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CAT #: 91000041

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

Intended Use

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 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 (IGH and IGK gene rearrangements) for post-treatment monitoring
  • Monitor and evaluate disease recurrence

Product Details

  • 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 heavy chain and kappa light chain genes.  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 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 testing of more than 400 clinical samples using the Revised European/American Lymphoma (REAL) classification performed at more than thirty prominent independent testing centers throughout Europe in a collaborative study known as the BIOMED-2 Concerted Action.  Results from this BIOMED-2 study indicate that testing for both IGH and IGK gene rearrangements may improve the reliability and sensitivity of the tests.2  In addition, testing for both IGH and IGK gene rearrangements led to 99% sensitivity, compared to 88% for IGH and 88% for IGK when tested alone, which may also increase the reliability of the tests, improving the likelihood of detecting clonal products in more than one tube.4

    The ABI detection based assays cannot reliably detect clonal populations comprising less than 1% of the total lymphocyte cell population.  Always interpret the results of molecular clonality tests in the context of clinical, histological and immunophenotypic data.

    This test kit includes six (6) master mixes.  The IGH Tube A, B, and C master mixes target the framework 1, 2, and 3 regions (respectively) within the variable region, and the joining region of the immunoglobulin heavy chain locus.  The IGK Tube A master mix targets the variable (V) and the joining (J) regions of the Ig kappa light chain locus.  Whereas the IGK Tube B master mix targets kappa deleting element (Kde) rearrangements with the variable (V) region and the intragenic Jϰ-Cϰ region.  The resulting Vϰ-Kde and Jϰ-Cϰ intron-Kde rearrangements are a result of unsuccessful rearrangements retained by the B-cell.  Lastly, 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 (bp) 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.  This improves consistency and facilitates cross training on a broad range of different assays.

  • 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) and joining (J) regions (IGH Tubes A, B, and C), the variable (V) and joining (J) regions (IGK Tube A) and the variable, Jϰ-Cϰ intron, and Kde regions (IGK Tube B).  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 and light chain 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 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.  DNA from samples containing a clonal population yield 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.  Note that the primers that amplify the different FR regions, which are located at three distinct sections along the heavy chain gene, produce a correspondingly different size-range of V-J products.  For rearrangements of the IGK gene, the length of the CDR3 region is limited and display significant skewing (platykurtosis).  Thus, PCR products display a very narrow Gaussian distribution and are most easily and reliably identified by heteroduplex analysis.

    Differential Fluorescence Detection

    Differential fluorescence detection is commonly used to resolve the different-sized amplicon products using a capillary electrophoresis instrument.  Primers can be conjugated with several different fluorescent dyes (fluorophors) so that they can produce different emission spectra upon excitation by a laser in the capillary electrophoresis instrument.  In this manner, different fluorescent dyes can correspond to different targeted regions.  This detection system results in unsurpassed sensitivity, single nucleotide resolution, differential product detection, and relative quantification. In addition, the use of agarose and polyacrylamide gels, as well as the use of carcinogens such as ethidium bromide, can virtually be eliminated.  Further, differential detection allows accurate, reproducible and objective interpretation of primer-specific products and automatic archiving of data.  Inter-assay and intra-assay reproducibility in size determination using capillary electrophoresis is approximately 1 to 2 nucleotides.  This reproducibility and sensitivity coupled with the automatic archiving of specimen data allows for the monitoring, tracking, and comparison of data from individual patients over time.

  • 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)
    • 3 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)


1.  Miller, JE et al. (1999). Molecular Diagnostics 4(2):101-117.

2.  Van Dongen, JJM et al. (2003). Leukemia. 17(12):2257-2317.

3.  Sandberg, Y, et al. (2005). J. Molecular Diagnostic 7(4):495-503.

4.  van Krieken, JHJM et al. (2007). Leukemia 21(3):201-206.


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

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