185例儿童急性白血病流式细胞术免疫分型研究

目的：探讨儿童急性白血病流式细胞术免疫分型的意义。方法：采用流式细胞术三色荧光标记技术和CD45／SSC双参数散点图设门,检测185例儿童急性白血病的免疫表型,对抗原表达情况进行分析。结果：流式细胞术免疫分型和FAB分型的符合率为89．19％。185例儿童急性白血病中,ALL为121例,占AL的65．41％,B—ALL为113例,主要表达B系的CD19（99．12％）、CD22（98．13％）、CD79a（96．19％）、CD10（86．73％）。T—ALL占8例;主要表达CD5（100％）、CD7（100％）、cCD3（100％）、CD8（87．5％）。AML为47例,占25．41％,主要表达CD33（93．62％）、CD15（78．72％）、CD64（76．6％）、MPO（76．6％）、CD13（74．47％）。在B—ALL,AML,T—ALL中,敏感性最高的抗体分别是CD19,CD33,CD5和CD7,特异性最强的抗体分别是CD79a,MPO,cCD3。AMLL为17例,占9．19％,其中B／M为9例,T／B为5例,T／M为3例。My＋-ALL为54例,占ALL的44．63％,表达的髓系抗原为CD13、CD15、CD33、CD64。Ly＋-AML为18例,占AML的38．30％,表达的淋系抗原为CD19、CD4、CD7。系列非相关抗原CD34的表达率为67．57％,HLA—DR的表达率为85．41％,CD38的表达率为80．59％,TdT的表达率为62．59％。结论：流式细胞术免疫分型在白血病分型中起重要作用,是FAB分型的补充和修正,提高了儿童急性白血病诊断的准确率。有必要进一步加强流式细胞术免疫分型的标准化工作。     

急性B淋巴细胞白血病儿童化疗后微小残留病灶免疫表型改变的分析

目的:分析儿童急性B淋巴细胞白血病(B-cell acute lymphoblastic leukemia,B-ALL)化疗过程中首次微小残留病灶(minimal residual disease,MRD)免疫表型的变化规律及特点,为临床诊断及后续微小残留病的监测提供依据。方法:回顾性分析我院2013年1月至2018年4月收治的393例B-ALL患儿的免疫分型结果及诱导化疗第15天首次MRD数据。结果:(1)在393例白血病中,B-ALL相关特征性免疫表型的出现频率为:CD19+/CD10+/34+64.4%;CD19+/CD10+/34部分表达,15.5%;CD19+/CD34+/CD20+,58.5%;CD19+/CD10+/CD13+,13.2%;CD19+/CD10+/CD33+,5.9%;CD19+/CD10+/CD117+,0.7%;CD19+/CD10+/CD123+,50.1%;CD19+/CD10-/34±,5.9%;CD19+/CD10-/CD20-,2.5%;CD34bright,12.2%;(2)共有285例首次MRD检测结果呈阳性,有181例(63.5%)MRD检测结果至少有1个抗原荧光强度发生改变,其中出现一个抗原强度变化的为83例(29.1%),2个抗原荧光强度变化的为57例(20.0%),3个抗原荧光强度变化为31例(10.9%),4个及4个以上抗原荧光强度变化为10例(3.5%)。抗原荧光强度变化频率最高的依次为CD45、CD34、CD20;(3)共有7例患儿复发,复发时行免疫分型检测,其中4例与初发时有抗原荧光强度变化。结论:(1)儿童B-ALL远高于其他白血病类型,且具有独特的相关特征性免疫表型。初发B-ALL免疫分型结果不仅可完善白血病MICM分型,更是化疗后MRD监测的线索及客观依据;(2)在儿童B-ALL化疗过程中,免疫表型极有可能会发生变化,在后续的微小残留病灶检测过程中应注意对变化抗原的判断和追踪。     

急性混合细胞白血病独特临床生物学特征及预后研究

目的:分析急性混合细胞白血病(HAL)独特的临床生物学特征及预后。方法:采用流式细胞术(FCM)分析白血病细胞的免疫表型,最终确诊56例HAL患者,对其骨髓标本进行细胞形态学及相关细胞化学染色分析,以确定其FAB分型,用聚合酶链反应检测骨髓细胞DNA IgH及TCRγ基因重排,采用兼顾急性髓细胞性白血病(AML)和急性淋巴细胞白血病(ALL)的方案治疗,同时结合多项临床生物学指标分析其转归和预后。结果:确诊的HAL其FAB分型以AML-M1/M2、ALL为主;其免疫分型以B系和髓系混合表达多见。CD34在HAL中呈高表达并且是对患者预后具有影响力的因素(P=0.03)。确诊的56例HAL患者中,28例出现IgH基因单克隆重排阳性(50.76%),22例出现TCRγ基因单克隆重排阳性(39.65%),其中2例IgH和TCRγ基因单克隆重排同时出现阳性。此类患者对治疗反应差、缓解率低为36%。结论:HAL属特殊类型白血病,有其独特的临床生物学特征,对化疗方案不敏感,预后较差。     

28例急性巨核细胞白血病实验室检查结果分析

目的:分析急性巨核细胞白血病(AMKL)患者实验室检查特点。方法:4管用8色抗体组合对28例AMKL患者的骨髓有核细胞进行免疫表型分析,同时结合分析患者骨髓细胞形态学、融合基因和染色体核型等检查结果。结果:28例AMKL患者中阳性表达率较高的是巨核细胞相关抗体:CD41a、CD61、CD42b、CD36,阳性率分别为81. 48%、92. 86%、72. 00%、70. 83%,其中,CD41a、CD61、CD42b三种抗体共表达的患者占53. 57%,至少表达两种抗体的患者占82. 14%。髓系祖细胞相关标志:CD117、CD34、CD38、HLA-DR阳性表达率分别为64. 29%、42. 86%、64. 29%和46. 15%,与非APL的AML患者相比表达率均较低(P 0.01);髓系全程抗原CD13、CD33在AMKL中阳性表达率与非APL的AML之间无统计学差异。髓系中后期抗原CD15及单核系抗原CD64、CD14、CD300e和胞浆抗原MPO、cCD79a和cCD3均阴性。与非Down综合征相关AMKL(non-DS-AMKL)相比,CD7与CD11b的表达在Down综合征相关AMKL(DS-AMKL)中较高(P 0.05)。AMKL患者中17例(65.4%)为复杂染色体核型,5例为+21染色体异常;仅5例患者核型正常。25例行白血病融合基因筛查,24例(96%)患者WT1基因表达增高(40.24±59.14%),12例患者(70.58%) EVI1基因表达增高(53.93±37.98%),4例患者融合基因阳性(2例MLL-AF9阳性,1例TLS-ERG,1例P210 BCL/ABL)。结论:AMKL中82.14%患者表达至少两种巨核细胞相关标志,髓系祖细胞标志表达相对较低,多为复杂染色体核型异常,WT1及EVI1异常表达率较高。     

正常人间围血及骨髓单个核白细胞和白血病肿瘤细胞的免疫分型

本文用10种单克隆抗体(McAb)分析了正常人周围血及有髓单个核细胞的免疫表型,以及急性淋巴细胞白血病(ALL)和急性髓细胞白血病(AML)的免疫表型,并以免疫双酶标记法观察了非T-ALL肿瘤细胞的肿瘤相关核仁抗原(HMNA)和细胞表面抗原的表达。结果:不同年龄正常群体周围血CD_4~+细胞数及CD_4/CD_8比值有差异;约3％的正常骨髓单个核细胞CD_(10)~+(ALL的抗原)。应用单克隆抗体对白血病的免疫分型,不仅能确诊肿瘤的谱系,还能了解细胞分化阶段。本文讨论了AML和ALL免疫分型中的鉴别性McAb。HMNA为多种肿瘤细胞的标记,本文观察到幼稚B细胞白血病及毛细胞白血病的肿瘤细胞中HMNA亦为阳性。     

Daxx在急性白血病的表达及意义

目的研究Daxx在急性白血病(AL)中的表达及其与AL的分型、临床特征、疗效及预后的关系.方法应用免疫组织化学方法检测88例初治AL骨髓细胞Daxx蛋白的表达情况,分析其与FAB分型、临床特征、疗效及预后的关系.结果急性髓细胞白血病(AML)骨髓细胞中Daxx的表达显著高于正常对照组(P<0.05)与急性淋巴细胞白血病(ALL)组(P<0.05).在AML亚型中,Daxx在M3中的表达显著高于M1,M2,M3,M4和M5.采用逐步回归法分析,校正其它参数,Daxx的表达与初诊时的白细胞数及骨髓原始细胞数目呈正相关,与疗效呈负相关. 结论 Daxx在急性髓细胞白血病中广泛表达,其表达的紊乱可能在AML的发病中起作用,还与AML的某些临床特征、疗效及预后密切相关.     

新融合蛋白CD19scFv/CD80抗白血病作用研究

B系急性淋巴细胞白血病(B-ALL)是一种十分常见的血液系统恶性肿瘤,尽管目前化疗效果显著,但仍有部分儿童和成人B-ALL患者疗效较差、预后不佳.研究表明,ALL细胞表面免疫共刺激分子CD80(B7.1)表达降低或不表达,导致白血病细胞不能有效地被细胞毒T细胞(CTL)识别杀伤.然而,几乎所有的B-ALL细胞均表达CD19表面抗原.因此,本研究利用前期构建的CD19单链抗体(scFv)/CD80融合基因及表达的融合蛋白,通过融合蛋白中抗CD19scFv,将CD80结合到B-ALL白血病细胞表面,靶向激活CTL细胞,起到杀伤白血病细胞的作用.在体外共培养实验中,该融合蛋白结合B-ALL细胞系Nalm-6细胞后,可诱导淋巴细胞增殖,分泌细胞因子并产生较显著的特异性细胞毒作用.以Nalm-6细胞在免疫缺陷小鼠(Mus musculus)中建立白血病动物模型,CD19scFv/CD80融合蛋白联合输注淋巴细胞可显著延长小鼠的生存时间.本研究表明,该融合蛋白可使逃避免疫监视的B-ALL细胞有效地转化成抗原提呈细胞,进而激活特异性免疫反应,具有潜在的临床应用价值.     

髓细胞性白血病患儿PTEN蛋白表达及其与免疫表型的关系

目的:探讨髓细胞性白血病(AML)患儿PTEN蛋白表达及其与免疫表型的关系。方法:选择AML患儿143例,根据免疫表型的不同分为两组:免疫分型为LY+AML型59例,LY-AML84例。所有患儿都给予免疫表型分析,检测PTEN蛋白表达情况并进行相关性分析。结果:LY+AML患儿CD34+阳性、CD117+阳性比例显著高于LY-AML患儿(P 0.05),染色体核型异常比例显著低于LY-AML患儿(P 0.05)。LY+AML患儿的PTEN蛋白表达量为(65.33±2.34)%,阳性表达率为94.9%;而LY-AML分别为(20.11±4.11)%和13.1%,与LY+AML患儿对比差异都有统计学意义(P 0.05)。在AML患儿中,Spearman相关分析显示PTEN蛋白表达水平与免疫分型呈现显著相关性(r=0.653,P=0.000)。多因素logistic回归方法显示PTEN蛋白表达、CD34+阳性、CD117+阳性、染色体核型异常为影响AML患儿免疫表型的主要独立危险因素(OR=1.098、1.045、1.092、0.294,P 0.05)。结论:AML患儿骨髓单个核细胞的PTEN蛋白表达上调使得LY+AML型发生风险显著增加,可作为AML患儿病情判断与预后预测的参考指标之一。     

急性髓系白血病免疫表型特征及遗传学特征分析

目的:研究急性髓系白血病免疫表型特征以及遗传学特征。方法:选取2011年1月到2014年5月我院收治的急性髓系白血病患者169例,采用流式细胞术和相关的单克隆抗体来分析所有患者的骨髓免疫表型,采用染G染色体显带技术分析患者的核型,根据淋系抗原(lym Ag)的表达将患者分为lym Ag+组和lym Ag-组。结果:抗原CD13、CD33、CD117以及MPO等髓系抗原最常在急性髓系白血病患者中表达,其中CD117在M3型病例中表达为85.7%(24/28),而CD34、HLA-DR双阴性、较强的自发荧光、CD13、CD33和MPO对M3型的诊断也具有一定的价值;其中47.9%(81/169)的患者伴随着淋系抗原表达,以CD7和CD56为主;60.4%(102/169)的患者伴随着核型异常;而伴随着t(8:21)的M2患者中的CD15、CD19和CD56的表达显著增强,而t(15:17)均发生于M3型患者中;而lym Ag+组患者CD34的阳性患者为77.8%(63/81)显著高于lym Ag-组的47.7%(42/88),两组比较差异具有统计学意义(P0.05)。结论:免疫表型对急性髓系白血病的诊断具有重要的意义,且免疫表型和异常核型存在密切的联系。     

CD47 在急性白血病干细胞中的表达及其对临床疗效及预后的影响

目的:探讨CD47在急性白血病患者骨髓白血病细胞的表达及其临床意义。方法:选择2013年5月-2015年5月在我院确诊的急性白血病患者101例作为研究对象,其中急性淋巴细胞白血病50例(ALL组),急性髓系白血病51例(AML组)。另选取同期在我院接受体检的健康志愿者39例作为对照组。采用流式细胞仪检测白血病细胞表面CD47的表达情况,并分析CD47表达与急性白血病患者临床疗效及复发情况的关系。结果:急性白血病患者白血病细胞CD47的阳性表达率明显高于健康对照组,差异具有统计学意义(P0.05);而ALL组与AML组患者白血病细胞CD47的阳性表达率比较差异无统计学意义(P0.05);CD47阴性表达的急性白血病患者CR率显著高于阳性表达者,差异具有统计学意义(P0.05);ALL组和AML组CD47阴性表达患者CR率显著高于CD47阳性表达患者,差异具有统计学意义(P0.05),但两组之间比较,差异无统计学意义(P0.05);CD47阳性表达的急性白血病患者复发率显著高于阴性表达患者,差异具有统计学意义(P0.05);ALL组和AML组CD47表达阳性患者复发率明显高于阴性患者,差异具有统计学意义(P0.05),但两组之间比较差异无统计学意义(P0.05)。结论:急性白血病患者白血病细胞表面CD47的表达异常升高,且与白血病患者的疗效和预后有关,CD47可能作为一种急性白血病的诊断及疗效和预后的辅助评估指标。     

Immunophenotyping of acute lymphoblastic leukemia using immunohistochemistry in bone marrow biopsy specimens

Flow cytometry is the preferred method of diagnosing and immunophenotyping acute lymphoblastic leukemia (ALL). However, there are situations in which immunohistochemical staining (IH) of bone marrow trephine biopsy specimens can be used to provide immunophenotypic information. To evaluate the use of IH and to confirm its value in diagnosing and typing of ALL, we studied 50 cases of denovo ALL that were previously classified into pre B, T and B by morphologic, cytochemical and FC methods. Paraffin embedded bone marrow trephine biopsies sections were stained using a panel of antibodies,namely, myeloperoxidase (MPO), terminal deoxynucleotidyl transferase (TdT), CD10, CD20, CD79a, CD3. The cases included 37 pre BALL, 10 T ALL and 3 mature BALL. TdT was the most commonly expressed antibody and was positive in 41 of 50 cases of ALL (82%) and in 95% of pre B ALL cases. CD79a and CD10 were positive in 68% and 65% of pre B ALL cases, respectively. CD79a showed similar positivity in B ALL cases (66%). CD 20 was positive in 66% of mature B ALL cases but less positive in pre B ALL (22%). CD3 was positive in 70% of T ALL cases and negative in other ALL subtypes. All of the cases were negative for MPO. Diagnosis and immunophenotyping of acute lymphoblastic leukemia is possible using immunohistochemical staining of bone marrow trephine biopsies.     

Flow cytometric characterization of phenotype,DNA indices and p53 gene expression in 55 cases of acute leukemia

OBJECTIVE: To characterize the phenotype of acute leukemia cases using flow cytometry, to detect mixed lineage cases and to use DNA index determination, including S-phase fraction (SPF) and p53 detection, to find if there was any correlation of SPF and p53 expression with outcome. STUDY DESIGN: Fifty-five cases of acute leukemia were enrolled in this study. A complete hemogram and routine bone marrow examination, including cytochemistry, was done. Mycloperoxidase-negative cases were evaluated on a flow cytometer using monoclonal antibodies. DNA indices were determined by flow cytometry in all cases, and p53 was detected immunohistochemically using the alkaline phosphatase/antialkaline phosphatase technique. RESULTS: Acute myeloblastic leukemia (AML) was diagnosed in 32 cases; acute lymphoblastic leukemia (ALL) was diagnosed in 18 (14 B lineage and 4 T line age). Four cases showed mixed lineage leukemia, and undifferentiated acute leukemia was diagnosed in one case. The mean/range of SPF for these groups were 3.76/0.33-6.91, 6.25/0.15-21.4, 2.89/0.35-10.64, 2.60/0.72-6.94 and 7.34, respectively. Aneuploidy was detected in two cases of B-lineage ALL and tetraploidy in a case of AML-M7, while all others were diploid p53. Was detected in 6 of 55 cases (10.90%). Follow-up was available for 24 patients. Five patients relapsed, and four had B-cell type ALL and were diploid and expressed no p53 gene. SPF% did not show any correlation with outcome. CONCLUSION: These data suggest that within acute leukemia subtypes, there is a wide variation in SPF. SPF does not seem to correlate with outcome. Immunophenotyping is essential to determine the lineage in myeloperoxidase-negative cases. It is perhaps the only way to diagnose mixed lineage leukemia and aberrant expression of markers presently. The p53 gene was detected less frequently. However, more studies are required from different centers with longer follow-up to evaluate prognostic significance.     

Distribution of the cytoskeletal protein,Nestin, in acute leukemia

Abstract

Nestin is a neuroepithelial stem cell marker that is expressed in some types of tumor cells. Recent reports suggest that Nestin may be closely related to malignant cell proliferation and migration. Acute leukemia (AL) is characterized by a lack of differentiation, which results in uncontrolled proliferation in the bone marrow and accumulation of immature cells. The expression and function of Nestin in AL is unclear. We investigated Nestin immunohistochemical patterns of 87 patients that included 47 cases of acute myeloid leukemia (AML) and 40 cases of acute lymphoblastic leukemia (ALL), and 20 patients in complete remission (CR) from AML or ALL. We also investigated the clinico-pathological features of 87 cases of AL and their CR and overall survival (OS). Nestin was expressed in leukemic blasts and mature granulocytic cells in most cases (39/47) of AML. Conversely, Nestin was expressed in mature granulocytic cells in fewer cases (6/40) of ALL, but not in blasts. Nestin expression appeared in leukemic blasts of AML, but not ALL. Nestin expression in AML blast cells was not associated with CR or OS. We provide evidence that Nestin is expressed in AL and might be a useful immunohistochemical marker for identifying AML and ALL.     

Requirement for NK cells in CD40 ligand-mediated rejection of Philadelphia chromosome-positive acute lymphoblastic leukemia cells.

We have previously developed a murine model of Philadelphia chromosome-positive acute lymphoblastic leukemia by i.v. injection of a pre-B ALL cell line (BM185) derived from Bcr-Abl-transformed BALB/c bone marrow. We are studying the potential to elicit autologous antileukemic immune responses by introducing genes encoding immunomodulators (CD40 ligand (CD40L), CD80, and GM-CSF) into leukemia cells. BM185 cells expressing CD40L or CD80 alone, when injected into BALB/c mice, were rejected in approximately 25% of mice, whereas cohorts receiving BM185 cells expressing two or more immunomodulator genes rejected challenge 50-76% of the time. The greatest protection was conferred in mice receiving BM185 cells expressing all three immunomodulators. Addition of murine rIL-12 treatments in conjunction with BM185/CD80/CD40L/GM-CSF vaccination allowed rejection of preestablished leukemia. BM185 cell lines expressing CD40L were rejected in BALB/c nu/nu (nude) mice, in contrast to cell lines expressing CD80 and/or GM-CSF. Nude mice depleted of NK cells were no longer protected when challenged with BM185/CD40L, demonstrating a requirement for NK cells. Similarly, NK cell depletion in immunocompetent BALB/c mice resulted in a loss of protection when challenged with BM185/CD40L, confirming the data seen in nude mice. The ability of CD40L to act in a T cell-independent manner may be important for clinical applications in patients with depressed cellular immunity following chemotherapy.     

Expression of CD13/aminopeptidase N in precursor B-cell leukemia: role in growth regulation of B cells

Expression of cell surface CD13 in acute B-cell leukemia (ALL-B) is often viewed, as an aberrant expression of a myeloid lineage marker. Here, we attempted to study the stage specific expression of CD13 on ALL-B blasts and understand its role in leukemogenesis as pertaining to stage of B-cell ontogeny. A total of 355 cases of different hematological malignancies were diagnosed by immunophenotyping. Among 68 cases of early B-cell ALL, 22 cases with distinct immunophenotype was identified as immature B-cell ALL. Blasts from these ALL-B patients demonstrated prominent expression of CD10, CD19, CD22, but neither cytoplasmic nor surface IgM receptors. This strongly indicates leukemogenesis at an early stage of B-cell development. We also identified, the existence of a subpopulation of cells with remarkably similar phenotype in non-leukemic marrow from healthy subjects (expressing CD10, CD19, CD22, CD24, Tdt together with the co-expression of CD13). This sub-population of B cells concomitantly expressing CD13 appeared to be a highly proliferating group. By blocking their cell surface CD13 in leukemic blasts with monoclonal antibody we were able to inhibit their proliferation. We hypothesized that neoplastic transformation at this stage may be facilitated by CD13. CD13 may thus be an important target for novel molecular therapy of early stage acute B-cell leukemia.