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CAT #: 11000041

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

Product Use

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

Product Details

  • Summary of Explanation of the Test

    SUMMARY:

    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.

    BACKGROUND:

    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.

Disclaimer

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

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