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患者骨髓凋亡细胞类型及TRAIL表达水平研究

目的探讨骨髓增生异常综合征(MDS)患者骨髓细胞凋亡累及的细胞类型,以及其与肿瘤坏死因子相关凋亡诱导配体(TRAIL)表达水平的关系。方法用流式细胞术检测骨髓有核细胞总凋亡率、CD3+、CD3+4细胞凋亡率和TRAIL表达水平,运用CellQuest及ModFit软件对所测得结果进行数据分析。结果MDS患者骨髓有核细胞总凋亡率为(17.39±10.17)%,CD+3、CD3+4细胞凋亡率分别为(20.41±11.61)%、(21.32±12.17)%,TRAIL表达水平为(7.55±3.10)%,均显著高于正常对照组[(5.88±1.38)%、(5.47±1.65)%、(4.92±2.11)%、(1.26±0.46%)],P<0.05。早期MDS有核细胞总凋亡率为(21.95±9.44)%、TRAIL表达水平为(9.00±2.47)%,均高于进展期[(8.26±2.27)%、(4.65±2.04)%],P<0.05。骨髓有核细胞总凋亡率与骨髓原始细胞数呈负相关(r=-0.65,P<0.05),与TRAIL表达水平呈正相关(r=0.85,P<0.05)。结论MDS存在过度凋亡,凋亡累及早期造血细胞和淋巴细胞;TRAIL可能是引起凋亡增加的原因之一。     

骨髓增生异常综合征患者骨髓CD34+细胞亚群及细胞周期分析

目的 探讨骨髓增生异常综合征(MDS)患者骨髓CD34+细胞亚群及细胞周期分布异常的临床意义.方法 采用流式细胞术检测50例(17例低危、33例高危)MDS患者、8例MDS转化的急性髓系自血病(MDS-AML)及25例对照组原代骨髓CD34+CD38+、CD34+CD38-细胞亚群及G0/G1期、S期和G2/M期细胞比例.结果 高危和MDS-AML组骨髓CD34+细胞比例[(2.29±2.17)%、(18.69±17.47)%]明显高于对照组[(0.36±0.49)%,P＜0.05].低危、高危及MDS-AML组CD34+CD38+细胞相对比例[(86.09±7.79)%、(81.68±11.82)%、(82.88±2.60)%]显著低于对照组[(92.21±3.85)%,P＜0.05],而CD34+CD38-细胞比例[(13.91±7.79)%、(18.32±11.82)%、(17.13±2.60)%]显著高于对照组[(7.79±3.85)%,P＜0.05].MDS组CD34+CD38-细胞比例与国际预后积分系统(IPSS)(r=0.493,P=0.001)、WHO分型预后积分系统(WPSS)积分(r=0.586,P=0.000)均呈正相关.低危、高危及MDS-AML组骨髓单个核细胞(BMMNC)中G0/G1期细胞比例[(94.52±4.32)%,(96.07±3.88)%,(94.65±4.55)%]明显高于对照组[(88.94±7.30)%,P＜0.01],而3组S期[(4.63±3.34)%,(3.45±3.80)%,(5.12±4.55)%]和G2/M期[(0.84±1.52)%,(0.48±0.74)%,(0.22±0.34)%]细胞比例明显低于对照组[(9.06±6.50)%,(2.00±2.93)%,P值均＜0.05],3组S+G2/M期细胞比例明显高于对照组(P＜0.01).CD34+CD38-细胞比例≤12.0%的MDS患者治疗有效率高于CD34+CD38-细胞比例＞12.0%的患者,但差异无统计学意义.结论 MDS患者原代骨髓CD34+细胞亚群分化异常,BMMNC存在G1期阻滞现象,提示CD34+细胞亚群和细胞周期测定可能有助于MDS患者的辅助诊断以及疗效和预后判断.     

校正后的流式细胞术原始细胞计数在骨髓增生异常综合征分型诊断中的应用

目的:探讨校正后的流式细胞术原始细胞计数在骨髓增生异常综合征(MDS)分型诊断中的应用价值.方法:采用流式细胞术多重逻辑设门技术计算73例MDS患者骨髓中CD16dim/-粒细胞和CD34+细胞比例,根据CD16dim/-粒细胞比例对骨髓外周血稀释进行校正,再计算骨髓中校正后的CD34+细胞数;采用校正后的CD34+细...     

87例骨髓增生异常综合征多参数流式细胞术免疫表型分析

目的：了解骨髓增生异常综合征（MDS）的免疫表型特征。方法：利用CD45／SSC参数散点图设门方法,对87例MDS患者的骨髓幼稚细胞群、成熟粒细胞群和成熟单核细胞群细胞表面分化抗原进行分析。结果：MDS的异常免疫表型可以分为抗原跨系列表达、抗原跨阶段表达、成熟粒细胞抗原分化异常、抗原表达缺失或表达增强、成熟粒细胞或幼稚细胞群CD45／SSC表达异常。78例（90．0％）MDS患者可以检测到明确的异常抗原表达,74例（85．1％）存在≥2个免疫表型异常,异常免疫表型不仅存在于幼稚细胞群（70．0％）,也存在于成熟粒细胞群（81．6％）和单核细胞群（39．1％）．常见的异常免疫表型包括：成熟粒细胞群CD13／CD16分化异常（41．3％）;幼稚细胞群CD45表达异常（34．5％）;成熟粒细胞群跨系列表达CD38（29．9％）;幼稚细胞群CD34＋CD11b＋跨阶段表达异常（24．1％）;成熟粒细胞群SSC减低（11．5％）;成熟粒细胞群跨系列表达CD56（8％）;单核细胞群跨系列表达CD56（8％）。随着患者骨髓幼稚细胞比例增高,异常免疫表型数量也逐渐增多。结论：在大多数MDS患者骨髓细胞中可以检测到明确的异常免疫表型,在此基础上应用多参数流式细胞仪可以有助于MDS的诊断和预后判断。     

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。     

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。     

骨髓粒细胞与红细胞表面CD55 CD59缺失对血液疾病的诊断意义研究

目的探讨骨髓粒细胞、红细胞表面糖基化磷脂酰肌醇(GPI)锚定蛋白CD55、CD59缺失(CD55-、CD59-,也称PNH细胞)在血液系统疾病中的意义。方法采用流式细胞仪检测中山大学附属第一医院血液科2008年9月至2010年11月诊治的正常人、阵发性睡眠性血红蛋白尿、再生障碍性贫血(AA)、骨髓增生异常综合征(MDS)、急性髓细胞白血病(AML)、多发性骨髓瘤(MM)及营养不良性贫血患者外周血及骨髓中红细胞和粒细胞CD55、CD59缺失,并对结果进行分析。结果正常人骨髓粒细胞CD55-高于外周血(P<0.05);PNH患者骨髓红细胞CD55-、CD59-高于外周血(P<0.05);正常人、AA、MDS、AML、MM及营养不良性贫血各组间骨髓粒细胞CD55-表达无显著差异(P>0.05)。结论单一骨髓粒细胞CD55-表达升高特异性差。     

骨髓增生异常综合征和再生障碍性贫血患者T淋巴亚群及CD34阳性细胞的研究

目的：对骨髓增生异常综合征（MDS）和再生障碍性贫血（AA）患者的外周血T淋巴细胞亚群、NK细胞及骨髓CD34阳性细胞占单个核细胞（MNC）的比率进行测定,探讨两者细胞免疫异常及骨髓CD34阳性细胞与发病机制的关系。方法：用流式细胞术（FCM）对15例MDS患者,11例AA患者,12例正常对照组T淋巴细胞亚群、NK细胞及CD34阳性细胞占MNC的比率进行测定。结果：MDS患者外周血CD3＋T、CD4＋ T、CD8＋T淋巴细胞均较正常人组明显降低,CD4＋/CD8＋倒置。NK细胞CD16＋56也较正常人组低。AA患者CD4＋ T淋巴细胞稍低于正常人组,但CD8＋T淋巴细胞明显升高,表现为CD4＋/CD8＋也倒置。NK细胞正常。MDS患者CD34＋占骨髓MNC的比率明显高于正常组,AA患者骨髓MNC的比率低于正常组。结论：MDS和AA患者均存在免疫功能状态紊乱,二者的发病机制不同。CD34＋率的测定有助于二者的鉴别诊断,同时是判断MDS预后的简便、可靠的方法。     

AA和MDS患者骨髓CD+34细胞及G-CSFR的表达及意义

目的检测再生障碍性贫血(AA)和骨髓增生异常综合征(MDS)患者骨髓CD+34细胞占单个核细胞(MNC)的比率及其表面粒细胞集落刺激因子受体(G-CSFR)的表达率,以探讨二者可能的发病机制.方法用流式细胞术(FCM)检测13例AA、22例MDS及12例非血液病患者骨髓CD+34细胞占MNC的比率及其表面G-CSFR的表达率.结果 AA组与对照组、AA组与MDS组、MDS-难治性贫血(RA)组与MDS-难治性贫血伴原始细胞增多(RAEB)组的骨髓MNC中CD+34细胞比率比较有显著性差异(P＜0.05),但G-CSFR的表达率比较无显著性差异(P＞0.05).大多数重型AA(SAA)患者(3/4)及很少慢性AA(CAA)患者(1/9)的骨髓MNC中CD+34细胞比率小于0.1%.大多数G-CSFR表达率低(＜14%)的MDS患者(7/9)外周血中性粒细胞减少;中性粒细胞减少在G-CSFR表达率正常(14%～28.9%)的患者(1/6)很少见;G-CSFR表达率高(＞28.9%)的患者(3/7)也存在中性粒细胞减少.结论骨髓CD+34细胞检测有助于判断AA患者病情及MDS患者的预后,亦可用于鉴别AA和MDS.     

Apoptosis in relation to CD34 antigen expression in normal and myelodysplastic bone marrow

An increased bone marrow (BM) apoptosis is one of the mechanisms responsible for the ineffective hematopoiesis of myelodysplastic syndromes (MDS). It is controversial whether the excessive apoptosis in myelodysplasia predominantly involves the subset of progenitor cells or of maturing cells. We investigated the degree of apoptosis in MDS BM and its differences from normal marrow in relation to CD34 antigen expression. A double-labelling technique that combined the Tdt-mediated dUTP nick end labelling (TUNEL) method with immunocytochemistry for CD34 antigen was used on BM slides of 18 MDS patients and 11 controls. The apoptotic rate (AR) appeared significantly higher in CD34-negative than in CD34-positive cell subsets both in myelodysplastic and in normal BM. When MDS and normal CD34-negative cell populations were compared, a greater AR in MDS CD34-negative cells was found, while no statistical difference in AR resulted from the comparison between MDS and normal CD34-positive cell populations. Our results suggest that in myelodysplastic as well as in normal BM the apoptotic phenomenon predominantly involves the maturing cells. The increase in apoptotic levels which can be observed in myelodysplastic compared to normal BM seems to be mainly due to an increase in apoptosis in the differentiated cell population.     

Evaluation of apoptosis as a prognostic factor in myelodysplastic syndromes

The myelodysplastic syndromes (MDS) are a group of disorders characterized by peripheral pancytopenia despite normo- or hypercellular bone marrow. This is thought to be as a result of the apoptosis of haematopoietic bone marrow cells resulting in ineffective haematopoiesis. To clarify the relationship between prognosis and apoptosis and/or cell proliferation in the bone marrow, we studied 51 cases with MDS. Bone marrow biopsies were stained immunohistochemically for MIB-1 (marker for proliferating cells) and CD34 (marker for stem cells). Apoptosis was visualized by detection of DNA fragmentation using TdT incorporation of nucleotides on 3' ends of DNA (TUNEL technique) and expressed as the apoptotic rate. MDS patients included 32 with refractory anaemia (RA), one RA with ringed sideroblasts (RARS) patient, seven RA with excess of blasts (RAEB) patients, eight patients with RAEB in transformation (RAEB-t) and three patients with chronic myelomonocytic leukaemia (CMMoL). In addition, we also studied six cases with acute myeloid leukaemia (AML) arising from MDS (AML-MDS) and ten control subjects. Fatal pancytopenia was the cause of death in 19 out of 51 patients. The apoptotic rate was higher in MDS patients (5.5%) than in control subjects (0.6%) and AML-MDS patients (0.4%). The percentage of MIB-1 positive cells was higher in MDS and AML-MDS than in control. The percentage of CD34-positive cells was higher in AML-MDS, RAEB, RAEB-t and CMMoL patients than control subjects and RA patients. Our findings indicate the activation of both the proliferative and apoptotic rates in MDS. Poor prognosis correlated significantly with higher apoptotic rates, but not with percentages of MIB-1 and CD34-positive cells. Our results suggest that apoptosis might be a useful prognostic factor and inhibition of apoptotic mechanisms may induce leukaemic transformation in MDS.     

扩张型心肌病的心肌组织细胞凋亡的研究

目的：研究扩张型心肌病（DCM）的心肌细胞凋亡及其与心功能的关系。方法：DCM组21例,其心肌组织14例来自右室心内膜心肌活检（EMB）亚组,7例来自死后尸检（尸检组）;对照组为5例死于非心血管疾病的尸检心肌组织。用原位细胞凋亡检测心肌组织凋亡细胞,计算凋亡指数（AI）。结果（1）DCM组的AI明显高于正常组（P＜0．01）,EMB亚组的AI明显低于尸检亚组（P＜0．01,但明显高于对照组（P＜0．01）。（2）DCM组中,心胸比（HTR）＜0．6、左室舒张末期内（LVDd)＜65mm和左室射血分数（LVEF）≥40％的患者AI均分别明显于低于HTR≥0．6、LVDd ≥65mm和LVEF＜40％的患者（P值均＜0．01）,但仍分别显著高于对照组（P值均＜0．01）。结论：DCM存在明显心肌细胞凋亡并随心功能恶化而程度加重,提示凋亡是DCM的心肌细胞丢失和心功能不全的重要机制。     

白血病心脏浸润所致心肌凋亡研究

目的 :研究白血病细胞心脏浸润时心肌细胞的凋亡情况。方法 :建立小鼠白血病模型 （ L6 1 5） ,采用流式细胞术（ FCM）检测小鼠心肌细胞的细胞周期和细胞凋亡率。结果 :白血病组小鼠的心肌凋亡率明显高于正常对照组 （ P<0 .0 1）。结论 :白血病细胞心脏浸润可使心肌细胞凋亡率增高。     

Hematopoietic features and apoptosis in the bone marrow of severe Plasmodium falciparum-infected patients: preliminary study

The mechanism of anemia in severe falciparum malaria is still not completely understood. The purpose of this study was to determine whether apoptosis in the erythroid lineage causes anemia in falciparum malaria. Bone marrow aspirated from 8 severe falciparum malaria patients, 3 normal volunteers and 5 retrospective normal bone marrow smears were investigated. By light microscopic study, 5 of 8 hyperparasitemic patients had hypocellular bone marrows and erythroid hypoplasia, whereas the other 3 patients had normal cellularity. The mean myeloid : erythroid ratio of these 5 patients was significantly (p < or = 0.05) higher than normal. Apoptosis of bone marrow nucleated cells (BMNC) could be determined from the exposure of phosphatidylserine (PS) on the cell membrane but not DNA fragmentation (180-250 bp) or ultrastructural morphology. The percentages of apoptotic BMNC and apoptotic erythroid cells in bone marrow from each patient and controls varied from low to high, and were not associated with parasitemia. This study suggests that destruction of erythroid lineage, particularly through apoptosis regulation, cannot solely account for anemia in falciparum malaria.     

Oxidative DNA damage in CD34+ myelodysplastic cells is associated with intracellular redox changes and elevated plasma tumour necrosis factor-α concentration

Ineffective haemopoiesis in the myelodysplastic syndromes (MDS) is mediated, at least in part, by apoptosis, though the mechanisms of apoptotic induction are unclear. Tumour necrosis factor-α (TNF-α) promotes apoptosis via intracellular oxygen free radical production, oxidation of DNA and proteins, and is increasingly implicated in the pathogenesis of MDS. Using single-cell gel electrophoresis, we have identified oxidized pyrimidine nucleotides in the progenitor-enriched bone marrow CD34⁺ compartment from MDS patients (P = 0.039), which are absent in both CD34^? MDS cells (P = 0.53) and also CD34⁺ cells from normal subjects (P = 0.55). MDS CD34⁺ blood cells also showed oxidized pyrimidine nucleotides compared with CD34^? cells (P = 0.029). Within normal subjects no differences were seen between CD34⁺ and CD34^? bone marrow cell compartments. CD34⁺ bone marrow cell oxidized pyrimidines were strongly associated with elevated plasma TNF-α and low bone marrow mononuclear cell glutathione concentrations (5/6 patients) and the inverse relationship was also found (3/4 patients). This data implies a role for intracellular oxygen free radical production, perhaps mediated by TNF-α, in the pathogenesis of ineffective haemopoiesis in MDS and provides a rationale for the bone marrow stimulatory effects of antioxidants such as Amifostine in MDS.     

Deviation of type I and type II T cells and its negative effect on hematopoiesis in myelodysplastic syndrome

Immunemediated hematopoietic suppression has been considered as one of significant pathophysiological changes in less‐advanced myelodysplastic syndrome (MDS). To explore deviation of T cell subsets and its relationship to marrow cells apoptosis, measurement of helper‐T (Th)/cytotoxic‐T (Tc) subsets as well as the deviation situation within this two subsets (Th1/Th2 and Tc1/Tc2) in marrow was performed by flow cytometry from 39 MDS patients and 13 normal controls. Interferon (INF)‐γ and tumor necrosis factor (TNF)‐α in marrow serum was simultaneously detected by ELISA (enzyme‐linked immunosorbent assay). Furthermore, apoptosis rate of marrow cells was demonstrated by TUNEL (TdT‐mediated dUTP nick end labeling). Results showed that Th and Tc subsets were unevenly activated, both deviating to type I response, which was especially obvious in patients with RCMD (according to WHO classification) and in lower‐risk cases defined by International Prognosis Scoring System (IPSS). Level of INF‐γ/TNF‐α in MDS marrow serum was markedly elevated, and so did the apoptosis rate of marrow cells. Although type I deviation was observed both in Th and Tc subsets, just Th1 cell percentage showed positive correlation with level of INF‐γ/TNF‐α and apoptotic index of nucleated cells. In addition, cytokines level in marrow serum presented positive correlation to apoptosis. We then deduced that the increased Th1 cells in marrow may account for nucleated cells apoptosis in MDS through overproduced proapoptotic cytokines such as INF‐γ and TNF‐α. Our results suggested that type I deviation of T cell subsets may play a role in pantocytopenia in MDS and the deviation pattern may be as a direct and effective parameter to predict response of immunosuppression therapy.     

The relative extent and propensity of CD34+ vs. CD34- cells to undergo apoptosis in myelodysplastic marrows

The paradox of peripheral cytopenias despite cellular bone marrow (BM) observed in myelodysplastic syndromes (MDS) has been associated with excessive intramedullary apoptosis of hematopoietic cells. Since MDS is regarded as a stem cell disorder, the present studies were undertaken to examine the relative susceptibility and propensity of early progenitor CD34+ cells to undergo apoptosis as compared to more maturing/matured CD34- cells. Five serial studies were performed on 4 independent groups of 36 newly diagnosed MDS patients. First, in 2 separate groups of 16 and 8 patients each, measurement of the extent of apoptosis in CD34+ and CD34- fractions of the BM aspirate mononuclear cells was carried out using independent biparametric flow cytometry methods, CD34 labeling/terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) (n = 16), and CD34 labeling/reduced uptake of nucleic acid staining dye LDS751 (n = 8). The difference in the median degrees of apoptosis in CD34+ vs. CD34- cells was not statistically significant by either technique (P = 0.583 and P = 0.674 for TUNEL and LDS751, respectively). In the next group of 4 MDS patients, a double-labeling was performed on plastic embedded marrow biopsy sections, to detect CD34 antigen with specific monoclonal antibody and apoptosis by in situ end labeling (ISEL) of fragmented DNA. Despite high overall apoptosis (56.2% +/- 18.4%), only an occasional CD34+ cell was found to be simultaneously labeled with ISEL. Finally, in the last group of 8 MDS patients, CD34+ cells were separated from CD34- cells on affinity column and cultured in serum containing medium for 4 hours. At 0- and 4-hour time points, ISEL was carried out to label apoptotic cells. In addition, a fluorometric assay was employed to estimate the activity of a proapoptotic enzyme, Caspase 3. Both the net increase in % ISEL labeled cells (apoptotic index or AI) and Caspase-3 activity were significantly lower in CD34+ cells as compared to CD34- cells (AI, 0.87% +/- 0.5% vs. 3.97% +/- 1.4%, n = 6, P = 0.028 and Caspase-3 Units/mg protein, 46.9 +/- 25.0 vs. 71.7 +/- 23.03, n = 5, P = 0.042, respectively). We conclude that when estimated in a total population of mononuclear cells, CD34+ cells and CD34- cells show comparable degrees of apoptosis. However, once separated the CD34+ fraction demonstrates lower propensity to undergo apoptosis, thereby suggesting the CD34- fraction as being a possible source for proapoptotic signaling.