNA
IVS-0029 Clonal Control DNA
IVS-0030 Clonal Control DNA
IVS-0031 Clonal Control DNA

4-089-1270
4-089-1720
4-089-2800
4-089-2810
4-089-2820
4-089-2830
4-089-2840
4-089-2850
4-089-3070

IVS-0015 Clonal Control RNA
IVS-0020 Clonal Control RNA
IVS-0032 Clonal Control RNA
10-1 IVS-0032 Clonal Control RNA
10-2 IVS-0032 Clonal Control RNA
10-3 IVS-0032 Clonal Control RNA
10-4 IVS-0032 Clonal Control RNA
10-5 IVS-0032 Clonal Control RNA
IVS-0035 Clonal Control RNA

Tissue DNA Control
4-092-0010

IVS-0000 Polyclonal Control DNA

LymphoTrack® Low Positive Controls
4-088-0098 LymphoTrack® B-cell Low Positive Control
4-088-0108 LymphoTrack® T-cell Low Positive Control

LymphoQuant® Internal Controls
4-088-0118
4-088-0128

LymphoQuant® B-cell Internal Control
LymphoQuant® T-cell Internal Control

Somatic Hypermutation
Sanger Sequencing Assays
5-101-0030
5-101-0031
5-101-0040
5-101-0041

IGH Somatic Hypermutation Assay v2.0 – Gel Detection
IGH Somatic Hypermutation Assay v2.0 – ABI Fluorescence Detection
IGH Somatic Hypermutation Assay v2.0 MegaKit – Gel Detection
IGH Somatic Hypermutation Assay v2.0 MegaKit – ABI Fluorescence

ABI Reagents
6-098-0061

HI-Deionized Formamide with ROX Size Standard (ABI 3100)

Taq DNA Polymerases
6-097-0130

FalconTaq DNA Polymerase

Next-Generation Sequencing
CE-IVD LymphoTrack® Dx Assays
9-121-0007
9-121-0009
9-121-0037
9-121-0039
9-121-0047
9-121-0057
9-121-0059
9-121-0069
9-121-0089
9-121-0099
9-121-0109
9-121-0119
9-121-0129
9-121-0139
9-122-0009
9-122-0019
9-122-0007
9-225-0009
9-225-0019
9-227-0007
9-227-0009
9-227-0019
9-500-0007
9-500-0009

LymphoTrack® Dx IGH FR1 Assay – S5/PGM™
LymphoTrack® Dx IGH FR1 Assay Kit A – MiSeq™
LymphoTrack® Dx IGH FR2 Assay – S5/PGM™
LymphoTrack® Dx IGH FR1 Assay Panel – MiSeq™
LymphoTrack® Dx IGH FR3 Assay – S5/PGM™
LymphoTrack® Dx IGH FR1/2/3 Assay – S5/PGM™
LymphoTrack® Dx IGHV Leader Somatic Hypermutation Assay Kit A – MiSeq™
LymphoTrack® Dx IGHV Leader Somatic Hypermutation Assay Panel – MiSeq™
LymphoTrack® Dx IGH FR2 Assay Kit A – MiSeq™
LymphoTrack® Dx IGH FR2 Assay Panel – MiSeq™
LymphoTrack® Dx IGH FR3 Assay Kit A – MiSeq™
LymphoTrack® Dx IGH FR3 Assay Panel – MiSeq™
LymphoTrack® Dx IGH FR1/2/3 Assay Kit A – MiSeq™
LymphoTrack® Dx IGH FR1/2/3 Assay Panel – MiSeq™
LymphoTrack® Dx IGK Assay Kit A – MiSeq™
LymphoTrack® Dx IGK Assay Panel – MiSeq™
LymphoTrack® Dx IGK Assay – S5/PGM™
LymphoTrack® Dx TRB Assay Kit A - MiSeq™
LymphoTrack® Dx TRB Assay Panel - MiSeq™
LymphoTrack® Dx TRG Assay – S5/PGM™
LymphoTrack® Dx TRG Assay Panel – MiSeq™
LymphoTrack® Dx TRG Assay Kit A – MiSeq™
LymphoTrack® Dx Software – S5/PGM™
LymphoTrack® Dx Software – MiSeq™

Cell Line RNA Controls
4-089-0100
4-089-0190
4-089-0200
4-089-0210
4-089-0220
4-089-0230
4-089-0240
4-089-0910
4-089-0920
4-089-0930
4-089-0940
4-089-0950
4-089-0960

IVS-0002 Clonal Control RNA
IVS-0003 Clonal Control RNA
10-1 IVS-0003 Clonal Control RNA
10-2 IVS-0003 Clonal Control RNA
10-3 IVS-0003 Clonal Control RNA
10-4 IVS-0003 Clonal Control RNA
10-5 IVS-0003 Clonal Control RNA
IVS-0011 Clonal Control RNA
10-1 IVS-0011 Clonal Control RNA
10-2 IVS-0011 Clonal Control RNA
10-3 IVS-0011 Clonal Control RNA
10-4 IVS-0011 Clonal Control RNA
10-5 IVS-0011 Clonal Control RNA

Product List by Catalog Number 153

Warranty and Liability Limits
Invivoscribe is committed to providing the highest quality products. Invivoscribe warrants that for products which are provided with Instructions forUse, these products meet or exceed the performance standards described in the Instructions For Use. If a product is covered by product specifications
and does not perform as specified, our policy is to replace the product or credit the full purchase price. No other warranties of any kind, expressedor implied, are provided by Invivoscribe. Invivoscribe liability shall not exceed the purchase price of the product. Invivoscribe shall have no liability fordirect, indirect, consequential or incidental damages arising from the use, results of use, or inability to use its products; product efficacy under purchaser
controlled conditions in purchaser’s laboratory must be established and continually monitored through purchaser defined and controlled processes
including but not limited to testing of positive, negative, and blank controls every time a sample is tested. Ordering, acceptance and use of productconstitutes purchaser acceptance of sole responsibility for assuring product efficacy and purchaser agreement to the limitation of liability set forthin this paragraph.

Intended Uses
Research Use Only products have not been approved by any regulatory agency for clinical use and are not for diagnostic or therapeutic use.
Our CE-marked assays, sold in Europe and not for sale or use in the United States, are in vitro diagnostic devices intended for detectionof clonal gene rearrangements and translocations for patients with suspected lymphoproliferations.

Patent Notice

Many of the products described herein are covered by one or more patents including European Patent No. 1549764, European Patent No. 2418287,
European Patent No. 2460889, European Patent No. 1633884, Japanese Patent No. 4708029, United States Patent No. 7,785,783, United States Patent No.
8859748 and United States Patent No. 10280462, Japanese Patent No. 6189600, Brazilian Patent No. PI0410283, Canadian Patent No. 2525122, Indian
Patent No. 243620, Mexican Patent No. 286493, Chinese Patent No. 1806051, and Korean Patent No. 10-1215194. All of these patents are licensed to
Invivoscribe Inc.
Use of these products may require the application of methods, such as Polymerase Chain Reaction (PCR), or the use of reagents or equipment coveredby third party patents. Any necessary license to use these third party rights is the responsibility of the user and no such license is granted by Invivoscribe,
expressly or by implication.

Notice©2024 Invivoscribe, Inc. All rights reserved. All trademarks mentioned herein are the property of Invivoscribe®, and/or its affiliates, or (as to thetrademarks of third parties used herein) their respective owners.
®
Invivoscribe®, Invivoscribe, LabPMM®, LymphoTrack®, LeukoStrat®, LymphoQuant®, IdentiClone®, Improving Lives with Precision Diagnostics® andStreamlined CDx® are registered trade marks of Invivoscribe Inc. FalconTaqTM is a trademark of Invivoscribe Inc.

ILLUMINA® and MISEQTM are registered trademarks of Illumina, Inc.
THERMO FISHER SCIENTIFIC® and ION PGM™ and ION S5™ are trademarks of Thermo Fisher Scientific or its subsidiaries.
AGILENT® and BIOANALYZER® are registered trademarks of Agilent Technologies, Inc.
KAPA™ is a trademark of Kapa Biosystems.
PERKIN ELMER® and LABCHIP® are registered trademark of Perkin Elmer.
MICROSOFT®, WINDOWS®, and EXCEL® are registered trademarks of Microsoft Corporation.
Roche® is a registered trademark of F. Hoffmann-La Roche Ltd.

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