Genomic DNA is extracted from all specimens.
In the PCR assay, a total of 34 upstream and 5 downstream primers are used (InVivoScribe IGH and IGK Gene Clonality Assay kits). The primers are designed to amplify fragments from all theoretical rearrangements of the immunoglobulin (Ig) heavy and Ig kappa light chain genes. Each unique rearrangement should produce PCR fragment(s) of unique size(s). The primers cannot amplify anything if the Ig genes are not rearranged because the distance is too great. The primers are labeled with a fluorescent tag so that the PCR product can be detected. The PCR fragments are analyzed by capillary gel electrophoresis using the Applied Biosystems (ABI) 3130XL machine for fragment size and amount.(van Dongen JJ, Langerak AW, Bruggemann M, 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 December;17:2257-2317; van Dongen JJ, Wolvers-Tettero IL: Analysis of immunoglobulin and T-cell receptor genes. Part 1: basic and technical aspects. Clin Chim Acta 1991 April;198[1-2]:1-92; package insert: IGH Gene Clonality Assay-ABI Fluorescence Detection and IGK Gene Clonality Assay-ABI Fluorescence Detection. InVivoScribe Technologies, San Diego, CA 2004; Gene Images AlkPhos Direct Labeling and Detection system. Amersham Biosciences, UK Limited)