【作者】 黄竞；

【导师】 杜欣；

【作者基本信息】 汕头大学 ， 临床医学， 2010， 硕士

【摘要】 背景和目的:骨髓增生异常综合征(myelodysplastic syndome,MDS)是一组异质性恶性克隆性造血干/祖细胞疾病。目前MDS诊断主要依赖于综合患者临床症状、血象、外周血及骨髓涂片和骨髓活检形态学以及细胞遗传学等的结果,然而MDS临床表现可以多种多样,形态学变化受主观因素干扰大,而且只有约40%～50%患者检出细胞遗传学异常,因此有时难以确定MDS诊断。近年来,流式细胞术(flow cytometry,FCM)已经广泛应用于血液系统肿瘤(如白血病和淋巴瘤)的免疫表型检测,为诊断、分型、治疗和预后提供客观的辅助依据。MDS作为血液系统肿瘤的一种,本研究利用多参数流式细胞术了解MDS患者骨髓细胞免疫表型特征,并通过初步构建流式积分系统,探讨其在诊断、鉴别诊断及预后中的临床意义。方法:收集41例MDS患者和30例非MDS患者骨髓标本,应用多参数流式细胞术,采取一系列的抗体组合和CD45/SSC设门策略,对骨髓细胞表面抗原表达及原始细胞数进行检测,采用两独立样本t检验和单方向方差分析比较MDS与non-MDS及MDS亚型间原始细胞数、各抗原表达情况及流式积分,分析其免疫表型特点,并据此构建流式积分系统,同时结合血液学、形态学及细胞遗传学等检查结果,采用ROC曲线分析和Spearman相关分析评价该积分系统的优缺点。用SPSS13.0统计软件对数据进行统计分析。结果:MDS与non-MDS相比,原始细胞、单核细胞、淋巴细胞及有核红细胞群比例均显著升高(P值分别为:0.000、0.000、0.001及0.029),而成熟粒细胞群比例显著下降(P=0.021);原始细胞群比例在RCMD、RAEB-I和RAEB-II亚型间逐渐增高,在RCMD与RAEB-I、RCMD与RAEB-II及RAEB-I与RAEB-II亚型间有显著差异(P值分别为:0.020、0.000及0.010),余细胞群比例亚型间无显著差异(P值均>0.05)。CD34、CD123和CD133在原始细胞群表达显著增高(P值分别为:0.000、0.003和0.000),CD38、HLA-DR、CD13、CD33、CD7和CD117表达均无显著差异(P值分别为:0.223、0.306、0.536、0.273、0.577和0.368);CD34在RCMD、RAEB-I和RAEB-II亚型间表达逐渐升高,在RCMD与RAEB-I、RCMD与RAEB-II及RAEB-I与RAEB-II亚型间表达有显著差异(P值分别为:0.022、0.000和0.000), CD133、CD117、CD13和HLA-DR在RAEB-II表达比RCMD显著增高(P值分别为0.001、0.000、0.001和0.047);其余抗原亚型间比较均无显著差异(P>0.05)。CD10、CD11b、CD15、CD16和CD66d在成熟粒细胞群表达显著增高(P值分别为:0.012、0.027、0.016、0.005和0.024);CD33、CD13、HLA-DR、CD56及CD117表达均无显著差异(P值分别为0.058、0.337、0.649、0.096和0.081);CD10在RAEB-II型表达显著低于RAEB-I型(P=0.001);CD13和HLA-DR在RCMD型表达显著低于RAEB-I和RAEB-II型(P分别为0.013、0.005和0.018、0.003);其余抗原亚型间比较均无显著差异(P>0.05)。CD15、CD33和CD64在单核细胞群表达均显著升高(P值分别为:0.001、0.007和0.000);CD7、HLA-DR、CD14、CD13、CD61和CD56表达均无显著差异(P值分别为:0.075、0.066、0.408、0.153、0.137和0.322);所有抗原亚型间比较均无显著差异(P>0.05)。在淋巴细胞群CD25+CD8+、CD7和CD8表达显著升高(P值分别为:0.000、0.002和0.004),CD5和CD19表达显著降低(P值分别为0.011和0.000);CD25+CD4+、CD4、CD25和CD56表达均无显著差异(P值分别为:0.524、0.508、0.942和0.978);CD5在RCMD和RAEB-II的表达均显著高于RAEB-I(P值分别为:0.006和0.037)。其余抗原亚型间比较均无显著差异(P>0.05)。GPA+CD71+、CD71、CD105和GPA在有核红细胞群表达均显著升高(P值分别为:0.026、0.020、0.033和0.024),GPA+CD105+和CD71+CD105+表达均无显著差异(P值分别为:0.050和0.938)。所有抗原亚型间比较均无显著差异(P>0.05)。在41例MDS患者中有7例检出复杂核型异常,且均为RAEB-II患者,占MDS患者的17%,占RAEB-II患者的47%;核型异常与IPSS积分呈显著正相关(r=0.703,P=0.000)。通过骨髓原始细胞数和各细胞群不同抗原表达情况初步构建流式积分系统,该积分系统用于MDS诊断有较高符合率(P=0.000),95%可信区间为(0.921,0.997),灵敏性和特异性分别为:87.8%和86.7%,MDS组比non-MDS组流式积分显著升高(P=0.000),RCMD、RAEB-I和RAEB-II亚型间,随着疾病进展,流式积分也显著升高(P=0.000)。流式积分与MDS分型、染色体核型、IPSS和WPSS呈显著正相关(r值分别为:0.656、0.410、0.791和0.425,P值分别为:0.000、0.008、0.000和0.006)。骨髓各细胞群流式积分与相应外周血细胞绝对值计数无显著相关性(P值均>0.05)。结论:本研究应用多参数流式细胞术分析了MDS患者骨髓细胞各细胞亚群不同抗原表达情况,能够对MDS免疫表型特征进行较全面了解,并通过初步构建流式积分系统,综合评价MDS免疫表型异常特点,为临床MDS诊断、分型、治疗和预后评价提供一种实用和客观的辅助方法。更多还原

【Abstract】 Background and Objective: Myelodysplastic syndromes (MDS) are a group of clonal stem/progenitor cells diseases.Currently, a combination of morphology, to detect multilineage dysplasia in the bone marrow and peripheral blood, and bone marrow biopsy, to detect the proliferation of bone marrow and cytogenetics, to detect characteristic clonal abnormalities are the main methods in establishing a diagnosis of MDS. However, the clinical manifestations are various and the various of morphology depend upon the experiences of haematologists, and only approximately 40%-50% patients were detected with abnormal cytogenetics. So, to make the diagnosis of MDS is difficulty sometimes. Recently, flow cytometry (FCM) is widely used to detect the immunophenotype of malignant hematological disease (such as leukemia and lymphoma), which can provide objective adjuvant evidence for diagnosis, classification, therapy and prognosis. MDS is a heterogeneous group of myeloid neoplasms. The aim of this study is to use multiparametric flow cytometry to analysis the immunophenotypic characteristic of MDS patients and establish the flow cytometry scoring system, explore the clinical significance of this scoring system for diagnosis, classification and prognosis.Methods: 41 bone marrow samples were collected from MDS patients and 30 bone marrow samples were obtained from non-MDS patients, applying multiparametric flow cytometry and adopting an extensive panel of monoclonal antibodies and CD45/SSC gating strategy, to detect the expressions of antigens of bone marrow cells and the quantity of blast. Comparing the quantity of blast, the expressions of antigens and the FCM scores between MDS and non-MDS with Independent-Samples T Test and among MDS subtypes with One-Way ANOVA, analyzing the feature of immunophenotyping and establishing FCM scoring system as well, combining the results of hematology, morphology, cytogenetics and other methods to evaluate this FCM scoring system with ROC Curve and Spearman rank order correlation analysis.Results: Comparing with the non-MDS group, the proportion of blast, monocyte, lymphocyte and nucleated erythrocyte were significantly increased in the MDS group (P=0.000, P=0.000, P=0.001, P=0.029, respectively), but percentage of granulocyte was significantly decreased (P=0.021); The proportion of blast was increased grandually in RCMD, RAEB-I and RAEB-II and there were significant differences in RCMD vs RAEB–I , RCMD vs RAEB–II and RAEB–I vs RAEB-II(P=0.020, P=0.000, P=0.010, respectively). Other cell populations were no significant differences(P>0.05).The expressions of CD34, CD123 and CD133 on blast cells were much higher than non-MDS group (P=0.000, P=0.003, P=0.000, respectively), the expressions of CD38, HLA–DR, CD13, CD33, CD7 and CD117 were no significant differences (P=0.223, P=0.306, P=0.536, P=0.273, P=0.577 and P=0.368, respectively). Between RCMD vs RAEB-I, RCMD vs RAEB-II and RAEB-I vs RAEB-II, the expressions of CD34 were significantly increased (P=0.022, P=0.000, and P=0.000, respectively), Meanwhile the expressions of CD133, CD117, CD13 and HLA-DR on blast of RAEB–II were significantly increased than RCMD(P=0.001, P=0.000, P=0.001, P=0.047, respectively). Other expressions of antigens were no significantly differences among the subtypes of MDS(P>0.05).The expressions of CD10, CD11b, CD15, CD16 and CD66d on granulocytes were much higher than non-MDS group(P=0.012, P=0.027, P=0.016, P=0.005, P=0.024, respectively); there were no significantly differences of the expressions of CD33, CD13, HLA-DR, CD56 and CD117(P=0.058, P=0.337, P=0.649, P=0.096, P=0.081, respectively); the expression of CD10 in RAEB-II was decreased significantly than RAEB-I(P=0.001); the expressions of CD13 and HLA-DR in RCMD were significantly decreased than RAEB-I and RAEB-II(P=0.013, P=0.005 and P=0.018, P=0.003, respectively). Other expressions of antigens were no significantly differences among the subtypes of MDS(P>0.05).The expressions of CD15, CD33 and CD64 on monocytes were significantly higher than non-MDS group(P=0.001, P=0.007, P=0.000, respectively), however, there were no significantly differences of the expressions of CD7, HLA-DR, CD14, CD13, CD61 and CD56 on monocytes(P=0.075, P=0.066, P=0.408, P=0.153, P=0.137, P=0.322, respectively). Expressions of all antigens were no significantly differences among the subtypes of MDS(P>0.05).The expressions of CD25+CD8+, CD7 and CD8 on lymphocytes were significantly increased than non-MDS group(P=0.000, P=0.002, P=0.004, respectively), while the expressions of CD5 and CD19 were significantly decreased(P=0.011, P=0.000, respectively); the expressions of CD25+CD4+, CD4, CD25 and CD56 were no significantly differences(P=0.524, P=0.508, P=0.942, P=0.978, respectively); the expressions of CD5 in RCMD and RAEB-II was significantly increased than RAEB-I(P=0.006, P=0.037, respectively). Other expressions of antigens were no significantly differences among the subtypes of MDS(P>0.05).The expressions of GPA+CD71+, CD71, CD105 and GPA on nucleated erythrocytes were significantly increased than non-MDS group(P=0.026, P=0.020, P=0.033, and P=0.024, respectively), while the expressions of GPA+CD105+ and CD71+CD105+ were no significantly(P=0.050, P=0.938, respectively). Expressions of all antigens were no significantly differences among the subtypes of MDS(P>0.05).7 out of 41 MDS patients had complicate chromosome karyotype and all these 7 patients were RAEB-II, account for 17% of MDS patients and 47% of RAEB-II patients. There was good positive correlation between abnormal chromosome karyotype and IPSS scores(r=0.703, P=0.000).Base on the proportion of blast and the expressions of antigens on cell populations, we establish this BM FCM scoring system preliminarily. Using this scoring system for MDS diagnosis had a good coincidence rate(P=0.000), 95% confidence interval was 0.921～0.997,sensitivity and specificity were 87.8% and 86.7%. The BM FCM scores of MDS group was significantly higher than non-MDS group(P=0.000),as disease progression, the score was increased(P=0.000). Meanwhile, there were good positive correlations between FCM scores and MDS classification, karyotype, IPSS scores and WPSS scores respectively(r=0.656, r=0.410, r=0.791 and 0.425, P=0.000, P=0.008, P=0.000 and 0.006 respectively). However, there were no significantly differences between FCM scores of BM cell populations and absolute counts of PB(P>0.05).Conclusions: Using multiparametric flow cytometry to analysis the expressions of antigens of bone marrow cells from MDS patients could help us know much more about the immunophenotypic characteristics of MDS. Through synthetical evaluation the immunophenotypic abnormalities of MDS base on our preliminary study on establishing this BM FCM score system, we may could provide a practical, objective, reliable and adjuvant approach.更多还原