CAT #: 11000041
IGH + IGK B-Cell Clonality Assay MegaKit ABI Fluorescence Detection
Immunoglobulin Heavy Chain and Kappa Light Chain Gene Rearrangement Assays are useful for the study of:
- Identifying clonal B-cell populations highly suggestive of B-cell malignancies
- Lineage determination of leukemias and lymphomas
- Monitoring and evaluation of disease recurrence
- Detection and assessment of residual disease
- Evaluation of new research and methods in malignancy studies
- Summary of Explanation of the Test
Invivoscribe’s Gene Rearrangement and Translocation Assays represent a new approach to PCR-based clonality testing. These standardized assays were carefully optimized testing positive and negative control samples using multiplex master mixes.
Polymerase chain reaction (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) and joining (J) regions (IGH Tubes A-C) of the immunoglobulin heavy chain (IGH), the variable (V) and joining (J) regions (IGK Tube A) of the immunoglobulin kappa light chain (IGK) and the variable, intragenic and Kappa Deleting Element (Kde) regions (IGK Tube B) of the immunoglobulin kappa light chain (IGK). 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 & light chains genes 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 this region is amplified using DNA primers that flank it, a clonal population of cells yields one or two prominent amplified products (amplicons) within the expected size range. Two products are produced in cases when the initial rearrangement was non-productive and was followed by rearrangement of the other homologous chromosome. In contrast, DNA from a normal or polyclonal (many clones) population produces a bell-shaped curve of amplicon products (Gaussian distribution) that reflect the heterogeneous population of V-J region rearrangements.
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 smear of different-sized amplicon products that form a Gaussian distribution around a statistically favored, average-sized rearrangement. As expected, primers that amplify from the different FR regions, which are located at three distinct regions along the heavy chain gene, produce a correspondingly different size-range of V-J products.
Gel electrophoresis is commonly used to resolve the different-sized amplicon products and ethidium bromide or other DNA intercalating dyes to stain and detect these products. A powerful alternative method is use of differential fluorescence detection with primers conjugated with fluorescent dyes that correspond to different targeted regions. Reaction products from several different master mixes can be pooled, fractionated using capillary electrophoresis, and detected simultaneously. This detection system results in unsurpassed sensitivity, single base resolution, differential product detection and relative quantification. In addition, the laboratory can eliminate the use of agarose and polyacrylamide gels, as well as the use of carcinogens such as ethidium bromide. Further, differential detection allows accurate, reproducible and objective interpretation of primer-specific products and automatic archiving of data. The limit of detection of this assay has been determined to be approximately 1 clonal cell in 100 hundred normal cells, and inter-assay and intra-assay reproducibility in size determination using capillary electrophoresis is approximately 1-2 basepairs. This reproducibility and sensitivity allows monitoring and tracking of individual tumors during research or methods development. The automatic archiving of specimen data allows comparison of data collected at different times.
This test kit includes 6 master mixes. IGH Tubes A, B, and C target the framework 1, 2, and 3 regions (respectively) within the variable region, and the joining region of the IGH locus. IGK Tubes A and B target the variable, intragenic and joining regions of the IGK locus. The last master mix, the Specimen Control Size Ladder, targets multiple genes and generates a series of amplicons of 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 thermocycler program and similar detection methodologies are used with all of the BIOMED tests. Many of our customers have remarked that this improves consistency and facilitates cross training on a broad range of different assays. These robust Invivoscribe assays can be used to test DNA extracted from virtually any source.
- Specimen Requirements
This assay tests genomic DNA
- 5cc of peripheral blood, bone marrow biopsy, or bone marrow aspirate anti-coagulated with heparin or EDTA. Ship at ambient temperature; OR
- Minimum 5mm cube of tissue shipped frozen; or at room temperature or on ice in RPMI 1640; OR
- 2µg of genomic DNA; OR
- Formalin-fixed paraffin embedded tissue or slides.
This assay is based on the EuroClonality/BIOMED-2 Concerted Action BMH4-CT98-3936.
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 for Research Use Only; not for use in diagnostic procedures.
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.
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