Lynas C, Howe D, Copplestone J A, Johnson S A, Phillips M J
Department of Haematology, Derriford Hospital, Plymouth PL6 8DH.
Clin Mol Pathol. 1995 Apr;48(2):M101-4. doi: 10.1136/mp.48.2.m101.
Aims-To establish a reverse transcription polymerase chain reaction (RT-PCR) for the detection of clonal T cell populations, and to evaluate the sensitivity and reliability of the technique.Methods-After reverse transcription of the target RNA with a consensus T cell receptor (TCR) beta constant (C) region primer, consensus C, variable (V), diversity (D) and joining (J) region primers were used to amplify across various portions of the TCRbeta V-D-J-C junction.Results-In normal T cells the polyclonal rearrangements produce a ladder of PCR bands representing the different sized junction fragments. The presence of a T cell clone leads to over-representation of one junction fragment, hence a disproportionately brighter band in the PCR ladder. In a series of 16 patients the RT-PCR detected nine of nine shown to have a clonal TCRbeta rearrangement by Southern blotting and for six of seven patients, it confirmed the presence of a clone indicated by histology or immunophenotyping with FACS analysis, but which was undetectable (five patients) or not investigated (two patients) by Southern blotting. Investigations mixing RNA from normal lymphocytes and the Jurkat TCR-Vbeta8 T cell line suggested that the method was more sensitive than Southern blotting.Conclusions-All PCR methods are faster and easier than Southern blotting, but RT-PCR also improves detection of clonal T cell populations, is reliable and produces results that are easy to interpret.
目的——建立用于检测克隆性T细胞群体的逆转录聚合酶链反应（RT-PCR），并评估该技术的敏感性和可靠性。方法——使用共有T细胞受体（TCR）β恒定（C）区引物对靶RNA进行逆转录后，使用共有C区、可变（V）区、多样性（D）区和连接（J）区引物扩增跨越TCRβ V-D-J-C连接的各个部分。结果——在正常T细胞中，多克隆重排产生代表不同大小连接片段的PCR条带梯。T细胞克隆的存在导致一个连接片段过度呈现，因此在PCR条带梯中出现不成比例的亮带。在一系列16例患者中，RT-PCR检测出9例经Southern印迹显示有克隆性TCRβ重排的患者，对于7例患者中的6例，它证实了组织学或流式细胞术免疫表型分析所指示的克隆的存在，但Southern印迹未检测到（5例患者）或未进行研究（2例患者）。将来自正常淋巴细胞的RNA与Jurkat TCR-Vβ8 T细胞系混合进行的研究表明，该方法比Southern印迹更敏感。结论——所有PCR方法都比Southern印迹更快、更容易，但RT-PCR也提高了克隆性T细胞群体的检测能力，可靠且产生易于解释的结果。
