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。     

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。     

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。     

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与免疫异常

骨髓增生异常综合征与免疫异常徐世荣骨髓增生异常综合征（ＭＤＳ）为造血干细胞疾病，不仅在细胞遗传学、分子生物学、细胞生物学、血细胞酶学等方面有异常，而且在免疫学方面也存在异常。近１０余年来，研究进一步证实免疫功能异常在ＭＤＳ发病、发展，向急性白血病（Ａ...     

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患者细胞遗传学检测临床意义

目的研究骨髓增生异常综合征患者细胞遗传学异常表型及其与MDS患者转归关系。方法采用骨髓细胞直接法或24~48小时培养法制备染色体,用RHG技术进行核型分析。结果MDS患者异常染色体出现率46%,主要有 8、-7、5q-、11q-、20q-,并有复杂异常核型。复杂异常核型患者急性白血病转化率明显高于核型正常者。结论MDS患者有异常核型者易转为急性白血病,异常染色体出现与该类患者转归有直接关系,核型分析对MDS患者预后判断有重要价值。     

2例骨髓巨核细胞胞浆表达CD34的骨髓增生异常综合征病例临床分析

<正>骨髓增生异常综合征( myelodysplastic syndrome,MDS)是一组起源于髓系定向造血干细胞或多能干细胞的异质性克隆性疾病,主要特征是无效造血和高危演变为急性髓系白血病(acute myeloid leukemia ,AML),临床表现为造血细胞在质和量上出现不同程度的异常变化~([1,2])。1982年FAB协作组提出以形态学为基础的MDS分型,主要依据MDS患者外周血和骨髓细胞发育异常的特征进行分类。2008年WHO推出     

Impaired generation of bone marrow CD34-derived dendritic cells with low peripheral blood subsets in patients with myelodysplastic syndrome

Myelodysplastic syndrome (MDS) is a stem cell disorder characterized by ineffective haematopoiesis and blood cytopenias. The present study investigated the potential of bone marrow CD34(+) progenitors in MDS patients to proliferate and differentiate into dendritic cells (DCs) in a cytokine-supplemented liquid culture system and analysed the status of blood DC subsets in these patients. CD34(+) progenitors had low potential to generate DCs in vitro, as the number of DCs obtained from one CD34(+) cell was significantly lower compared with controls (median value 0.2 vs. 4, P = 0.003). In patients, the survival and proliferation of CD34(+) cells in culture was not correlated to the degree of apoptosis. Phenotypically and functionally CD34(+)-derived DCs were similar in MDS patients and normal subjects. The percentage of both circulating DC subsets in patients was extremely diminished compared with controls (myeloid DC: 0.10 +/- 0.10% vs. 0.35 +/- 0.13%, P < 0.001; plasmacytoid DC: 0.11 +/- 0.10% vs. 0.37 +/- 0.14%, P < 0.001). In cases with the 5q deletion both CD34-derived DCs and blood DCs harboured the cytogenetic abnormality. Our results indicate that, in MDS, the production of DCs is affected by the neoplastic process resulting in ineffective 'dendritopoiesis' with low blood DC precursor numbers. This quantitative DC defect probably contributes to the poor immune response against infectious agents and to the escape of the malignant clone from immune recognition with disease progression towards acute leukaemia.     

AA和MDS患者骨髓CD+34细胞表面GM-CSFR的表达及意义

目的 探讨再生障碍性贫血（AA）和骨髓增生异常综合征（MDS）患者骨髓CD34^＋细胞表面粒细胞-巨噬细胞集落刺激因子受体（GM—CSFR）的表达情况。方法 用流式细胞术（FCM）和单克隆抗体技术检测14例AA、23例MDS和8例非血液病患者骨髓CD34^＋细胞表面的GM—CSFR表达率。结果 MDS组骨髓CD34^＋细胞表面GM-CSFR的表达率明显高于对照组及AA组（P〈O．05）,而AA组与对照组间差异无统计学意义（P〉0．05）。结论 AA患者造血干细胞没有质的缺陷;造血干细胞异常可能是MDS的主要发病机制之一。     

CD34+ cell selection is required to assess HOXA9 expression levels in patients with myelodysplastic syndrome

Overexpression of HOXA9 is linked to the molecular pathogenesis of acute myeloid leukaemia (AML) and myelodysplastic syndrome (MDS), conferring a poor prognosis. HOXA9 expression levels were analysed in the diagnostic bone marrow (BM) samples of 13 MDS patients. HOXA9 was expressed by CD34(+) BM cells at median levels 3.1-fold higher than in CD34(-) cells from the same patient and at median levels 4.3-fold higher than in CD34(+) cells from healthy donors. These results indicate that CD34(+) cell selection is required to accurately assess the expression levels of HOXA9 and related genes in the multipotential malignant progenitor cells of MDS patients.     

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.     

Analysis of CD34-positive cells in bone marrow from patients with myelodysplastic syndromes and acute myeloid leukemia and in normal individuals: a comparison between FACS analysis and immunohistochemistry

Abstract: In patients with myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), expression of the hematopoietic stem cell marker CD34 has been associated with a poorer prognosis. CD34 is usually analyzed by flow cytometry (FC), but may also be analyzed using immunohistochemistry (IH). The present study was undertaken to compare these 2 methods. Bone marrow from 16 patients with MDS and 12 with AML and from 12 healthy young volunteers was studied. The expression of CD34 was analyzed with FC on fresh bone marrow cells and with IH on sections of paraffin-embedded bone marrow. The correlation between FC and IH was good both for patients with MDS (p<0.0001) and AML (p<0.01). However, in patients with a high number of CD34-positive cells, the FC method seemed to result in a higher percentage of positive cells compared to the IH method. In normal bone marrow, the ratio between the percentage of CD34-positive cells and the percentage of bone marrow blasts was approximately 0.8. In the whole group of MDS patients, this ratio was 1:1, while in patients with refractory anemia (RA) and ring sideroblastic anemia (RAS) it was 1.6. Patients with MDS differed significantly from patients with de novo AML, who showed a ratio of only 0.23 (p<0.01). We conclude that the FC and IH methods for measuring expression of CD34 are well-correlated in MDS and reasonably well correlated in AML. A stem cell phenotype is more commonly expressed on precursor cells from patients with MDS than from patients with AML.     

Detection of paroxysmal nocturnal hemoglobinuria clones in patients with myelodysplastic syndromes and related bone marrow diseases, with emphasis on diagnostic pitfalls and caveats

Background

The presence of paroxysmal nocturnal hemoglobinuria clones in the setting of aplastic anemia or myelodysplastic syndrome has been shown to have prognostic and therapeutic implications. However, the status of paroxysmal nocturnal hemoglobinuria clones in various categories of myelodysplastic syndrome and in other bone marrow disorders is not well-studied.

Design and Methods

By using multiparameter flow cytometry immunophenotypic analysis with antibodies specific for four glycosylphosphatidylinositol-anchored proteins (CD55, CD59, CD16, CD66b) and performing an aerolysin lysis confirmatory test in representative cases, we assessed the paroxysmal nocturnal hemoglobinuria-phenotype granulocytes in 110 patients with myelodysplastic syndrome, 15 with myelodysplastic/myeloproliferative disease, 5 with idiopathic myelofibrosis and 6 with acute myeloid leukemia.

Results

Paroxysmal nocturnal hemoglobinuria-phenotype granulocytes were detected in nine patients with low grade myelodysplastic syndrome who showed clinicopathological features of bone marrow failure, similar to aplastic anemia. All paroxysmal nocturnal hemoglobinuria-positive cases demonstrated loss of the four glycosylphosphatidylinositol-anchored proteins, with CD16⁻CD66b⁻ clones being larger than those of CD55⁻CD59⁻ (p<0.05). Altered glycosylphosphatidylinositol-anchored protein expression secondary to granulocytic hypogranulation, immaturity, and/or immunophenotypic abnormalities was present in a substantial number of cases and diagnostically challenging.

Conclusions

These results show that routine screening for paroxysmal nocturnal hemoglobinuria clones in patients with an intrinsic bone marrow disease who show no clinical evidence of hemolysis has an appreciable yield in patients with low grade myelodysplastic syndromes. The recognition of diagnostic caveats and pitfalls associated with the underlying intrinsic bone marrow disease is essential in interpreting paroxysmal nocturnal hemoglobinuria testing correctly. In our experience, the CD16/CD66b antibody combination is superior to CD55/CD59 in screening for subclinical paroxysmal nocturnal hemoglobinuria because it detects a large clone size and is less subject to analytical interference.     

CD34 immunohistochemistry of bone marrow biopsies: Prognostic significance in primary myelodysplastic syndromes

Bone marrow (BM) biopsies from 58 patients with primary myelodysplastic syndrome (MDS) were studied using QBEND10, a monocional antibody that recognizes the human progenitor CD34 antigen in routine aldehyde-fixed paraffin-embedded samples. FAB subtypes were RA (5 patients), RARS (9 patients), RAEB (20 patients), RAEBt (11 patients), CMML (3 patients). In addition, 10 MDS patients whose BM biopsies revealed heavy reticulum fibrosis were included. Neither the percentage of CD34⁺ cells nor the number of CD34⁺ aggregates (defined as clusters of 3 or more cells) correlated with the presence and morphology of abnormal localizations of immature precursors (ALIP). When all patients were considered, median survival was 69 months in those with less, and 25 months in patients with more than 1% CD34⁺ cells (P < 0.05). Median survival was 15 months in patients with CD34⁺ aggregates and 41 months in those without aggregates (P = 0.0017). When RAEB patients were considered median survival was 41 months in those with less than 1%, and 29 months in those with more than 1% CD34⁺ cells; the 4-year survival chance was 45% in the former and 18.3% in the latter group. Therefore, CD34 positivity of more than 1% identifies a subset of RAEB patients with shorter life expectancy. In addition, leukemic transformation was observed in 11 of 35 patients (31%) with no CD34 aggregates, but in 14 of 23 patients (60%) with aggregates (P < 0.05). CD34 immunostaining, which can be easily performed on routinely prepared BM biopsies, was found to be a powerful prognostic tool for predicting survival and outcome in MDS. © 1994 Wiley-Liss, Inc.     

Donor leucocyte transfusions for relapse in myelodysplastic syndrome after allogeneic bone marrow transplantation

A 31-year-old man with refractory anaemia of excess blasts, which had karyotypic abnormalities, received an allogeneic bone marrow transplant (BMT). At time of relapse, 3 months after BMT, he was treated with donor leucocyte transfusions (DLT). Grade III acute GVHD (graft-versus-host disease) occurred 35 d after DLT which was fully reversed with cyclosporin and prednisolone. His condition was complicated by a herpes zoster infection. 2 months after DLT, neutrophil and platelet count were increased and karyotypic abnormalities disappeared. This observation demonstrates that DLT is an effective treatment for relapse of myelodysplastic syndrome (MDS) after BMT.     

GPI-anchored protein-deficient T cells in patients with aplastic anemia and low-risk myelodysplastic syndrome: implications for the immunopathophysiology of bone marrow failure

Glycosylphosphatidylinositol-anchored protein-deficient (GPI-AP(-) ) T cells can be detected in some patients with bone marrow failure (BMF), but the link between these cells and BMF pathophysiology remains to be elucidated. To clarify the significance of GPI-AP(-) T cells in BMF, peripheral blood from 562 patients was examined for the presence of CD48(-) CD59(-) CD3(+) cells using high-resolution flow cytometry (FCM), and the GPI-AP(-) T cells were characterized with regard to their phenotype and sensitivity to inhibitory molecules, including herpesvirus entry mediator (HVEM) and a myelosuppressive cytokine, TGF-β. A multi-lineage FCM analysis detected CD48(-) CD59(-) CD3(+) T cells in 72 (12.8%) of the patients, together with GPI-AP(-) myeloid cells. Unexpectedly, 12 patients (10 with aplastic anemia and 2 with myelodysplastic syndrome-refractory anemia, 2.1%), who showed clinical features similar to those of other BMF patients with GPI-AP(-) myeloid cells, such as a good response to immunosuppressive therapy, displayed 0.01-0.3% GPI-AP(-) cells exclusively in T cells. The CD48(-) CD59(-) T cells consisted of predominantly effector memory (EM) and terminal effector cells, while CD48(-) CD59(-) T cells from non-BMF patients who had received anti-CD52 antibody only showed EM and central memory phenotypes. TGF-β and HVEM capable of inhibiting T-cell proliferation via its GPI-AP CD160 ligation suppressed the in vitro proliferation of GPI-AP(+) T cells more potently than that of GPI-AP(-) T cells from the same patients. The presence of GPI-AP(-) T cells, as well as GPI-AP(-) myeloid cells, may therefore reflect the immunopathophysiology of BMF in which cytokine-mediated suppression of hematopoietic stem cells via GPI-AP-type receptors takes place.     

再生障碍性贫血和骨髓增生异常综合征患者骨髓CD34+细胞及其粒细胞集落刺激因子受体的研究

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CD34 细胞占骨髓单个核细胞(BMMNC)的比率及其表面粒细胞集落刺激因子受体(G-CSFR)的表达率。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CD34 细胞占BMMNC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)组与难治性贫血伴原始细胞增多(RAEB)组BMMNC中CD34 细胞的比率比较差异有统计学意义(P<0.05),但G-CSFR的表达率差异无统计学意义(P>0.05)。多数重型AA(SAA)患者(3/4)及少数慢性AA(CAA)患者(1/9)BMMNC中的CD34 细胞少于0.1%。多数G-CSFR表达率低(<14%)的患者(7/9)外周血中性粒细胞减少;而表达率正常(14.0%~28.9%)的患者(1/6)很少见;表达率高(>28.9%)的患者(3/7)也可存在中性粒细胞减少。结论:造血干细胞减少是AA的主要发病机制之一,其表面G-CSFR的表达率不是影响AA的主要因素;MDS患者CD34 细胞比率升高是一个预后不良的指标,G-CSFR的检测可部分解释MDS患者外周血中性粒细胞减少的原因。