流式细胞术检测Cyclin D1和BCL-2在B细胞淋巴瘤诊断中的意义
本文关键词:流式细胞术检测Cyclin D1和BCL-2在B细胞淋巴瘤诊断中的意义,由笔耕文化传播整理发布。
目的:探索流式细胞术检测Cyclin D1和BCL-2在B细胞淋巴瘤诊断中的方法、可行性及临床意义。方法:1)收集我院血液内科收治确诊的血液病患者45例,其中男22例,女23例,年龄8-84岁,其中HL3例,CLL15例,DLBCL11例,MCL3例,MZL2例,FL2例,NK-T细胞淋巴瘤4例,,T细胞淋巴瘤5例,收治后详细记录治疗经过,并进行后期随访和疗效评定。根据2008年WHO淋巴瘤分类标准,将淋巴瘤患者分类。根据张之南《血液病诊断与疗效指南》疗效判定,将接受治疗的患者分为完全缓解(CR),不确定的完全缓解(CRu),部分缓解(PR),疾病稳定(SD),复发(适用于取得CR/Cru者)和疾病进展(PD)。2)按照年龄、性别相匹配的原则,选取在我院接受常规体检且各项检验指标均在正常范围内的健康体检者25名作为健康对照组。3)选取病理科已确诊的Cyclin D1或BCL-2阳性病例组为阳性对照组(8例),病理已确诊的Cyclin D1或BCL-2阴性的病例组为阴性对照组(7例)。采用四色直接免疫荧光素法标记细胞抗原,包括细胞膜表面和胞核抗原,如:CD5、CD19、CD20、CD23、CD10、 Cyclin D1、BCL-2等,以CD19+细胞设门,多参数流式细胞术检测各亚型淋巴瘤患者外周血、骨髓或淋巴结Cyclin D1及BCL-2表达水平。所有数据用SPSS13.0统计软件处理分析;各计量资料采用均数±标准差((?)±s)表示,两组数值符合正态分布及方差齐性采用t检验,否则采用非参数检验;多组之间的比较,数值符合正态分布和方差齐性采用方差分析,否则采用非参数检验,P<0.05为差异具有统计学意义。比较两种检测方法的差异采用配对卡方检验,P<0.05为差异具有统计学意义。结果1)本课题对25例正常成人志愿者Cyclin D1MFI及BCL-2MFI进行了分析后得出x±s值及95%可信区间,从而得出流式检测正常参考值范围,并由此得出判断Cyclin D1的阳性阈值为Cyclin D1MFI>0.5;同样,判断BCL-2的阳性阈值为BCL-2MFI>2.7。2)根据FCM确定的Cyclin D1及BCL-2阴阳性诊断标准,单纯与病理免疫组化结果进行对比发现,FCM检测MCL患者的Cyclin D1灵敏度及特异性为100%,检测FL患者的BCL-2灵敏度及特异性也为100%,且FCM检测Cyclin D1及BCL-2结合免疫表型等对病理不能分类淋巴瘤明确了亚型5例。3)比较健康对照组与全体B细胞淋巴瘤患者组外周血Cyclin D1和BCL-2MFI显示:B细胞淋巴瘤患者Cyclin D1(1.824±0.312)和BCL-2(4.259±0.541)的表达水平较正常对照组(分别为0.356±0.159,1.938±0.324)明显升高,两者均具有统计学显著性差异(p<0.0001),4)不同亚型淋巴瘤患者外周血Cyclin D1MFI的表达有差异,霍奇金淋巴瘤患者Cyclin D1MFI(0.386±0.112)明显低于非霍奇金淋巴瘤患者(1.623±1.987)(p<0.001),而在非霍奇金淋巴瘤患者中,套细胞淋巴瘤患者CyclinD1阳性最高(100%),其余亚型均为阴性;不同亚型淋巴瘤患者外周血BCL-2的表达情况有差异,霍奇金淋巴瘤患者BCL-2(2.045±0.877)明显低于非霍奇金淋巴瘤(4.045±0.499)患者(p<0.001),在非霍奇金淋巴瘤中,T细胞淋巴瘤均为阴性,FL、MCL表达阳性率最高(100%),其次为MZL(50%)、CLL(30%)、DLBCL(45%)。5)在Cyclin D1或BCL-2阳性的淋巴瘤患者中,对治疗后临床判断达部分缓解以上的患者进行流式细胞仪检测,发现Cyclin D1(治疗前3.099±0.349;治疗后1.008±0.279)或BCL-2(治疗前7.814±1.030;治疗后3.131±0.522)的平均荧光强度均有显著降低(p<0.001)。结论:1)流式细胞术检测淋巴细胞中Cyclin D1及BCL-2的表达方法简便可行;2)建立流式细胞术检测淋巴细胞中Cyclin D1及BCL-2的参考值;3)FCM检测CyclinD1及BCL-2对B细胞淋巴瘤的诊断分型有意义,具有很好的灵敏度和特异性;4)淋巴瘤患者治疗后Cyclin D1或BCL-2表达下调,其表达变化与治疗效果有关,可以作为临床观察治疗效果的指标之一。
Objective: To explore the method of detecting Cyclin D1and BCL-2by flowcytometry, as well as feasibility and significance.Methods:1) Patients with hematologic diseases in final diagnosis werecollected and recorded with45cases in detail,22were male,23female,age8-84, including HL3cases, CLL15cases, DLBCL11cases, MCL3cases,MZL2cases,FL2cases, NK-T cell lymphoma4cases, T cell lymphoma5cases,including treatment, follow-up and evaluation of curative effect.According to the2008WHO lymphoma classification criteria, patients wereclassified. According to Zhang Zhinan 《blood disease diagnosis andtreatment guide》 to judge the curative effect, treated patients weredivided into complete remission (CR), does not determine the completeremission (CRu), partial remission (PR), stable disease (SD),recurrence (for CR/Cru) and disease progression (PD).2)Accordingto the age and gender matching principle, selected routine physicalexamination and all tests were within the normal range in20healthy personsas a healthy control group.3)Select the pathology confirmed Cyclin D1or BCL-2positive cases as positive control group (8cases), select thepathology confirmed Cyclin D1or BCL-2negative cases as negative controlgroup (7cases). Four color monoclonal antibodies directly labeled withimmunofluorescence were used to analyze the surface and cytoplasma antigens,such as CD5, CD19, CD23, CD20, Cyclin D1, BCL-2and so on, CD19+cells wereset as door, we used multi-parameter flow cytometry to investigate peripheral blood Cyclin D1and BCL-2subsets in lymphoma patients.All datawas analyzed by statistical software SPSS13.0; the measurement data wasexpressed as mean±standard deviation ((?)±s). We used t test when twosets of values in line with normal distribution and homogeneity of variance,or using non-parametric test; we used analysis of variance when multiplesets of comparisons between values in line with normal distribution andhomogeneity of variance, or we used non-parametric tests,P<0.05meant thedifference was statistically significant. We used paired chi-square testto compare differences in detection methods, P<0.05meant the differencewas statistically significant.Results:1), The subject of the25normal adult volunteers Cyclin D1MFIwere analysed x±s values and95%confidence interval, so that the CyclinD1MFI>0.5is positive, thus establishing the Cyclin D1diagnosticcriterion; BCL-2MFI were also analysised x±s values and95%confidenceinterval in25cases of normal adult volunteers, so that the BCL-2MFI>2.7is positive, thus establishing the BCL-2diagnostic criterion;2),Cyclin D1and BCL-2diagnosed by FCM criteria of positive and negative,simplely compared with immunohistochemistry results of pathological in MCLpatients, FCM analysis of Cyclin D1sensitivity and specificity of100%,the detection of patients with FL BCL-2sensitivity and the specificityis100%.FCM detection of Cyclin D1and BCL-2with the combination of immunephenotype clear5cases of pathology can not be classified by lymphomasubtype.3) Compared with the healthy control group, all the B-cell lymphomain patients with peripheral blood of Cyclin D1(1.824±0.312) and BCL-2MFI (4.259±0.541) was significantly much more increased expressionlevels than normal control group (0.356±0.159,1.938±0.324), bothstatistically significant difference (p <0.0001).4) Different subtypes of lymphoma in peripheral blood of patients express differences of CyclinD1MFI.In patients with Hodgkin’s lymphoma of Cyclin D1MFI (0.386±0.112)was significantly lower than non-Hodgkin’s lymphoma patients (1.623±1.987)(p <0.001).In non-Hodgkin’s lymphoma patients, mantle cell lymphomapatients with Cyclin D1-positive (100%), and the remaining subtypes werenegative; different subtypes of lymphoma in peripheral blood of patientswith BCL-2expression differences, in patients with Hodgkin’s lymphomaBCL-2(2.045±0.877) was significantly lower than non-Hodgkin’s lymphoma(4.045±0.499) patients (p <0.001), in non-Hodgkin’s lymphoma, T celllymphoma express lowest, positive rate of MCL and FL was the highest (100%),followed by MZL (50%), CLL (30%), DLBCL (45%).5) lymphoma patients withCyclin D1or BCL-2-positive, more than patients detected the clinicaljudgment of partial remission after treatment by flow cytometry and foundthat Cyclin D1(before treatment3.099±0.349; after treatment,1.008±0.279)or expression of BCL-2(before treatment7.814±1.030; aftertreatment,3.131±0.522) mean fluorescence intensity are significantlylower (p <0.001).Conclusion:1, the method of flow cytometry for detection of Cyclin D1andBCL-2is feasible;2, Establishment of Cyclin D1and BCL-2reference valuein flow cytometry;3, FCM analysis of cyclin D1and BCL-2Diagnosticclassification of B-cell lymphoma meaningful, with good sensitivity andspecificity.4. Cyclin D1or BCL-2of Lymphoma patients after treatmentdownregulation, and its expression change with treatment, can be used asa clinical observation on evaluate the treatment.
流式细胞术检测Cyclin D1和BCL-2在B细胞淋巴瘤诊断中的意义
缩略词表5-6摘要6-8Abstract8-10引言11-13材料方法13-18统计学方法18-19结果19-28讨论28-34结论34-35参考文献35-38附录38-40致谢40-41综述41-49 参考文献46-49
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本文关键词:流式细胞术检测Cyclin D1和BCL-2在B细胞淋巴瘤诊断中的意义,由笔耕文化传播整理发布。
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