Simultaneous flow cytometric analysis of cell surface markers and telomere length: analysis of human tonsilar B cells

Telomere Flow FISH is a recently developed method which allows the measurement of telomere length in purified subsets of cells using flow cytometry. However, the harsh conditions required for flow FISH have precluded its use with conventional cell surface staining, thus limiting its utility for large scale clinical studies. We have now developed a method which permits simultaneous analysis of cell surface markers along with telomere length estimation by flow cytometry. This new assay employs the covalent crosslinking of monoclonal antibodies conjugated with a heat stable fluorochrome to the cell surface prior to flow FISH. Using this technique we have confirmed that human germinal center B cells (IgD(-)/CD38(+)) have dramatically longer telomeres than pre-germinal center founder B cells (IgD(+)/CD38(+)). This approach simplifies the analysis of complex cell populations and will facilitate widespread investigation of telomere length in health and disease states.     

Analysis of cell division among subpopulations of lymphoid cells in sheep. I. Thymocytes

The number, distribution and surface phenotype of dividing cells in the thymus, and differences between the cell cycle status of thymocyte subpopulations, were studied in fetal and post-natal lambs using double-labelling techniques. Dividing cells were labelled in vivo for various periods with 5-bromo-2-deoxyuridine (BrdU). The proportions of constituent thymocyte subpopulations that had synthesized DNA during the labelling period were measured by flow cytometry or immunohistochemistry using a panel of monoclonal antibodies (mAbs) specific for sheep lymphocyte differentiation antigens and MHC class I and class II antigens in conjunction with an anti-BrdU mAb. The proportion of thymocytes that incorporated BrdU during a 1-hr labelling period varied with age, and levels of 30%, 13% and 9% were measured, respectively, in 40- and 125-day-old fetuses and 8-week-old lambs. Eight percent of the thymocytes in lambs were synthesizing DNA, with 4% entering the G2 phase per hour, and a substantial number of thymocytes (21%) had a G2 + M phase DNA content. A small subset of thymocytes (1-3%) recognized by mAb E-79 localized to the subcapsular region of the cortex and displayed the highest level of BrdU incorporation. Cortical-type thymocytes (CD1+) comprised 50-70% of thymocytes; however, few of these incorporated BrdU and the proportion in the G2 + M phase of the cell cycle was higher than for other thymocyte subpopulations. The 197+CD4-CD8- T cells also showed no evidence of in vivo division.     

Flow cytometry: an 'old' tool for novel applications in medical genetics

Flow cytometry was originally established as an automated method for measuring optical or fluorescence characteristics of cells or particles in suspension. In the meantime, flow cytometers have become user-friendlier, less expensive instruments with an increasing importance in clinical diagnostics. Besides the classical fields of application, such as immunophenotyping blood cells or analyzing the cell cycle status by measuring the DNA content, novel flow cytometric methods have been developed to identify and to quantify disease-related gene sequences. Here we give an overview of current and future applications, including the detection of viral sequences via microsphere-based PCR assays and the analysis of single nucleotide polymorphisms, reflecting individual phenotypic traits. Furthermore, flow cytometry allows the quantification of gene expression changes as well as the isolation of differentially expressed gene sequences. Flow cytometry is also convenient for multiplex analyses, e.g. when hybridizing DNA samples to a mixture of various microsphere populations each coated with different DNA probes. Last but not least, the use of magnetic beads in combination with flow cytometers coupled with automated devices enables molecular diagnostics on a large scale. Overall, this review demonstrates flow cytometry as a rapid, sensitive, and reproducible tool applicable to a wide range of medical genetic approaches.     

Flow cytometric analysis of bromodeoxyuridine-induced micronuclei

The effects of DNA substitution by the thymidine analogue 5-bromodeoxyuridine(BrdU) on cell cycle progression and micronucleus inductionwere studied in different mammalian cell cultures. Simultaneousflow cytometric measurements of DNA content and side scatterof nuclei in Chinese hamster embryo (CHE) cells revealed a concentration-dependenttemporary block in the G₂/M phase of the first cell cycle. NTH3T3 cells and human amniotic fluid fibroblast-like cells, onthe contrary, did not show any cell cycle disturbances in thepresence of BrdU. Micronucleus frequency increased as soon asCHE cells started to divide and reached a plateau when all cellshave divided. The height of this plateau was almost equal for60 and 100 µM BrdU. This saturation of micronucleus inductionwas due to a saturation of BrdU incorporation into DNA alreadyat a dosis of 60 µM as shown by the BrdU/Hoechst quenchingtechnique. Indirect immunofluorescent staining of kinetochoreswith CREST antibodies revealed that nearly all BrdU-inducedmicronuclei were kinetochore-negative suggesting the presenceof acentric chromosome fragments in these micronuclei. DNA distributionsof micronuclei measured by flow cytometry showed several peaksrepresenting micronuclei which contain DNA fragments of definedsizes induced by non-random breakage of chromosomes 1 and Xas verified by flow karyotyping and C-banding.     

Analysis of cell division among subpopulations of lymphoid cells in sheep. II. Peripheral lymphocytes

The number, distribution and surface phenotype of dividing cells in lymph nodes and blood and differences between the cell-cycle status of lymphocyte subpopulations were studied in lambs using double-labelling techniques. Dividing cells were labelled in vivo for various time periods with 5-bromo-2-deoxyuridine (BrdU). After removal of lymphoid tissues, the proportions of constituent lymphocyte subpopulations which had synthesized DNA during the labelling period were measured by flow cytometry or immunohistochemistry using a panel of monoclonal antibodies (mAbs) specific for sheep lymphocyte differentiation antigens and MHC class I and class II antigens in conjunction with an anti-BrdU mAb. There was a higher overall level of cell division in the ileocaecal lymph node than in either the prescapular or parathymic lymph nodes. In all three lymph nodes, the majority of lymphocytes which incorporated BrdU occurred in B-cell follicles or germinal centers. CD4+ and CD8+ T cells had a higher level of cell division (LI 5-14%) than those recognized by mAb 197 (CD4- CD8- subset) (LI less than or equal to 3%).     

Cell surface and intracellular marker analysis as a laboratory test for the diagnosis of hematological disorders

Immunophenotyping of hematopoietic malignancies is representative application of cell (surface) marker analysis by flow cytometry and monoclonal antibodies in the clinical laboratory. The multitude of available monoclonal antibodies demands a standardization of the selection and combination of antibodies. Therefore, some international committee or working group proposed the panels or guidelines for the selection of antibodies. Intracellular antigens are of major importance for immunophenotyping of hematological malignancies, and flow cytometric detection of intracellular antigens was improved by the development of new permeabilization/fixation solutions. Recently new gating method was recommended for better isolating the target cells in the flow cytometric analysis. Finally CD55 and CD59 assay for the diagnosis of PNH was mentioned.     

Medicaid and Medicare reimbursement for flow cytometry

Medicaid, a program administered by individual states but involving federal funding, is the source of medical coverage for many low-income patients. This method of reimbursement is crucial for many flow cytometry laboratories, but is not well understood by many laboratory professionals. Conversely, flow cytometry often is not well understood by administrators in Medicaid offices. The potential exists for great variation in Medicaid reimbursement for flow cytometry services from state to state. As a first step toward elucidating the extent of this variation and bringing more information about Medicaid to laboratory professionals, state Medicaid offices were asked to provide the fee-for-service reimbursement for flow cytometry services. These services included Current Procedural Terminology (CPT) codes 85045 (reticulocyte counts), 86359 (total T-cell count), 86360 (absolute CD4 and CD8 counts, with ratio), 86361 (absolute CD4 count), 86812 (HLA typing, single antigen [B27]), 88180 (immunophenotyping, per surface marker), and 88182 (DNA, cell cycle analysis). Data were collected on technical and professional components and on global reimbursement. Wide variation exists in reimbursement amounts for these tests. Variation for CPT code 88180 was markedly pronounced.     

Analyses of cell cycle and DNA]

Cell cycle and DNA index have been analyzed mainly by flow cytometry by staining cells with propidium iodide (PI) and by analyzing obtained data by computer. On the other hand, as bromodeoxyuridine (BrdU) is incorporated into single-stranded DNA, cells labeled by BrdU are in the S phase. Double staining with PI and BrdUrd enabled us to distinguish early S from G0G1 phase and late S from G2M phase. However, recent developments in molecular biology have afforded us evidences that many kinds of proteins, such as products of oncogene and suppressor gene and cell cycle-regulating and suppressing proteins, have important roles in cell cycle progression. In this paper, I would like to introduce our recent results concerning to the cell cycle, especially evaluation of cell cycle by BrdU-staining method, relationship between prognoses of patients with lung cancer after complete resection and cell cycle factors, an association of Ras family with farnesylation inhibitors, effects of microtubules on transportation of MAPK into the nucleus, and localization of Grb2.     

Flow cytometry and its application in small animal oncology

Flow cytometry measures multiple characteristics of single cells. The use of flow cytometry in the veterinary clinical laboratory has increased considerably during the past decade. The most common applications of flow cytometry in small animal oncology are measurement of DNA content in tumours and immunophenotyping of haematopoietic malignancies. DNA ploidy and S-phase rate of various tumours in dogs have been found to be independent predictor of patient outcome. In dogs with lymphomas immunophenotyping is recommended as a part of the patient work-up. Flow cytometry has shown to be a suitable method for immunophenotyping of canine lymphomas. However, it has not become a routine technique in small animal oncology yet. This report reviews the applications of flow cytometry in small animal oncology. Besides basic principles and technical aspects, the clinical relevance of DNA-analysis and immunophenotyping are discussed.     

Immunophenotyping of lymphocyte subsets in bronchoalveolar lavage fluid. Comparison of flow cytometric and immunocytochemical techniques.

In order to compare flow cytometry with the conventional peroxidase anti-peroxidase method for the immunophenotyping of bronchoalveolar lavage fluid (BALF) lymphocytes, we studied BALF samples from 27 patients with various interstitial lung diseases. The results achieved with both methods were consistent concerning CD3+ pan T cells, CD4+ T helper/inducer, CD8+ T suppressor/cytotoxic and CD57+ natural killer cells. In contrast, a statistically significant lower anti-HLA-DR positive subset was obtained with flow cytometry than with the immunoperoxidase method (p less than 0.005). Since regression analyses and reliability counts showed further agreement between the methods, we conclude that flow cytometric immunophenotyping of BALF lymphocytes leads to similar, if not better, subset analyses than the immunoperoxidase method.     

Dual‐labelled antibodies for flow and mass cytometry: A new tool for cross‐platform comparison and enrichment of target cells for mass cytometry

Antibody conjugates applicable in both conventional flow and mass cytometry would offer interesting options for cross‐platform comparison, as well as the enrichment of rare target cells by conventional flow cytometry (FC) sorting prior to deep phenotyping by mass cytometry (MC). Here, we introduce a simple method to generate dual fluorochrome/metal‐labelled antibodies by consecutive orthogonal labelling. First, we compared different fluorochrome‐conjugated antibodies specific for CD4, such as FITC, Vio667, VioGreen or VioBlue for their compatibility with the conventional secondary MAXPAR^® labelling protocol. After labelling with ¹⁴¹Pr, the fluorescence emission spectra of all fluorochromes investigated retained their characteristics, and CD4 dual conjugates (DCs) provided consistent results in immune phenotyping assays performed by FC and MC. The phenotypical composition of CD4⁺ T‐cells was maintained after enrichment by FC sorting using different CD4 DCs. Finally, magnetic cell depletion was combined with FC sorting using CD19‐VioBlue‐¹⁴²Nd, CD20‐VioGreen‐¹⁴⁷Sm, CD27‐Cy5‐¹⁶⁷Er and CD38‐Alexa488‐¹⁴³Nd DC to enrich rare human plasmablasts to purities >80%, which allowed a subsequent deep phenotyping by MC. In conclusion, DCs have been successfully established for direct assay comparison between FC and MC, and help to minimise MC data acquisition time for deep phenotyping of rare cell subsets.     

人脐带间充质干细胞的富集及向神经细胞的诱导分化

目的:寻找一种稳定、高效的分离人脐带间充质干细胞(MSCs)的方法,并探讨脐带MSCs向神经细胞方向分化的可能性。方法:分别采用组织块贴壁法和双酶消化法分离人脐带MSCs,对比其培养成功率;BrdU掺入实验检测脐带MSCs的增殖能力;流式细胞仪检测脐带MSCs表面分子标志;采用丹参联合生长因子的方法诱导其向神经细胞分化,免疫荧光方法检测神经元特异性烯醇化酶(NSE)、微管相关蛋白2(MAP2)、胶质纤维酸性蛋白(GFAP)的表达。结果:组织块贴壁法获得脐带MSCs成功率高;BrdU阳性标记率达90%以上;流式细胞仪检测显示细胞表达CD29、CD44和CD90,不表达CD34;脐带MSCs经诱导分化,伸出长突起,呈神经元样细胞改变,且表达神经元标志性蛋白NSE、MAP2。神经胶质细胞标志性蛋白GFAP表达较少。结论:成功建立了高效、稳定的脐带MSCs分离培养方法。脐带MSCs经诱导可向神经细胞方向分化,为临床移植治疗神经系统疾病提供了理想的细胞来源。     

Measurement of lymphocyte subset proliferation by three-color immunofluorescence and DNA flow cytometry

We developed a method for simultaneous flow cytometric analysis of three-color immunofluorescence and DNA content. We show here that staining with 7-amino-actinomycin D (7-AAD) at 10 microg/ml using a phosphate-citrate buffer at low pH containing saponin for cell membrane permeabilization yields good resolution DNA histograms with low coefficients of variation. Furthermore, light scatter properties of cells are preserved after permeabilization; this permits gating on cell populations that differ in scatter signals on the flow cytometer. Because of the low pH of the phosphate-citrate staining buffer, Alexa488, a pH-independent green-fluorescent fluorochrome is used instead of fluorescein-isothiocyanate (FITC) for cell surface staining in combination with phycoerythrin (PE) and with allophycocyanin (APC) which are both pH insensitive. Removal of 7-AAD after staining and replacing it with non-fluorescent actinomycin D (AD) retains DNA staining and allows detection of Alexa488, PE and APC cell surface immunofluorescence without interference from fluorescent 7-AAD in solution for clear identification of cell subpopulations even after prolonged stimulation in culture. Thus, using a four-color benchtop flow cytometer, measurement of Alexa488, PE and APC three-color immunofluorescence can be combined with 7-AAD DNA content analysis. Furthermore, we demonstrate that sample storage overnight without fixation for later analysis on the flow cytometer is possible without compromising results. Application of the method to the assessment of the differential proliferative responses of lymphocyte subsets of human peripheral blood mononuclear cells (PBMC) that were costimulated with CD3 and with CD28.2 is presented.     

An automated method to prepare cell suspensions from human biopsy samples for immunophenotyping by flow cytometry

A comparison was made between a manual and automated method for preparing single cell suspensions from various types of human biopsy samples. The automated method uses the shearing action of fluid movement generated by a reciprocating paddle system. When compared on 11 samples, the automated instrument always isolated cells in less time, usually requiring only 15-30 seconds in contrast to 10-15 minutes for the manual method. The time comparisons involved "set up" time of the required minor equipment as well as the time to make a complete single cell suspension from the portion of the biopsy sample sent to the Flow Cytometry Lab so extra cells could be used for other purposes and cryogenically stored for future reference. Cells isolated by the automated method from various B-lymphocytic and T-lymphocytic malignancies were still viable and could be successfully immunophenotyped by flow cytometry. The immunophenotyping results were compared for both cell isolation methods on seven of these samples, and the results were comparable. The automated technique has now been used to satisfactorily immunophenotype more than 50 biopsy samples. The automated method should represent a significant aid for clinical flow cytometry laboratories performing immunophenotyping tests by efficiently preparing single cell suspensions in a few seconds instead of minutes. Furthermore, the automated method uses a closed sterile bag system that helps minimize the exposure of personnel to potential infectious material present in biopsy samples and prevents external contamination of cell suspensions. The automated technique proven successful for immunophenotyping may also be helpful for related procedures involving hematopoietic malignancies such as DNA content analysis and cell functional assays by flow cytometry, as well as other assays such as tissue typing, gene probe, and in vitro chemosensitivity assays.     

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。     

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。     

Flow cytometric DNA-histogram analysis: non-stoichiometric fluorochrome binding and pseudoaneuploidy.

Detection of aneuploid subpopulations using flow cytometry requires stoichiometric binding of nucleic acid-specific fluorochromes onto DNA. It is shown that parameters like cell type specificity and differentiation stage, cell cycle stage, loss of DNA-integrity, cell preparation, and cytochemistry affect fluorochrome binding to DNA and give rise to the appearance of pseudo-aneuploid cell populations. Intercalating as well as non-intercalating fluorochromes show non-stoichiometric DNA-labelling in cell populations with identical DNA content, and pseudo-aneuploidy was found in flow cytometers equipped with either arc lamps or argon lasers. Pseudo-aneuploidy was never observed with intercalating and non-intercalating fluorochromes within identical specimens, consisting of cells of various differentiation states (e.g., bone marrow) or containing large numbers of dead cells. Therefore, fluorochromes exhibiting different base-pair specificities or steric binding modes should be applied to be sure of the correct interpretation of small levels of hypo- or hyper-diploidy (+/- 20 per cent).     

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.     

Circulating CD14-CD36+ peripheral blood mononuclear cells constitutively produce interleukin-10

The impact of immune regulatory imbalance covers surprising physiological breadth. Although dominance of anti-inflammatory cytokines such as IL-10 is associated with reduced immune responsiveness and susceptibility to persistent infection, conditions such as cardiovascular disease and diabetes are linked to chronic inflammation and lower IL-10 levels. An appropriate threshold for immune activation is critical for optimal protection from infection and conversely, from short- and long-term side-effects of immune effector mechanisms. To assess the possibility that IL-10 plays a role in setting this threshold and that healthy maintenance of immune silence may involve low-level immune suppression, we sought out and characterized human peripheral blood cells constitutively producing the immunosuppressive cytokine IL-10. We determined the surface phenotype of circulating PBMC constitutively producing IL-10 by surface and intracellular flow cytometry and visualized their ultrastructure by electron microscopy. The frequency of IL-10-producing and -secreting cells was estimated by ELISPOT and flow cytometry. Up to 1% of PBMC constitutively produce IL-10. These CD14(-)CD36(+)CD61(+) nonadherent cells expressed general markers of hematopoietic and progenitor cells (CD45 and CD7) but no stem cell, T cell, B cell, NK cell, monocytes or dendritic cell markers. Inflammation-associated TLRs were also absent. The IL-10-producing cells had prominent nuclei, multiple mitochondria, and abundant rough endoplasmic reticulum. Healthy individuals have PBMC constitutively producing IL-10. Although the lineage of these cells remains unclear, their properties and frequency suggest a potential role in homeostatic or innate immune suppression.