B-Cell Clonality – IgH and IgK Rearrangement Testing

Test Description

Detection of monoclonal, polyclonal or oligoclonal populations of B-cells by PCR and fragment sizing analysis of the IgH and IgK genes

Background

A multimodality approach is used in the diagnosis of lymphomas. Diagnosis may be particularly challenging in some cases solely based on morphology and immunophenotyping. The molecular testing exploits the rearrangement of the immunoglobulin heavy chain and kappa genes. These genes rearrange over kilobases of genetic sequence and are unique for each normal mature B-cell and plasma cell. A reactive process, thus will demonstrate a polyclonal expansion of B-cells with different sixe rearrangements whereas a malignant process will often demonstrate expansion of a B-cell population that originates from a single cell and hence all will have the same rearrangement. The detection of a clonal immunoglobulin heavy chain / kappa gene rearrangement by polymerase chain reaction (PCR) can be extremely helpful as an aid in the diagnosis of malignant B-cell lymphoma.

Clinical Utility

If the nature of the B-cell lymphoproliferative process cannot be accurately determined by morphology and immunophenotyping, PCR-based studies for B cell gene rearrangements can help in establishing clonality. The presence of a clonal immunoglobulin (B cell) or T cell receptor (T cell) gene rearrangement is usually (but not always) indicative of a neoplasmic process. This test can also be used to follow-up the patients post treatment to see if the clone still persists and compare the lesions from two different sites to determine if this is the same or a different clonal process.

Methodology

Loci Tested: IgH / IgK. B-cell clonality is determined by PCR and fragment sizing analysis of fluorescently labeled products. Master mixes are commercially provided by InVivoScribe in a BIOMED-2 Assay for IgH and IgK. Patterns consistent with polyclonal, oligoclonal and monoclonal B-cell populations are interpreted from fragment-sizing electropherograms.

Specimen Requirements

Peripheral Blood: 3-5 ml, collected in EDTA (purple top) tube, store at room temperature 24 hours
Bone Marrow: 0.5-1 ml, collected in EDTA (purple top) tube, store at room temperature, 24 hours
Tissue: Frozen or fresh tissue may be used. A minimum of 2 x 2 x 2 mm is required (5 x 5 x 5 mm is preferred). For local facilities, we prefer the sample to be sent fresh on wet ice to arrive in the lab the same day. For outside facilities, the preferred approach is to snap freeze tissue directly or as cell pellets at -20 or -70°C and mail with sufficient dry ice to prevent thawing before arrival at our laboratory.
Paraffin Sections: 10 sections on glass slides. More sections may be required if the tissue is small. Please call the lab if you have questions. Include % tissue area involved by neoplasm and the pathologist’s signature.
Unacceptable Specimens: Decalcified specimen, Frozen blood or bone marrow specimens are unacceptable as are tissue samples that have undergone a freeze/thaw cycle(s).

Turnaround Time

Performed twice each week

Reported

Within 3-7 business days of receipt

CPT

IGH 81263; IGK 81264

Shipment Must Include

Specimen

Requisition form

Patient pathology report

References

van Dongen, JJM et al. Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: Report of the BIOMED-2 Concerted Action BMH4-CT98-3936, Leukemia (2003) 17, 2257–2317.
Sandberg, Y et al, BIOMED-2 Multiplex Immunoglobulin/T-Cell Receptor Polymerase Chain Reaction Protocols Can Reliably Replace Southern Blot Analysis in Routine Clonality Diagnostics, Journal of Molecular Diagnostics, 2005, Vol. 7, No. 4, 495.
Evans, P.A., C. Pott, et al, (2007). Significantly improved PCR-based clonality testing in B-cell malignancies by use of multiple immunoglobulin gene targets: Report of the BIOMED-2 concerted action BHM4-CT98-3936. Leukemia 21(2): 207-214.
Vargas, R.L., R.E.Felgar, et al, (2008), Detection of clonalityin lymphoproliferations using PCR of the antigen receptor genes: Does size matter? Leukemia Research 32(2): 335-338.