骨髓增生异常综合征骨髓单个核细胞内负调控因子的研究

为研究MDS患者细胞凋亡与骨髓内不同T细胞亚群胞内负调控因子的关系，采用胞内细胞因子染色技术对骨髓内不同亚群T细胞胞内的TNF—α和IFN-γ水平进行检测和分析，用RT—PCR技术测定BMMNC的凋亡基因Fas的mRNA水平。结果表明，MDS患者骨髓内产生TNF-α增多的淋巴细胞为CD4^ CD45RO^ 、CD8^ CD45RO^ 细胞，产生IFN-γ增多的为CD4^ CD45RO^ 、CD8^ CD45RO^ 、CD8^ CD45RA^ 细胞，但均以CD8^ CD45RO^ 细胞为主；MDS患者T淋巴细胞产生的IFN-γ与Fas mRNA存在相关性，而TNF—α与Fas mRNA相关性不强。结论：MDS患者造血微环境中不仅T淋巴细胞亚群失调，而且各亚群分泌负调控因子增多；MDS患者髓内IFN-γ主要来自于T淋巴细胞。     

骨髓增生异常综合征克隆细胞生物学特征的初步研究

目的 研究骨髓增生异常综合征（MDS）克隆细胞和急性髓系白血病（AML）克隆细胞的部分生物学差异。方法 对经核型分析证实有克隆标志的51例MDS和11例AML患者骨髓中克隆细胞（荧光原位杂交分析）和原始细胞百分比进行比较；检测MDS患者骨髓中晚期红系（血型糖蛋白A阳性的晚幼红细胞）、粒系（DAPI复染可分辨的分叶核细胞）和巨核系（CD61阳性磁珠分选，CD41阳性的形态学成熟的巨核细胞）中克隆细胞比例；同步检测MDS患者骨髓及外周血中克隆细胞比例；用流式细胞术检测含有克隆细胞的MDS患者外周血中性粒细胞的吞噬和氧化功能并与正常人和AML患者标本比较。结果 MDS患者骨髓克隆细胞百分比（平均48．2％）均高于原始细胞（平均6．7％）（P〈0．01）。进展型MDS与非进展型相比，克隆细胞与原始细胞数更接近。单纯染色体＋8异常与单纯5q-患者比较，克隆细胞与原始细胞数量更接近。而在11例AML患者，克隆细胞与原始细胞百分比的平均差距接近零。在MDS患者骨髓细胞中检出相当数量带有克隆标志的晚期造血细胞：分叶核细胞中平均为45．9％；晚幼红细胞中为46．0％；成熟巨核细胞中为38．0％。MDS患者外周血中检出与骨髓中核型一致的克隆细胞，数量与骨髓中克隆细胞数相关（骨髓中平均为48．6％，外周血中为37．3％）。功能性检测显示MDS患者外周血中的中性粒细胞具有与正常对照者几乎一致的对DHR的摄取和氧化功能（P〉0．05）。结论 MDS克隆细胞与AML克隆细胞具有明显的生物学差异。     

Increased immature hematopoietic progenitor cells CD34+/CD38dim in myelodysplasia

BACKGROUND: Myelodysplastic syndromes (MDS) are clonal disorders affecting hematopoietic progenitor cells (HPC). Despite the relevance of clonal CD34+ cells in developing MDS, only few studies analyze the phenotype of this cell population. The aim of this study was to evaluate phenotypic changes on HPC in MDS that could reflect abnormalities in the differentiation process of stem cells. METHODS: We analyzed the expression of CD38 and HLA-DR on CD34+ cells by flow cytometry in 36 patients with MDS, as well as in healthy donors (n = 12) and patients with other hematological disorders: non-Hodgkin lymphomas and multiple myeloma, both in complete remission (CR) (n = 32); acute lymphoblastic leukemia in CR (n = 17); de novo acute myeloblastic leukemia (AML) at diagnosis (n = 22) and in CR (n = 37); and AML secondary to MDS at diagnosis (n = 19). Cases with available karyotype were grouped according to the International Prognostic Scoring System (IPSS). RESULTS: Compared to normal BM, the fraction of immature HPC, characterized as CD34+bright, intermediate FSC/SSC, and CD38dim, was significantly increased in high risk MDS and secondary AML, but not in low risk MDS, (P < or = 0.001, P = 0.03, and P = 0.7). De novo AML showed decreased immature HPC. High numbers of immature HPC correlated with higher IPSS risk groups (P = 0.05) and showed significant impact on disease progression (P = 0.03). CONCLUSION: Our study confirms that evaluation of CD38 expression pattern on HPC is an easy and reproducible test that allows evaluating the immature subset of progenitor cells. Increased immature HPC in high risk MDS and secondary AML may reflect blocked differentiation of CD34+ cells in these diseases.     

再生障碍性贫血患者的淋巴细胞表型变化

目的研究再生障碍性贫血（AA）患者骨髓（BM）及外周血（PB）淋巴细胞及其活化相关分子的表达及临床意义。方法采用双色免疫荧光标记、流式细胞仪分析AA患者的BM和PB中淋巴细胞膜分子表达。结果AA患者BM和BP中CD8＋细胞增加，CD4／CD8比例下降，BM中CD25＋细胞和HLA-DR＋细胞增多，急性AA增加尤为显著（P（0．01），BM中CD16＋或CD56＋细胞也明显增多（P〈0．05）；双标记分析提示T细胞主要为CD8＋细胞；急性AA患者CD8＋-CD25＋细胞显著增多（P〈0．01），AA患者BM中淋巴细胞活化相关分子表达增多，尤其是4—1BB＋、CD95L＋和CD40L＋细胞显著增多（P〈0．01）。结论AA患者BM中淋巴细胞活化相关膜分子增多，是AA免疫功能异常及最终导致造血功能衰竭的原因之一。     

PNH患者外周血及骨髓正常和异常造血干/祖细胞组成分析

目的　研究阵发性睡眠性血红蛋白尿症 (PNH)患者外周血及骨髓造血干 祖细胞的组成及含量。方法　用流式细胞仪测定 2 1例PNH患者外周血及骨髓造血干 祖细胞 (以CD3 4+为标志 )锚连蛋白CD59的表达 ,并将患者造血干 祖细胞总数与正常人进行比较。结果　PNH患者骨髓及外周血CD3 4+细胞总量均明显低于正常人 (P <0 .0 0 1) ,但在疾病缓解时总量与正常对照差异无显著性 (P >0 0 5 )。PNH患者外周血CD3 4+细胞主要以CD59+表型为主 [CD3 4+CD59+细胞占总CD3 4+细胞的 (81.4±16 1) % ],临床未缓解或骨髓中CD3 4+细胞以CD59- 表达为主的患者 ,其外周血CD3 4+细胞仍以CD59+表型为主。结论　PNH患者造血干 祖细胞含量明显减少 ,但在外周血中以正常表型为主     

骨髓增生异常综合征中基质细胞衍生因子1与血管新生及细胞凋亡的相关性研究

本研究探讨骨髓增生异常综合征(MDS)中骨髓基质细胞衍生因子-1(stromal cell-derived factor-1,SDF-1)的表达,及其与骨髓CD34+细胞的凋亡和骨髓血管新生的关系。搜集40例MDS病例,根据IPSS积分分为低危和高危2组,采集患者的骨髓穿刺物和活检标本。检测骨髓血浆中SDF-1的含量,骨髓CD34+细胞的凋亡率,骨髓活检标本的微血管密度(MVD)。结果显示:SDF-1α在低危组和高危组的表达量分别为(2313±417)pg/ml,(1241±501)pg/ml,前者显著高于后者(p<0.05);且2组的表达率均显著高于健康正常者(710±153)pg/ml(p<0.05);Annexin-Ⅴ/PI双染色显示,低危MDS患者CD34+细胞的凋亡率为(54.75±14.15)%,显著高于对照组的(18.51±8.66)%和高危组的(23.96±12.20)%(p<0.05),后两者相比差异无显著性(p>0.05);相关分析显示:低危组中骨髓SDF-1含量与CD34+细胞凋亡率呈正相关(p<0.05);在高危组中骨髓SDF-1含量与MVD呈现正相关(p<0.05)。结论:骨髓SDF-1含量、CD34+细胞凋亡和MVD在MDS中存在显著异常,在不同风险度的病例中也有明显的不同,且SDF-1水平与骨髓细胞的凋亡和血管新生具有相关性。     

骨髓增生异常综合征患者骨髓细胞周期及CD34+细胞增殖特征的研究

目的　探讨骨髓增生异常综合征 (MDS)患者骨髓细胞周期分布及CD34 细胞增殖特征。方法　碘化丙锭细胞核染色分析MDS、MDS转化的急性髓系白血病 (MDS AML)和原发性AML患者骨髓G0 /G1期、S期和G2 /M期细胞比例 ;免疫荧光双标法分析 3组患者骨髓CD34 细胞中增殖性抗原Ki6 7的表达。结果　与正常组相比 ,MDS患者和原发性AML患者骨髓单个核细胞 (BMM NC)G0 /G1期细胞比例呈明显增高趋势 [(92 .4 8± 4 .4 8) %、(96 .71± 2 .75 ) %对 (86 .94± 6 .77) % ](P<0 .0 5 ) ,而MDS组与正常组相比 ,S期和G2 /M期比例呈明显降低趋势 [(6 .79± 3.98) %、(0 .86±0 .82 ) %对 (11.97± 7.0 0 ) %、(1.10± 0 .98) % ](P值均 <0 .0 5 )。MDS AML患者与原发性AML相比 ,G0 /G1期细胞为 (91.16± 7.0 9) %对 (96 .71± 2 .75 ) % ,S期为 (7.90± 6 .70 ) %对 (2 .87± 2 .4 9) %、G2 /M期为 (0 .96± 0 .99) %对 (0 .4 3± 0 .4 2 ) % ,S G2 /M期为 (8.84± 7.0 9) %对 (3.34± 2 .83) % (P值均 <0 .0 5 )。MDS患者骨髓CD34 Ki6 7 细胞明显高于正常组 [(1.13± 1.10 ) %对 (0 .2 4±0 .2 2 ) % ](P <0 .0 1) ,MDS低危组患者CD34 Ki6 7 细胞为 (0 .5 4± 0 .4 9) % ,明显低于高危组 [(1.6 9± 1.6 6 ) % ](P <0 .0     

Phenotypic characterization and preclinical production of human lineage-negative cells for regenerative stem cell therapy

BACKGROUND: Regenerative stem cell therapy (SCT) is currently being tested in clinical trials. The ideal type and source of cells have not yet been defined. Lineage (Lin) depletion is an experimental procedure capable of enriching all recently recognized SC types with regenerative potency. This study was performed to define a practicable monoclonal antibody (MoAb) cocktail for Lin depletion and to test whether clinical-scale Lin depletion is possible. STUDY DESIGN AND METHODS: MoAbs (CD2/14/15/19/41/56/glycophorin A) were selected to mark seven mature hematopoietic lineages. Lin7-negative (Lin7NEG) cells were analyzed in peripheral blood (PB, n = 9), mobilized PB (MPB, n = 5), umbilical cord blood (UCB, n = 5), and marrow aspirates (BM, n = 4) by flow cytometry. Preclinical Lin depletion was tested with leukapheresis products from PB following good manufacturing practice (GMP) principles. RESULTS: Lin7NEG cells comprised 0.23 +/- 0.04, 0.27 +/- 0.03, 0.53 +/- 0.07, and 0.49 +/- 0.03 percent of PB, MPB, UCB, and BM, respectively. Basophils, CD34+, and dendritic cells constituted the major Lin7NEG subpopulations (84 +/- 2, 90 +/- 3, 40 +/- 3, and 80 +/- 3% in PB, MPB, UCB, and BM, respectively). Minor populations included CD7- and CD45- cells. Preclinical CD2/14/15/19/56 (Lin5) depletion after automated red blood cell and platelet reduction resulted in up to a 16.7-fold enrichment of CD34+ and CD34-/Lin5NEG cells. CONCLUSIONS: A seven-MoAb cocktail is sufficient to label more than 99 percent of nucleated cells in PB, MPB, UCB, and BM. Preclinical Lin depletion can be performed under GMP conditions from PB apheresis procedures.     

Minimal residual disease testing to predict relapse following transplant for AML and high-grade myelodysplastic syndromes

Evaluation of: Lange T, Hubmann M, Burkhardt R et al. Monitoring of WT1 expression in PB and CD34(+) donor chimerism of BM predicts early relapse in AML and MDS patients after hematopoietic cell transplantation with reduced-intensity conditioning. Leukemia 25, 498-505 (2011). Early detection of relapse is critical for patients who have undergone hematopoietic stem cell transplantation (HSCT) for acute myeloid leukemia (AML) or high-grade myelodysplastic syndromes (MDS), since therapy can be initiated while disease burden remains low. As these neoplasms represent a heterogeneous group of malignancies with distinct underlying mutations, no single genetic marker exists that both defines AML/MDS and can be exploited for sensitive detection of neoplastic cells prior to overt hematologic relapse. Conversely, the Wilms' tumor gene (WT1) expression level is increased in blasts of most AML/MDS patients, and quantitative analysis of WT1 expression has been used to predict relapse following myeloablative HSCT. In this article, we review a recently published study evaluating the usefulness of multiple markers, including WT1 expression, for predicting relapse in AML/MDS patients following reduced-intensity conditioning nonmyeloablative HSCT.     

骨髓增生异常综合征患者骨髓CD34+细胞 Fas,FasL及Bcl-2的表达和凋亡

为了观察骨髓增生异常综合征 (MDS)患者骨髓CD34+ 细胞Fas ,FasL和Bcl 2的表达和凋亡情况并探讨这些抗原的表达和细胞凋亡的关系。采用流式细胞术测定了 2 6例MDS和 10例急性髓系白血病 (AML)患者及 6例非血液病患者 (对照 )骨髓CD34+ 细胞的Fas,FasL和Bcl 2表达率和细胞凋亡率。结果显示 ,各型MDS患者CD34+ 细胞Fas和FasL表达率较对照组明显增加 (P <0 .0 1) ,Bcl 2的表达率除难治性贫血 /环形铁粒幼细胞性难治性贫血 (RA/RAS)与对照组无显著差异 (P >0 .0 5 )外 ,难治性贫血伴有原始细胞增多 (RAEB)和转变中的难治性贫血伴原始细胞增多 (RAEB t)患者均明显高于对照组 (P <0 .0 1) ;各型MDS间CD34+ 细胞Fas的表达率相近 ,而Bcl 2的表达率却存在非常显著性差异 ,即RA/RAS 相似文献   

610例急性髓系白血病免疫表型和白血病相关免疫表型分析

目的探讨急性髓系白血病（AML）患者免疫表型和白血病相关的免疫表型（LAIP）特征及在微量残留病（MRD）检测中的意义。方法采用CD45／SSC设门四色流式细胞术，检测610例AML患者和20名健康志愿者下列抗原的表达：CD7、CDll7、CD33、CD34、CDl0、CDl9、CD56、CD38、CDl3、CDl4、CD64、CD9、CDl6、CD2、CD5、CDllb、CDl23、HIA-DR。结果正常骨髓中CD34^＋和CDll7^＋SSC值低（SSC^low）的细胞比例分别为（0．35±0，15）％和（0．76±0．31）％。CD34^＋SSC^low细胞中绝大多数细胞共表达CDl3、CD33、CD38、CDll7、HLA-DR（84％～94％）。CD9和CDl5的相对比例为20％和33％，而CDllb、CD56、CDl9和CD64的相对比例均低于10％，CD7为12％。CDll7^＋SSC^low细胞中，CDl9^＋、CD11b^＋、CD56^＋和CD7^＋细胞相对比例低于10％，CD9^＋细胞为14％，CD38^＋和HIA-DR^＋细胞的相对比例为87％和91％，其余抗原（CDl5、CDl3、CD33、CD34）均在30％～50％。AML患者中CDll7、CD38表达率约为95％，CD33、CD9表达率约为84％。HLA-DR和CDl3表达率分别为77．23％和75．25％。CD64和CD34的表达率分别为64．41％和59．51％。CDl5表达率为43．06％，CDllb表达率为22，07％。86．39％的AML患者LAIP阳性，以AML-M1和M3最高，AML-M4E0最低。LAIP主要表现为交叉系列抗原表达和非同期共抗原表达，前者以CD34与CD7、CDl9、CD56同时阳性为主。在非同期抗原表达中，CD34^＋CD64^＋、CDll7^＋CDllb^＋、CDll7^＋CD38^-/dim、CDll7^＋HLA-DR-／^dim正常值在0．01％左右，与AML之间的对数差大于3，为灵敏度较高的LAIP。结论多参数流式细胞术适于80％以上AML患者MRD检测。     

多发性骨髓瘤细胞系CZ-1的建立及其生物学特性

目的 建立多发性骨髓瘤（MM）细胞系并鉴定其生物学特性。方法 分离1例晚期λ轻链型MM患者外周血和骨髓的单个核细胞，用液体培养法进行培养，用瑞特-姬姆萨染色和细胞化学染色确定细胞形态；用免疫固定电泳技术检测细胞是否分泌单克隆λ轻链，用荧光定量PCR法检测细胞是否被EB病毒感染；用流式细胞仪分析细胞的免疫表型；用染色体G分带法研究细胞的遗传学特征。结果 所得细胞系在饲养细胞或条件培养液存在时，可以持久增殖，该细胞分泌λ轻链，EB病毒阴性；细胞在瑞特-姬姆萨染色下呈典型的原，幼浆细胞形态，细胞化学染色，酸性磷酸酶染色（ ），髓过氧化物酶（-）；细胞免疫表型为CD10^ ,CD28^ ,CD38^ ,CD138^ ,CD56^ ,CD49d^ ,CD54^ ,CD44^ ,CD58^ ，而不表达CD19，CD40，CD95，CD95L，CD34，CD2，CD5，骨髓来源和外周血来源的细胞系免疫表型无明显不同，核型分析结果均为i(lq ),8q 13q ,i(17q),i(18q), M。结论 用1例MM患者的外周血和骨髓细胞同时建立了一个生物学特性相同的分泌λ轻链的MM细胞系CZ-1。     

基质细胞衍生因子-1及其受体在G-CSF诱导的造血干/祖细胞动员中的作用

目的探讨基质细胞衍生因子-1（SDF-1）及其特异性受体CXCR4在G-CSF诱导的造血干／祖细胞（HSPC）动员中的作用。方法应用酶联免疫吸附实验（ELISA）、免疫组织化学、流式细胞术等方法检测健康供者稳态及G-CSF动员过程中骨髓、外周血SDF-1／CXCR4的变化，并应用SDF-1中和性抗体阻断BALB／c小鼠SDF-1信号通路，进一步验证SDF-1／CXCR4在动员中的作用。结果G-CSF动员前骨髓和外周血的SDF-1浓度分别为（7．23±0．66）μg/L和（5．43±0．35）μg／L，动员后分别为（5．88±1．03）μg／L和（5．42±0．52）μg/L。动员后骨髓SDF-1蛋白水平下降（P＜0．05），骨髓和外周血之间的SDF-1浓度梯度消失（P＞0．05）；稳态骨髓、动员后骨髓和动员后外周血的CD34^＋ CXCR4^＋细胞在CD34^＋细胞群中的比例分别为（40．98±21．56）％、（65．80±24．68）％和（27．54±26．03）％。动员后CXCR4在骨髓CD34^＋胞上表达增加（P＜0．05），而外周血CD34^＋细胞CXCR4表达降低（P＜0．05）。SDF-1中和性抗体可降低G-CSF动员的BALB／c小鼠外周血成熟白细胞和祖细胞集落数量（P＜0．05）。结论骨髓中SDF-1水平的降低以及CXCR4在HSPC上表达的下降促进了G-CSF介导的动员的发生。     

VLA-4整联蛋白和造血细胞迁移之间的相关关系研究

为了阐明VLA-4(CD49d)和造血细胞迁移方向之间的相关关系，将重组人G—CSF稀释后注射于小鼠皮下，以流式细胞仪检到不同时间后Sca-1^ 细胞表面CD49d的表达，并分析Sca-1^ 细胞计数与CD49d表达之间的相关关系。结果表明，注射G—CSF后骨髓和外周血的CD49d的表达都明显下降，同时在第7—9天外周血Sca-1^ 细胞数达到高峰，骨髓有核细胞数下降。而随着CD49d的表达回升，外周血Sca-1^ 细胞数下降，骨髓细胞数却又有增多趋势。结论：VLA-4介导造血细胞与骨髓微环境的粘附，调节CD49d的表达可能合导致造血细胞在骨髓和外周血之间相互迁移。     

间充质干细胞与人脐血CD34+细胞共移植对NOD/SCID小鼠造血重建的影响

目的 观察间充质干细胞(MSC)与不同比例脐血CD34+细胞共移植对NOD/SCID小鼠造血重建的影响,明确MSC与脐血CD34+细胞共移植的最适数量.方法 给60Coγ射线照射的雌性NOD/SCID小鼠共移植人MSC和不同比例的脐血CD34+细胞,观察共移植后42 d内小鼠外周血白细胞和血小板变化,并于移植后42 d处死小鼠,用流式细胞术检测外周血、骨髓和脾脏人源细胞含量.结果 与单纯脐血CD34+细胞移植相比较:①脐血CD34+细胞与1、5和10倍数量的MSC共移植时,可明显减轻外周血白细胞和皿小板的下降幅度(P<0.01),提前1周使白细胞和血小板恢复至正常水平(P<0.05),三组间差异无统计学意义(P>0.05);②MSC与不同比例的脐血CD34+细胞共移植均可明显提高外周血、骨髓和脾脏造血细胞植入率.比例为10:1时,外周血、骨髓和脾脏中的人源细胞(huCD45+细胞)含量分别增加了(2.8±0.6)倍、(3.5±0.9)倍和(5.2±0.6)倍,增加倍数差异均有统计学意义(P<0.01),达到了最佳的植入效果.结论 脐血CD34+细胞与10倍数量的MSC共移植可达到最佳的促进造血重建作用.     

骨髓增生异常综合征患者骨髓CD34-和CD34+细胞凋亡与增殖的意义及对生存的影响

本研究分析骨髓增生异常综合征（MDS）患者骨髓CD34^＋和CD34-细胞凋亡和增殖情况,探讨MDS的发病机制,并判断其与疾病预后的关系。流式细胞术分析20例高危MDS、20例低危MDS患者及10例正常对照者骨髓CD34^＋细胞的比例、CD34^＋细胞和CD34-细胞凋亡、增殖的百分率,计算各组中的凋亡／增殖（A／P）比。并用单变量和多变量生存分析法分析CD34^＋细胞和CD34-细胞增殖和凋亡对预后的影响。结果表明：①MDS高危组患者骨髓CD34^＋细胞的比例明显高于低危组（P〈0．05）,而低危组与对照组比较无显著差异;②CD34^＋,CD34-细胞的凋亡率在MDS低危组中均为最高,明显高于MDS高危组和对照组。在低危组中,CD34-细胞的凋亡率为（80．36±1．82）％,明显高于CD34^＋细胞的（54．75±2．18）％（P〈0．05）,而在高危组中,CD34^＋,CD34-细胞的凋亡率无显著差异;③CD34^＋细胞的增殖率在MDS高危组中最高,明显高于低危组和对照组,而CD34-细胞的增殖率在MDS高危和低危组间无显著差异。高危组CD34^＋细胞的增殖率为（50．67±3．37）％,明显高于CD34-细胞的（30．99±1．96）％（P〈0．05）;④CD34^＋、CD34-细胞的A／P值在MDS低危组均明显高于高危组和正常对照组,而CD34-细胞的A／P值在MDS高、低危组均明显高于CD34^＋细胞的A／P值（P〈0．05）。此外,CD34^＋细胞的凋亡率与生存及预后明显相关。结论：CD34^＋细胞百分率随MDS危险度增加而逐渐增加,在低危组中以CD34-细胞的凋亡占主导,随着病情进展,在高危组中则以CD34^＋细胞的增殖占主导,提示异常的凋亡和增殖在MDS的发生和发展中起重要作用。此外,CD34^＋细胞的凋亡率可能是独立的预后不良因素,抑制凋亡可能诱导MDS向白血病的转化。     

骨髓增生异常综合征患者外周血树突细胞数量、亚群及其表面协同刺激分子表达的研究

目的 检测骨髓增生异常综合征（MDS）患者外周血树突细胞（DC）总量、亚群（pDC和mDC）比例及其表面协同刺激分子（CD80、CD86和CIMO）表达，探讨MDS患者细胞免疫异常的形成机制。方法 选取38例MDS患者及19名正常对照，采用荧光素单克隆抗体标记法和流式细胞术检测MDS患者外周血中Lin1^—HLA-DR^＋细胞（DC）、Lin1^-HLA—DR^＋CD123^＋细胞（pDC）、Lin1—HLA—DR^＋CD11c^＋细胞（mDC）的数量以及CD80、CD86和CIMO在DC膜上的表达。结果低危MDS组、高危MDS组和正常对照组外周血DC总量分别为（33．7±7．0）×10^6／L、（56．3±29．0）×10^6／L和（12．1±1．4）×10^6／L（P〈0．05），pDC数量分另U为（12．6±4．1）×10^6／L、（3．6±1．0）×10^6／L和（6．6±0．7）×10^6／L（P〉0．05），mDC数量分别为（16．7±6．3）×10^6／L、（28．7±17．6）×10^6／L和（5．5±0．9）×10^6／L（P〈0．05）。低危MDS组、高危MDS组和正常对照组DC占外周血单个核细胞（PBMNC）百分比分别为（2．37±0．53）％、（3．58±1．39）％和（0．68±0．08）％（P〈0．05），pDC占PBMNC百分比分别为（0．82±0．29）％、（0．31±0．06）％和（0．37±0．04）％（P〉0．05），mDC占PBMNC百分比分别为（0．96±0．35）％、（1．51±0．70）％和（0．32±0．05）％（P〈0．05）。低危MDS组、高危MDS组和正常对照组外周血中CD80^＋DC分别为（30．6±11．8）×10^6／L、（2．3±0．9）×10^6／L和（2．3±0．6）×10^6／L（P〈0．05），CD86^＋DC分别为（25．1±7．4）×10^6／L、（12．4±6．3）×10^6／L和（6．2±3．2）×10^6／L（P〈0．05），CD40^＋DC分别为（2．8±1．0）×10^6／L、（1．5±0．9）×10^6／L和（3．2±2．3）×10^6／L（P〉0．05）。结论 MDS患者外周血中DC数量增多、比例异常；以mDC增多为主，pDC无明显增多；MDS患者DC高表达CD80和CD86，CD40表达不增高。提示MDS患者诱导抗肿瘤细胞免疫的抗原呈递细胞（pDC）数量及功能不足；而与正常造血克隆炎性损伤有关的抗原呈递细胞（mDC）却增多。这可能是导致MDS患者细胞免疫异常的重要机制之一。     

Pharmacodynamic monitoring of BAY 43-9006 (Sorafenib) in phase I clinical trials involving solid tumor and AML/MDS patients, using flow cytometry to monitor activation of the ERK pathway in peripheral blood cells

BACKGROUND: We previously reported a flow cytometry technique to monitor pharmacodynamic effects of the raf kinase inhibitor BAY 43-9006 based on the ability of phorbol ester (PMA) to phosphorylate extracellular-regulated kinase (ERK) in peripheral blood (Chow et al., Cytometry 2001;46:72-78). In this article, we describe its application to phase I trials of BAY 43-9006 in solid tumor and AML/MDS patients. METHODS: The previously described whole blood lysis method was used to monitor BAY 43-9006 effects on peripheral T-cells of solid tumor patients. A modified whole blood fixation protocol was developed for the AML/MDS trial, using the c-kit ligand stem cell factor (SCF) to activate ERK as an alternative to PMA, and incorporating immunophenotypic markers to identify leukemic blasts. RESULTS: At all dose levels of BAY 43-9006 used to treat solid tumor patients, ERK could be activated by PMA in peripheral T-cells and we were not able to show inhibition of raf kinase. A similar effect was seen in the lymphocytes of AML/MDS patients during treatment with BAY 43-9006. However, we found strong inhibition when ERK was activated via c-kit using SCF. Furthermore, normal donor CD34+ve stem cells were much more sensitive to BAY 43-9006 when ERK was activated by SCF, compared to PMA. CONCLUSIONS: These findings support the further development of flow cytometry applications to monitor signal transduction inhibitors during early phase clinical trials.     

In vivo and vitro effects of interferon-alpha 2b on functional activity of T-lymphocytes from patients with rheumatoid arthritis

AIM: To investigate the effect of recombinant alpha 2b-interferon (r alpha 2b-IFN) on functional capacity of peripheral blood (PB) T cells in rheumatoid arthritis (RA) patients and the relationship between functional characteristics of T lymphocytes and the disease activity. MATERIALS AND METHODS: PB mononuclear cells (PBMC) were separated by Ficoll-Verografine++ gradient centrifugation from 24 healthy donors (HD) and 75 RA patients 19 of which were treated with r alpha 2b-IFN (realdiron, Biofa, Lithuania) in the dosage 1 million IU i.m. each other day for 20 days, 10 injections a course. Cell surface markers (CD3, CD4, CD8) and adhesion molecules (CD18, CD54, CD2) were analyzed using specific monoclonal antibodies (MoAbs) and flow cytometry on the PBMC, freshly isolated and treated for 72 hours with medium alone, PHA, r alpha 2b-IFN and their combination. The proliferative response of PBMC to MoAbs for CD3, PHA and r alpha 2b-IFN were assessed by 3H-thymidine incorporation. The percentage of spontaneous and inducing apoptosis was quantified by flow cytometry using propidium iodide staining. RESULTS: The expression of CD18 was lower on RA PB lymphocytes compared to HD PB lymphocytes (p < 0.05). After stimulation of PBMC in both RA patients and HD with PHA, percentages of CD2+, CD3+, CD4+, CD18+ cells significantly diminished (p < 0.05), whereas the percentages of CD54+ and CD18+ (p < 0.05) cells increased. We have found three types of RA PB lymphocytes response to complex factors in vitro: 1) the presence of the proliferative response to T-mitogens but not to r alpha 2b-IFN (56% of the patients); 2) the presence of the increased proliferative response to T-mitogens and r alpha 2b-IFN (17% of the patients); 3) the absence of the proliferative response to T-mitogens and r alpha 2b-IFN (27% of the patients). PBMC of HD demonstrate only the first type of the response. R2 alpha b-IFN demonstrated own mitogenic effect and increased mitogen-induced proliferation in PBMC cultures with a high proliferative response to T-mitogens. The levels of spontaneous and inducing apoptosis were increased in RA PB lymphocytes compared to HD. After stimulation with PHA, RA PB lymphocytes preferentially underwent apoptosis whereas cells of HD proliferated. High disease activity correlated positively with an increase of a proliferative response to mitogens and apoptosis and a decrease in the percentage of lymphocytes, expressed adhesion molecules. The treatment with r alpha 2b-IFN induces changes in T-cell response to mitogens similarly to those after incubation with r alpha 2b-IFN in vitro before treatment. CONCLUSION: Functional capacity of RA PB lymphocytes relates to the disease activity. Inhibitory or stimulatory effects of r alpha 2b-IFN depend on functional activity of RA lymphocytes. Using the test with alpha 2b-IFN incubation, we may predict changes of apoptosis and proliferation levels caused by different agents in RA lymphocytes after treatment with r alpha 2b-IFN.     

Peripheral blood MDS score: a new flow cytometric tool for the diagnosis of myelodysplastic syndromes

BACKGROUND: Myelodysplastic syndromes (MDS) are a heterogeneous group of hematopoietic disorders diagnosed using morphologic and clinical findings supported by cytogenetics. Because abnormalities may be subtle, diagnosis using these approaches can be challenging. Flow cytometric (FCM) approaches have been described; however the value of bone marrow immunophenotyping in MDS remains unclear due to the variability in detected abnormalities. We sought to refine the FCM approach by using peripheral blood (PB) to create a clinically useful tool for the diagnosis of MDS. METHODS: PB from 15 patients with MDS was analyzed by multiparametric flow cytometry using an extensive panel of monoclonal antibodies. Patterns of neutrophil antigen expression were compared with those of normal controls (n = 16) to establish light scatter and/or immunophenotypic abnormalities that correlated with MDS. A scoring algorithm was developed and validated prospectively on a blinded patient set. RESULTS: PB neutrophils from patients with MDS had lower side scatter and higher expression of CD66 and CD11a than did controls. Some MDS PB neutrophils demonstrated abnormal CD116 and CD10 expression. Because none of these abnormalities proved consistently diagnostic, we sought to increase the power of the assay by devising a scoring system to allow the association of multiple abnormalities and account for phenotypic variations. The PB MDS score differentiated patients with MDS from controls (P < 0.0001) in the test set. In a prospective validation, the PB MDS score successfully identified patients with MDS (sensitivity 73%, specificity 90%). CONCLUSIONS: FCM analysis of side scatter and only four additional immunophenotypic parameters of PB neutrophils using the PB MDS score proved more sensitive than standard laboratory approaches and may provide an additional, more reliable diagnostic tool in the identification of MDS.