骨髓增生异常综合征患者骨髓细胞免疫表型特点分析

目的:探讨流式细胞术(FCM)检测骨髓增生异常综合征(MDS)患者骨髓细胞免疫表型在MDS诊断及预后中的价值。方法:用FCM分析44例初诊MDS患者骨髓细胞免疫表型,分析MDS患者免疫表型的表达及分布情况;建立流式积分系统(FCSS)分析其与WHO分型及国际预后积分系统(IPSS)、WHO分型预后积分系统(WPSS)积分的相关性。结果:MDS患者骨髓细胞免疫表型存在多种异常:①骨髓原始细胞表达成熟抗原CD11b、CD15,及淋巴细胞相关抗原CD2、CD5、CD7、CD19或CD56,原始、幼稚细胞比例增高。②成熟粒细胞CD13/CD16、CD11b/CD16关系模式异常,表达CD56,中性粒细胞颗粒性减低。③单核细胞有CD56和CD34异常表达,单核细胞比例增高等。④红系Gly表达减弱,CD71表达减少,有核红细胞比例增高。⑤MDS患者骨髓细胞FCSS积分与WHO分型、IPSS积分、WPSS积分呈正相关。结论:MDS患者骨髓细胞存在多种免疫表型异常,FCM分析MDS患者骨髓细胞免疫表型异常可以为MDS的诊断和预后提供参考。     

骨髓增生异常综合征免疫表型分析

目的：探讨免疫表型测定在骨髓增生异常综合征（MDS）诊断及分型中的价值。方法：采用一组系列相关单克隆抗体和流式细胞术对19例MDS患者免疫表型进行检测，并对其中的10例进行了细胞遗传学检查。结果：MDS患者骨髓单个核细胞（MNC）CD13，CD33抗原表达率平均分别为36．69％和41．86％，而T淋巴系抗原CD3的表达平均仅为14．49％，且随着低危的难治性贫血（RA）向高危的难治笥贫血伴原始细胞增多（RAEB）或难治性贫血伴原始细胞增多－转变型（RAEB-t)的进展，较早期的髓系抗原CD13，CD33及干（祖）细胞抗原CD34的表达升高，并伴有T淋巴系抗原CD3的表达降低。10例进行了细胞遗传学检查的患者中，5例有染色体核型异常，染色体核型异常的患者与染色体核型正常的患者在抗原表达上存在区别。结论：对MDS患者进行免疫表型检查有助于MDS的诊断分型研究。     

低危骨髓增生异常综合征免疫表型分析

目的探讨低危骨髓增生异常综合征（MDS）分型诊断及鉴别诊断的临床意义。方法选用多种单克隆抗体,应用直接免疫荧光流式细胞术CD45/SSC设门提取骨髓的原始细胞对27例低危MDS患者进行免疫表型检测,并与对照组及再生障碍性贫血（AA）组进行比较分析。结果与对照组、AA组比较,低危MDS患者CD34＋表达明显增高（P均〈0.05）,各系免疫表型多重表达异常发生率增高。结论 MDS骨髓原始细胞免疫异常表型检测有利于MDS的诊断和鉴别诊断;流式细胞术可检测到MDS免疫表型的微小变化,比细胞遗传学检测更敏感。     

骨髓增生异常综合征的免疫表型和SURVIVIN、P15INK4B、TRF1基因表达

目的探讨骨髓增生异常综合征（MDS）特征性免疫表型,SURVIVIN、P15INK4B、TRF1基因表达程度,及其与临床病情进展的关系。方法抽取50例MDS患者骨髓细胞,用流式细胞学技术检测免疫表型;抽取单个核细胞,用RT-PCR方法检测SURVIVIN、P15INK4B、TRF1基因mRNA表达程度。结果与对照组相比,MDS患者骨髓细胞原始群CD7和CD34表达增多;粒细胞群CD34表达增多,CD13、CD33、CD11b表达下降;单核细胞表达CD117和CD34。与对照组相比,MDS患者骨髓单个核细胞SURVIVIN和TRF1基因mRNA表达增加,P15INK4B基因mR-NA表达下降（P均〈0.05）。从低危组到高危组,SURVIVIN和TRF1基因mRNA表达增加,P15INK4B基因mRNA表达下降（P均〈0.05）。结论 MDS免疫表型有其特征性,SURVIVIN、P15INK4B和TRF1基因表达可能与其发病进展有关。     

多参数流式细胞术分析难治性贫血伴原始细胞增多患者骨髓细胞的免疫表型特点

目的探讨骨髓增生异常综合征（MDS）亚型中难治性贫血伴原始细胞增多（RAEB）患者骨髓细胞免疫表型特点及其在诊断、鉴别诊断及预后中的临床意义。方法应用多参数流式细胞术,对35例RAEB患者和20例非MDS患者骨髓细胞表面抗原表达进行检测。结果RAEB组与非MDS组相比,原始细胞比例明显升高（P〈0．01）,成熟粒细胞比例显著下降（P〈0．01）,而淋巴、单核及有核红细胞比例无显著差异（P〉0．05）;RAEB组CD34、CD7和CD56在原始细胞表达明显增高（P〈0．01）;CD19在淋巴细胞亚群表达降低（P〈0．05）,而CD,。表达升高（P〈0．05）,CD,表达无差异;HLA—DR、CD56及CD15^＋ CD11b^-在粒-单核细胞亚群表达明显升高（P〈0．01）,而CD10、CD15^＋ CD11b^-表达明显下降（P〈0．01）,CD13和CD33表达无差异;RAEB组CD34及CD7高表达者的生存期短于低表达者。结论多参数流式细胞术可用于检测RAEB骨髓细胞异常免疫表型特点,为临床对三系减少或者贫血患者的诊断、鉴别诊断及预后评价提供新方法。     

骨髓增生异常综合征的免疫表型分析

目的:探索骨髓增生异常综合征(MDS)的特征性免疫表型,评估流式细胞术(FCM)在其诊断中的价值。方法:采用FCM检测20例MDS患者及10例对照骨髓单个核细胞的免疫表型。结果:①MDS患者CD34+细胞比例明显增加,且高度MDS患者CD34+细胞比例高于低度患者,而低度MDS患者CD34+细胞比例与对照组相近。②MDS患者粒系SSC的平均荧光强度明显低于对照组。③MDS患者粒细胞CD13/CD16分布明显异常,且与形态学粒系病态造血无关。④MDS患者粒细胞CD56表达频率增高。⑤MDS患者红系CD71平均荧光强度低于对照组。⑥MDS患者红系CD71/GlyA分布可见明显异常,且与形态学红系病态造血无关。结论:FCM在MDS的诊断方面有着重要意义,可以作为重要的补充手段。     

24例大颗粒淋巴细胞白血病患者免疫表型和临床特征分析

目的：探讨大颗粒淋巴细胞白血病（LGLL）患者免疫表型与临床特征的关系。方法：收集24例LGLL患者流式细胞术免疫分型和临床资料,比较T／NK不同亚型间的差异。结果：在CD45／SSC点图上分析,24例LGLL患者骨髓／外周血均可见大颗粒淋巴细胞群。T-LGLL表达特异性T系抗原及大多数泛T抗原,均为CD8＋CD4-亚型,侵袭性,T-LGLL可见泛T抗原丢失,并伴T系抗原表达强度改变。NK—LGLL主要表达泛T标志CD2、CD7及CD56和CD11C,不表达T系特异性标志CD3、CD5,但在惰性和侵袭性病程中免疫表型无显著差异。T-LGLI,惰性与侵袭性患者组间比较,平均年龄、WBC计数、淋巴细胞比例、血红蛋白含量和B症状差异有显著意义（P〈0．05）;NK-LGLL惰性和侵袭性患者组间比较在年龄、血红蛋白含量、肝脾淋巴结肿大和B症状间有显著差异（P〈0．05）。结论：骨髓细胞或血细胞免疫表型分析可有助于诊断LGLL及T／NK亚型,而结合临床特征则有助于判断其侵袭性或惰性。     

骨髓增生异常综合征骨髓细胞免疫表型的探讨

用流式细胞术分析45例原发性骨髓增生异常综合征（MDS）患者和10例良性血液病病人的骨髓细胞免疫表型。结果：88．9％MDS表现二系或二系以上免疫标志异常，基中CD2、CD71、CD13、CD33、HLA－DR、CD34免疫标志变化最大，明显高于对照组（P＜0．01）。RA组：CD2、CD71（P＜0．05）、HLA－DR（P＜0．01）明显高于对照组；RAEB：除上述标志同RA外，髓系相关标志CD13、CD33（P＜0．01）和干／祖细胞标志CD34明显增高（P＜0．05）；RAEB－t组：髓系相关标志CD13（P＜0．05）、CD33（P＜0．01）和干／祖细胞标志CD34、HLA-DR（P＜0．01）表达率增高。CD15：HLADR与CD15：CD34比值在RAEB－t最低。     

细胞免疫表型分析诊断骨髓增生异常综合征临床意义

骨髓增生异常综合征(MDS)是克隆性造血干细胞疾病,其基本病理特征是无效造血,并表现为骨髓细胞病态造血和细胞表面标记的混杂表达。对MDS患者进行细胞免疫表型检测可以分析原始细胞群、粒细胞群和单核细胞群的表达异常,有助于MDS的诊断。由于对MDS细胞免疫表型分析是一个非常复杂的过程,规范与统一标准、使检测结果具有可比性和可信性是关键问题。     

骨髓增生异常综合征患者骨髓单个核细胞免疫表型特点及临床意义

目的:探讨骨髓增生异常综合征(MDS)患者骨髓单个核细胞免疫表型特点及临床意义。方法:回顾性分析48例MDS患者免疫表型,对比各亚型间免疫表型表达阳性率的高低,并评估其与IPSS积分的相关性。结果:48例MDS患者骨髓单个核细胞表达CD34、CD117、CD11b、CD33、CD13为主,RAEB1及BAEB2患者CD34、CD117及早期髓系抗原CD33、CD13阳性表达率较RCMD患者增高(P<0.05);骨髓原始细胞比例与CD34、CD117、CD13及CD33阳性表达率呈正相关;对这些患者进行IPSS积分系统评估,高危组CD34及CD117表达阳性率较中危1组升高(P<0.05),CD34表达阳性率与IPSS积分呈正相关。结论:MDS患者进行骨髓单个核细胞免疫表型检测对病情评估及预后判断有重要价值。     

The value of dot plot patterns and leukemia-associated phenotypes in AML diagnosis by multiparameter flow cytometry

The immunophenotypic features in patients with acute myeloid leukemia (AML) were investigated at diagnosis using a wide antibody panel including progenitor-associated, myeloid and lymphoid markers in quadruple combinations. Analyzed were bone marrow samples from 37 adult and pediatric patients for exact identification of AML blasts according their localization on CD45/SSC dot plots and aberrant immunophenotypes in various subtype of AML. We found the localization of AML blasts on CD45/SSC dot plots, which in combination with immunophenotype profile of blasts allow discrimination of several AML subtypes (M0-M2, M3, M4/M5 and other types). In 27/37 AML patients (73%) at least one leukemia-associated phenotype (LAP) was found, two or more aberrancies coexisted in more than a half of them (78%). Asynchronous expression was the most frequent type of LAP (77.8%, 21/27) followed by coexpression of lymphoid-associated antigens, which occurred in 18/27 (66.7%) patients. Presented study showed that leukemic cells of each AML patient had a unique antigenic profile and could be discriminated from their normal counterparts based on typical light scatter profiles and aberrant antigen expression that could further be used for detection of minimal residual disease.     

Immunophenotypic clustering of myelodysplastic syndromes

Myelodysplastic syndromes (MDSs) are heterogeneous diseases of bone marrow (BM) cell precursors for which immunophenotypic characterization is still considered irrelevant despite the accuracy and sensitivity of flow cytometry techniques. The aim of this study was to determine whether immunophenotypic abnormalities could be defined in MDSs and could correlate with the French-American-British classification and cytogenetics. Analysis was performed on 275 BM samples (207 MDS patients, 68 controls) and 25 control blood samples. Immunophenotyping was based on a primary gating of blast cells, monocytes, and granulocytes according to CD45 antigen expression and side scatter light diffraction. Immunophenotypic hierarchical clustering was performed to analyze the results. The data obtained show that (1) immunophenotypic clustering partly discriminates patients with refractory anemia with excess blasts/refractory anemia with excess blasts in transformation (RAEB/RAEB-T), chronic myelomonocytic leukemia (CMML), and refractory anemia/refractory anemia with ring sideroblasts (RA/RARS) for CD45(lo) blast cells and patients with RA/CMML, RARS, and RAEB/RAEB-T for CD45(hi)/side scatter(hi) (SS(hi)) granulocytes; (2) the most discriminating markers were CD16, CD34, CD36, CD38, CD71, and HLA-DR for blast cells and CD11b, CD13, CD33, CD36, CD38, CD71, and HLA-DR for CD45(hi)/SS(hi) granulocytes; (3) clusters related to CD34 expression were associated with high levels of blast cells on BM smear; (4) clusters related to high levels of CD36 expression on CD45(lo) blast cells and CD45(hi)/SS(hi) granulocytes were associated with a poor International Prognosis Scoring System score; and (5) high levels of CD71 expression on CD45(hi)/SS(hi) granulocytes were associated with the RARS category. These results show a close relationship between immunophenotypic abnormalities and BM dysplasia and suggest that flow cytometry could be a future tool for the characterization of MDSs.     

Flow cytometric scoring system (FCMSS) assisted diagnosis of myelodysplastic syndromes (MDS) and the biological significance of FCMSS‐based immunophenotypes

In the myelodysplastic syndromes (MDS), the haematopoietic cells show various levels of abnormal maturation and differentiation, which can be detected by flow cytometry. Testing the anomalies of stage‐ or lineage‐specific surface antigens in CD34⁺ blasts can distinguish MDS from non‐clonal cytopenic diseases, and also reflect the pathological characteristics of MDS as a class of clonal diseases for providing new clues to basic research. The present study established a flow cytometric scoring system (FCMSS) based on theproportion and antigenic co‐expression of CD34⁺ blasts. This FCMSS showed good sensitivity and specificity (77·8% and 100%) in the assisted diagnosis of low‐risk MDS without chromosome anomalies, ringed sideroblasts and excess marrow blasts. Moreover, we explored and reported different modes of abnormal expression of CD34⁺ blasts antigens in different disease stages and analyzed the biological significance of the immunotypes for the first time. We found expression of mature myeloid antigens and lymphoid antigens gradually decreased, and early functional antigens gradually increased from low‐risk MDS with normal karytype to low‐risk MDS with abnormal karyotype then to high‐risk MDS. The patients with higher FCM scores were generally accompanied with HLA‐DR15 allele or hypocellular marrow. Evolution of clones and immunological factors might have influence on expression of antigens in CD34⁺ blasts.     

Patterns of membrane CD45 isoform expression by leukaemic blasts and normal mature myeloid cells.

The membrane expression of CD45R isoforms by leukaemic blasts from 92 cases of acute myeloid (AML) and 12 cases of lymphoblastic leukaemia was analysed by single and two-colour flow cytometry. Compared to normal mature granulocytes, which invariably expressed UCHL1 (CD45RO) but not 2H4 (CD45RA), leukaemic myeloid blasts showed 2H4+ UCHL1- and 2H4- ++UCHL1- composite patterns of expression with the first of these phenotypes being associated in particular with the most immature AML subtype (M0). Similar analyses of blast cell fractions from monocytic AML variants, revealed wide variation in CD45R expression in cases of M4, whereas M5 leukaemias were typically 2H4+ ++UCHL1+ with minor 2H4- UCHL1- components. As most normal mature monocytes were also 2H4+ ++UCHL1+, this suggested that monocytic myeloid differentiation was primarily associated with the "acquisition" of UCHL1 and the development of 2H4/UCHL1 co-expressing cells. The expression of membrane CD45RA by leukaemic blasts is an abnormal characteristic and may be related to the maintenance of an undifferentiated state by malignant myeloid precursors.     

流式细胞术在难治性血细胞减少伴多系发育异常和再生障碍性贫血的鉴别诊断中的价值

目的 评价流式细胞术(FCM)在骨髓增生异常综合征(MDS)亚型难治性血细胞减少伴多系发育异常(RCMD)和再生障碍性贫血(AA)的鉴别诊断中的价值.方法 回顾性盲法分析168例RCMD和77例AA患者的骨髓流式数据,比较流式分析结果与诊断金标准进行诊断试验评价.结果 单个免疫表型的异常用于鉴别诊断RCMD和AA,特异度较高(75.3%～100.0%),但灵敏度较低(5.4%～50.0%).用提高灵敏度的平行试验重新进行评价,CD+34细胞≥1%、髓系原幼细胞≥3%、粒细胞CD117异常表达这3项指标联合髓系原幼细胞CD13表达缺失或粒细胞CD33表达增强,诊断的灵敏度有了较大提高(>62%),而特异度没有明显降低(>92%).根据上述评价得出的不同诊断价值,对8个原幼细胞和髓系异常抗原表达指标给以不同评分,以积分值≥1.5分判断为RCMD,<1.5分判断为AA,灵敏度和特异度均较高,在RCMD与AA组,分别为64.9%和93.5%.结论 单个免疫表型指标鉴别诊断RCMD和AA,特异度高,但灵敏度较低.CD+34细胞≥1%、髓系原幼细胞≥3%和粒细胞CD117异常表达这3项指标与其他几项组合可获得较好的灵敏度和特异度.积分法可以更精确地鉴别RCMD和AA.     

Immunophenotypic characterization of myelomonocytic cells in patients with myelodysplastic syndrome

Summary. Infections, an important determinating factor in the clinical course of myelodysplastic syndromes (MDS), result in activation of myelomonocytic cells. In this study we demonstrate activation-associated immunophenotypic changes of cell surface antigens on monocytes and granulocytes observed in two groups of MDS patients, one with low and another one with high clinical risk, and compared them to healthy individuals. Significantly changed expression of the complement receptors 1 (CD35) and 3 (CD11b), the Fcγ receptor I (CD64), the leucocyte-homing receptor (CD44) and the activation associated membrane proteins CD67 and M5 were found on monocytes and/or granulocytes of MDS patients. In low-risk MDS patients we observed activation-associated phenotypic changes only in monocytes, whereas in high-risk MDS patients, both monocytes and granulocytes showed such changes. Additionally, we performed respiratory burst experiments and observed an impaired response of monocytes and granulocytes derived from MDS patients. Despite the fact that all patients were free of infection by clinical criteria, cell surface phenotyping as well as the reduced respiratory burst capacity of myelomonocytic cells suggests in vivo preactivation of these cells.     

未经治疗的潜伏梅毒患者外周血淋巴细胞亚群的变化及意义

目的了解未经治疗的潜伏梅毒患者外周血淋巴细胞亚群的变化,并探讨其临床意义。方法对41例未经治疗的潜伏梅毒患者及29例健康对照者的抗凝全血,采用流式细胞仪检测相关淋巴细胞亚群（包括T淋巴细胞亚群、NK细胞及B细胞）,比较两组间的差异,并对其血清学滴度的变化进行相关性分析。结果潜伏梅毒患者CD4＋T淋巴细胞数、CD4＋/CD8＋比值均低于正常对照组（P〈0.01）,CD8＋T淋巴细胞数明显高于对照组（P〈0.01）;CD3＋、CD1＋9、CD1＋6CD5＋6与正常对照组相比无显著性差异（P〉0.05）;梅毒血清学滴度与CD4＋T淋巴细胞数量呈负相关。结论未经治疗的潜伏梅毒患者淋巴细胞亚群失衡,存在细胞免疫抑制。     

Flow cytometry in myelodysplastic syndrome: analysis of diagnostic utility using maturation pattern-based and quantitative approaches

Flow cytometry (FCM) is being increasingly evaluated for the diagnosis of myelodysplastic syndrome (MDS). We employed multiple FCM approaches to assess MDS. Five-color FCM, morphology blind, was done on bone marrow aspirates of 57 suspected MDS and 31 normal controls. Maturation pattern, quantitative FCM for low-grade MDS that awards FCM score, and expression of selected antigens on erythroid cells and CD34(+) blasts were evaluated. FCM results were correlated with clinical and laboratory workup. Patients (n = 57) included proven MDS (n = 14), suspected MDS (n = 13), and non-MDS (n = 30). By pattern-based approach, all proven cases were FCM positive. In suspected MDS, 11 (84.61 %) were positive including morphology-negative cases, and two (15.38 %) were intermediate. In non-MDS cases, 27 of 30 (90 %) were FCM negative, 2 of 30 (6.67 %) intermediate, and 1 of 30 (3.33 %) a hematinic-responsive case, positive. Quantitative parameters that characterized MDS included FCM score of >3, percentage CD34(+) B cells, and expression of CD11b, CD15, and CD56 on myeloblasts. CD71 MFI on CD235a(+) erythroblasts and CD38 MFI on myeloblasts were significantly lower in MDS. The former was present in FCM-intermediate suspected MDS but not FCM-intermediate non-MDS cases. Used in the overall clinical context, both maturation pattern recognition and quantitative approaches, the latter for low-grade MDS, are sensitive methods of diagnosing MDS, including cases negative by morphology and cytogenetics, especially if combined with evaluation of selected antigens, CD71 on CD235a(+) cells and CD38 on CD34(+) cells. The value of FCM in morphology-negative cases needs better definition of specificity through more extensive evaluation of secondary dyspoiesis.