三色荧光标记抗体在急性白血病免疫分型中的应用

目的：探讨多参数分析流式细胞术白血病免疫分型的方法。方法：用自行设计的七组三色荧光标记抗体组合，共含15种抗体，对200例急性白血病患者骨髓或外周血进行流式细胞术免疫分型。结果：应用本组抗体组合可对T-急性淋巴细胞白血病、B-急性淋巴细胞白血病和急性粒细胞白血病-M1-M7患者作出免疫学诊断，并可诊断形态学难以辨认的及一般免疫分型法较难诊断的单核细胞白血病。结论：三色荧光标记抗体组合可用较少的抗体，特异地对急性白血病各亚型进行多参数免疫表型分析，提高了免疫分型的准确性。     

细胞表面分化抗原和胞浆内抗原在急性白血病免疫分型中的应用

目的　探讨细胞表面分化抗原CD117及胞浆内抗原CD79a、CD3、MPO在急性白血病细胞的表达规律及意义。方法　对2001-02~2003 -12哈尔滨医科大学附属第二医院血液科39例初诊急性白血病病人,采用CD45 /SSC双参数散点图设门方法,运用直接免疫荧光技术,进行三色流式细胞术免疫表型分析。结果　双表型急性白血病(BAL)CD117表达率为87. 5%。急性髓系白血病(AML)CD117表达率为88%、cMPO表达率为70%,cCD79a及cCD3 在AML中未见表达。6例急性淋巴细胞白血病(ALL)中2例B ALL,cCD79a均为阳性,CD117均为阴性; 3例T ALL,cCD3 均为阳性,其中1例CD117阳性; 1例T、B混合型ALL,cCD79a及cCD3 均为阳性,并伴有CD117阳性。结论　检测CD117、cMPO、cCD79a、cCD3 有助于鉴别AML、ALL、与BAL,且采用CD45 /SSC双参数散点图设门方法,能排除骨髓中正常细胞的干扰,并可直接地证明双表型或双克隆型急性白血病。     

白血病免疫表型分析

目的：应用流式细胞术研究白血病免疫表型的特点.方法：采用单克隆抗体直接免疫荧光标记法，对209例白血病患者进行免疫表型分析.结果：CD13、CD33是急性髓细胞白血病特异和敏感的指标，CD2、CD7是T-淋巴细胞白血病（T-ALL）特异和敏感的指标，CD19、CD10是B-ALL特异和敏感的指标.结论：运用流式细胞术对白血病进行免疫分型可以极大提高诊断的准确性，指导临床治疗和判断预后。     

91例急性白血病FCM免疫分型分析

目的：研究急性白血病免疫分型及临床意义。方法：采用流式细胞术检测91例急性白血病患者免疫分型情况。结果：①80％以上急性非淋巴细胞白血病(ANLL)患者主要表达CD13、CD33。②按免疫表型的特征可将急性白血病分为3类：系列专一性表达；交叉表达；“裸细胞”型。ANLL中，以系列专一性比例最高，交叉表达在ALL中占有一定比例，“裸细胞”型在ANLL和ALL中比例均最少。交叉表达的病例中，CD7^ ANLL患者CR率低于系列专一性表达者。结论：AL免疫分型可出现系列专一性表达；交叉表达；“裸细胞”型3种类型，CD7^ ANLL交叉表达的患者完全缓解率低于系列专一性表达者。     

116例急性髓系白血病免疫表型特点分析

目的探讨免疫表型检测对急性髓系白血病(AML)的诊断、治疗、预后的临床意义。方法回顾性分析已经确诊的116例AML患者的免疫表型结果。免疫表型检测采用流式细胞术对AML患者的骨髓样本进行多种抗原的检测。结果 116例AML白血病患者主要表达CD33(91.4%)、CD13(94%)、CD117(69%)、HLA-DR(70.7%)、CD34(71.6%),M3较少表达CD34及HLA-DR。淋系抗原在AML患者中也有阳性表达,其中CD56、CD7、CD19阳性率最高,分别为17.2%、15.5%、9.5%。伴有CD56、CD7阳性的AML患者CR率低于CD56、CD7阴性的AML患者。结论白血病免疫表型检测对AML分型诊断、预后判断有重要的指导意义。     

微量APAAP法与常规APAAP法在急性白血病免疫分型中应用对比

用常规APAAP法进行急性白血病免疫学分型需血量较多,对小儿患者及需反复抽血的成人采血痛苦也大,微量采血法较方便、经济,且可减少采血量。本文就两种方法的染色结果进行了对比研究。1 材料和方法1.1 病例急性淋巴细胞性白血病(ALL)10例,年龄2～60岁,中位年龄6岁。急性非淋巴细胞性白血病(ANLL)10例,年龄3～70岁,中位年龄26岁。以上病例外周血幼稚细胞均高于50%。1.2 器材与试剂微试管,内径5mm,长3～     

自配试剂在Coulter流式细胞仪检测急性白血病免疫分型中的应用

目的 分析自配Coulter流式细胞仪试剂在检测急性白血病免疫分型中的应用效果.方法 将自配全血溶解试剂和鞘液及清洗液的理化性质、准确度和精密度以及对检测急性白血病免疫表型抗原的应用效果与原装试剂进行比较分析.结果 自配试剂与原装试剂理化性质基本一致;两种试剂分别测定同一白血病免疫分型标本20次,结果各胞膜抗原批内CV值均＜2%;检测急性白血病免疫分型胞膜和胞浆多种抗原,分别与原装试剂比较,两者差异均无统计学意义(P＞0.05),且检测胞浆抗原cMPO和cCD79a结果均呈高度正相关(r＞0.970).结论 自配Coulter流式细胞仪试剂对白血病免疫分型的检测效果好,可代替原装试剂进行应用并推广.     

急性髓系白血病112例流式细胞术检测免疫表型分析

目的探讨流式细胞术对急性髓系白血病（AML）不同亚型抗原表达检测的作用。方法采用流式细胞术CD45／SSC双参数散点图设门方法对112例初治AML患者骨髓细胞进行12种抗原检测,结合FAB分类,比较不同亚型中抗原表达差异。结果在112例初治AML中,CD13、CD33、CD34的表达常见,分别为100％、94．6％、77．8％;在淋系抗原中,CD2的表达最为常见（30．4％）,其次为CD7（26．8％）;且某些免疫表型与FAB分类具有相关性。结论流式细胞术有助于AML的准确诊断及分型,对于AML患者治疗方案选择及预后的判断具有重要价值。     

急性淋巴细胞白血病免疫分型的特点及其临床意义

目的:为了探讨急性淋巴细胞白血病(ALL)各亚型免疫分型的特点及其临床意义。方法:采用CD45/SSC双参数散点图设门,应用三色流式细胞术,对81 例ALL的初诊患者骨髓标本进行免疫分型,并对其中45例进行核型分析。结果:①B细胞系列的ALL(B ALL)中CD19表达最常见(阳性率为100%),而T细胞系列的ALL(T ALL)中CD5和CD7表达阳性率最高,均为90%;B -ALL和T- ALL都存在抗原交叉表达的现象;两组患者的完全缓解(CR)率差异无统计学意义(P>0.05);②伴髓系抗原表达的急性淋巴细胞白血病(My+ALL)比较常见,本组达到39.5%,常累及B淋巴系统(占My+ ALL的84.4%);各髓系抗原中以CD13 表达阳性率最高;此类患者的CR率较高,儿童CR率为72.2%,成人为78.6%;③急性杂合性白血病(HAL)的发病率为19.8%,以髓系、B系共同表达者居多;并且CD34表达阳性率较高(81.3%),该类患者CR率较低(儿童和成人分别为50%和40%);④CD34在B ALL,My+ALL和HAL中表达阳性率较高,而T ALL中少见(P<0.05)。结论:免疫分型在诊断特殊类型的ALL(如HAL,My+ALL)中具有显著优势;CD19和CD5诊断B- ALL和T- ALL的灵敏度较好,但特异性不高,存在抗原交叉表达;CD34和髓系抗原的表达与CR率无相关性,但在HAL,CD34的表达与CR率成负相关。     

较难分类的急性白血病的免疫分型研究

报告２７例接传统方法较难分类的急性白血病（ＡＬ）的免疫分型结果，其中２６例ＰＯＸ阴性。结合免疫表型等检查，证实为急性双表型白血病（ＡＢＬ）１５例、急性未分化白血病（ＡＵＬ）４例，急性髓系白血病（ＡＭＬ）Ｍ０型４例、急性淋巴细胞白血病（ＡＬＬ）２例、生全髓细胞白血病（ＡＰＭ）和急性巨核细胞白血病（ＡＭＫＬ）各１例。论文该类病例进行髓系过氧化物酶（ＭＰＯ）和分化抗原的免疫学检查有重要意义。     

细胞分化抗原胞浆CD79a在急性白血病细胞上的表达

目的探讨细胞分化抗原胞浆CD79a(CyCD79a)在各型急性白血病细胞上的表达情况及其意义。方法选取2003年1月至2004年4月河北医科大学第二医院急性白血病患者214例及外院选检病例标本7例,采用CD45/SSC双参数散点图设门方法和三色流式细胞术,对急性白血病患者221例各型急性白血病的细胞免疫表型CyCD79a等B系分化抗原进行检测,比较各型急性白血病患者的表达率。结果急性B淋巴细胞白血病(B-ALL)患者CyCD79a表达率为100%(57/57),高于CD10、CD19、CD20和胞浆CD22(CyCD22)等其他B系分化抗原表达率;急性髓细胞白血病(AML)患者CyCD79a表达率为0.7%(1/134),低于或等于上述其他B系分化抗原表达率;急性T淋巴细胞白血病(T-ALL)患者CyCD79a表达率则为13.3%(2/15)。结论Cy-CD79a对B-ALL具有较高的敏感性和较好的特异性。因此,CyCD79a可以作为诊断B-ALL的一线和首选免疫标志,并可用于B-ALL与T-ALL和AML的鉴别诊断。     

Proposed minimal panel of antibodies for cost-effectiveness and accuracy in acute leukemias immunophenotyping: Prospective study at a tertiary care center

Introduction: Flowcytometry has an essential role in the diagnosis and classification of acute leukemias. However, there exists a great degree of inter-laboratory variability on issues like panel selection, antibody combinations, gating strategies, fluorochromes, and clonal selection.

Aim: The primary aim of this study was to derive a minimal panel of antibodies and evaluate its diagnostic usefulness in acute leukemias by flowcytometry by using the detailed immune-phenotype of different lineage-specific or non-specific markers.

Materials and methods: This prospective observational study involved 400 newly diagnosed cases of acute leukemias. Bone marrow aspirate samples were subjected to morphological evaluation, cytogenetics and flow cytometric immunophenotyping.

Results: A minimal panel of eight antibodies comprising of CD45/CD34/CD19/MPO/cytoCD3/CD64/CD117/CD79a was derived by applying different permutations and combinations with a diagnostic yield of 97.5%. The minimal panel was further validated by testing in an independent cohort of patients with similar demographic characteristics, where it showed a high diagnostic yield of 98% in comparison with the screening panels proposed by other recently published studies.

Conclusion: It may be concluded that the diagnostic performance of the eight antibody panel is better than most other panels used across the different laboratories in terms of yield, number of antibodies used and the scientific approach used to derive and validate the results and so henceforth may be applied in any setting with limited resources for better diagnostic accuracy.     

Flow cytometry in acute leukemia

Immunophenotyping of acute leukemia is one of the most important clinical application of Flow cytometery. The aim of this work is to review recent advances in flow cytometery methods, quality control, troubleshooting and its prevention and data analysis of acute leukemia. Multiparameter flow cytometery is a useful adjunct to morphology and cytochemistry and it is an invaluable tool in the diagnosis of acute leukemia.     

成人双表型急性白血病的临床特征、生物学特性及疗效的研究

目的：探讨成人双表型急性白血病(BAL)的临床特征、生物学特性、治疗效果及预后。方法：采用流式细胞仪分析急性白血病的免疫表型，根据EGIL标准诊断BAL。结果：成人BAL的构成比为8．9％，CD34表达阳性率为75.0％，P70表达阳性率为70.0％，明显高于同期非BAL(均P＜0.05)；12例患者治疗后完全缓解率为33．3％，12个月总生存率为8．3％，明显低于同期非BAL(P＜0．05，P＜0．01)；其疗效与CD34和P70的表达呈负相关。结论：BAL常伴有较多的不良预后因素，治疗效果差，生存期短，合适的治疗方案还有待于进一步探讨。     

Flow cytometry study of cell cycle, apoptosis and drug resistance in acute leukemia

The response to therapy of leukemic cells is largely determined by their capacity of proliferation and apoptosis in presence of the administered drugs. We describe here the main markers used in flow cytometry (FCM) and involved in the assessment of cell cycle parameters: single labeling by Propidium Iodide (PI) and double labeling anti-Bromodeoxyuridine (BrdUrd)/PI which, both in vitro and in vivo, gives cell percentages in the different cell cycle phases. The markers of cell cycle progression can be divided into proliferation markers such as PCNA (proliferating cell nuclear antigen) or Ki-67 and cell cycle progression markers. The latter, which are the core of the cell cycle machinery, are molecules recently characterized (Cyclins, CDKs (cell dependent kinases), CDIs (cyclin-dependent kinase inhibitors)) and their cell expression can be analyzed using FCM. FCM is also one of the best means to detect and quantitate appoptotic cells. Several techniques are described: Nuclear labeling using Hoechst 33342: mitochondrial labeling using DiOC6(3): detection of DNA fragmentation using 1) labeling of fixed and permeabilized cells with a DNA marker or 2) labeling of the free 3 DNA ends using incorporation of labeled deoxynucleotides; detection of antigens in apoptotic cells (Bcl-2, Fas, phospholipids...). At last, we analyzed flow cytometry methods to study the cell resistance to Ara-C and anthracyclins. In combination with cell kinetic studies and detection of apoptotic cells, they should increase the efficiancy of the acute leukemia treatment.     

急性白血病患者周期素D_1及其激酶抑制蛋白p15检测的意义

目的 :探讨细胞周期素 D1 及其激酶抑制蛋白 p15表达在急性白血病 (AL)中的临床意义。方法 :用间接免疫荧光标记流式细胞术研究 46例初治 AL 细胞治疗前后 D1 、p15、细胞周期及 DNA倍体与疗效间关系。结果 :2 8例急性髓系白血病 (AML)中 2 2例 p15阳性 ,6例阴性。 18例急性淋巴细胞白血病 (AL L)中 p15均阳性。AML与 AL L D1 均阳性。高 p15者 G1 期细胞百分率 (G1 % )增多 ,S期细胞百分率 (S% )减少。低 p15者 G1 %减少 ,S%增多。 DNA非整倍体者均高表达 p15和 D1 ,完全缓解率低 (30 % )。高 p15低 D1 者 2 0例中 18例 (80 % )完全缓解 (CR) ;低 p15高 D1 者 10例 ,8例 (80 % ) CR;高 p15高 D1 者 14例中 10例 (71.4% ) CR,低 p15低 D1 者 2例均 CR。结论 :p15和 D1 在 AL 中表达有异质性。高 p15者显示 G1 期阻滞 ,低 p15显示 G1 / S期转化 ,p15和 D1表达有助于指导治疗     

Surface markers in acute non-lymphoid leukemia: Analysis with a panel of 36 monoclonal antibodies

Summary The reactivity of a panel of monoclonal antibodies was studied in fifty-four cases of acute myeloid (AML) or undifferentiated (AUL) leukemias. Thirty-six antibodies from the Myeloid section of the Second Workshop on Human Leukocyte Differentiation Antigens were used in an indirect immunofluorescence assay. The antibodies could be classified into three groups recognizing respectively granulocytic, monocytic or granulomonocytic leukemias. Most antibodies stained erythroblastic and megakaryoblastic leukemias. In each group, it was possible to define antibodies staining either the less differentiated forms (FAB M 1 and M 5 a) or the more differentiated forms (M 2, M 3, M 4 and M 5 b). Six out of eight AUL were stained by some of the antibodies (mainly from the monocytic group). However, a heterogeneity of stainings in a same blast population was observed.     

Myeloid lineage specificity and high sensitivity of monoclonal antibody GM 58/8 proves its usefulness as a diagnostic reagent in acute leukemia

Monoclonal antibody (MoAb) GM 58/8 was earlier reported to be directed against an antigen expressed by myeloid progenitors (CFU-GM), myeloid precursors, granulocytes, and monocytes. Immunophenotyping of 216 cases of acute leukemia [acute myeloblastic leukemia (AML) = 147 and acute lymphoblastic leukemia (ALL) = 69] and 18 cases of chronic granulocytic leukemia in blast crisis (CGLBC) with this antibody showed that GM 58/8 reacted with 92% of AML cases (M1-M5) and 100% of myeloblastic crisis in CGL cases. All cases of ALL, lymphoblastic crisis in CGL, erythroleukemia, and erythroblastic crisis in CGL were unreactive with GM 58/8. The antibody revealed the myeloid phenotype in an additional 15 cases of otherwise unclassifiable acute leukemia and six cases of CGLBC. Eleven cases of acute "mixed lineage" leukemia were also diagnosed with the help of GM 58/8. The high specificity (100%) and sensitivity (92%) of MoAb GM 58/8 for myeloblastic leukemia is unmatched by almost all previously described myeloid MoAb and proves its usefulness as a single diagnostic reagent for AML and myeloblastic crisis in CGL.     

Treatment with monoclonal antibodies in acute lymphoblastic leukemia: current knowledge and future prospects

After rapid improvement of treatment results in adult acute lymphoblastic leukemia (ALL) from less than 10% to 30–40% in the past decades, more recently stagnation has been observed. In addition, a borderline for further intensification of chemotherapy appears to be reached in adult ALL patients. New, preferably non-chemotherapy, approaches are therefore urgently required. One of those is targeted therapy with monoclonal antibodies (MoAbs). ALL blast cells express a variety of specific antigens which may serve as targets, such as CD19, CD20, CD22, CD33, and CD52. Published results of MoAbs in ALL are reviewed. Most experience is available for anti-CD20 (rituximab) which led to a significant improvement of the outcome in B-cell non-Hodgkins lymphoma (NHL). In ALL, rituximab is combined with chemotherapy mainly in mature B-ALL and Burkitts lymphoma and preliminary results are promising. In the future, studies will also be done in B-precursor ALL. Another promising B-cell antibody is anti-CD22. Several CD19 MoAbs were also tested in phase I studies. However, results are not conclusive and these MoAbs are not generally available. Far less experience with MoAb therapy is available for T-ALL, but clinical studies are on the way with anti-CD52 and anti-CD25 in adult T-cell leukemia/lymphoma. Overall, it can be stated that MoAb therapy in ALL is a promising treatment approach. Monotherapy with MoAbs in relapsed ALL occasionally led to responses, but higher effectivity can be expected from a combination with chemotherapy and treatment in the state of minimal residual disease. Well-designed studies and joint efforts are required to explore optimal combinations, timing and dosage of MoAb therapy in ALL.