Role of flow cytometry in diagnostics of myelodysplastic syndromes--beyond the WHO 2008 classification

Multiparameter flow cytometry (FCM) is an excellent method to follow the expression patterns of differentiation antigens using monoclonal antibodies to surface and cytoplasmic proteins. Although several authors described various aberrant immunophenotypic features in the bone marrow of patients with myelodysplastic syndromes (MDS), the World Health Organization 2008 classification recommended that, only if 3 or more phenotypic abnormalities are found involving 1 or more of the myeloid lineages can the aberrant FCM findings be considered suggestive of MDS. In the absence of conclusive morphologic and/or cytogenetic features, FCM abnormalities alone were considered not sufficient to establish MDS diagnosis and further follow-up of the patients was recommended. Review of the literature gives accumulating evidence that FCM has become an important part of the integrated diagnostic work-up of patients with suspected MDS. Several studies have also reported FCM findings significant for prognosis and therapy choice in MDS patients. Technical progress in multicolor FCM and new analysis programs, together with ongoing efforts to standardize the methodology, will make it possible to apply FCM in individual risk assessment and choice of best therapy for MDS patients.     

多参数流式细胞术在非霍奇金淋巴瘤诊断中的应用

目的探讨流式细胞术在非霍奇金淋巴瘤（NHL）诊断中的应用意义。方法用多参数流式细胞术对40例淋巴组织增生性疾病的细针穿刺或手术切除淋巴结新鲜标本的活细胞表面抗原进行检测,同时结合细胞涂片的形态学观察,分析淋巴细胞的免疫表型特征,并与病理诊断及免疫组织化学结果进行比较。结果40例诊断为NHL的标本,经过流式细胞免疫分型联合细胞涂片观察细胞形态进行分析,37例（92．5％）符合NHL,其中病理组织学诊断为B—NHL的20例,经过流式细胞仪检测均为B—NHL,符合率为100％;病理组织学诊断为T—NHL的17例中,经过流式细胞仪检测符合T—NHL的12例（70．6％）;2例（11．8％）修正为B—NHL;3例（17．6％）未能明确诊断。结论流式细胞免疫分型有助于提高NHL诊断的正确性和亚型分类的准确性。     

Evaluation of canine bone marrow proliferative disorders by use of flow cytometric analysis of CD45 expression and intracytoplasmic complexity

Bone marrow from 14 dogs with proliferative disorders was evaluated by labeling cells with anti-CD45 and analyzing them by use of flow cytometry. Bone marrow from five healthy dogs was also evaluated to identify normal cell labeling patterns. The cells were displayed as the logarithm of red fluorescence intensity vs side-angle light scatter plots (CD45 scatter plots). For healthy dogs, the CD45 scatter plot technique identified four discrete cell populations. The immunophenotype of these cells identified these populations as granulocytes, erythroid cells, lymphocytes, and monocyte/macrophages. The CD45 scatter plot technique identified discrete populations of atypical cells in all dogs with proliferative disorders. Immunophenotyping of the atypical populations resulted in diagnoses of myelodysplastic syndrome, acute myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, metastatic lymphosarcoma, megakaryoblastic leukemia, and metastatic mast cell tumor. Possible clinical applications of the CD45 labeling technique include detection of malignant cell populations for immunophenotyping, early detection of metastasis of lymphosarcoma to bone marrow, detection of residual malignant cells after chemotherapy, and early detection of recurrence of leukemias.     

Flow cytometry: methodology and applications in pathology

Flow cytometry offers pathologists a powerful new method which can be applied especially for diagnosis, prediction of prognosis and measurement of response to therapy of tumours. It also has a wide variety of other applications in research and its use for investigation of non-neoplastic disease has only just begun. The object of this brief review is to stimulate interest in this technique and to outline some of the most interesting applications that may be of use to pathologists in the near future.     

Immunophenotyping of cytologic specimens by flow cytometry

Immunophenotyping by flow cytometry is well established as an ancillary technique in the diagnosis of hematopoietic neoplasms. However, flow cytometry is rarely performed on cytologic specimens because most cytologists are more comfortable with direct microscopy and believe that there is inadequate cellularity for analysis. Paradoxically, cytologic material is usually cell suspensions making it ideal for flow cytometry. In order to evaluate the usefulness of immunophenotyping cytologic specimens by flow cytometry, we retrospectively reviewed all cytologic specimens submitted to our flow cytometry unit from 1988 to 1991. Thirty-one cerebrospinal fluid specimens were analyzed. There were inadequate cells for analysis in 15 cases. Five showed a monoclonal proliferation; 11 were nondiagnostic. A range (r) of one to six cell surface markers were performed. Thirty-two body cavity fluids were analyzed: 7 peritoneal, 19 pleural, 2 pericardial, and 4 bronchoalveolar lavage. There were cells to analyze in all cases. Seven had a monoclonal proliferation; 25 were nondiagnostic (r = 4-21 markers performed). One hundred eighteen fine needle aspirates (FNA) were reviewed; 58 FNA were radiologically guided, 60 were superficial lesions. There were inadequate cells for analysis in two cases. Sixty-one demonstrated a monoclonal proliferation; 55 were nondiagnostic (r = 1-22 markers performed). We conclude that immunophenotyping by flow cytometry is of limited value for cerebrospinal fluid analysis and that knowledge of previous immunophenotyping studies is essential for correct analysis; analysis of body cavity fluids is easily performed but less often demonstrates a monoclonal proliferation. Immunophenotyping by flow cytometry is a valuable adjunctive technique for FNA and yields adequate cells for analysis.     

Clinical applications of flow cytometry and cell immunophenotyping to companion animals (dog and cat)

Clinical applications of flow cytometry to certain diseases of the dog and cat are now possible. The utility of such applications for diagnosis, prognosis and follow-up are illustrated here by a number of examples: feline AIDS resulting from FIV infection, Leukocyte Adhesion Deficiency in Irish setters, deep pyoderma in German shepherds, Immune-mediated Thrombocytopenia, canine Systemic Lupus Erythematosus and Leishmaniasis, Leukemia and Lymphoma.     

Flow cytometry as a tool to discriminate respiratory-competent and respiratory-deficient yeast cells

Summary The cationic lipophilic dye Rhodamine 123 (Rh123) is selectively enriched in mitochondria in a membrane potential-dependent manner. Application of drugs which interfere with the electron flow of the respiratory chain lead to a severe reduction of mitochondrial dye uptake. In this communication we show that the same effect is observed after Rh123-staining of respiratory-deficient yeast mutants. Based on this observation we used flow cytometry to discriminate respiratory-compentent and respiratory-deficient yeast cells. Combined with a cell sorter we were able to selectively enrich respiring and non-respiring yeast cells, repectively, from a mixture of cells.     

Flow cytometry assay for rapid analysis of DNA strand breaks

A method is described of analyzing breaks within nuclear DNA using flow cytometry of extracted nucleoids. Breaks in the DNA molecule are detected by the relaxation of supercoiled topological domains and may be observed at a frequency of 1:5 × 10⁵ bp or better. This represents an improvement over previous methods of such analyses in that both the speed of data accumulation is increased due to the elimination of potentially confounding factors of extended lysis incubation and the use of DNA intercalators. Such a technique may find application in studies which require very rapid assessment of rare breaks within the linear DNA molecule, data may be recorded with minimal cell processing approximately 60–90 seconds after lysis of live cellular material.     

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。     

直肠癌细胞的流式细胞仪分析及临床意义

目的:为了探讨直肠癌细胞增殖情况与其预后及淋巴结转移的关系。方法:本文通过对30例Ⅲ期直肠癌(DUKS C期)石蜡包埋组织块及10例正常组织对照石蜡块,进行细胞悬液制备,并采用B-D公司生产的FACS Calibur型流式细胞仪,进行细胞获取和分析。结果:①正常10例直肠组织细胞G₀-G₁期细胞峰值道数为49.82±1.80。②DI(DNA指数)、SPF(S期细胞百分率)、PI(细胞增殖指数)与淋巴结转移情况无关(P>0.05)。③SPF与直肠癌5年生存率关系密切(P<0.01)。结论:S期细胞百分率越高预后越差,而DI、PI与预后无关。     

流式细胞术检测抗生素最低抑菌浓度

目的　建立一种流式细胞术 (FCM )快速检测抗生素最低抑菌浓度 (MIC)的方法 ,与常规药敏试验结果进行比较 ,探讨其应用价值。方法　取 2 0株肠杆菌科细菌 ,用碘化丙啶 (PI)染色 ,分别检测阴性对照管和MIC浓度抗生素作用管的平均荧光强度 (MFI) ,计算两者比值 ,据此 ,设定流式细胞药敏试验 (FCST)检测MIC的判断值 ,根据设定的判断值对 2 8株临床分离细菌同时进行常规药敏和FCST双盲测定。结果　 2 0株肠杆菌科细菌测得的MFI比值 x为 2 .0 7,CV为 0 .3 1;将MFI大于阴性对照细菌 2倍 (增加 10 0 % )的最低药物浓度定义为该菌的MIC ,2 8株临床菌株FCST所测得的MIC与常规药敏试验相比r=0 .92 ,(P <0 .0 0 1) ,回归方程为 :Y =1.4X -0 .48。结论　FCST与常规药敏试验结果有显著相关性 ,并且具有快速、稳定、准确、便于自动化等优点 ,有着较大的临床应用前景。     

白血病免疫分型诊断技术的进展

白血病是造血系统的恶性疾病,俗称“血癌”,是国内十大高发恶性肿瘤之一.目前,免疫分型已是当今白血病诊断分型、预后判断和残留病检测的一个有力手段.免疫分型诊断的主要方法有免疫细胞化学技术、流式细胞术、细胞芯片技术,也有个别学者在尝试压电免疫传感器.临床上仍以流式细胞术(FCM)方法为主,目前一次最多能检测到15种抗原,且昂贵.而细胞免疫芯片检测技术,一次可以检测数十种乃至上百种细胞抗原,如能应用于临床,将是一种方便,快速,简单,经济实用的白血病免疫分型检测手段.     

人子宫内膜干细胞的分离、培养及鉴定

目的 分离、培养子宫内膜干细胞并进行鉴定.方法 采用酶消化和机械方法相结合分离人子宫内膜细胞,两次滤网过滤分离基质细胞和上皮细胞.采用有限稀释法培养基质细胞和上皮细胞,观察细胞的形态及生长特性,免疫荧光检测波形蛋白和细胞角蛋白表达情况.原代培养15 d后,计算细胞克隆形成率,流式细胞术检测抗原CD133、CD34、CD45、CD90、CD73及CD29表达.结果 基质细胞呈纤维样细胞形态,排列呈辐射状,免疫荧光显示其波形蛋白表达阳性;原代培养15 d后,细胞克隆形成率达(1.34±0.44)％.上皮细胞呈多角形或椭圆形,免疫荧光显示其细胞角蛋白表达阳性;原代培养15d后,未发现克隆形成.流式细胞术检测克隆形成的基质细胞CD29、CD90、CD73表达阳性,CD34、CD45、CD133表达阴性.结论 人体存在少量具有克隆形成能力和高度增生潜能的子宫内膜干细胞,这些干细胞也可能来源于骨髓间充质干细胞.     

Flow cytometry in diagnostic cytology

Flow cytometry (FCM) is a useful adjunct to cytologic examination, because the quantitative biochemical information it provides complements the morphologic information gained during visual examination. It aids in the interpretation of bladder washings, and is particularly useful for the assessment of lymphoid lesions, whether they originate from fine-needle aspiration, cerebrospinal fluid, or effusions. Optimal use of FCM frequently requires assessment of more than one parameter; simultaneous use of cell differentiation markers and nuclear DNA quantitation is often significantly more useful than either alone. Despite the utility of FCM, however, the potential for future development appears to be limited. Improvements in image cytometry allow reasonable assessment of ploidy and S-fraction to be made from specimens prepared on glass slides. Multiparameter measurements may also be accomplished with imaging techniques, which allow the further advantage of visual identification of cells with equivocal morphologic changes. The development of artificial intelligence methods for use with imaging technology has also significantly exceeded that of FCM. Finally, image cytometry is often more useful for samples with few cells. Other challenges are posed by immunocytochemical methods which compete with flow cytometry as tools for assessment of proliferation. Given the relatively high cost of FCM instrumentation, survival of FCM as an ancillary technique in cytopathology will require further technical refinements to offset the advantages currently associated with image cytometry and immunocytochemistry. Diagn. Cytopathol. 1998;18:41–46. © 1998 Wiley-Liss, Inc.     

Flow cytometry and its use in the diagnosis and management of mature lymphoid malignancies

The last decade has seen major advances in flow cytometric immunophenotyping and this has expanded the utility of flow cytometry to investigate the antigens present on normal and neoplastic haematopoietic cells. This review summarizes how flow cytometry is used currently in the diagnosis and management of mature lymphoid malignancies. The establishment of disease-specific phenotypes allows the creation of assays which can detect neoplastic cells with high specificity and sensitivity. Certain lymphoid neoplasms are well defined immunophenotypically, while others are more heterogeneous. The availability of more sophisticated cytometers and a wider selection of antibodies in routine diagnostic laboratories will lead to the resolution of these more complex disease entities.     

蚕蚀性角膜溃疡患者外周血淋巴细胞免疫表型和T细胞活化的研究

目的：观察蚕蚀性角膜溃疡患者外周血淋巴细胞免疫表到和T细胞活化的变化,进一步探讨本病患者的免疫功能。方法：用流式细胞术检测8例活动性蚕蚀性角膜溃病患者外周血淋巴细胞对项免疫表型变化,用植物凝集素和沸被醉酯作为刺激剂,以HLA－DR,CD69、CD71、CD25的表达为T细胞活化的指标,用流式细胞术分析。结果：蚕蚀性角膜溃疡患者外周血CD4、CD5、CD8、CD10、CD11b、CD15、CD16、CD19、CD23、CD25、CD41、CD69、HLA-DP、HLADR抗原表达阳性淋巴细胞以及HLA－DR、CD69、CD71、CD25在CD3和CD8细胞的表达均明显高于正常对照组。结论：蚕蚀性角膜溃疡患者具有增强的细胞免疫和体液免疫,外周血T细胞及其亚群出现异常活化,反映患者体内存在自身免疫反应而支持该病发病之自身免疫学说。     

流式细胞仪分离人早孕绒毛外滋养细胞及鉴定

目的用流式细胞仪分离早期绒毛外滋养细胞，以便对其功能进行进一步研究。方法用胰蛋白酶消化法得到早期绒毛细胞，用流式细胞仪分离纯化绒毛外滋养细胞。所得的细胞用免疫细胞化学，光镜，透射电镜检以及Westen-blot方法鉴定。结果使用流式细胞仪成功的分离了绒毛外滋养细胞，纯度超过98％。结论此方法可快速准确大量获得绒毛外滋养细胞。     

Flow cytometry and staining kinetics to monitor the G0/G1 transition

The phenanthridine dyes propidium iodide (PI) and ethidium bromide (EB) have contributed to make DNA quantitation a routine measurement by flow cytometry. As planar molecules they easily intercalate between base pairs of double stranded nucleic acids. When the quantity of dye exceeds the amount of DNA, the intensity of emitted fluorescence can be related to the amount of DNA. This is, in fact, the basic requirement of quantitative measurement. When staining is performed at low dye concentration and there are no sufficient molecules to saturate all the available DNA-binding sites, the emitted fluorescence, other than the true DNA quantity, may be dependent on the state of chromatin condensation. A staining procedure aimed to stress the influence of nuclear structure on the emitted fluorescence of PI is described. This is achieved by means of a very low dye concentration (<0.01 g/ml PI) to guarantee staining far below saturation condition. In this particular condition the staining rate slows down to be monitored by flow cytometry. As different experimental models the leucocytes of the normal human peripheral blood have been used taking into account the relatively condensed chromatin of granulocytes with respect to that of the mononuclear cells. Significantly higher fluorescence intensity have been obtained from mononuclear cells compared to that of the polymorphonuclear ones. Quiescent versus exponentially proliferating cell cultures had also been tested. In this staining condition low fluorescence intensity have been obtained by condensed chromatin structure (G₀) in comparison to the more decondensed (G₁) one.Abbreviations PI propidium iodide - PBS phosphate buffered saline - BrdU bromodeoxyuridyne - FCM flow cytometry - PMNC polymorphonuclear cells - MNC mononuclear cells     

流式细胞术检测细胞毒方法的建立

目的：建立一种用流式细胞仪测定细胞介导细胞毒活性的方法。方法：用羧基荧光素二醋酸盐琥珀酰亚胺酯(CFSE)标记靶细胞，再用碘化丙碇(PI)标记DNA筛选细胞膜已破坏的靶细胞。效靶比为100：1—6．25：1，共孵育时间为1-6小时，流式细胞仪收集1000个靶细胞并测定被杀细胞的百分率。结果：CFSE是合适的标记靶细胞的荧光染料，18小时后也能很好地区分效靶细胞；靶细胞的自然死亡率低于5％；杀伤百分率随着效靶比和共孵育时间的增加而增加。最佳效靶比为50：1—25：1，共孵育时间为2—4小时。结论：CFSE／PI双荧光染料标记的流式细胞分析检测细胞毒具有多个优点：避免放射性试剂的应用，敏感性增强，单细胞水平的分析。     

Identification of CD56 and CD57 by flow cytometry in Ewing’s sarcoma or primitive neuroectodermal tumor

 CD56 and CD57 are commonly considered as natural killer and neuroectodermal markers, but their expression has been identified in a wide spectrum of neoplasms including some cases of Ewing’s sarcoma (ES) and primitive neuroectodermal tumor (PNET). We report two cases of small, round blue cell tumor (SRBCT), in which flow cytometry immunophenotyping (FCI) detected strong expression of CD56 and CD57 (one case). Immunohistochemical staining with Leu-19 and Leu-7 confirmed the FI results. Although CD56 and CD57 expression is consistent with ES/PNET, it can be potentially misleading if results of FCI are interpreted in the absence of other findings. These cases suggest the utility of FCI in undifferentiated SRBCT. The literature on CD56 and CD57 expression in ES/PNET is reviewed and discussed. Received: 5 January 1998 / Accepted: 19 February 1998