The IdentiClone™ IGK Gene Clonality Assay is an in vitro diagnostic product intended for PCR-based detection of clonal immunoglobulin kappa light chain gene rearrangements in patients with suspect lymphoproliferations.

Specifically, the IGK Gene Clonality Assay can be used to:

1. Identify clonality in atypical lymphoproliferative disorders
2. Support a differential diagnosis between reactive lesions and hematologic malignancies
3. Assign presumptive lineage in mature monoclonal lymphoproliferative disorders
4. Identify tumor-specific markers (IGK and IGK-Kde rearrangements) for post-treatment monitoring
5. Monitor and evaluate disease recurrence

IdentiClone™ assay kits are in vitro diagnostic products. These standardized PCR-based tests come with a Standard Operating Procedure (SOP), an interpretative guide, master mixes, and controls. Master mixes are composed of a buffered magnesium solution, deoxynucleotides, and multiple primers that target the gene segments of interest. Multiple primers are used to ensure a more comprehensive testing approach necessary to reliably identify clonal rearrangements. These assay kits are complete with the exception of Taq DNA polymerase, which is not provided. A single thermocycler program and similar detection methods are used within each series of kits to improve consistency, reduce human error, and facilitate cross training.

Standard Protocol:

1. Using gloved hands, remove the master mixes from the freezer. Allow the tubes to thaw; then gently vortex to mix.
2. In a containment hood or dead air box remove an appropriate aliquot to clean, sterile microfuge tube (one tube for each of the master mixes). Aliquot volumes should be 45ul for each sample + 135ul for the positive, negative and no template controls. We recommend adding an additional 20ul to correct for pipetting errors.
3. Add the appropriate amount of either AmpliTaq Gold or AmpliTaq DNA polymerase (0.25ul of either AmpliTaq Gold or AmpliTaq @ 5U/ul per 50ul total PCR reaction volume) to each of the master mixes and gently mix by inverting several times or gently vortexing.
4. Aliquot 45ul of master mix to individual wells of a PCR plate.
5. Add 5ul of DNA from the unknown and control samples to individual tubes or wells containing the respective master mix reactions, and pipette up and down several times to mix. Amplify target DNA using the universal thermocycler program.

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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. This IdentiClone™ PCR assay employs multiple consensus DNA primers that target conserved genetic regions within the immunoglobulin heavy chain 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.

Gene rearrangement analysis can also be performed by Southern Blot (SB)-based techniques. Although SB analysis is very reliable, it is increasingly replaced by PCR techniques because of the greater efficiency and sensitivity of PCR. Moreover, PCR is relatively easy, less labor intensive, and requires much lower quantities of high molecular weight DNA than SB tests. In addition, PCR can often be performed on DNA isolated from paraffin-embedded tissue, whereas SB cannot be performed because the DNA is often degraded. Therefore there is a strong need to replace SB analysis with reliable PCR techniques.

InVivoScribe Technologies’ IdentiClone™ 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. 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 done 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 appear in Leukemia, a leading peer-reviewed journal.

The ABI detection based assays cannot reliably detect clonal populations comprising less than 1% of the total lymphocyte cell population. It should be emphasized that the results of molecular clonality tests should always be interpreted in the context of clinical, histological and immunophenotypic data.

This test kit includes 3 master mixes. 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 Jk-Ck region. The resulting Vk-Kde and Jk-Ck intron-Kde rearrangements are a result of unsuccessful rearrangements retained by the B-cell. The third master mix, the Specimen Control Size Ladder, targets multiple genes and generates a series of amplicons of 96, 199, 299, 399, and 608 basepairs 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 our Gene Clonality Assays. This improves consistency and facilitates cross training on a broad range of different assays. Click to read more

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 variable (V) and joining (J) regions (IGK Tube A), or the variable, Jκ-Cκ intron, and Kde regions (IGK Tube B). The conserved V and J regions lie on either side of the hypervariable, complementarity-determining region 3 (CDR3) 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 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. On a gel, this distribution of products is seen as a smear. 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. For Vκ-Jκ rearrangements, the length of the CDR3 is limited and rearrangements in this region 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. Click to read more

This product tests genomic DNA. DNA can be extracted from the following specimens:

1. 5cc of peripheral blood, bone marrow biopsy, or bone marrow aspirate anti-coagulated with heparin or EDTA; OR,
2. Minimum 5mm cube of tissue shipped frozen, at room temperature, or on ice in RPMI 1640; OR,
3. Formalin-fixed paraffin embedded tissue or slides.

This product is covered by European Patent EP 1549764B1, European patent application 03756746.8 and divisional applications thereof, Japanese Patent Number P04708029B2, and United States Patent Application Number 10/531,106. All of these patents and patent applications are licensed exclusively to Invivoscribe Technologies, Inc.

This product requires nucleic acid amplification methods such as Polymerase Chain Reaction (PCR). No license under these patents to use amplification processes or enzymes.