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。     

急性髓细胞白血病免疫表型分析

目的：分析成人初治急性髓细胞白血病（AML）髓系抗原、CD34及Pgp表达特点及其与预后的关系。方法：采用流式细胞仪免疫荧光标记法检测65例初治成人AML的免疫表型。结果（1）髓系抗原阳性率依次为CD33＞CD15＞CD13＞CD14,CD33阳性率高达98．46％。CD14在M5、M6表达率较高;（2）CD34及HLA－DR阳性表达率以M1、M5较高,M3最低;（3）P－糖蛋白（PG)阳性率58．33％,Pgp阳性AML的CR率28．57％,明显低于Pgp阴性的CR率（63．63％,P＜0．005）。结论AML的免疫表型检测髓系单抗可选择CD33、CD15和（或）CD14。CD34和HLA－DR低表达为M3的特征。Pgp表达与CR率有密切的关系。     

骨髓增生异常综合征骨髓细胞凋亡和TNF-α关系的研究

目的：探讨骨髓增生异常综合征(MDS)骨髓细胞调亡特征以及与肿瘤坏死因子α(TNF—α)的关系。方法：分别采用形态学和dUTP末端缺口标记(TUNEL)方法检测骨髓细胞调亡；采用ELISA方法检测骨髓单个核细胞培养后上清液中TNF—α的浓度，并与缺铁性贫血(IDA)患者进行比较。结果：形态学方法检测骨髓单个核细胞培养后细胞调亡指数，MDS明显高于IDA(P＜o．01)，MDS各分型(RA／RAS、RAEB／RAEB—t)之间细胞调亡指数差异无显著性意义(P＞0．05)。同一个体TUNEL法测得的骨髓细胞调亡指数均高于形态学观测的结果。TUNEL检测MDS细胞调亡指数RA／RAS＞RAEB／RAEB—t，但差异无显著性意义(P＞0．05)。MDS骨髓单个核细胞上清中TNF-α浓度高于IDA组，差异有显著性意义(P＜0．05)；MDS各分型之间RA／RAS显著高于RAEB／RAEB—t(P＜0．05)。结论：MDS骨髓细胞确实凋亡过度，早期MDS重于晚期MDS，并与TNF—α呈线性关系；采用TUNEL法检测凋亡细胞较形态学方法敏感。     

类风湿关节炎中HLA-DR分子表达量的改变

目的 探讨外周血淋巴细胞表面HLA－DR分子表达量在类风湿关节炎（RA）发病中的作用。方法 选择RA患者活动期34例,静止期40例;正常对照（NC）43例,急性风湿性关节炎（ARA）21例及系统性红斑狼疮（SLE）28例,分别检测外周血淋巴细胞表面的HLA－DR分子表达量。同时在活动期RA治疗3个月前后,分别检测HLA－DR分子表达量、类风湿因子（RF）及对症状计分,进行统计学分析。结果 ①RA活动组HLA－DR分子表达水平最高（P＜0．05）,ARA组及RA静止组较NC组明显增高（P＜0．05）,而SLE组与NC组无明显差别（P＞0．05）;②RA活动组HLA－DR分子表达量及RF的阳性率均在治疗后显著下降,临床症状明显改善。结论 ①检测患者外周血淋巴细胞表面HLA－DR表达量有助于RA的诊断及监测RA活动度;②HLA－DR分子表达量与RF的动态变化相平行;③动态观察HLA－DR分子表达量变化有助于判断RA病情及其预后。     

骨髓增生异常综合征骨髓细胞DNA倍体和增殖活性的研究

目的：探讨DNA倍体和增殖活性在骨髓增生异常综合征（MDS）的变化和意义。方法：用流式细胞仪（FCM）DNA单参数和DNA／膜抗原双参数法分析了41例MDS患者和10例良性血液病患者的骨髓细胞DNA倍体和增殖活性。结果：10例良性血液病无一例FMC0－DNA非整倍体检出，MDSFCM－DNA非整倍体检出，MDSFCM－DNA非整倍体检出率（63．4％）明显高于对照组，MDS骨髓细胞G0／G1期百分比明显高于对照组（P＜0．05），S＋G2／M期百分比明显低于对照组（P＜0．05），CD71^ ,CD15^ ,CD33^ ,HLA-DR^ 免疫标志阳性细胞多处于增殖期。结论：FCM－DNA非整倍体是MDS一个有用的诊断和预后指标，MDS造血细胞增殖分化异常。     

检测降钙素基因鉴别难治性贫血和AA的研究

目的：探讨降钙素（CT）基因高甲基化能否作为鉴别骨髓增生异常综合征-难治性贫血，（MDS－RA）和再生障碍性贫血（AA）的分子标志。方法：采用设有内、外参照的聚合酶链反应（PCR），结合限制性内切酶和激光扫描技术，检测25例MDS－RA和25例AA患者骨髓细胞CT基因5′端甲基化率（CTMR）。结果；25例MDS－RA患者CTMR为22．8±17．68％，显著高于对照组（P＜0．05），25例AA患者CTMR为9．46±14．84％，与对照组差异无显著性（P＞0．05）。25例MDS－RA中6例已随访3～10年未转化为白血病，CTMR正常，余19例中1例l．5个月后转化为原始细胞过多性难治性贫血（RAEB），3例2～9个月后转化为急性髓性白血病AML。25例再障中3例CTMR＞30％，其中2例2～4．5个月后转化为AML。结论：CTMR对鉴别MDS－RA或AA可能是一个有用的分子标志。     

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。     

骨髓增生异常综合征的染色体检测与临床分期,预后的关系

近2年半我们检测了42例骨髓增生异常综合征（MDS）患者的染色体，发现其中10例患者存在染色体畸变类型的数量异常为主。其中，低危组（RA和RAS）的染色体畸变率14．8％，临床治疗效果好，预后佳；高危组（RAEB、RAEB－T和CMML）的染色体畸变率40％，临床治疗效差，预后欠佳。两级的中位生存期、死亡率和转白率差异显著（P＜0．05）。     

骨髓增生异常综合征(WHO分型)细胞的生物学特征及其意义

目的：本研究分析骨髓增生异常综合征（MDS）的WHO诊断和分型特点，了解其细胞形态学特征，国际预后积分系统（IPSS）和染色体变化的特点以及免疫学表型特征。方法：采用回顾性研究收集近6年来我院122例确诊为MDS患者的临床资料、实验室检查资料、染色体及免疫表型结果。采用SPSS11．5软件包进行统计学分析。结果：MDS中位发病年龄为41．5岁，初诊时59．84％的患者有全血细胞减少。WHO-RCMD和FAB-RA患者比例较高，各占33．61％和50．82％。骨髓细胞形态学提示各系均有不同程度病态造血，以3系病态造血最多见（55．74％）。81例骨髓活检患者中60．49％出现病态造血，46．9％出现ALIP现象。51例患者进行细胞遗传学检查，染色体异常率为47．1％，染色体异常发生率在WHO各亚型中差异无统计学意义（P〉O．05）。IPSS积分以中危-I组最多见，染色体异常发生率随危险度上升而增高（P〈0．05）。流式细胞术检测中MDS患者CD34^＋阳性率为75％，高于同组AMLM1和AML—M2（P〈0．05）；CD33叶。阳性率为62．5％，CD13^＋阳性率为56．25％，低于同组AML-M1和AML-M2（均P〈0．05）。结论：WHO分型对MDS的早期诊治及其预后具有临床指导意义，优于FAB分型。骨髓细胞学、骨髓活检、细胞遗传学及免疫表型的联合诊断，可以减少WHO-RA假阳性率发生。如将免疫学指标列入IPSS系统，对MDS预后判断更科学。     

Clinical significance of phenotypic features of blasts in patients with myelodysplastic syndrome

Knowledge of the blast phenotype in myelodysplastic syndrome (MDS) would be valuable, as in other malignancies, but remains sparse. This is mainly because MDS blasts are a minor population in clinical samples, making analysis difficult. Thus, for this blast phenotype study, we prepared blast-rich specimens (using a new density centrifugation reagent for harvesting blasts) from blood and marrow samples of 95 patients with various MDS subtypes and 21 patients with acute leukemia transformed from MDS (AL-MDS). Flow cytometry revealed that a high proportion of the enriched blast cells (EBCs) from almost all patients showed an immunophenotype of committed myeloid precursors (CD34(+)CD38(+)HLA-DR(+)CD13(+)CD33(+)), regardless of the disease subtype. The cytochemical reaction for myeloperoxidase was negative in 58% of the cases. Thus, the EBC phenotype is more immature in MDS than in de novo acute myeloid leukemia. MDS EBCs often coexpressed stem cell antigens and late-stage myeloid antigens asynchronously, but rarely expressed T- and B-lymphoid cell-specific antigens. Markers for myeloid cell maturation (CD10 and CD15) were more prevalent on EBCs from low-risk MDS (refractory anemia [RA] and RA with ringed sideroblasts), whereas markers for myeloid cell immaturity (CD7 and CD117) were more prevalent on EBCs from high-risk MDS (chronic myelomonocytic leukemia, RA with excess blasts [RAEB], and RAEB in transformation) and AL-MDS. A shift to a more immature phenotype of EBCs, accompanying disease progression, was also documented by sequential phenotyping of the same patients. Further, CD7 positivity of EBCs was an independent variable for a poor prognosis in MDS. These data represent new, valuable information regarding MDS.     

Bmi-1 is useful as a novel molecular marker for predicting progression of myelodysplastic syndrome and patient prognosis

The International Prognostic Scoring System (IPSS) has been widely used to predict the prognosis of patients with myelodysplastic syndrome (MDS). However, IPSS does not always provide a sufficiently precise evaluation of patients to allow the appropriate choice of clinical interventions. Here, we analyzed the expression of Bmi-1, which is required to regulate the self-renewal in CD34+ cells from 51 patients with cases of MDS and acute myeloid leukemia preceded by MDS (MDS-AML). Higher positivity rate of Bmi-1 was preferentially seen in refractory anemia with excess blasts (RAEB), RAEB in transformation (RAEB-T), and MDS-AML compared with refractory anemia (RA) and RA with ringed sideroblasts (RARS). IPSS score was positively correlated with the percentage of Bmi-1 expression. Patients with RA and RARS with a higher percentage of Bmi-1+ cells showed disease progression to RAEB. Here, we propose Bmi-1 as a novel molecular marker to predict the progression and prognosis of MDS.     

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.     

Immunotyping of blasts in refractory anaemia with excess of blasts

Summary. A study of immunological markers was performed in 16 patients with newly diagnosed refractory anaemia with excess of blasts (RAEB) and RAEB in transformation (RAEB-T) and in 12 other patients with acute myeloid leukaemia evolving from RAEB or RAEB-T. Immunocytochemical investigation of bone marrow blasts was done using a modified indirect immunoperoxidase technique. This method permitted accurate morphological identification of blasts and other cells in bone marrow. The monoclonal antibodies used in RAEB and RAEB-T samples were anti-CD34, -c-kit, -HLA-DR and -CD13.
The range of CD34 expression of blasts in RAEB samples was 1–14% (mean 6·2%) and in RAEB-T samples 29–48% (mean 35·5%). CD34 positivity was detected in 3·94% (mean 47·4%) of the bone marrow blasts in acute myeloid leukaemia evolving from RAEB and RAEB-T. Expression of c-kit was demonstrated only in a low percentage of blast cells in RAEB, RAEB-T and acute myeloid leukaemia following myelodysplasia. A high percentage (> 30%) of blasts in most patients with RAEB, RAEB-T and acute myeloid leukaemia was HLA-DR and CD13 positive. We observed the transformation from RAEB to acute myeloid leukaemia in three patients. The proportion of CD34 positive blasts increased to 25% and 32% in two patients. The third patient showed an unchanged percentage of CD34 positivity of blasts.
These findings indicate that the CD34 positivity of blasts increases with the progression of myelodysplasia to RAEB-T and acute myeloid leukaemia demonstrating the instability of the clonal defect in myelodysplasia.     

Expression and prognostic significance of Bcl-2 family proteins in myelodysplastic syndromes

Excessive apoptosis is implicated in the pathogenesis of myelodysplastic syndromes (MDS). We assessed by flow cytometry the expression of several members of the Bcl-2 family in bone marrow mononuclear cells (BMMNC) of 168 MDS samples at diagnosis. The proteins studied were Bcl-2, Bcl-xL (anti-apoptotic), Bax, Bad, Bak, and Bcl-xS (pro-apoptotic). The percentage of BMMNC expressing Bcl-2 and Bcl-xL was higher in refractory anemia with excess of blasts (RAEB), RAEB in transformation (RAEB-T), and chronic myelomonocytic leukemia (CMML) than in refractory anemia (RA) and RA with ringed sideroblasts (RAS). Conversely pro-apoptotic proteins Bad, Bak, and Bcl-xS were detected in a higher percentage of cells in RA and RAS. RA and RAS were associated with an increased Bcl-xS/Bcl-xL ratio. The expression of anti-apoptotic proteins was also correlated with that of CD34 and P170 and with the percentage of blast cells. Two-color analyses demonstrated that CD34 and Bcl-2 were usually expressed in the same cells. No significant correlation was found with cytogenetic abnormalities. Higher expression of pro-apoptotic Bcl-2-family proteins (Bak, Bad, Bcl-xS) and higher Bcl-xS/Bcl-xL ratio were associated with longer survival and decreased risk of leukemic transformation in univariate analysis, whereas expression of anti-apoptotic proteins was associated with decreased survival. Consequently Bcl-2 proteins expression was well correlated with the International Prognostic Scoring System (IPSS). Our data confirm that the control of apoptosis is deregulated in MDS cells. Moreover, the study of markers such as CD34 (or Bcl-2), Bcl-xL, and Bcl-xS provides additional prognostic information.     

Deficiency of lymphocyte lectin-dependent cytotoxicity in myelodysplastic syndromes

We studied a group of patients with myelodysplastic syndromes (MDS) for surface markers and cytotoxic activities of peripheral blood mononuclear cells (PBMNC). The results indicate a significant increase in the total count of CD11b+, Leu7+ and CD16+ with a percent reduction in CD4+. A reduction in PHA-induced cellular cytotoxicity (PHA-ICC) and NK activity were found. A similar phenotype was found both in refractory anemia (RA) and (RA) with excess of blasts (RAEB/RAEB-t). However, the functional activities reached the normal level only in RA patients; while in RAEB/RAEB-t patients a significant reduction was detected in PHA-ICC and NK activity.     

The proliferative activity of myelopoiesis in myelodysplasia evaluated by multiparameter flow cytometry

Double staining of bone marrow cells for CD13 and CD33 leucocyte differentiatian antigens and for DNA content has allowed us to evaluate the proliferative capacity of myelopoiesis in patients with myelodysplastic syndromes (MDS) using flow cytometry. By analysing 39 patients (15 RA/RAS, 14 RAEB and 10 RAEB-t) and eight normal controls, we found significant differences in both the percentage of cells positive for these immature myeloid antigens between the FAB groups as well as in the fractions of CD13 and CD33 positive cells in S or S-G₂M phase of the cell cycle. Moreover, a clear decrease in the immature myeloid cell proliferative activity upon progression within the FAB groups was evident. Finally, we found a significant negative association between the percentage of myeloblasts in the bone marrow and the proliferative activity of the immature myeloid cells. indicating that the block in differentiation in MDS patients might be coupled to a simultaneous block in proliferation, especially in advanced stages. These data suggest that the use of double parameter assays in the longitudinal follow-up of MDS patients might yield new information about the biology of MDS.     

HLA-DR15 (DR2) is overrepresented in myelodysplastic syndrome and aplastic anemia and predicts a response to immunosuppression in myelodysplastic syndrome

The extent and importance of autoimmune mechanisms in myelodysplastic syndrome (MDS) and the role of immunosuppression in the treatment of this disease are not well defined. We report overrepresentation of HLA-DR2 and its serologic split HLA-DR15 in both MDS and aplastic anemia (AA). Four clinically and ethnically defined patient groups were analyzed. The HLA-DR15 antigen frequencies among North American white MDS patients (n = 72) and AA patients (n = 59), who received immunosuppressive treatment at the National Institutes of Health (NIH), were 36% and 42%, respectively. These antigen frequencies were significantly higher than that of the control population of 240 North American white NIH blood donors typed for HLA antigens by the same molecular technique (HLA-DR15, 21.3%, P =.01 for MDS, P <.001 for AA). Among North American white patients reported in the International Bone Marrow Transplant Registry (IBMTR), 30% of 341 MDS patients and 33% of 364 AA patients were positive for HLA-DR2. These antigen frequencies were higher than those reported for the general North American white population (HLA-DR2, 25.3%, P =.089 for MDS, P =.01 for AA). The DR15 and DR2 frequencies were significantly increased in MDS refractory anemia (RA) (P =.036 and P =.01, respectively) but not MDS refractory anemia with excess blasts. In the NIH MDS patients, HLA-DR15 was significantly associated with a clinically relevant response to antithymocyte globulin (ATG) or cyclosporine immunosuppression (multivariate analysis, P =.008). In MDS with RA, DR15 may be useful as a guide to pathophysiology, prognosis, and treatment.     

Immunophenotype of myeloid granulocytes: a pilot study for distinguishing myelodysplastic syndrome and aplastic anemia by flow cytometry

It is often difficult to distinguish myelodysplastic syndrome (MDS) from aplastic anemia (AA) because of the considerable clinical, cytologic histologic similarities between these two disorders; however, distinguishing between AA and MDS is of great importance because there is a higher risk of progression to acute leukemia in patients with MDS compared with AA. Up to now, CD34⁺ cells in MDS and AA patients have been studied extensively; however, little information is available on myeloid granulocytes. The aim of this study was to determine whether immunophenotype of myeloid granulocytes in AA patients was different from that of MDS. Flow cytometry was used to assess the immunophenotype of myeloid granulocytes in 22 patients with MDS, 12 with AA, and 10 normal subjects. Our data showed that the percentages of CD13⁺ granulocytes, CD33⁺ granulocytes, CD34⁺ granulocytes, and HLA-DR⁺ granulocytes were significantly higher in patients with MDS than in AA patients and normal subjects (P < 0.05). The percentages of CD15⁺ granulocytes and CD10⁺ granulocytes were significantly lower in patients with MDS than in AA patients and normal subjects (P < 0.05). There were no significant differences in the expression of these markers between patients with AA and normal subjects (P > 0.05). As refractory anemia progressing to refractory anemia with excess blasts, the percentages of CD13⁺ granulocytes, CD33⁺ granulocytes, CD34⁺ granulocytes and HLA-DR⁺ granulocytes were significantly increased, whereas, the percentage of CD15⁺ granulocytes was significantly decreased (P < 0.05). These data suggest that immunophenotype of myeloid granulocytes may be a useful parameter for the differential diagnosis of MDS and AA.