Cytometry aims to analyze cells, of any type, using dedicated instruments. The quantitative aspect makes flow cytometry (FCM) a good complementary tool for morphology. Most of the identification tools are based on immunostaining of cell structure details and more and more tools are available in terms of specificities and labels. FCM is under exponential development thanks to technical, immunological and data analysis progresses. Actual generations are now routinely using 6 to 10 simultaneous immuno-labeling on 20 to 100,000 cells, at high speed and short sample preparation and can easily detect rare events at frequency below 10-4 cells. Data interpretation is complex and requires expertise. Mathematical tools are available to support analysis and classification of cells based. Cells from tissues can also be analyzed by FCM after mechanical and or enzymatic separation, but in situ cells can also be analyzed with the help of cytometry. Very new instruments bring spectral analysis, image in flow and mass spectrometry. Medical applications are very broad, notably in hemopathies, immunology, solid tumors, but also microbiology, toxicology, drug discovery, food and environmental industry. But, the limit of FCM is its dependence on operator from sample preparation, instrument settings up to data analysis and a strong effort is now under progress for standardization and constitution of international data bank for references and education.

The estrogen receptors (ESR alpha and beta) and the androgen receptor (AR) mediate genomic and non-genomic effects on breast tumor growth and proliferation. We analyzed 101 breast cancer patients for allelic loss in microsatellites located in regulatory regions of the ESRs and AR genes in breast cancer tumors. The loss of heterozygosity (LOH) at these loci was found in 36.2% of tumor tissues (ductal carcinoma cases), for 19% of cases at the ESRa locus, for 16% at the ESR beta locus, and for 10% at the AR locus. The LOH in at least one of the two ESR loci was correlated to poor prognosis criteria: ESR-negative status (P = 0.007), PR-negative status (P = 0.003), high Scarff-Bloom-Richardson (SBR) grade (P = 0.0007), high MIB-1 proliferation index (P = 0.02), and diminished apoptosis potential (TP53-positive status, P = 0.018). When AR was also considered, the LOH in at least one of the three loci was associated with ESR-negative status (P = 0.036), PR-negative status (P = 0.027), high SBR grade (P = 0.005), high mitotic index (P = 0.0002), TP53-positive status (P = 0.029), and proliferating index (high MIS-1, P = 0.03). Allelic loss was observed in 26% of normal tissue adjacent to tumor with LOH at the ESR alpha locus and in 7.1% of tumors with LOH at the ESR beta locus. The LOH in tumor tissue in the regulatory regions of ESR alpha, ESR beta, and AR genes has potentially synergistic effects on tumor proliferation, histological aggressiveness, down-regulation of ESR alpha and progesterone receptor (PR) genes, and is an early genetic alteration in cancer that is possibly involved in passage to estrogen independence.

Usually, the blood cell counting activity in haematology laboratory uses the comparison of IQC values to the target values proposed by the manufacturer. We intended to improve the monitoring of the proper functioning of our analytical measure system for 17 main haematologic parameters. To set the allowable critical limits of IQC, we propose our reflection based on several elements: benchmark and expert recommendation, clinical requirements, statistical indicators of the laboratory calculated using IQC values (3 levels, 2 different lots, 2 haematology analysers and 2 passage modes) and the EEQ values, during four months. We exploited the reports obtained from the middleware (our own IQC values), and the interlaboratory comparison reports (obtained from SNCS and EuroCell websites) and we compared our performances to the Ricos objectives, to set clearly argued allowable limits for IQC values. Finally, the allowable limits correspond to the imprecision limits stated by Ricos for 14 parameters (desirable for 11 parametres and minimal for 3 parameters) and personalized limits (more exigent than desirable Ricos limits) for 3 parameters of blood cell counting.

La cytometrie (cyto = cellule ; metrie = mesure) consiste en l'analyse objective, quantitative et multiparametrique des cellules. Elle utilise la fluorescence, des moyens fluidiques, optiques et le soutien informatique pour le traitement des signaux ou des images. Ses performances sont exceptionnelles (analyse 4, 6, 8, a tres grande vitesse (de 500 a 10 000 cellules par seconde). La cytometrie en image est tres utilisee en recherche. En analyse medicale, la cytometrie en flux est de plus en plus utilisees, pour le typage des leucemies, la numeration des tres nombreux sous-types cellulaires par exemple pour le suivi du SIDA ou des traitements immunosuppresseurs et greffes. De plus, l'etat d'activation, de maturation et de proliferation des cellules peuvent etre mesures. La haute precision et large utilisation de la cytometrie en routine a deja permis de decrire des nouveaux sous-types cellulaires tels que les lymphocytes T gamma delta, muqueux, les cellules TNK, les lymphocytes regulateurs dont l'action est ciblee au sein de la cellule immune. Des proliferations monoclonales de signification indeterminees mais potentiellement evolutives ont ete observees. Les desordres proliferatifs et des heteroploidies peuvent etre rapidement analyses. D'autres applications en cancerologie et microbiologie sont en cours de developpement. En conclusion, l'apport majeur de la cytometrie est de pouvoir aborder les populations cellulaires dans leur grande diversite et complexite. Il serait en effet ridicule de limiter ces systemes a des ensembles homogenes et uniformes. La « sociologie » des populations cellulaires est un nouveau champ encore a defricher. ================================================== Cytometry (cyto = cell; metry = measuring) consist in an objective, quantitative et multiparametric analysis of cells. It is based on fluorescence, fluidic and optical tools with the help of signal or image computer treatment. It is a high performance system allowing simultaneously analysis 4, 6, 8 or more parameters, at very high speed from 500 to 10 000 cells per second). Image cytometry is largely used in research. In medical analysis, Flow-cytometry is more and more used for leukaemia typing, lymphocyte counting, among them HIV monitoring or immunological treatments follow-up. Identification of numerous cell subtypes and their activation, maturation or proliferation status is now possible. Its high precision and wide use have lead to the description of new cell subtypes (e.g. gamma delta, mucosal, T.NK or regulatory T cells...) that have targeted activity. Recently, oligoclonal clonopathies of undetermined significance but with risks for pathological development have been described. Proliferative or heteroploidy disorders can also be evaluated. Applications in solid tumor and microbiology are under development. In conclusion, the major point of cytometry is to bring tools to approach cell populations in their diversity and complexity. It would be ridiculous to consider them as homogeneous, uniform systems. In other words, cell « sociology » is a wide new field that remains to be explored.

Background: alpha beta T cells have a wide distribution of CD3 membrane density. The aim of this article was to evaluate the significance of the CD3 differential expression on T cell subsets.Analysis was performed on healthy donors and renal transplant patients by flow cytometry.The results obtained are: (1) CD3 expression was widely distributed (CV = 38.3 +/- 3.1 to 43 +/- 2.3%). (2) The CD4, CD8, CD45 and forward scatter were similarly distributed. (3) The diversity of CD3 expression was directly related to the clonotypes: gamma 9, non gamma 9 from gamma delta T cells and V beta clonotype from alpha beta T cells (e.g., V beta 3FITC 7,980 +/- 1,628 V beta 8PE: V beta 20-FITC 11,768 +/- 1,510). (4) Using a computer simulation, we could confirm differential kinetics of T cell activation according to the initial parameters. Finally, in vitro activation was significantly higher on v beta 8 and V beta 9 (high CD3) compared with V beta 2 and v beta 3 (low CD3, P = 0.040-0.0003).In conclusion, T cells have highly heterogeneous CD3 expression, possibly predetermined and with clear functional significance. (C) 2009 Clinical Cytometry Society

La cytométrie (cyto = cellule ; métrie = mesure) consiste en l’analyse objective, quantitative et multiparamétrique des cellules. Elle utilise la fluorescence, des moyens fluidiques, optiques et le soutien informatique pour le traitement des signaux ou des images. Ses performances sont exceptionnelles et permettent l’analyse simultanée de 4, 6, 8 paramètres, voire plus, à très grande vitesse (de 500 à 10 000 cellules par seconde). La cytométrie en image est encore une technique de recherche. En analyse médicale, la cytométrie en flux est de plus en plus utilisée, pour le typage des leucémies, la numération des très nombreux sous-types cellulaires, par exemple pour le suivi du sida ou des traitements immunosuppresseurs et des greffes. De plus, l’état d’activation, de maturation et de prolifération des cellules peut être mesuré. La haute précision et large utilisation de la cytométrie en routine a déjà permis de décrire de nouveaux sous-types cellulaires tels que les lymphocytes T gamma delta, muqueux, les cellules TNK, les lymphocytes régulateurs, dont l’action est ciblée au sein de la réponse immune. Des proliférations monoclonales de signification indéterminées mais potentiellement évolutives ont pu être observées. Les désordres prolifératifs et des hétéroploïdies peuvent être rapidement analysés. D’autres applications en cancérologie et microbiologie sont en cours de développement. En conclusion, l’apport majeur de la cytométrie est de pouvoir aborder les populations cellulaires dans leur grande diversité et complexité. Il serait en effet aujourd’hui ridicule de limiter ces systèmes à des ensembles homogènes et uniformes. La « sociologie » des populations cellulaires est un nouveau champ encore à défricher.

OBJECTIVE Macrophage-derived tumor necrosis factor alpha (TNFalpha) is a dominant mediator of synovitis in rheumatoid arthritis (RA). This study was undertaken to assess whether and how immune complexes (ICs) formed by the interaction of disease-specific autoantibodies to citrullinated proteins (ACPAs) with their main synovial target antigen, citrullinated fibrin, contribute to TNFalpha production by macrophages. METHODS An in vitro human model was developed in which monocyte-derived macrophages were stimulated with ACPA-containing ICs that were generated by capturing ACPAs from RA sera on immobilized citrullinated fibrinogen. Cellular activation was evaluated by TNFalpha assay in culture supernatants. Selective blockade of IC interactions with the 3 classes of Fcgamma receptors (FcgammaR) was used to assess the contribution of each receptor to macrophage activation. In addition, 2 citrullinated fibrin-derived peptides bearing major ACPA epitopes were tested for their capacity to inhibit formation of macrophage-activating ACPA-containing ICs. RESULTS ACPA-containing ICs induced a dose-dependent TNFalpha secretion by macrophages from 14 of 20 healthy donors. The macrophage response was systematically higher than that of the paired monocyte precursors. TNFalpha secretion was not reduced by blockade of FcgammaRI or FcgammaRIII, but was strongly repressed when interaction of ICs with FcgammaRII was prevented. The 2 citrullinated peptides significantly inhibited ACPA reactivity to citrullinated fibrinogen and, when tested together, almost completely abolished formation of macrophage-activating ICs, thereby diminishing the secreted TNFalpha levels. CONCLUSION Our model demonstrates the inflammatory potential of ACPA-containing ICs via engagement of FcgammaRIIa at the surface of macrophages, strongly supporting their pathophysiologic involvement. Continuing dissection of these molecular pathways could open the way to new therapeutic approaches in patients with RA.

Given that breast cancer is depending on multiple hormonal influences, the nuclear receptors, estrogen receptor alpha, estrogen receptor beta and androgen receptor, are candidates for cancer susceptibility markers. We conducted an association study in a case-control population (139 cases and 145 controls) by genotyping three potentially functional microsatellites (TA)n, (CA)n and (CAG)n in the ERa, ERb and AR genes respectively. For (CAG)n polymorphism, a significant difference was observed using a cut-off 15 repeats CAG between genotypes short-short/short-long/long-long in cases and control subjects (p = 0.009) and also between the distribution of short/long allele in the two groups of individuals (p = 0.001). Genotypes comprising one or two short (CAG)n sequences had higher risk of breast cancer compared to genotypes with two long allele (odds ratio = 1,93; confidence interval = 1.05-3.55; p = 0.03). No significant difference was observed in allele frequency or in short/long allele percentage for (CA)n or (TA)n polymorphism (cut-off 22 CA and 19 TA repeats), neither in genotype frequencies (short-short, short-long or long-long). When the three microsatellite genotype were taken in analysis, the profile short CA-long TA-short CAG could clearly discriminate between cases and controls (p = 0.006). Also, this combined genotype profile has greater predictive values for breast cancer than (CAG)n genotype alone (predictive positive value 57,1% versus 53,7% and predictive negative value 53% versus 23% respectively). Our results sustain a polygenic model of breast cancer with gene-gene interactions; combined effects of three low-risk polymorphisms conferred significant genetic predisposition. Genotyping hormonal receptor genes ERa, ERb and AR could be a useful genetic marker for defining disease risk.

Technological advances in instruments allow the evaluation of many lymphocyte subsets in one step. The aim of this study was to evaluate the new FACSCanto flowcytometer in routine conditions, using a 6 color combination, single platform, whole blood, lysis, no wash protocol.

Background: Routine CD4/CD8 T-cell phenotyping may shows a small fraction of CD4 (dim)CD8(+) T cells with a homogeneous appearance as described for lymphoproliferative syndromes or chronic infections. The aim of this study was to elucidate the significance of CD4(dim)CD8(+) T cells and their degree of diversity.Methods: Phenotyping was performed in 272 samples from healthy donors, elderly patients, and immunocompromised (human immunodeficiency virus or renal transplantation) patients.Results: The CD4(dim)CD8(+) T cells had decreased fluorescence intensity for CD4 but not for CD8. The frequency of patients with CD4(dim)CD8(+) T cells (> 20 cells/mu l; 10.3% of patients with human immunodeficiency virus and 7.7% with renal transplantation) was not significantly different when compared with healthy donors (9.7%). The CD4(dim)CD8(+) T cells did not express the activation marker CD69. The CD8 of CD4(dim)CD8(+) T cells expressed the heterodimeric (alpha beta) isoform. In 13 of 26 samples, the apparently highly homogeneous CD4(dim)CD8(+) T cells expressed one predominant T-cell receptor V beta clonotype. These predominant clonotypes were widely distributed among patients: V beta 5.2, 17, 2, 3, 5.1, 13.1, 14, and 20.Conclusions: Whether these findings demonstrate an oligoclonal reaction to chronic inflammation or an emerging lymphoproliferative disorder must be elucidated in a long-term longitudinal study. By analogy to monoclonal gammopathy, we propose to name this phenomenon "oligoclonal clonopathy of undetermined significance." (c) 2005 Wiley-Liss, Inc.

Routine T cells phenotyping occasionally reveals a CD4+CD8dim T cell subset with an apparently homogeneous dot plot. The aim of this study was to elucidate their immunological significance from analysis of 31 healthy donors, 21 elderly and 220 immune deficient patients. CD4+CD8dim T cells expressed reduced levels of CD8 (11-17,000 compared to 96-128,000 mol/cell on CD8+ T Cells). CD4 was expressed at the same level as on CD4+ T cells. The occurrence of raised CD4+CD8dim T cells (> 20 cells/muL) was similar in kidney transplant recipients (28.4%) and healthy donors (26%). It was somewhat lower in HIV+ patients (19.7%) possibly due to virally induced CD4+ T lymphopenia. However, an age effect is possible because the occurrence was raised (33.3%) in 70 volunteers (chi2 test NS). On the other hand, the size of the CD4+CD8dim subset was not correlated with age. CD4+CD8dim T cells did not express the activation markers CD69 (n = 220) or CD25 (n = 10) and expressed the homodimeric (alphaalpha) isoform of CD8, suggesting they are related to mucosal immunity (MALT). We selected 29 patients with unambiguous dot plots. In 26 of them one predominant TCR Vbeta clonotype was expressed on 18 to 94% of CD4+CD8dim T cells and never on more than 10% of conventional T cells. The predominant clonotypes were Vbeta8 (n = 5), Vbeta2 (n = 4), Vbeta13.1 and Vbeta 21 (n = 3 each). Whether this reveals a chronic stimulation or an emerging lymphoproliferative disorder must be elucidated. We propose to name this entity: "Oligoclonal Clonopathy of Undetermined Significance (OCUS)."