High-resolution analysis of gene rearrangements in lymphoid malignancies.
As T-cell receptor and immunoglobulin gene rearrangements provide specific clonal markers for lymphoid cell proliferations, analysis of these genes is useful for distinguishing between reactive and malignant disease. We have developed an automated, high-resolution analysis of PCR fragments to identify clonally rearranged TCR-gamma (TCR gamma) genes and IgH genes. Consensus primers are used to detect the majority of possible rearrangements in multiplex PCR assays, and the PCR products are fluorescently labelled for visualisation with ABI Genescan software. Polyclonal populations of lymphoid cells are represented by a spectrum of fragments, whereas a monoclonal population of cells is represented by one or two discrete bands, indicating rearrangement of one or both alleles. For TCR-gamma PCR, the rearranged DNA fragment from a monoclonal population of T-cells diluted to 0.1% in DNA from a polyclonal population of cells is still readily distinguishable from the polyclonal background. Similarly, for IgH PCR, the gene rearrangement from a monoclonal population of B-cells is still distinguishable to 0.5% in a polyclonal background. As this technique allows semi-quantitative resolution of fragments one base different in size, it is ideal for detecting monoclonal and oligoclonal populations of B- and T-cells. The accurate size determination of PCR fragments also minimises the risk of false positives resulting from contamination, as individual monoclonal rearrangements are frequently patient-specific on the basis of size alone.
Ayling
J F
JF
Kanematsu Laboratories, Royal Prince Alfred Hospital, Sydney, Australia.
Iland
H J
HJ
D002999:Clone Cells; D005453:Fluorescence; D015326:Gene Rearrangement, B-Lymphocyte, Heavy Chain; D015333:Gene Rearrangement, beta-Chain T-Cell Antigen Receptor; D006801:Humans; D007938:Leukemia / Q000235:genetics*; D008223:Lymphoma / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D012751:Sezary Syndrome / Q000235:genetics