运用BIOMED-2 PCR检测脑脊液中的恶性B淋巴细胞

Detection of Malignant B Lymphocytes in Cerebrospinal Fluid by using BIOMED-2 PCR

本研究旨在建立一种检测弥漫性大B细胞淋巴瘤(DLBCL)中枢神经系统(CNS)累及患者脑脊液(CSF)中恶性B淋巴细胞的敏感的方法。对9例考虑有CNS累及风险的DLBCL患者采集其CSF,离心获取细胞沉淀,在直接裂解后运用BIOMED-2 PCR检测免疫球蛋白重链(IgH)基因重排(恶性B淋巴细胞特征性改变),并将此方法的敏感性与细胞学检测及流式细胞术进行比较。此外,通过一系列数量/浓度的肿瘤细胞,分析两种样品处理方式(细胞直接裂解法和传统DNA提取法)导致的敏感度差异,并评估直接裂解法联合BIOMED-2 PCR方案的敏感度。结果显示,BIOMED-2 PCR检测到5例DLBCL患者的CSF中存在克隆性IgH基因重排(恶性B淋巴细胞"阳性"),而细胞学检测和流式细胞术均只能明确检测2例为"阳性",表明BIOMED-2 PCR敏感性高于细胞学检测和流式细胞术。另外,经过改进的样品处理方式——细胞直接裂解法比传统的DNA提取能获得更高的BIOMED-2PCR敏感度,前者可以检测到浓度低至1%、细胞数低至20个的肿瘤细胞。结论:细胞直接裂解联合BIOMED-2PCR是一种敏感的、非常适用于CSF(细胞数少)的检测方法,可辅助诊断DLBCL病例的CNS累及。

The purpose of this study was to develop a sensitive method for the detection of malignant B lymphocytes in cerebrospinal fluid （CSF） from patients with diffuse large B-cell lymphoma （DLBCL） who were considered as risk of central nervous system （CNS） involvement. Nine CSF samples were collected and then centrifuged. The cell precipitate was lysed directly. The supernatant was used to detect immunoglobulin heavy chain （IgH） gene rearrangement （ characteristic changes of malignant B lymphocytes ） by BIOMED-2 PCR. The sensitivity of this method was compared with that of cytology defection and flow cytometry. In addition, through a series of quantity / concentration of tumor cells, the sensitivity differences caused by two sample handling methods （direct cell lysis vs traditional DNA extraction） were analyzed, and the sensitivity of direct cell lysis combined with BIOMED-2 PCR was evaluated. The results showed that the positive clonality of IgH gene rearrangement were detected by BIOMED-2 PCR in 5 cases, but the positive were detected by cytology defection/flow cytometry only in 2 cases, which indicated that the BIOMED-2 PCR assay gives a better yield. In addition, when combined with BIOMED-2 PCR, direct cell lysis produced sensitivity much higher than DNA extraction. The former can enable clonality detection from a minimum of 0. 1%/20 tumor cells. It is concluded the method of direct cell lysis combined with BIOMED-2 PCR is sensitive and suitable for paucicellular CSF detection. It may aid the diagnosis of CNS involvement in patients with DLBCL.