Outside North America 
CAT #: 91030010
IdentiClone® IGL Gene Clonality Assay - Gel Detection
Intended Use
The IdentiClone® IGL Gene Clonality Assay is an in vitro diagnostic product intended for PCR-based detection of clonal immunoglobulin lambda light chain gene rearrangements in patients with suspect lymphoproliferations and 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
Product Details
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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 that are unique in length and sequence for each cell. Therefore, 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 PCR assay employs multiple consensus DNA primers that target conserved genetic regions within the immunoglobulin lambda light chain (IGL) gene. This test is used to detect the vast majority of clonal B-cell malignancies from DNA. Test products can be analyzed using a variety of detection formats, including gel and capillary electrophoresis.
Invivoscribe’s IdentiClone assays represent a simple 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 more than thirty prominent independent testing centers throughout Europe in a collaborative study known as the BIOMED-2 Concerted Action.2
This test kit includes two (2) master mixes. The IGL Tube master mix targets the variable (V) and joining (J) regions of the immunoglobulin lambda light chain locus. 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. A single thermal cycler program and similar detection methodologies are used with all of our Gene Clonality Assays which improves consistency and facilitates cross training on a broad range of different assays.
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Principles of the Procedure
Polymerase Chain Reaction (PCR)
PCR assays are routinely used for the identification of clonal B-cell populations. These tests amplify the DNA between primers that target the conserved framework (FR) of the variable (V) regions and the conserved joining (J) regions (IGL Tube). These conserved regions lie on either side of an area within the V-J region where programmed genetic rearrangements occur during maturation of all B and T lymphocytes. The antigen receptor genes that undergo rearrangement are the immunoglobulin heavy chain andlight chains in B-cells, and the T cell receptor genes in T-cells. Each B- and T-cell has a single productive V-J rearrangement that is unique in both length and sequence. Therefore, when DNA from a normal or polyclonal population is amplified using DNA primers that flank the V-J region, a bell-shaped curve (Gaussian distribution) of amplicon products within an expected size range is produced. This Gaussian distribution reflects the heterogeneous population of V-J rearrangements. (In certain cases, where lymphocyte DNA is not present, no product is seen.) For DNA from samples containing a clonal population, the yield is one or two prominent amplified products (amplicons) within a diminished polyclonal background.
Since the antigen receptor genes are polymorphic (consisting of a heterogeneous population of related DNA sequences), it is difficult to employ a single set of DNA primer sequences to target all of the conserved flanking regions around the V-J rearrangement. N-region diversity and somatic mutation further scramble the DNA sequences in these regions. Therefore, multiplex master mixes, which target several FR regions, are required to identify the majority of clonal rearrangements. As indicated, clonal rearrangements are identified as prominent, single-sized products within the background of different-sized amplicon products that form a Gaussian distribution around a statistically favored, average-sized rearrangement.
Gel Detection
Gel electrophoresis, such as agarose gel electrophoresis or non-denaturing polyacrylamide gel electrophoresisis (PAGE), is commonly used to resolve the different amplicon products based on their size, charge, and conformation. Since DNA is negatively charged, when an electrical potential (voltage) is applied across the gel containing PCR products, the electrical field causes the amplicons to migrate through the gel. Smaller DNA fragments are able to easily migrate through the gel matrix, whereas larger DNA fragments migrate more slowly. This causes a separation of the amplicon products based on size. Ethidium bromide or other DNA intercalating dyes can then be used to stain and detect these products in the gel.
A heteroduplex analysis can also be performed and run on a polyacrylamide gel to differentiate clonal and non-clonal PCR products. A heteroduplex analysis involves denaturing the PCR products at a high temperature, then quickly re-annealing the DNA strands by suddenly reducing the temperature. This causes a large portion of DNA strands to incorrectly bind to other non-homologous strands creating loops in the DNA. These loops cause a significant reduction in the ability of the DNA to migrate through a polyacrylamide gel. However, if the majority of the PCR products are clonal, when a heteroduplex analysis is performed, most of these PCR products will correctly re-anneal with a homologous strand. These PCR products will run normally through the polyacrylamide gel. Therefore, in a clonal sample with a polyclonal background, a heteroduplex analysis will cause most of the polyclonal product to run much slower through the polyacrylamide gel, thereby increasing their separation and the ability to identify the clonal band(s).
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Specimen Requirements
This assay tests genomic DNA (gDNA) from the following sources:
- 5 cc of peripheral blood, bone marrow biopsy, or bone marrow aspirate anti-coagulated with heparin or EDTA (stored at 2°C to 8°C and shipped at ambient temperature)
- Minimum 5 mm cube of tissue (stored and shipped frozen; or stored and shipped in RPMI 1640 at ambient temperature or on ice)
- 2 µg of gDNA (stored at 2°C to 8°C and shipped at ambient temperature)
- Formalin-fixed paraffin embedded tissue or slides (stored and shipped at ambient temperature)
References
1. Miller, JE, et al. (1999) Molecular Diagnostics. 4(2):101-117.
2. Van Dongen, JJM et al. (2003) Leukemia. 17(12):2257-2317.
Disclaimer
This assay is based on the EuroClonality/BIOMED-2 Concerted Action BMH4-CT98-3936.
Legal Notice
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 the Instructions 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 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.
This product is an in vitro diagnostic product is not available for sale or use within North America.
This product is covered by one or more of the following: European Patent Number 1549764, European Patent Number 2418287, European Patent Number 2460889, Japanese Patent Number 4708029, United States Patent 8859748, United States Patent 10280462, and related pending and future applications. All of these patents and applications are licensed exclusively to Invivoscribe®. Additional patents licensed to Invivoscribe ®covering some of these products apply elsewhere. Many of 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 this product.
IdentiClone® is a registered trademark of Invivoscribe®.
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