多色流式细胞术对40例急性白血病免疫分型的研究

白血病的形态学、免疫学、细胞遗传学和分子生物学分型已成为现代白血病诊断所必需,其中流式细胞术免疫分型已成为急性白血病(AL)诊断和分型的重要依据,具有重要的临床诊断意义.我们采用流式细胞仪四色荧光标记技术,CD45/SSC双参数散点图设门方法,可全面地与特异地检测出白血病细胞抗原的分布,使白血病免疫分型的结果更为准确和客观[1].     

急性白血病患者骨髓细胞形态和免疫表型同步检测的比较分析

目的 探讨骨髓细胞免疫表型在急性双表型白血病中的诊断价值以及与骨髓细胞形态学的比对研究.方法 利用流式细胞仪四色免疫荧光直接标记技术对230例急性白血病患者进行细胞免疫表型、骨髓细胞形态学分型检测及分析.采用流式细胞术四色免疫荧光直接标记技术与CD45/SSC设门分析技术检测13例双表型急性白血病患者,根据欧洲白血病免疫分类组(EGIL)积分标准进行免疫学分型.结果 230例急性白血病患者免疫分型诊断结果与骨髓细胞形态学和组织化学诊断具有高符合率,其中免疫分型诊断为急性双表型白血病的13例(5.7％).此外,急性双表型白血病患者高表达CD34胞膜抗原(84.6％),提示预后不良.结论 急性双表型白血病发病率较低,骨髓细胞免疫表型对诊断及鉴别双表型急性白血病极为特异,以髓系和B淋巴系抗原共表达为主.应用骨髓细胞形态学和流式细胞术联合检测急性双表型白血病可以提高诊断的准确性,有效地指导临床制定治疗方案和预后判断.     

新疆乌鲁木齐地区142例急性白血病流式细胞术免疫分型研究

目的:研究新疆乌鲁木齐地区急性白血病(AL)患者免疫表型特征及分布特点。方法:选用细胞表面分子CD20、CD14、CD3、CD2、CD33、HLA-DR、CD15、CD10、CD5、CD22、CD7、CD13、CD34、CD11b、CD19、CD117等的单克隆抗体,采用流式细胞仪CD45/SSC双参数散点图设门法对142例AL患者进行免疫表型分析。结果:35例急性淋巴细胞白血病(ALL),其中6例伴有髓系抗原的表达(14.3%)且表达最频繁的为CD13;96例急性髓系白血病(AML),其中21例伴有淋系抗原的表达(21.9%)且表达最频繁的为CD7;11例为FAB难以分类的急性白血病(UAL),兼有淋系和髓系抗原的表达。ALL免疫分型特点在新疆汉族和维吾尔族(简称维族)中差异无统计学意义(P>0.05),在AML中,汉族髓系抗原的表达率依次为CD13>CD33>CD15,维族髓系抗原的表达率依次为CD13>CD15>CD14,且维族患者多伴有淋系抗原CD7的表达。结论:FCM免疫分型是在细胞形态学和细胞染色基础上对AL诊断与分型的重要补充,免疫表型的检测对AL的诊断和治疗有重要意义。     

141例白血病三色流式细胞术免疫分型研究

白血病是常见的恶性肿瘤之一，其分型80年代以前主要是依靠形态学，自单克隆抗体与流式细胞术联合应用以来，使诊断白血病的准确率提高到90％以上，从而为临床合理治疗、判断预后提供了重要的科学依据。为了克服流式细胞术单参数免疫标记测定易将正常细胞包括在内从而影响结果分析的不足，我们采用以CD45和侧向角散射（side scatter，SSC）双参数散点图设门，应用三色流式细胞术对141例白血病患者进行免疫分型研究，现将结果报道如下。     

多参数流式细胞术分析急性白血病免疫表型

为了探讨急性白血病免疫表型特征,利用流式细胞术三色荧光直接标记技术CD45/SSC多参数散点图设门方法,对162例急性白血病患者幼稚细胞表面及胞浆内分化抗原进行分析.结果提示:急性非淋巴细胞白血病(ANLL)患者主要表达CD117(94.9%)、CD13(88.5%)和CD33(70.5%);急性淋巴细胞白血病(ALL)患者中B-ALL主要表达cCD79a(100%)、CD19(92.1%),T-ALL表达cCD3(100%)、CD2(83.3%);ANLL伴淋系抗原表达(Ly ANLL),以CD7(56.2%)、CD19(31.2%)多见;ALL伴髓系抗原表达(My ALL),以CD13(88.9%)、CD33(27.8%)多见.为此用多参数流式细胞术三色荧光直接标记法进行免疫分型,对急性白血病的确诊、治疗和预后有重要的临床意义.     

急性髓系白血病177例免疫分型特点及预后分析

目的:探讨急性髓系白血病(AML)患者免疫分型的特点及临床意义.方法:采用CD45/SSC双参数散点图设门方法对177例急性髓系白血病患者进行三或四色流式细胞术免疫分型分析.结果:177例AML显示,CD33,CD38,CD117,CD13及HLA-DR高度表达,阳性率分别为92.66%,81.36%,75.14%,68.93%和67.80%.40.7%的AML患者伴有淋系抗原表达,最常见的是CD7(22.6%),其次是CD19(7.91%).结论:多色流式细胞仪术免疫分型对AML的诊断及预后具有重要意义.     

195例急性白血病免疫表型分析

应用流式细胞技术可快速确定白血病细胞胞膜或胞质内的抗原，通过分析自血病细胞的抗原表达，来了解白血病细胞所属细胞系列及其分化程度，这就是白血病免疫分型。本文通过流式细胞术对195例急性白血病（AL）的免疫表型进行了分析。     

热变性对血细胞流式细胞术分析结果的影响

目的 初步探讨热变性对外周血细胞在流式细胞术分析结果中的影响,从而提高流式-荧光原位杂交（Flow-FISH）技术的应用.方法 收集5例非骨髓造血干细胞疾病患者肝素抗凝外周血标本,以CD45-Alexa Fluor（R）647标记细胞表面抗原,高温变性后,采用流式细胞术分析热变性前后外周血有核细胞的散射光信号和荧光信号变化.结果 热变性后,外周血粒细胞的侧向散射光明显缩小;单核细胞不易通过散射光被区分和设门.所有细胞CD45表达强度均减弱,以淋巴细胞为著;通过侧向散射光和CD45设门虽能大致区分各群细胞,但不及未热变性细胞清晰.结论 热变性后,外周血细胞在流式细胞术中的散射光信号和荧光信号均发生了变化,按照常规FSC/SSC和CD45/SSC方法设门进行细胞亚群分析的结果不精确,利用系列特异性荧光抗体标记是一种有前景的方法.     

34例急性白血病免疫表型与疗效关系

为提高急性白血病 (AL )疗效和生存期 ,探讨AL免疫表型与疗效关系 ,对 34例AL进行了免疫表型检测 ;采用单克隆抗体 (McAb )和流式细胞仪 (FCM )检测 ;在急性非淋巴细胞白血病中所检测的各种抗原的阳性表达率依次为CD33>CD13>CD11b >CD14,干 /祖细胞分化抗原CD34的表达率为 2 8% ,M3 不表达CD34和HLA DR。CD34+的完全缓解 (CR )率明显低于阴性组 ;免疫表型的检测和研究将有助于指导临床治疗及判断预后。     

急性白血病免疫表型分析

目的 用免疫细胞化学法检测急性白血病(AL)的免疫表型特征,从而研究其在AL诊断及分型中的意义.方法 取70例AL患者骨髓或外周血涂片,采用生物素-亲和素桥联碱性磷酸酶酶标法(ABC-AP法)检测14种单克隆抗体(McAb)的表达情况.结果 形态学无法分型的病例通过免疫细胞化学法获得明确诊断.抗原MPO、CD68、CD14、CD41均具有较强的系列特异性.HLA-DR表达率在B细胞急淋中高于T细胞急淋,在M3中低表达.CD7发生交叉表达率最高.伴有淋系抗原表达的急性髓细胞白血病(Ly+AML),易见于年龄轻的男患且贫血程度重.结论免疫表型的测定可提高AL诊断的准确性,有助于AL分型,并为治疗及判断预后提供重要依据.     

Incidence, sensitivity, and specificity of leukemia-associated phenotypes in acute myeloid leukemia using specific five-color multiparameter flow cytometry

We assessed the usefulness of 5-color multiparameter flow cytometry to detect leukemia-associated phenotypes (LAPs) in the bone marrow of patients with newly diagnosed acute myeloid leukemia (AML) and determined its usefulness for detection of minimal residual disease (MRD). Overall, 94% of patients (51/54) with AML had LAPs at diagnosis. The frequency of leukemic bone marrow/median frequency of LAPs in normal or regenerating bone marrow samples using maximum log difference statistics revealed that CD2, CD56, CD11b, CD7, and CD19 expression on AML blasts represented the most sensitive and reliable markers for detection of MRD. Serial dilutional experiments showed that the sensitivity level of immunophenotyping was between 10-4 and 10-5 and that the approach was highly reproducible.Immunophenotypic analysis using a CD45 gating strategy, 5-color staining, and an extensive panel of monoclonal antibodies allowed the identification of LAPs in 94% of AML cases, and these immunophenotypes can be used for MRD monitoring with a sensitivity limit of 10-4 to 10-5.     

Expression of Leukocyte Common Antigen (CD45) on Various Human Leukemia/Lymphoma Cell Lines

CD45 antigen (leukocyte common antigen), a unique and ubiquitous membrane glycoprotein with a molecular mass of about 200 kDa, is expressed on almost all hematopoietic cells except for mature erythrocytes. However, the biological function of this glycoprotein still remains to be resolved. In order to clarify the role of CD45 antigen in hematopoietic cell differentiation and function, its expression on human leukemia lymphoma cell lines was studied by membrane immunofluorescence. Thirty eight established cell lines were analyzed using T29–33, a monoclonal antibody (MoAb) that recognizes the common epitopes of this glycoprotein molecule. Conventional cell marker studies were also carried out on these cell lines to compare their CD45 expression. It was shown that CD45 expression varies among B lineage cells depending on cell differentiation, in contrast to its stable expression on leukemic T cell (6/6, positive) and myeloid (5/5, positive) lineage cell lines. On the other hand, only two out of six histiomonocytoid lineage cell lines were positive. Human T cell leukemia lymphoma virus type I (HTLV-I) associated T cell lines derived from peripheral blood leukocytes of patients with adult T cell leukernia/ lymphoma (ATL/L) in Japan did not express CD45 on their cell surface. Taken together, these observations suggest that CD45 has a functional role in hematopoietic cell activation and differentiation. Acta Pathol Jpn 40: 107–115, 1990.     

流式细胞术在我院急性白血病免疫分型中的应用

目的探讨本实验室所用13种单抗对白血病免疫分型的应用价值。方法应用流式细胞术（FCM）使用13种单抗对99例急性白血病进行免疫分型,与形态学分型相比较。结果使用13种单抗从99例急性白血病中分出髓系白血病（AML）59例、淋系白血病（ALL）27例、混合型（MAL）5例、未分化型（AUL）2例,诊断率达93.9%（93/99）。AML中各抗原表达情况：CD13〉CD33〉CD14、CD7〉CD2、CD19〉CD10〉CD5〉CD20,其中多表达CD13、CD33;ALL中各抗原表达情况：CD19〉CD13〉CD10〉CD20〉CD33〉CD5〉CD7〉CD2、CD14,其中CD10、CD13、CD19、CD20表达率较高。免疫分型与形态学分型结果相比较,AML二者的吻合率为90.5%（57/63）、ALL二者的吻合率为76.5%（26/34）;其中1例M0免疫分型为B/M混合、1例M1免疫分型为T/M混合、1例M2a免疫分型为T/M混合、1例L1免疫分型为B/M混合、1例L2免疫分型为T/B混合、1例L2免疫分型为Ly＋-AML、2例L2免疫分型为AUL;1例形态学为浆细胞白血病的免疫分型为AML、1例淋单混合型白血病CD7、CD13、CD14阳性。结论白血病细胞、免疫分型各具特征,形态学相同的白血病免疫分型未必相同,形态学不同的白血病免疫分型未必不同;本实验室所选单抗组合基本能满足白血病的免疫分型,对于一些混合型和未分化型白血病的诊断亦能明确。     

Expression of leukocyte common antigen (CD45) on various human leukemia/lymphoma cell lines

CD45 antigen (leukocyte common antigen), a unique and ubiquitous membrane glycoprotein with a molecular mass of about 200 kDa, is expressed on almost all hematopoietic cells except for mature erythrocytes. However, the biological function of this glycoprotein still remains to be resolved. In order to clarify the role of CD45 antigen in hematopoietic cell differentiation and function, its expression on human leukemia/lymphoma cell lines was studied by membrane immunofluorescence. Thirty-eight established cell lines were analyzed using T29/33, a monoclonal antibody (MoAb) that recognizes the common epitopes of this glycoprotein molecule. Conventional cell marker studies were also carried out on these cell lines to compare their CD45 expression. It was shown that CD45 expression varies among B-lineage cells depending on cell differentiation, in contrast to its stable expression on leukemic T cell (6/6, positive) and myeloid (5/5, positive) lineage cell lines. On the other hand, only two out of six histiomonocytoid lineage cell lines were positive. Human T cell leukemia/lymphoma virus type I (HTLV-I)-associated T cell lines derived from peripheral blood leukocytes of patients with adult T cell leukemia/lymphoma (ALT/L) in Japan did not express CD45 on their cell surface. Taken together, these observations suggest that CD45 has a functional role in hematopoietic cell activation and differentiation.     

Large cell anaplastic lymphoma: Evaluation of immunophenotype on paraffin and frozen sections in comparison with ultrastructural features

Summary Eleven cases of large cell anaplastic lymphoma (T typen=5, B typen=4, 0 typen=2) were investigated using electron microscopy and immunophenotyping on formalin-fixed paraffin sections and frozen sections of fresh tissue, to determine whether morphological criteria exist for the discrimination of T, B, and 0 phenotypes. Tumour cell lineage could not be established from ultrastructural features. On paraffin material monoclonal B-cell markers Ki-B5 and L-26 served as reliable tools for recognizing the B phenotype of large cell anaplastic lymphomas (previously determined on fresh material), whereas monoclonal antibodies MT1 (CD43) and UCHL1 (CD45RO) were of limited value in lineage determination.     

Discrepancies between morphologic, cytochemical, and immunologic characteristics in acute myeloblastic leukemia

This study was designed to compare the cytochemical pattern with the immunologic phenotype in 108 cases of acute myeloblastic leukemia (AML) classified according to the French-American-British (FAB) criteria. Special attention was paid to the cases where discrepancy existed between these approaches and to a group of 11 patients considered as unclassifiable mainly because a second cell population--megakaryoblastic--was detected. Three types of discrepancies were observed: cases with typical morphologic characteristics and cytochemistry but devoid of lineage-specific antigens; these mainly include poorly differentiated leukemias (eight M1, four M2, and eight M5a), suggesting that the cytochemical enzymes are earlier myeloid markers than the currently available monoclonal antibodies; cases in which immunologic characteristics were discordant with morphologic characteristics and cytochemistry; these include two M2 cases positive for monocytic monoclonal antibodies (CD14); six M5b cases positive for granulocytic monoclonal antibodies (CD15); and seven M4 cases lacking in CD14 or CD15 antigens; cases with discrepancies between morphologic characteristics and cytochemistry and in which the immunologic markers permitted the correct assessment of cell lineage (six cases). These results show that the classification of these patients is better achieved by a combined morphologic, cytochemical, and immunologic approach.     

急性淋巴细胞性白血病微小残留病变筛选指标的特点分析

目的 对儿童急性淋巴细胞性白血病微小残留病变(MRD)筛选指标进行分析并评估其意义和表达特点.方法 分离35例初发B-急性淋巴细胞性白血病(ALL)患儿的单个核细胞,对符合CD38、CD45弱表达,CD58、CD21、CD22强表达,CD34和Cu同时表达,染色体相关抗原CD66c表达的,与CD10/CD34/CD19进行四色抗体组合,应用流式仪检测,如在双参数点图上所选择的四色抗体组合出现的位置明显有别于正常骨髓相应位置的,则认定该抗体组合为有效的筛选标记并进行随后的MRD监测.结果 35例患儿中31例存在至少一个MRD标记,覆盖率为88.6%;21/35例患儿(60%)存在2个或2个以上的筛选标记;TdT/CD10/CD34/CD19为最常见的四色组合.结论 TdT/CD10/CD34/CD19作为四色MRD筛选标记覆盖率高,应作为常规和首选的筛选标记;免疫表型中Pro-B缺乏有效的筛选标记,出现2个或以上的筛选标记,对提高MRD的精确度具有重要意义.     

Utilization of monoclonal antibodies and immuno-scanning electron microscopy for the positive identification of human leukemic cells

Monoclonal antibodies generated against normal and leukemic human leukocytes were tested for their differential reactivity with leukemia and lymphoma cell lines as well as with circulating lymphoid and myeloid leukemic cells by means of immuno-scanning electron microscopy (immuno-SEM). Anti-T (OKT3), anti-mu-chain, anti-CALLA (J5), anti-BA-1, anti-BA-2, and anti-nonlymphoid (Mol) monoclonal antibodies were covalently conjugated to polystyrene latex microspheres (immunolatex), using a two-step glutaraldehyde reaction, and subsequently incubated with the various cell types. Cultured B-type Burkitt lymphoma cells (Daudi) and chronic lymphocytic leukemia (CLL) cells displayed extensive labeling with monoclonal anti-mu, anti-B1, and anti-BA-1 immunolatex conjugates, while cultured malignant T cells (HD-Mar) showed positive labeling with OKT3 immunolatex alone. Cultured myelomonocytic cells (GDM-1) and cells obtained from patients with acute myeloblastic (AML) and monoblastic leukemia (AMoL) labeled only with anti-Mol immunolatex, while cultured promyelocytic cells (HL-60) displayed far less labeling with this conjugate. Common-type acute lymphoblastic leukemia (C/ALL) cells were labeled predominantly with the J5 (anti-CALLA) and anti-BA-2 immunolatex conjugates. Evidence is presented indicating that immuno-SEM employing monoclonal antibodies is a reproducible technique which may be used in the study of leukocyte maturation and may provide additional information in the classification of poorly differentiated leukemias.