BackgroundFlow cytometry (FCM) aids the diagnosis and prognostic stratification of patients with ... more BackgroundFlow cytometry (FCM) aids the diagnosis and prognostic stratification of patients with suspected or confirmed myelodysplastic syndrome (MDS). Over the past few years, significant progress has been made in the FCM field concerning technical issues (including software and hardware) and pre‐analytical procedures.MethodsRecommendations are made based on the data and expert discussions generated from 13 yearly meetings of the European LeukemiaNet international MDS Flow working group.ResultsWe report here on the experiences and recommendations concerning (1) the optimal methods of sample processing and handling, (2) antibody panels and fluorochromes, and (3) current hardware technologies.ConclusionsThese recommendations will support and facilitate the appropriate application of FCM assays in the diagnostic workup of MDS patients. Further standardization and harmonization will be required to integrate FCM in MDS diagnostic evaluations in daily practice.
Multiple myeloma (MM) is still considered incurable and new therapeutic approaches are therefore ... more Multiple myeloma (MM) is still considered incurable and new therapeutic approaches are therefore needed. Deep‐sequencing analysis revealed the presence of BRAF mutations in up to 15% of patients. The clinical experience of BRAF‐targeted therapy in myeloma patients harboring BRAF mutation is still limited. We here report the case of a patient with penta‐refractory (bortezomib, lenalidomide, carfilzomib, pomalidomide, and daratumumab) MM with extramedullary BRAF‐mutated disease that achieved clinical response to dual BRAF and MEK inhibition. At the time of disease progression, gene sequencing analysis of the tumor at the time of progression demonstrated a clonal evolution with emergence of a NRAS mutation and persistence of BRAF and TP53 mutations. Backtracking of the NRAS mutation was performed by digital polymerase chain reaction on the baseline biopsy and identified the pre‐existence of the NRAS at a subclonal level. This observation is the first report of acquired NRAS mutation le...
International Journal of Laboratory Hematology, 2019
Monoclonal antibodies have become daily partners of both biologists and clinicians, as reagents a... more Monoclonal antibodies have become daily partners of both biologists and clinicians, as reagents and therapeutic agents. Behind their odd names and incredible diversity lies an amazing story of inventiveness and daring. This review tries to retrace the major steps of this saga, initiated by the search for anti‐rabbit red blood cell antibodies and currently culminating in amazing molecular constructions saving lives. After some historical and basic reminders, the fields of reagents and drugs will be addressed. This invaluable contribution of immunology to the understanding of both physiology and treatment clearly deserves to be fully recognized.
Lymphocyte multiplication can be inducedin vitroby mitogens or specific antigens, and is usually ... more Lymphocyte multiplication can be inducedin vitroby mitogens or specific antigens, and is usually measured using isotopic methods involving tritiated thymidine. Cellular proliferation can also be analyzed by flow cytometry techniques based on cell cycle analysis through the measurement of DNA content. We applied this method to lymphocytes from 113 individuals, to evaluate lymphocyte proliferation after stimulationin vitroby a mitogen (phytohaemagglutinin, PHA) or a recall antigen (tetanus toxoid), using a kinetic approach with four points sequential measurements of the S and G2 phases over six days of culture. The proportion of cells in S phase after PHA stimulation was significantly higher than in controls overall and as early as on day three of the culture. Activation with a recall antigen significantly induced increasing S phase cell proportions up to day six. These data suggest that flow cytometric assessment of the S phase could be a useful alternative to isotopic methods measur...
ABSTRACTThe triggering receptor expressed on myeloid cell type 1 (TREM-1) is a cell surface molec... more ABSTRACTThe triggering receptor expressed on myeloid cell type 1 (TREM-1) is a cell surface molecule that has been identified on both human and murine polymorphonuclear neutrophils and mature monocytes. The activation of TREM-1 in the presence of microbial components amplifies the inflammatory response and may be responsible for the hyperresponsiveness observed during the initial stage of sepsis. To investigate the effect of the modulation of the TREM-1 pathway during experimental murine sepsis, we used analogue synthetic peptides derived from the extracellular moiety of TREM-1. The TREM-1 ligand was expressed on both peritoneal and peripheral neutrophils during experimental peritonitis in mice. The TREM-1 peptides inhibited the recognition by TREM-1 of its ligand and protected endotoxinic mice from death. In septic rats, the TREM-1 peptides improved the hemodynamic status, attenuated the development of lactic acidosis, modulated the production of such proinflammatory cytokines as t...
SUMMARYThe origin of mesangial IgA deposits in IgA nephropathy (IgAN) remains obscure. A signific... more SUMMARYThe origin of mesangial IgA deposits in IgA nephropathy (IgAN) remains obscure. A significant proportion of deposited immunoglobulin is dimeric (J chain-positive). Previous studies of J chain expression within lymphoid tissue in IgAN have utilized antibodies which other investigators have found to be non-specific. To address this problem, we have developed an in situ hybridization (ISH) method for the detection of J chain mRNA within IgA plasma cells. Tonsils from 12 patients with IgAN and 12 controls were studied using (i) non-isotopic ISH for J chain mRNA, and (ii) combined immunofluorescence (IF) and fluorescent ISH. J chain mRNA-positive cells were identified in germinal centres, and within the subepithelial and interfollicular zones. A greater number of J chain mRNA-positive cells were found in the germinal centres of patients (mean 57.7±4.6 cells/105μm2) compared with controls (mean 36.9±3.5 cells/105μm2) (P < 0.001). Combined IF and fluorescent ISH showed a greater ...
Summary This study was designed to investigate the immunological properties of stroma reaction T ... more Summary This study was designed to investigate the immunological properties of stroma reaction T cells and tumoral cells by comparison with non-tumoral lung tissue and local lymph nodes in order to explore interactions between tumour cells and the immune system. Immunodetection of major histocompatibility complex (MHC) molecules, CD3/T cell receptor (TCR) complex and T cell subsets markers was carried out in situ on frozen sections, and the semi-quantitative expression of CD3, CD4 and CD8 was examined in flow cytometry on lymphocytes of nodal, tumoral and healthy lung tissue from 62 patients with non-small cell lung cancer. This study showed alterations on lymphocytes and tumour cells in lung cancer, consistent with an impairment of T cell activation. CD3, TCRαβ and accessory molecules expression is down-modulated on peri- or intra-tumoral lymphocytes. MHC class I and class II molecules are down-modulated significantly on tumour cells. Other differences were noted, such as the rever...
Cytokines are key modulators of the immune system and also contribute to regulation of the ovaria... more Cytokines are key modulators of the immune system and also contribute to regulation of the ovarian cycle. In this study, Bender MedSystems FlowCytomix technology was used to analyze follicular cytokines (proinflammatory: IL-1β, IL-6, IL-18, IFN-γ, IFN-α, TNF-α, IL-12, and IL-23;, and anti-inflammatory: G-CSF), chemokines (MIP-1α, MIP-1β, MCP-1, RANTES, and IL-8), and other biomarkers (sAPO-1/Fas, CD44(v6)) in 153 women undergoingin vitrofertilization (IVF). Cytokine origin was studied by mRNA analysis of granulosa cells. Higher follicular MIP-1αand CD44(v6) were found to correlate with polycystic ovary syndrome, IL-23, INF-γ, and TNF-αwith endometriosis, higher CD44(v6) but lower IL-βand INF-αcorrelated with tubal factor infertility, and lower levels of IL-18 and CD44(v6) characterized unexplained infertility. IL-12 positively correlated with oocyte fertilization and embryo development, while increased IL-18, IL-8, and MIP-1βwere associated with successful IVF-induced pregnancy.
gamma-Irradiation of acrolein and other acrylic monomers allowed the synthesis of spherical polyf... more gamma-Irradiation of acrolein and other acrylic monomers allowed the synthesis of spherical polyfunctional hydrophilic microparticles in the size range of 50 to 300 nm, on which antigens (immunoglobulins G, chorionic gonadotropin hormone, prealbumin) could be covalently bound. Microsphere-antigen conjugates clustered together in the presence of specific antiserum or monoclonal antibodies and their agglutination was quantified by light-scattering measurement performed with a specially designed nephelometer. Essential factors concerning the conjugate agglutination and its quantitation (size of microsphere, amount of antigen bound on microsphere, concentration of conjugate, concentration of agglutinating reagent, angle of light-scattering observation) were successively studied. A microparticle-enhanced nephelometric immunoassay for prealbumin was finally developed as an example of application. It was based on the inhibition of the immunoagglutination of microspheres-prealbumin conjugate by free prealbumin. This prealbumin immunoassay was easy to perform (one-step assay without washing or phase separation), fast (30 min), reliable (variation coefficients ranged from 3.6% to 7.5% for within- and between-assay determination), and sensitive (1 microgram/L detected). It was correlated with conventional immunonephelometry and radial immunodiffusion (correlation coefficients, 0.98). Microparticle-enhanced nephelometric immunoassay offered many advantages over the last two methods. Its better sensitivity allowed a lower reagent consumption and a larger sample dilution (contrary to the conventional immunonephelometry, sample pretreatment and sample blank measurement were unnecessary). Its inhibition mode induced a total accuracy for sample with high analyte concentration (a risk of underevaluation in antigen excess conditions existed in all method based on a noncompetitive antigen-antibody reaction) and provided the possibility to quantify haptens.(ABSTRACT TRUNCATED AT 250 WORDS)
BackgroundFlow cytometry (FCM) aids the diagnosis and prognostic stratification of patients with ... more BackgroundFlow cytometry (FCM) aids the diagnosis and prognostic stratification of patients with suspected or confirmed myelodysplastic syndrome (MDS). Over the past few years, significant progress has been made in the FCM field concerning technical issues (including software and hardware) and pre‐analytical procedures.MethodsRecommendations are made based on the data and expert discussions generated from 13 yearly meetings of the European LeukemiaNet international MDS Flow working group.ResultsWe report here on the experiences and recommendations concerning (1) the optimal methods of sample processing and handling, (2) antibody panels and fluorochromes, and (3) current hardware technologies.ConclusionsThese recommendations will support and facilitate the appropriate application of FCM assays in the diagnostic workup of MDS patients. Further standardization and harmonization will be required to integrate FCM in MDS diagnostic evaluations in daily practice.
Multiple myeloma (MM) is still considered incurable and new therapeutic approaches are therefore ... more Multiple myeloma (MM) is still considered incurable and new therapeutic approaches are therefore needed. Deep‐sequencing analysis revealed the presence of BRAF mutations in up to 15% of patients. The clinical experience of BRAF‐targeted therapy in myeloma patients harboring BRAF mutation is still limited. We here report the case of a patient with penta‐refractory (bortezomib, lenalidomide, carfilzomib, pomalidomide, and daratumumab) MM with extramedullary BRAF‐mutated disease that achieved clinical response to dual BRAF and MEK inhibition. At the time of disease progression, gene sequencing analysis of the tumor at the time of progression demonstrated a clonal evolution with emergence of a NRAS mutation and persistence of BRAF and TP53 mutations. Backtracking of the NRAS mutation was performed by digital polymerase chain reaction on the baseline biopsy and identified the pre‐existence of the NRAS at a subclonal level. This observation is the first report of acquired NRAS mutation le...
International Journal of Laboratory Hematology, 2019
Monoclonal antibodies have become daily partners of both biologists and clinicians, as reagents a... more Monoclonal antibodies have become daily partners of both biologists and clinicians, as reagents and therapeutic agents. Behind their odd names and incredible diversity lies an amazing story of inventiveness and daring. This review tries to retrace the major steps of this saga, initiated by the search for anti‐rabbit red blood cell antibodies and currently culminating in amazing molecular constructions saving lives. After some historical and basic reminders, the fields of reagents and drugs will be addressed. This invaluable contribution of immunology to the understanding of both physiology and treatment clearly deserves to be fully recognized.
Lymphocyte multiplication can be inducedin vitroby mitogens or specific antigens, and is usually ... more Lymphocyte multiplication can be inducedin vitroby mitogens or specific antigens, and is usually measured using isotopic methods involving tritiated thymidine. Cellular proliferation can also be analyzed by flow cytometry techniques based on cell cycle analysis through the measurement of DNA content. We applied this method to lymphocytes from 113 individuals, to evaluate lymphocyte proliferation after stimulationin vitroby a mitogen (phytohaemagglutinin, PHA) or a recall antigen (tetanus toxoid), using a kinetic approach with four points sequential measurements of the S and G2 phases over six days of culture. The proportion of cells in S phase after PHA stimulation was significantly higher than in controls overall and as early as on day three of the culture. Activation with a recall antigen significantly induced increasing S phase cell proportions up to day six. These data suggest that flow cytometric assessment of the S phase could be a useful alternative to isotopic methods measur...
ABSTRACTThe triggering receptor expressed on myeloid cell type 1 (TREM-1) is a cell surface molec... more ABSTRACTThe triggering receptor expressed on myeloid cell type 1 (TREM-1) is a cell surface molecule that has been identified on both human and murine polymorphonuclear neutrophils and mature monocytes. The activation of TREM-1 in the presence of microbial components amplifies the inflammatory response and may be responsible for the hyperresponsiveness observed during the initial stage of sepsis. To investigate the effect of the modulation of the TREM-1 pathway during experimental murine sepsis, we used analogue synthetic peptides derived from the extracellular moiety of TREM-1. The TREM-1 ligand was expressed on both peritoneal and peripheral neutrophils during experimental peritonitis in mice. The TREM-1 peptides inhibited the recognition by TREM-1 of its ligand and protected endotoxinic mice from death. In septic rats, the TREM-1 peptides improved the hemodynamic status, attenuated the development of lactic acidosis, modulated the production of such proinflammatory cytokines as t...
SUMMARYThe origin of mesangial IgA deposits in IgA nephropathy (IgAN) remains obscure. A signific... more SUMMARYThe origin of mesangial IgA deposits in IgA nephropathy (IgAN) remains obscure. A significant proportion of deposited immunoglobulin is dimeric (J chain-positive). Previous studies of J chain expression within lymphoid tissue in IgAN have utilized antibodies which other investigators have found to be non-specific. To address this problem, we have developed an in situ hybridization (ISH) method for the detection of J chain mRNA within IgA plasma cells. Tonsils from 12 patients with IgAN and 12 controls were studied using (i) non-isotopic ISH for J chain mRNA, and (ii) combined immunofluorescence (IF) and fluorescent ISH. J chain mRNA-positive cells were identified in germinal centres, and within the subepithelial and interfollicular zones. A greater number of J chain mRNA-positive cells were found in the germinal centres of patients (mean 57.7±4.6 cells/105μm2) compared with controls (mean 36.9±3.5 cells/105μm2) (P < 0.001). Combined IF and fluorescent ISH showed a greater ...
Summary This study was designed to investigate the immunological properties of stroma reaction T ... more Summary This study was designed to investigate the immunological properties of stroma reaction T cells and tumoral cells by comparison with non-tumoral lung tissue and local lymph nodes in order to explore interactions between tumour cells and the immune system. Immunodetection of major histocompatibility complex (MHC) molecules, CD3/T cell receptor (TCR) complex and T cell subsets markers was carried out in situ on frozen sections, and the semi-quantitative expression of CD3, CD4 and CD8 was examined in flow cytometry on lymphocytes of nodal, tumoral and healthy lung tissue from 62 patients with non-small cell lung cancer. This study showed alterations on lymphocytes and tumour cells in lung cancer, consistent with an impairment of T cell activation. CD3, TCRαβ and accessory molecules expression is down-modulated on peri- or intra-tumoral lymphocytes. MHC class I and class II molecules are down-modulated significantly on tumour cells. Other differences were noted, such as the rever...
Cytokines are key modulators of the immune system and also contribute to regulation of the ovaria... more Cytokines are key modulators of the immune system and also contribute to regulation of the ovarian cycle. In this study, Bender MedSystems FlowCytomix technology was used to analyze follicular cytokines (proinflammatory: IL-1β, IL-6, IL-18, IFN-γ, IFN-α, TNF-α, IL-12, and IL-23;, and anti-inflammatory: G-CSF), chemokines (MIP-1α, MIP-1β, MCP-1, RANTES, and IL-8), and other biomarkers (sAPO-1/Fas, CD44(v6)) in 153 women undergoingin vitrofertilization (IVF). Cytokine origin was studied by mRNA analysis of granulosa cells. Higher follicular MIP-1αand CD44(v6) were found to correlate with polycystic ovary syndrome, IL-23, INF-γ, and TNF-αwith endometriosis, higher CD44(v6) but lower IL-βand INF-αcorrelated with tubal factor infertility, and lower levels of IL-18 and CD44(v6) characterized unexplained infertility. IL-12 positively correlated with oocyte fertilization and embryo development, while increased IL-18, IL-8, and MIP-1βwere associated with successful IVF-induced pregnancy.
gamma-Irradiation of acrolein and other acrylic monomers allowed the synthesis of spherical polyf... more gamma-Irradiation of acrolein and other acrylic monomers allowed the synthesis of spherical polyfunctional hydrophilic microparticles in the size range of 50 to 300 nm, on which antigens (immunoglobulins G, chorionic gonadotropin hormone, prealbumin) could be covalently bound. Microsphere-antigen conjugates clustered together in the presence of specific antiserum or monoclonal antibodies and their agglutination was quantified by light-scattering measurement performed with a specially designed nephelometer. Essential factors concerning the conjugate agglutination and its quantitation (size of microsphere, amount of antigen bound on microsphere, concentration of conjugate, concentration of agglutinating reagent, angle of light-scattering observation) were successively studied. A microparticle-enhanced nephelometric immunoassay for prealbumin was finally developed as an example of application. It was based on the inhibition of the immunoagglutination of microspheres-prealbumin conjugate by free prealbumin. This prealbumin immunoassay was easy to perform (one-step assay without washing or phase separation), fast (30 min), reliable (variation coefficients ranged from 3.6% to 7.5% for within- and between-assay determination), and sensitive (1 microgram/L detected). It was correlated with conventional immunonephelometry and radial immunodiffusion (correlation coefficients, 0.98). Microparticle-enhanced nephelometric immunoassay offered many advantages over the last two methods. Its better sensitivity allowed a lower reagent consumption and a larger sample dilution (contrary to the conventional immunonephelometry, sample pretreatment and sample blank measurement were unnecessary). Its inhibition mode induced a total accuracy for sample with high analyte concentration (a risk of underevaluation in antigen excess conditions existed in all method based on a noncompetitive antigen-antibody reaction) and provided the possibility to quantify haptens.(ABSTRACT TRUNCATED AT 250 WORDS)
Uploads
Papers by marie c bene