Acute lymphoblastic leukemia: diagnosis and detection of minimal residual disease following therapy

Flow cytometric immunophenotyping (FCI) is an important diagnostic modality in the evaluation of patients who have suspected or known acute lymphoblastic leukemia (ALL). It enables rapid identification, quantification, and immunophenotypic characterization of leukemic blasts, permitting accurate and timely diagnosis. Beyond facilitating the classification of ALL into fundamental diagnostic categories, FCI may anticipate recurrent cytogenetic and molecular abnormalities. FCI permits the detection of leukemic blasts after therapy at a level lower than that achievable by conventional microscopic examination. Flow cytometric detection of minimal residual disease is among the strongest prognostic factors in patients who have ALL and may provide an opportunity for more precise risk-adapted therapies.     

流式细胞术-白血病免疫分型规范化

近十年来流式细胞术已被我国各级医院较广泛采用,主要用途之一是测定血液恶性肿瘤的免疫表型。流式细胞术的免疫分型（FCM－IM）可为白血病与淋巴细胞增殖性疾病（LPO）提供准确的数据。国际上公认是诊断血液系统恶性疾病一项不可缺少的标准,还可提供指导治疗的相关信息。图1与图2示某些白血病与LPDs典型的免疫表型。值得注意的是变异是常见的。现提出以下几点注意事项以使实验结果更为可靠并减少实验室间的差异：①用直接免疫标记法及多色分析法。②CD45／SSC设门法优于FSC／SSC设门法,可更清楚地显示不正常细胞群体。在原／幼细胞少时尤为明显。③结论中详细描述不正常细胞群的表型较计算各标志细胞的百分数更为重要。④用直接标记,多色分析法时不再以20％为正常值界线。⑤由于抗体／抗原的多向性,白血病细胞抗原表达的不保真性以及白血病细胞生物学的多变性,单纯依靠FCM术数据勿轻易诊断杂合性或双表型白血病。流式细胞仪不是细胞计数器,其实验结果须结合临床和其它有关信息综合判断。     

免疫分型在骨髓增生异常综合征的诊断和预后判断中的作用研究进展

骨髓增生异常综合征（myelodysplasticsyndromes,MDS）是一种以一系或多系病态造血为特征的髓系肿瘤,具有很高的向急性白血病转化的风险。随着对MDS认识的深入,MDS的诊断标准经历了由形态学单一诊断指标向多指标综合的质的飞跃。在2007维也纳标准和2008WHO分型标准中,免疫分型均被作为次要诊断指标。MDS患者骨髓细胞免疫表型在整个疾病过程中呈现出紊乱及异常表达,其中一些改变对其诊断、分型、预后和治疗等方面具有重要意义,而且基于流式细胞术检测而建立的流式积分系统（FCSS）更是可以客观、量化评价这些免疫表型的异常,能弥补目前国际上通用的国际预后积分系统（international prognostic scoring system,IPSS）及WHO预后积分系统（WHO classification-based prognostic scoring system,WPSS）评价MDS患者预后的缺陷,对临床上MDS的诊断和治疗具有重要的指导作用。本文从MDS的异常免疫表型和流式积分系统对MDS免疫分型在MDS诊断和预后中的作用作一综述。     

Quantitative analysis of cytoplasmic myeloperoxidase positive leukemic cells by FCM]

Development of a fixation allowed flow cytometric analysis of nuclear and intracellular antigen in leukemic cells. In this paper the analyzing procedure of cytoplasmic myeloperoxidase by FCM was described. This procedure is more sensitive than cytochemical staining of fixed cell smears and more specific than cell surface immunophenotyping by CD13, CD14, CD33. So, this technique is useful for classification of myelogenous leukemic cells especially MO type leukemia by FAB classification. This technique also allowed two color analysis of cell surface antigens and cytoplasmic antigens. Mixed lineage leukemia can be easily and accurately classified by using of this procedure.     

流式细胞术表型分析的质量控制

FCM表型分析已经从外周血淋巴细胞的双参数定量测定发展到当今对血液细胞病理学包括骨髓、淋巴结分析的5个或更多参数的定性测定,其中白血病和淋巴瘤的免疫表型分析在血液淋巴系统恶性疾病诊断、分类及病情监测上都是对形态学检验的一种重要补充.FCM 5个或更多参数分析的复杂性以及数据的解释依赖于仪器、试剂、程序的标准化及校准.此...     

四色流式细胞术检测B细胞急性淋巴细胞白血病微量残留病的临床意义

目的探讨利用四色流式细胞术（FCM）检测B细胞急性淋巴细胞白血病（B-ALL）微量残留病（MRD）的临床意义.方法采用以抗CD34/CD10/CD45/CD19为主的两种四色荧光标记抗体组合,对98例B-ALL患者的671份骨髓标本和1份脑脊液标本进行FCM多参数MRD检测,98例随访患者中26例无发病初期的免疫分型资料.结果FCM检测显示白血病细胞＜0.0001（MRD阴性）的标本为579份;白血病细胞＞0.0001的样本数为93份,其中64份骨髓标本白血病细胞比例＜0.05,29份标本白血病细胞比例＞0.05,我们将其归为复发病例（包括治疗后未达CR的病例）.共20例患者复发,其中19例血液学复发,1例中枢神经系统复发.15例血液学复发者在复发前7～17周发现MRD阳性,包括发病时免疫分型资料不明者6例,MRD水平均＞0.0001;2例分别在复发前3个月和9个月检测MRD为阴性,此后中断检测.在诱导治疗结束和治疗3个月时,如果MRD水平＞0.0001,复发率为50%（12例中有6例复发）,而MRD阴性组复发率为7.5%（40例中有3例复发）（P=0.000）.结论利用FCM进行MRD跟踪监测可预测复发,治疗初期患者MRD＞0.0001,复发的危险性较高.而在掌握了正常B祖细胞抗原表达规律的基础上,可不完全依赖于发病时的免疫表型资料.     

多发性骨髓瘤免疫表型流式细胞术研究新进展

多发性骨髓瘤(multiple myeloma,MM)是一种常见的血液系统恶性肿瘤,以浆细胞恶性增殖、溶骨破坏为特征,表现为M蛋白、骨骼破坏、贫血、肾功能受损和免疫功能异常.通过流式细胞术(flow cytometry,FCM)检测其免疫表型特点,已经作为血液系统疾病的常用检测手段,在辅助诊断、评估预后以及监测微小残留病灶方面的价值,已经被越来越多的学者认同.本文就将免疫表型分析在MM中的应用进展方面作一个综述,包括流式细胞术在MM中的应用和MM中流式免疫表型的特点.     

Drug resistance in B-cell chronic lymphocytic leukemia: predictable by in vitro evaluation with a multiparameter flow cytometric cytotoxicity assay

BACKGROUND: Patients with B-cell chronic lymphocytic leukemia (B-CLL) often demonstrate variable responses to similar treatments. It would be highly desirable to develop a personalized therapeutic strategy for selection of appropriate drugs or regimens based on the drug sensitivity profiles of leukemic cells from individuals. METHODS: We applied a multiparameter flow cytometric drug cytotoxicity assay to evaluate drug effects specifically on B-CLL cells from 43 individuals after leukemic cells were incubated in vitro with fludarabine, chlorambucil, cladribine, or prednisolone. RESULTS: We demonstrated that different B-CLL cell populations from 43 individuals showed a marked variability in drug sensitivity. In vitro resistance to fludarabine was greatest in B-CLL cells with deletions of p53, a cytogenetic abnormality that is almost invariably associated with a poor therapeutic response clinically. CONCLUSIONS: In vitro drug sensitivity profiles analyzed by a multiparameter flow cytometric cytotoxicity assay may serve as a tool to facilitate individualized selection of appropriate drugs for treatment in B-CLL. Prospective trials will be needed to validate the clinical utility of this flow cytometric cytotoxicity assay.     

Immunophenotyping leukemias: the new force in hematology.

OBJECTIVE: To review basic concepts of immunophenotyping leukemias and introduce clinical laboratory scientists (CLSs) to the technological changes utilized in this laboratory methodology. DESIGN: Literature review. DATA SYNTHESIS: Immunophenotyping in the clinical laboratory is emerging as an advantageous way to separate and classify leukemic malignancies. Immunophenotyping involves the use of flow cytometers and immunofluorescence in order to achieve great sensitivity and specificity for malignant cells. A basic understanding of components of the flow cytometer and how it works is necessary to understand immunophenotyping. Monoclonal antibodies specific to the malignant cells of question play an essential part in this technique. Various fluorescent dyes and cell panels also must be incorporated into the system. Analysis is done and statistics are plotted on dot plots that can be read by the CLS to give helpful insight into the etiology of disease process. Immunophenotyping is a very powerful tool that has the ability to revolutionize the clinical laboratory setting. The CLS working in hematology must become aware of and comfortable with this methodology.     

Flow cytometric measurement of cell surface and intracellular antigens present in leukemia cells]

Recent advances in flow cytometric technology has enabled us to perform multiparameter analysis of cell surface and intracellular antigens. The clinical application of such analyses requires to establish the procedures of sample preparation, fixation, staining, instrument calibration and data analysis. This article describes the basic elements involved in establishing such procedures and explores the use of multiparameter flow cytometric analysis in the characterization of human leukemia cells. Flow cytometric measurements of cell surface and intracellular antigens of leukemia cells can provide important insights into the biologic features of these cells as well as significant diagnostic information.     

Preliminary results of evaluation of progress in chemotherapy for childhood leukemia patients employing Fourier-transform infrared microspectroscopy and cluster analysis

Acute lymphoblastic leukemia (ALL) is the most common malignancy in children, but remarkable progress in methods of chemotherapy has increased the cure rate to 80%. The leukemic cells called blasts are eliminated within 7 days of chemotherapy. Clinically, the blast count is monitored directly with the use of blood smears on the basis of specific genetic markers and immunophenotyping methods such as flow cytometry. In this article, we present preliminary results, obtained with the use of Fourier-transform infrared microspectroscopy and cluster analysis, of an approach to monitoring the progress made with chemotherapy in 1 B-cell and 2 T-cell pediatric ALL patients. Our results indicated that the biological marker derived from the spectra did not provide accurate prediction of the progress made with chemotherapy. However, cluster analysis of FTIR-MSP spectra provided good classification of the samples with and without blasts, which correlate satisfactorily with clinical data. Extensive studies are required to substantiate our findings statistically which may have potential application of FTIM in the diagnosis and follow-up of various types of malignancies.     

细胞因子诱导杀伤细胞过继免疫治疗血液系统恶性肿瘤的研究进展

细胞因子诱导杀伤(CIK)细胞具有T淋巴细胞与自然杀伤(NK)细胞的表型及功能特征.其体外培养细胞扩殖率高、不受主要组织相容性复合物(MHC)限制,在体外对血液系统恶性肿瘤细胞有显著的细胞毒性.这些特性均使得CIK细胞过继免疫治疗抗血液系统恶性肿瘤活性逐步成为相关研究的热点.多项临床试验结果表明,CIK细胞过继免疫治疗在血液系统恶性肿瘤患者中具有可行性及有效性;通过CIK细胞过继免疫治疗结合标准治疗方案已被证实在抗血液系统恶性肿瘤中发挥协同作用.CIK细胞过继免疫治疗在血液系统恶性肿瘤的治疗中能够提高患者完全缓解(CR)率、延长生存时间并提高生活质量.笔者拟就CIK细胞过继免疫治疗血液系统恶性肿瘤的相关研究进展进行综述.     

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.     

Flow Cytometry Immunophenotyping Evaluation in Acute Lymphoblastic Leukemia: Correlation to Factors Affecting Clinic Outcome

The authors conducted a flow cytometry immunophenotyping study in patients with acute lymphoblastic leukemia (ALL) from Natal, Rio Grande do Norte, Brazil. The patients (n = 126) were newly diagnosed using a panel of monoclonal antibodies: CD1a, CD2, CD3, CD4, CD7, CD8, CD10, CD13, CD33, CD14, CD19, CD22, CD79a, CD117, CD34, anti‐IgM, anti‐TdT, anti‐HLA‐Dr, and anti‐human kappa and lambda light chains. Additional data, such as patients' age and gender, clinical and laboratory findings such as presence of tumor masses, lymphadenopathy, hepatomegaly, splenomegaly, leukemic infiltration in the central nervous system (CNS) were also investigated. Results showed that 56.7% of the cases were B‐lineage ALL and 55% were T‐cell ALL. Also, we found that males were more affected by the disease, regardless of immunological classification. The correlation between age and immunological subtypes showed that the B‐lineage ALL occurred more frequently in patients aged under 15while the T‐cell ALL subtype was more frequent in adults. Immunophenotypic profiles and morphological subtypes showed a direct correlation between L3 subtype and B‐lineage ALL, while L1 and L2 subtypes correlated more often with B‐cell lineage and T‐cell ALL, respectively. Correlation analysis between immunophenotypic and clinical profiles showed that T‐cell ALL was more associated with a higher incidence of lymphadenopathy, hepatomegaly, splenomegaly and CNS leukemic infiltration, also showing a greater blast cell count in peripheral blood than the other subgroups. The presented data suggest that immunophenotyping is an important method in the diagnosis, monitoring and prognostic assessment in determining the pathological mechanisms of evolution of ALL.     

Flow cytometric immunophenotypic profiles of mature gamma delta T-cell malignancies involving peripheral blood and bone marrow

BACKGROUND: In this study we compared clinical findings with flow cytometric immunophenotypic results in a series of patients with aggressive and indolent gamma delta T-cell malignancies with peripheral blood and/or bone marrow involvement. METHODS: Gamma delta T-cell malignancies were detected based on flow cytometric demonstration of an abnormal T-cell population staining positive with T-cell receptor gamma delta and confirmed by morphologic and clinical reviews. Clinical data were obtained through chart review and discussion with the patients' physicians. RESULTS: Blood or bone marrow involvement was present in all patients. Hepatosplenic and cutaneous gamma delta T-cell lymphomas had an aggressive clinical course, whereas the gamma delta T-cell large granular lymphocyte (LGL) leukemias had an indolent course. Expressions of CD5, CD8, CD16, and CD57 differed in gamma delta T-cell LGL leukemia compared with hepatosplenic and cutaneous gamma delta T-cell lymphomas. CONCLUSIONS: Gamma delta T-cell malignancies have a poor prognosis with the exception of gamma delta T-cell LGL leukemia (indolent process). Because CD57 expression is specific for gamma delta T-cell LGL leukemias, expression of this antigen may be associated with a more indolent clinical course. Because cutaneous gamma delta T-cell lymphoma can present with peripheral blood involvement, flow cytometric evaluation of peripheral blood is important in staging these patients.     

2006 Bethesda International Consensus recommendations on the flow cytometric immunophenotypic analysis of hematolymphoid neoplasia: medical indications

The clinical indications for diagnostic flow cytometry studies are an evolving consensus, as the knowledge of antigenic definition of hematolymphoid malignancies and the prognostic significance of antigen expression evolves. Additionally the standard of care is not routinely communicated to practicing clinicians and diagnostic services, especially as may relate to new technologies. Accordingly there is often uncertainty on the part of clinicians, payers of medical services, diagnostic physicians and scientists as to the appropriate use of diagnostic flow cytometry. In an attempt to communicate contemporary diagnostic utility of immunophenotypic flow cytometry in the diagnosis and follow-up of patients with hematolymphoid malignancies, the Clinical Cytometry Society organized a two day meeting of international experts in this area to reach a consensus as to this diagnostic tool. This report summarizes the appropriate use of diagnostic flow cytometry as determined by unanimous approval of these experienced practitioners.     

淋巴结细针穿刺标本的流式细胞术分析

目的 评价淋巴结细针穿刺标本流式细胞术分析方法在诊断淋巴结病变,以及在恶性淋巴瘤和淋巴结反应性增生鉴别诊断中的应用价值.方法 对99份疑为淋巴结病变的淋巴结针吸标本涂片进行常规细胞学分析,并结合病理活组织检查确诊分型.使用三色标记的流式细胞术方法分析针吸标本中各细胞免疫表型(CD3、CD3、CD4、CD5、CD10、CD19、CD20、CD23、CD45、K、λ、FMC7、 CD34),确定标本中各细胞组成及有无异常表型细胞.对于淋巴瘤病例,则按照WHO分型标准,根据免疫表型进一步确定其亚型,并对各类病例流式细胞术分型结果和细胞学结果进行比较.结果 99份标本中,细胞涂片检出淋巴瘤40例,转移癌29例,反应性增生、坏死、结核30例,有2例非霍奇金淋巴瘤(NHL)被误诊为反应性增生;对其中18例NHL进行了病理活组织检查,其中包括B淋巴细胞非霍奇金淋巴瘤(B-NHL)16例,T淋巴细胞非霍奇金淋巴瘤2例.流式细胞术分析结果显示,99份标本中检出淋巴瘤35例(淋巴母细胞淋巴瘤4例,T淋巴细胞病变1例,其余30例为B-NHL);有28份B-NHL检测到K或λ轻链限制性表达,其K:λ或λ:K大于3:1,B淋巴细胞所占比例为(73.2±27.2)%,其中26份能根据免疫标志物的表达确定其亚型.经病理活组织检查确定为B-NHL的16份标本中,仅2例滤泡淋巴瘤与流式细胞术分型结果不一致.对于反应性增生及转移癌等标本,流式细胞术分析未查见异常淋巴细胞,其k:λ或λ:k均小于3:1.结论 淋巴结细针穿刺标本的流式细胞术分析有助于淋巴结病变的诊断和鉴别诊断,并可确定NHL的亚型.     

骨髓增生异常综合征免疫表型研究进展

骨髓增生异常综合征（MDS）是一组血液系统异质性的疾病,表现为骨髓原始细胞的形态和数目异常,无效造血及不同程度的外周血细胞减少并易转变为急性髓系白血病。本文就MDS疾病的骨髓形态学检查,骨髓活检,染色体核型分析,免疫分型等问题,特别是流式细胞术免疫分型技术检测对MDS诊断和预后意义的研究进展作一综述。     

71例急性早幼粒细胞白血病患者白血病细胞免疫表型分析

本研究通过回顾性分析急性早幼粒细胞白血病(APL)患者骨髓异常早幼粒细胞的免疫表型及患者初诊资料,探讨其免疫表型特点及其意义。利用常规6色免疫分型方法对71例APL患者的白血病细胞进行免疫表型分析。结果发现:MPO、CD33和CD13在所有患者的APL细胞中都有较强表达,其平均阳性细胞比例达到88%以上。CD117的阳性表达率为50.7%,其平均阳性细胞比例为52.5%。约10%患者的白血病细胞表达CD15,但大部分病例的阳性细胞率都集中在20%-40%的弱表达范围内,其平均阳性细胞比例为42.5%。少数患者的异常细胞表达CD34和HLA-DR,且表达强度较弱。约25%患者的APL细胞跨系表达了CD2、CD56,大部分也都集中于20%-60%的低表达范围内,其平均阳性细胞比例分别为39.3%和42.3%。由此认为,APL的典型免疫表型为MPO+CD13+CD33+CD117±CD15±CD34-HLA-DR-。CD2和CD56在CD34+或HLA-DR+组(包括CD34+HLA-DR+、CD34+HLA-DR-和CD34-HLA-DR+)的阳性比例明显高于CD34-和HLA-DR-组。初诊患者外周血白细胞计数、血小板计数、外周血中异常早幼粒细胞比例及CD13的阳性比例在CD15<10%、10%20%3组均出现显著的统计学差异。结论:APL患者的异常早幼粒细胞的免疫表型具有独特的特征,多色流式细胞术检测可辅助APL的快速诊断,对分析白血病细胞的来源和判断患者预后亦可能有着重要意义。     

Survivin在造血系统肿瘤中的研究进展

Survivin是一种新的凋亡抑制蛋白(inhibitor of apoptosisprotein，IAP)，具有细胞周期调控和凋亡抑制双重功能。在多种造血系统肿瘤组织中高表达，与诊断、预后和耐药密切相关。利用survivin致敏的树突状细胞疫苗、survivin反义核酸及Survivin阴性突变体可有效抑制肿瘤细胞生长，为采用生物学策略治疗造血系统肿瘤开辟了新途径.本综述重点阐述了survivin在造血系统肿瘤中的表达，survivin与造血系统肿瘤预后和耐药的关系及survivin在造血系统肿瘤治疗中的应用。