应用逆转录聚合酶链反应检测原发性肝癌患者外周血肿瘤细胞

目的寻求检测循环血中肝癌细胞的灵敏方法。方法采用逆转录聚合酶链反应（ＲＴ－ＰＣＲ）技术，对肝癌患者外周血有核细胞人甲胎蛋白ｍＲＮＡ（ＡＦＰｍＲＮＡ）进行检测。结果３１例原发性肝癌中有１５例检出ＡＦＰｍＲＮＡ，阳性率为４８．４％。良性肝病、肝转移癌患者和健康对照组均为阴性。外周血细胞ＡＦＰｍＲＮＡ的存在与肝内转移、门静脉癌栓和远处转移密切相关。结论外周血细胞ＡＦＰｍＲＮＡ是反映原发性肝癌患者有无血行播散的重要标志，ＲＴ－ＰＣＲ技术是检测循环血中原发性肝细胞癌的灵敏方法     

Real-time polymerase chain reaction in multiple myeloma: quantitative analysis of tumor contamination of stem cell harvests

OBJECTIVE: Autologous transplantation of bone marrow (BM) and peripheral blood progenitor cells (PBPC) is commonly used for treatment of multiple myeloma (MM). Although both stem cell sources harbor residual clonal cells, a quantitative evaluation of their level of tumor contamination (LTC) still needs to be performed through highly accurate and reproducible approaches. In this study, we used a validated real-time polymerase chain reaction (PCR) strategy to evaluate LTC of BM and PBPC samples obtained from MM patients. MATERIALS AND METHODS: The patients underwent two different mobilization courses (defined as early or late course) following two cycles of cyclophosphamide 5 g/m(2). LTC was evaluated by measuring the number of clonal immunoglobulin heavy-chain rearrangements followed by normalization of samples using the GAPDH gene. RESULTS: Overall, 26 PBPC and 12 BM samples were analyzed. Main results are as follows. 1) PBPC harvests are less contaminated than BM samples taken immediately after each mobilization course (median difference 2.68 logs; range 1.7 to 4.6) (p < 0.0001). 2) LTC of PBPC harvests has only minimal variation among different leukaphereses performed during the same mobilization course (median difference 0.45 logs; range 0.22 to 1.2). 3) No difference was observed among PBPC and BM samples obtained after the late mobilization course as compared to the early mobilization course (median reduction 0.21 logs; range -0.39 to 1.3) (p = 0.84). 4) In PBPC but not in BM samples, there is a clear overestimation of the percentage of plasma cells when flow cytometric evaluation of CD38(bright) cells is compared to real-time PCR results. This suggests that in PBPC, most CD38(bright) cells do not belong to the neoplastic clone. CONCLUSIONS: Real-time PCR using the IgH rearrangement proved an effective tool for monitoring LTC in stem cell harvests from MM patients. The smaller LTC of PBPC harvests supports the role of PBPC as stem cell rescue for MM patients compared to BM cells.     

Comparison of myeloma cell contamination of bone marrow and peripheral blood stem cell harvests

It could be speculated for patients with myeloma and other lymphoproliferative disorders that peripheral blood stem cells may be preferable to bone marrow for autologous transplantation because they may be less contaminated by neoplastic cells. To test this possibility, the immunoglobulin heavy chain gene rearrangement and limiting dilution polymerase chain reaction were used to sensitively quantify myeloma cells in bone marrow and peripheral blood stem cell collections, taken at a similar time, from eight patients with multiple myeloma. Levels of residual disease in the peripheral blood stem cell harvests were variable and did not reflect the tumour burden in the marrow. Peripheral blood stem cells contained 1.7 to 23 700-fold fewer myeloma cells compared with the bone marrow and would have resulted in reinfusion of 0.08 to 59 480-fold fewer myeloma cells based on total reinfused CFU-GM and 0.24 to 24 700-fold fewer myeloma cells based on total reinfused nucleated cells. Assuming that the proportion of clonogenic myeloma cells is equivalent, peripheral blood stem cells may be better than bone marrow as a source of haemopoietic stem cells for transplantation in multiple myeloma. The clinical follow-up suggested that patients transplanted with peripheral blood stem cells containing a low number of myeloma cells had better disease control than those transplanted with peripheral blood stem cells containing a high number.     

Contamination of peripheral blood stem cell harvests by circulating neuroblastoma cells

Peripheral blood stem cells (PBSC) are being used as one alternative to autologous marrow rescue for patients with neuroblastoma and other solid malignancies. Some physicians prefer use of PBSC because less risk of tumor contamination is believed to exist. This hypothesis was evaluated by immunocytologic analysis of blood samples and concurrently drawn bone marrow (BM) samples and of PBSC harvests obtained from 31 patients with disseminated neuroblastoma. We found circulating neoplastic cells in 75% of specimens analyzed at diagnosis, in 36% during therapy, and in 14% of PBSC harvests. Tumor cells in blood obtained during therapy did not appear until 3 months after the time of diagnosis. Clearance of circulating neuroblastoma cells was documented after two courses of induction chemotherapy. Six of 13 patients with minimal or no BM disease had positive blood specimens. We conclude that substantial risk of tumor contamination of PB harvests exists and recommend that induction chemotherapy be administered before hematopoietic progenitor cells are collected from blood.     

Detection of rotaviruses in the day care environment by reverse transcriptase polymerase chain reaction.

Group A rotavirus is an important cause of morbidity among infants and toddlers in day care centers. Transmission by the fecal-oral route is well established, but fomites and environmental surfaces may also play an important role in transmission. A highly sensitive polymerase chain reaction (PCR) assay was used to detect rotavirus RNA in day care environments. Areas sampled included floors, diaper change areas, toy balls, and other surfaces. In two centers undergoing outbreaks of rotavirus, 7 (39%) of 18 toy balls had detectable rotavirus as did 8 (21%) of 39 swabs from environmental surfaces. By comparison, only 1 (5%) of 21 toy balls and 1 (2%) of 44 environmental surface swabs had detectable rotavirus in centers without rotavirus outbreaks (P = .0001). Thus, rotaviruses are highly prevalent in day care centers during outbreaks of diarrhea. The monitoring of environments by sensitive nucleic acid amplification techniques may lead to strategies for the diminution of disease transmission within the day care environment.     

茎环结构的逆转录实时定量PCR对Huh7细胞microRNAs的检测

目的 建立一种简便检测人肝癌细胞株Huh7细胞microRNAs的实时定量PCR方法,并探讨其敏感性和特异性.方法 提取Huh7细胞总RNA,通过microRNA芯片检测出3个分别代表高、中、低拷贝的microRNA 122、24、146a,并用Northern blot证实.然后分别采用poly(A)加尾和茎环结构的逆转录实时定量PCR方法检测上述3个microRNAs.用Quantity One软件和7500系统软件进行分析.结果 Huh7细胞芯片microRNA 122、24、146a的信号强度分别为2201.49、410.20、4.70,Northern blot证实相对表达量分别为0.0383、0.0249、0.0001.poly(A)加尾逆转录实时定量PCR方法只能检测出microRNA 122,而茎环结构的逆转录实时定量PCR方法对microRNA 122、24,146a均能检测出,其相对于U6平均dCt值分别为2.5、5.8、12.1,与MicroRNA芯片和Northern blot结果一致.结论 应用茎环结构的逆转录实时定量PCR方法能够特异、敏感地检测出Huh7细胞高、中、低拷贝的microRNAs.     

巢式聚合酶链反应检测慢性粒细胞白血病bcr／abl融合基因

目的 探讨Ph染色体和bcr/abl融合基因在慢性粒细胞白血病（CGL）的发病机制、诊断、治疗、预后判断的价值。方法 对46例CGL患者作巢式逆转录聚合酶链反应（RT-PCR）检测bcr/abl融合基因,同时对其中28例作细胞遗传学检查。结果 46例CGL患者中,44例bcr/abl融合基因阳性,阳性率为95.7%;28例CGL患者作细胞遗传学检查。26例Ph染色体阳性,阳性率为92.9%;2例Ph染色体阴性的CGL患者,用RT-PCR检测出ber/abl融合基因。结论 巢式RT-PCR是一种快速、敏感而准确的检测方法,可以为部分Ph染色体阴性的CGL患者提供分子生物学的诊断依据。