Ståhlberg Anders, Aman Pierre, Ridell Börje, Mostad Petter, Kubista Mikael
Department of Molecular Biotechnology, Chalmers University of Technology, 405 30 Gothenburg, Sweden.
Clin Chem. 2003 Jan;49(1):51-9. doi: 10.1373/49.1.51.
An abnormal IgLkappa:IgLlambda ratio has long been used as a clinical criterion for non-Hodgkin B-cell lymphomas. As a first step toward a quantitative real-time PCR-based multimarker diagnostic analysis of lymphomas, we have developed a method for determination of IgLkappa:IgLlambda ratio in clinical samples.
Light-up probe-based real-time PCR was used to quantify IgLkappa and IgLlambda cDNA from 32 clinical samples. The samples were also investigated by routine immunohistochemical analysis and flow cytometry.
Of 32 suspected non-Hodgkin lymphoma samples analyzed, 28 were correctly assigned from real-time PCR measurements assuming invariant PCR efficiencies in the biological samples. Four samples were false negatives. One was a T-cell lymphoma, one was a diffuse large B-cell lymphoma, and one was reanalyzed and found lymphoma-positive by in situ calibration, which takes into account sample-specific PCR inhibition. Twelve of the samples were fine-needle aspirates, and these were all correctly assigned.
This work is a first step toward analyzing clinical samples by quantitative light-up probe-based real-time PCR. Quantitative real-time PCR appears suitable for high-throughput testing of cancers by measuring expression of tumor markers in fine-needle aspirates.
长期以来，异常的IgLκ:IgLλ比值一直被用作非霍奇金B细胞淋巴瘤的临床诊断标准。作为基于定量实时PCR的淋巴瘤多标志物诊断分析的第一步，我们开发了一种在临床样本中测定IgLκ:IgLλ比值的方法。
采用基于发光探针的实时PCR对32份临床样本中的IgLκ和IgLλ cDNA进行定量分析。这些样本还通过常规免疫组织化学分析和流式细胞术进行了研究。
在分析的32份疑似非霍奇金淋巴瘤样本中，假设生物样本中PCR效率不变，通过实时PCR测量，28份样本被正确分类。4份样本为假阴性。其中1份是T细胞淋巴瘤，1份是弥漫性大B细胞淋巴瘤，1份重新分析后通过考虑样本特异性PCR抑制的原位校准发现为淋巴瘤阳性。12份样本为细针穿刺抽吸物，这些样本均被正确分类。
这项工作是通过基于发光探针的定量实时PCR分析临床样本的第一步。定量实时PCR似乎适用于通过测量细针穿刺抽吸物中肿瘤标志物的表达来对癌症进行高通量检测。
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