Correlation analysis between peripheral blood dendritic cell subsets and PD-1 in patients with peritoneal adenocarcinoma

Abstract The aim of this study was to explore the association between differential percentages of dendritic cell (DC) subsets in peripheral blood and malignancy (grade and lymph node metastasis) of peritoneal adenocarcinoma patients and the frequencies of dendritic cell subsets in the normal controls. The peripheral blood of 30 patients with peritoneal adenocarcinoma and 12 healthy controls were collected for multicolor flow cytometry analysis. Peritoneal adenocarcinoma patients were grouped according to the malignant degree (grade and lymph node metastasis). Percentages of myeloid DCs (mDCs) and its subsets MDC1 and MDC2 in DCs were lower in peripheral blood of patients with peritoneal adenocarcinoma than in normal controls. The percentages of plasmacytoid dendritic cells (pDCs) and CD16+mDCs in DCs were higher than in normal controls. Compared with poor differentiation grade, patients with well/moderate differentiation grade had an increased percentage of CD16+mDCs. Contrary to CD16+mDCs, the percentage of MDC1 was lower in the well/moderate differentiation grade group. In patients with no lymph node metastasis, pDCs and CD16+mDCs levels were higher compared with patients with lymph node metastasis. mDCs and MDC1 levels had opposite results. pDCs were positively correlated with CD16+mDCs in peripheral blood of peritoneal patients, as was mDCs and MDC1. CD16+mDCs were negatively correlated with MDC1. The percentages of pDCs and CD16+mDCs in DCs were positively correlated with CD3+CD8+T cells, and pDCs also positively correlated with CD8+PD-1+T cells. Our results revealed that DCs subsets correlated with peritoneal adenocarcinoma malignancy. Dendritic cells play an independent role in the immune function of peritoneal adenocarcinoma.

Subpoblaciones de linfocitos T en adultos mayores cubanos con leucemia linfoide crónica / Subpopulations of T lymphocytes in Cuban older adults with chronic lymphoid leukemia

Introducción: la inmunosenescencia está asociada con un mayor riesgo de desarrollo de cáncer. Dentro de las hemopatías malignas que afectan a este grupo de edad, está la leucemia linfoide crónica (LLC), caracterizada por trastornos en la inmunidad adaptativa que incluye las subpoblaciones de linfocitos T. Objetivo: Determinar la frecuencia de las subpoblaciones de linfocitos T en los pacientes adultos mayores con leucemia linfoide crónica evaluados en el Instituto de Hematología e Inmunología de Cuba. Métodos: Se realizó un estudio transversal en 30 adultos mayores con leucemia linfoide crónica. Se cuantificaron los linfocitos TCD3+CD4+ y TCD3+CD8+ en sangre periférica por citometría de flujo. Para la lectura y el análisis de los datos se empleó un citómetro de flujo Beckman Coulter Gallios. Se utilizaron los valores porcentuales, la media y la desviación estándar. Se consideró estadísticamente significativo si p≤0.05. Resultados: Hubo un predominio de hombres que representaron el 56,7 por ciento y del grupo de 70-79 años de edad. No se reportó ningún adulto mayor con LLC con valores altos ni normales de linfocitos TCD3+CD4+. Predominaron los hombres con valores bajos porcentuales de linfocitos TCD3+CD4+, TCD3+CD8+ e inversión del índice CD4/CD8 en relación con las mujeres. Conclusiones: Los adultos mayores con LLC presentan alteraciones en el número de las subpoblaciones de linfocitos T. La acción de estas células en relación al crecimiento de células B malignas aún es desconocido y resulta importante determinar si esto puede reflejar un intento de evasión de las células tumorales al control inmunológico(AU)

Introduction: Immunosenescence is associated with an increased risk of cancer development. Among the malignant hemopathies that affect this age group, it is chronic lymphoid leukemia (CLL), characterized by disorders in adaptive immunity, which include subpopulations of T lymphocytes. Objective: To determine frequency of T lymphocyte subpopulations in older adult patients with chronic lymphoid leukemia evaluated at the Institute of Hematology and Immunology of Cuba. Methods: A cross-sectional study was conducted in 30 older adults with chronic lymphoid leukemia. TCD3+CD4+ and TCD3+CD8+ lymphocytes were quantified in peripheral blood by flow cytometry. A Beckman Coulter Gallios flow cytometer was used to read and analyze the data. The percentage values, the mean and the standard deviation were used. It was considered statistically significant if p≤0.05. Results: There was a predominance of men who represented 56.7 percent and the age group of 70-79 years. No older adults with CLL with high or normal values of TCD3+CD4+ lymphocytes were reported. Men predominated with low percentage values of TCD3+CD4+, TCD3+CD8+ lymphocytes and inversion of the CD4/CD8 ratio in relation to women. Conclusions: Older adult with CLL present alterations in the number of T lymphocyte subpopulations. The role of these cells in relation to the growth of malignant B cells it is unknown and it turns out important to determine if this may reflect an attempt to evade tumor cells from immune control(AU)

Natural regulatory T cells increase significantly in pediatric patients with parasitic infections: Flow cytometry study

Background: The most accepted definition of regulatory T cells (Tregs) relies on the expression of several biomarkers, including CD4, CD25, and transcription factor, Foxp3. The Tregs maintain tolerance to self-antigens and prevent autoimmune diseases. Aim: The purpose of this study was to determine the difference in natural Treg levels in Entamoeba histolytica, Schistosoma mansoni, Giardia lamblia, Enterobius vermicularis, and Hymenolepis nana infected patients. Setting and Design: Fifty-one pediatric subjects (29 males and 22 females) were recruited from a tertiary care hospital, and were divided into infected and non-infected (control) groups. The mean age of the subjects was 8.7 years. Materials and Methods: Blood samples were collected from infected and non-infected groups, and change in the level of Tregs in these subjects was investigated by flow cytometry. Statistical Analysis Used: The statistical analysis of data was performed by SPSS software. Quantitative data used in this study included mean and standard deviation. Data from the two groups were compared by the Student's t-test. The age of the patient and infection status were used for multivariate logistic regression analysis. Odds ratios (ORs) were estimated within a 95% confidence interval, and a P value of <0.05 was considered significant. Results and Conclusions: The levels of natural regulatory T cells, indicated by the biomarkers, CD4+, CD25+, and Foxp3+, increase significantly in patients infected by Entamoeba histolytica, Schistosoma mansoni, Giardia lamblia, Enterobius vermicularis, and Hymenolepis nana as compared to controls. They also increase in cases of mixed infection as compared to infection by a single parasite.

Evaluation of lymphocyte count, T-cell subsets and neutrophil-to-lymphocyte ratio as early predictors for severity and outcome of COVID-19 disease-a report from a highly complex hospital in Brazil

ABSTRACT Introduction: Lymphopenia is a laboratory marker of poor prognosis and severity of disease in the SARS-CoV-2 infection. This study aims to describe the immune profile of a Brazilian population. Methods: A total of 121 consecutive patients with severe acute respiratory syndrome (SARS) were analyzed between April and June 2020. Routine peripheral blood counts and multiparametric flow cytometry were performed on admission to assess lymphocytes and subsets (CD3, CD4, CD8). Demographic, clinical and laboratory data were collected from hospital sources. Results: The total of 116 patients included 63 (54.3%) males; 76 (62.8%) COVID-19 patients were divided, based on clinical characteristics and mechanical ventilation (MV) use, into moderate (n = 41; no MV) and severe (n = 35; MV) groups. The control group (n = 40) was comprised of patients with SARS of different etiologies. All patients had lymphopenia, with overall lymphocyte counts and their subsets considerably lower in severe patients, when compared to the moderate and controls. Patients with a high neutrophil-to-lymphocyte ratio (> 15.2) and T-cell lymphopenia (CD3 < 593 cells/μL, CD4 < 326 cells/μL, CD8 < 121 cells/μL) had a higher risk of being intubated and progressing to death. A total of 39 patients (95.1%) in the moderate group and 54.3% (n = 19) in the severe group were discharged; 28 patients died. Conclusion: Laboratory assessment of the neutrophil/lymphocyte ratio and T-cell subsets may be predictive of mortality and may be useful for stratifying COVID-19 patients.

Linfocitosis monoclonal de células B en familiares de pacientes colombianos con síndromes linfoproliferativos crónicos B

Introducción. La linfocitosis monoclonal de células B, generalmente, precede la leucemia linfocítica crónica y afecta alrededor del 12 % de la población adulta sana. Esta frecuencia se incrementa en familiares de pacientes con síndromes linfoproliferativos crónicos de células B. Objetivo. Determinar la frecuencia de linfocitosis monoclonal B en familiares de pacientes con síndromes linfoproliferativos crónicos B, sus características inmunofenotípicas y citogenéticas, posible relación con agentes infecciosos, y seguimiento a corto plazo de población colombiana. Materiales y métodos. Se estudiaron 50 adultos sanos con antecedentes familiares de síndromes linfoproliferativos crónicos de célula B, empleando citometría de flujo multiparamétrica, pruebas citogenéticas y serológicas, encuesta de hábitos de vida y seguimiento a dos años. Resultados. La frecuencia encontrada de linfocitosis monoclonal B fue del 8 %, con predominio del sexo femenino y edad avanzada, incrementándose al 12,5 % en individuos con antecedentes familiares de leucemia linfocítica crónica. Tres de cuatro individuos presentaron inmunofenotipo de tipo leucemia linfocítica crónica, todas con bajo recuento. A su vez, en estos individuos se observa de manera significativa un mayor número de células/ µl en subpoblaciones linfocitarias T, junto con mayor predisposición a la enfermedad. Las poblaciones clonales descritas aumentan a lo largo del tiempo de manera no significativa. Conclusiones. La frecuencia y comportamiento de la linfocitosis monoclonal de célula B en pacientes con antecedentes familiares de síndromes linfoproliferativos crónicos B es similar a lo encontrado en estudios relacionados, lo que sugiere que no existe afectación de genes de mayor relevancia que puedan desencadenar una proliferación clonal descontrolada, pero que generan desregulación inmunológica que podría indicar un mayor riesgo de infección grave en estos individuos.

Introduction. Monoclonal B-cell lymphocytosis generally precedes chronic lymphocytic leukemia, affecting about 12% of the healthy adult population. This frequency increases in relatives of patients with chronic B-cell lymphoproliferative disorders. Objective. To determine the frequency of monoclonal B-cell lymphocytosis in relatives of patients with chronic B-cell lymphoproliferative disorders, their immunophenotypic/ cytogenetic characteristics, a possible relationship with infectious agents, and short-term follow-up in the Colombian population. Materials and methods. Fifty healthy adults with a family history of chronic B-cell lymphoproliferative disorders were studied using multiparametric flow cytometry, cytogenetic/serological testing, lifestyle survey, and 2-year follow-up. Results. The frequency of monoclonal B-cell lymphocytosis found was 8%, with a predominance of female gender and advanced age, increasing to 12.5% for individuals with a family history of chronic lymphocytic leukemia. Three out of four individuals presented chronic lymphocytic leukemia-type immunophenotype, all with low counts. In turn, a significantly higher number of cells/µΙ is observed in these individuals in T lymphocyte subpopulations, together with a greater predisposition to the disease. The described clonal populations increase over time in a non-significant manner. Conclusions. The frequency and behavior of monoclonal B-cell lymphocytosis in patients with family history of chronic B-cell lymphoproliferative disorders are like those found in related studies, which suggests that there is no involvement of more relevant genes that can trigger uncontrolled clonal proliferation, but that generates immunological deregulation that could justify a greater risk of serious infection in these individuals.

Enfermedad mínima residual en leucemia mieloide aguda no promielocítica: Resultados preliminares de un estudio colaborativo internacional / Minimal residual disease in non-myelocytic acute myeloid leukemia: preliminary results of an international collaborative study

A pesar de los avances en los protocolos de tratamiento y en las medidas de soporte en pacientes con Leucemia Mieloide Aguda (LMA), 27% presentan recaídas de la enfermedad. Esto se debe, entre otras causas, a la persistencia de pequeñas cantidades de células malignas (blastos) resistentes a la terapia. Estas pequeñas cantidades de blastos remanentes se denominan Enfermedad Mínima Residual (EMR). La determinación de EMR requiere de técnicas no solo muy sensibles, sino también específicas, y permite evaluar la respuesta individual a la terapia. La introducción de la EMR como parámetro de respuesta y estratificación está bien definida en Leucemia Linfoblástica Aguda (LLA). Por el contrario, aunque existen publicaciones sobre el impacto pronóstico de la EMR en LMA, aún no se encuentra incluida en forma sistemática en los protocolos nacionales actuales, entre otros motivos, por lo laborioso de la determinación y por la necesidad de validación de la misma. Debe tenerse en cuenta que el inmunofenotipo de los blastos mieloides suele ser más heterogéneo que el de los blastos en LLA, presentando, en muchos casos, subpoblaciones diferentes entre sí, lo cual dificulta su detección certera y no hay consenso definido en cuanto a la metodología más eficaz. En este trabajo describimos una nueva estrategia de marcación y análisis estandarizada en un estudio multicéntrico internacional para LMA y la utilidad de la EMR como parámetro de respuesta y de estratificación. Asimismo, detallamos los resultados preliminares de nuestra cohorte de pacientes (AU)

Despite the improvement in treatment and supportive care of patients with Acute Myeloid Leukemia (AML), 27% of them relapse. This is due to the persistence of small amounts of malignant cells (blasts) resistant to therapy, among other causes. These small amounts of blasts are called Minimal Residual Disease (MRD). The determination of MRD requires not only techniques with high sensitivity but also with high specificity, and allows to evaluate the individual response to treatment. The introduction of MRD as a response parameter is well established in Acute Lymphoblastic Leukemia (ALL), and it is used in current stratification protocols. On the other hand, even though there are some reports regarding the prognostic impact of MRD in AML, it is still not included in the current national protocols due to the lack of validation of the determination, among other causes. This is due to the fact that the immunophenotype of myeloid blasts is more heterogeneous than in ALL, presenting different subpopulations, which difficults their accurate detection. Thus, there is still no consensus regarding the most effective approach. In this article, we describe a new staining and analysis strategy standardized by an international multicentric study, and the utility of EMR as a response and stratification parameter. Additionally, we show the preliminary results of our patient cohort. (AU)

Immunophenotypic characterization of normal and abnormal plasma cells in bone marrow of newly diagnosed multiple myeloma patients

Background: Identification of plasma cells into abnormal (APC) and normal (NPC) compartments is of utmost importance in flow cytometric (FC) analysis of multiple myeloma (MM) and related plasma cell dyscrasias for diagnosis, prognosis, and follow-up. No single phenotypic marker is sufficient to distinguish NPC from APC. Materials and Methods: 43 newly diagnosed cases of MM and 13 controls were included in the study. Bone marrow (BM) samples from the 2nd pass were processed on the same day with antibodies against CD38, CD138, CD19, CD81, CD45, CD117, CD200, CD56, cytoKappa, and cytoLambda in a 4-color experiment with CD38 and CD138 as gating antibodies. Results: Mean APC% in cases was 96.5%. The expected Immunophenotype (IP) of APC which is CD19-/56+/45-/81-/117+/200+ was found in only 13/43 MM cases. In 30/43 cases, APC revealed deviation from expected IP either for single or a combination of markers. Sensitivity for APC detection was highest for CD19 (95.2%) followed by CD56 (90.4%) and CD81 (83.7%). Specificity was highest for CD19 (100%), CD56 (100%), and CD81 (100%) followed by CD117 (92.3%). Combination of markers with maximum sensitivity to detect APC (97.6%) was CD81- or CD19- and CD200+ or CD56+ (two markers); and for NPC (92.3%) was CD81+ and CD19+ and CD56- (three markers). Conclusion: Plasma cell IP can be highly variable with multiple minor subpopulations in both cases and normal controls. CD 19 and CD56 are highly informative markers for a 4-color experiment. Assessment of multiple markers in an 8–10 color experiment is more informative but the lack of advanced flow cytometers should not limit the use of FC in a 4-color approach. Our results emphasize that even basic equipment with limited fluorochrome can provide meaningful information if used appropriately.

The dynamics of genome size and GC contents evolution in genus Nicotiana / A dinâmica do tamanho do genoma e a evolução dos conteúdos de GC no gênero Nicotiana

Abstract Hybridization and Polyploidization are most common of the phenomenon observed in plants, especially in the genus Nicotiana leading to the duplication of genome. Although genomic changes associated with these events has been studied at various levels but the genome size and GC content variation is less understood because of absence of sufficient genomic data. In this study the flow cytometry technique was used to uncover the genome size and GC contents of 46 Nicotiana species and we compared the genomic changes associated with the hybridization events along evolutionary time scale. The genome size among Nicotiana species varied between 3.28 pg and 11.88 pg whereas GC contents varied between 37.22% and 51.25%. The tetraploid species in genus Nicotiana including section Polydiclae, Repandae, Nicotiana, Rustica and Sauveolentes revealed both up and downsizing in their genome sizes when compared to the sum of genomes of their ancestral species. The genome sizes of three homoploid hybrids were found near their ancestral species. Loss of large genome sequence was observed in the evolutionary more aged species (>10 Myr) as compared to the recently evolved one's (<0.2 Myr). The GC contents were found homogenous with a mean difference of 2.46% among the Nicotiana species. It is concluded that genome size change appeared in either direction whereas the GC contents were found more homogenous in genus Nicotiana.

Resumo A hibridização e a poliploidização são os fenômenos mais comuns observados em plantas, principalmente no gênero Nicotiana, levando à duplicação do genoma. Embora as mudanças genômicas associadas a esses eventos tenham sido estudadas em vários níveis, o tamanho do genoma e a variação do conteúdo de GC são menos compreendidos devido à ausência de dados genômicos suficientes. Neste estudo, a técnica de citometria de fluxo foi usada para descobrir o tamanho do genoma e o conteúdo de GC de 46 espécies de Nicotiana, e comparamos as mudanças genômicas associadas aos eventos de hibridização ao longo da escala de tempo evolutiva. O tamanho do genoma entre as espécies de Nicotiana variou entre 3,28 pg e 11,88 pg, enquanto os conteúdos de GC variaram entre 37,22% e 51,25%. As espécies tetraploides do gênero Nicotiana, incluindo as seções Polydiclae, Repandae, Nicotiana, Rustica e Sauveolentes, revelaram aumento e redução do tamanho do genoma quando comparados à soma dos genomas de suas espécies ancestrais. Os tamanhos do genoma de três híbridos homoploides foram encontrados perto de suas espécies ancestrais. A perda da grande sequência do genoma foi observada nas espécies evolutivas mais velhas (> 10 Myr) em comparação com as que evoluíram recentemente (< 0,2 Myr). Os teores de GC foram homogêneos com diferença média de 2,46% entre as espécies de Nicotiana. Conclui-se que a mudança no tamanho do genoma apareceu em ambas as direções, enquanto os conteúdos de GC foram encontrados mais homogêneos no gênero Nicotiana.

Clinical significance of next-generation sequencing-based IGH/IGK gene rearrangement analysis in the diagnosis of minimal residual disease of children with acute B-cell lymphoblastic leukemia / 中华实用儿科临床杂志

Objective:To assess the clinical significance of next-generation sequencing (NGS)-based IGH/ IGK gene rearrangement analysis versus flow cytometry (FCM) in diagnosing minimal residual disease (MRD) of children with acute B-cell lymphoblastic leukemia (B-ALL). Methods:Clinical data, NGS-MRD and FCM-MRD findings at the initial diagnosis and after induction chemotherapy of 85 children diagnosed as B-ALL in Children′s Hospital of Nanjing Medical University from July 2019 to July 2021, were retrospectively analyzed.The sensitivity of the two methods, and the positive rate were compared by χ2 test or Fisher′ s test.The correlation was identified by Spearman correlation analysis. Results:Dominant clone sequences were detected in all children at the initial diagnosis by NGS, while selection markers were identified by FCM in 75(88.2%) patients.Positive MRD rate detected by NGS-MRD was significantly higher than that of FCM-MRD at the same time point after induction chemotherapy[31.8%(27/85) vs.9.4%(8/85), P<0.001]. Compared with those of FCM-MRD, NGS-MRD had good sensitivity (100.0%), specificity (75.3%) and negative predictive value (100.0%), and the positive predictive value was 29.6%.MRD results detected by NGS were consistent with that of FCM ( r=0.569, P<0.001). By July 27, 2022, 2 patients with NGS-MRD (+ )FCM-MRD (-)relapsed during maintenance chemotherapy. Conclusions:NGS is highly consistent with FCM in the detection of MRD in children with B-ALL, which is more sensitive.The combination of NGS-MRD and FCM-MRD benefits more in monitoring MRD in children with B-ALL after induction chemotherapy.

Research progress of imaging flow cytometry in automatic analysis of radiation biodosimetric indicators / 中国辐射卫生

@#A large number of people would be exposed to irradiation in large-scale nuclear and radiation accidents or nuclear terrorist attacks. Therefore, it is urgent to establish rapid and high-throughput biodosimetry for in triage, providing a basis for emergency management. Imaging flow cytometry (IFC) possesses the high through put advantages of traditional flow cytometry and the sensitivity and specificity of microscope, and has a good application prospect in the research and development of rapid, automated, and high-throughput biological dose estimation technology. This article reviews the application progress of IFC in biodosimetry, and provides a reference for the development of biological dose estimation and detection equipment for large-scale nuclear and radiation accidents.

Microfluidic Chip and Flow Cytometry for Examination of the Antiplatelet Effect of Ticagrelor / 中国医学科学院学报

Objective To examine the antiplatelet effect of ticagrelor by microfluidic chip and flow cytometry under shear stress in vitro. Methods Microfluidic chip was used to examine the effect of ticagrelor on platelet aggregation at the shear rates of 300/s and 1500/s.We adopted the surface coverage of platelet aggregation to calculate the half inhibition rate of ticagrelor.The inhibitory effect of ticagrelor on ADP-induced platelet aggregation was verified by optical turbidimetry.Microfluidic chip was used to construct an in vitro vascular stenosis model,with which the platelet reactivity under high shear rate was determined.Furthermore,the effect of ticagrelor on the expression of fibrinogen receptor (PAC-1) and P-selectin (CD62P) on platelet membrane activated by high shear rate was analyzed by flow cytometry. Results At the shear rates of 300/s and 1500/s,ticagrelor inhibited platelet aggregation in a concentration-dependent manner,and the inhibition at 300/s was stronger than that at 1500/s (both P<0.001).Ticagrelor at a concentration ≥4 μmol/L almost completely inhibited platelet aggregation.The inhibition of ADP-induced platelet aggregation by ticagrelor was similar to the results under flow conditions and also in a concentration-dependent manner.Ticagrelor inhibited the expression of PAC-1 and CD62P. Conclusion We employed microfluidic chip to analyze platelet aggregation and flow cytometry to detect platelet activation,which can reveal the responses of different patients to ticagrelor.

Correlation Analysis between Cerebrospinal Fluid Status and Prognosis in Childhood with Acute Lymphoblastic Leukemia by Flow Cytometry / 中国实验血液学杂志

OBJECTIVE@#To study the cerebrospinal fluid (CSF) status and prognosis value in patients with newly diagnosed acute lymphoblastic leukemia (ALL) by flow cytometry (FCM).@*METHODS@#The clinical features of the 75 newly diagnosed ALL patients from September 2020 to December 2021 in our centre were retrospective analyzed, as well as the bone marrow (BM) and CSF minimal residual disease (MRD) data, and the CSF conventional cytology data. Central nervous system infiltration(CNSI) positive was as CSF MRD positive by FCM or leukemia cells detected by conventional cytology. The status of CSF were compared and analyzed by FCM and conventional cytology, the clinical features and the prognosis value of different CNSI status in these patients were analyzed.@*RESULTS@#Among 75 newly diagnosed ALL, 16 cases (21%) with CNSI positive (CNSI+) were detected by FCM, while only 2 positive cases (3%) were detected by conventional cytology. The CNSI+ rate detected by FCM was significantly higher than conventional cytology(P<0.05). Compared with CNSI- ALL patients, the median age of CNSI+ ALL patients was significantly younger, and the median platelet count was significantly lower, the difference was statistically significant (P<0.05). Up to follow-up time (August 31, 2022), four ALL patients were died, including 3 patients were CNSI- and 1 patient was CNSI+. Furthermore, three cases were primary disease relapse, including 1 case was CNSI+. There was no significant difference in overall survival (OS) rate and relapse-free survival (RFS) rate of the patients with different CNSI status.@*CONCLUSION@#Compared with conventional cytology, FCM is a more sensitive assay to evaluate the central nervous system status in ALL patients. After active treatment, there was no significant difference in OS and RFS between patients with different CNSI status at diagnosis.

Development of flow cytometry and its application in plant research / 生物工程学报

Flow cytometry is a multi-parameter, rapid and efficient method for qualitative analysis and quantitative determination of various fluorescently labeled particles in liquid flow. Flow cytometry has been applied in multiple disciplines such as immunology, virology, molecular biology, cancer biology and infectious disease monitoring. However, the application of flow cytometry in plant research is hampered due to the special composition and structure of plant tissues and cells, such as cell walls and secondary metabolites. In this paper, the development, composition and classification of flow cytometry were introduced. Subsequently, the application, research progress and application limitations of flow cytometry in plant field were discussed. At last, the development trend of flow cytometry in plant research was prospected, which provides new perspectives for broadening the potential application scope of plant flow cytometry.

Development of a flow cytometry method for detection of bovine multi-cytokines / 生物工程学报

This study aims to develop a method to detect bovine multi-cytokines based on flow cytometry. Previously we have prepared and screened monoclonal antibodies against bovine cytokines IFN-γ, IL-2, TNF-α, IP-10 and MCP-1. These bovine cytokine monoclonal antibodies were fluorescently labeled, and the combination of antibody and cell surface molecules were used to develop the method for detecting bovine multi-cytokines. Subsequently, the developed method was used to determine the cytokine expression profile of Mycobacterium bovis BCG infected bovine peripheral blood mononuclear cells in vitro, and evaluate the cytokine expression level of peripheral blood CD4+ T cells of tuberculosis-positive cattle. The bovine multi-cytokine flow cytometry detection method can effectively determine the cytokine expression of BCG-infected bovine peripheral blood T lymphocytes. Among them, the expression levels of IFN-γ, IL-2, and TNF-α continue to increase after 40 hours of infection, while the expression levels of IP-10 and MCP-1 decreased. The combined detection of IFN-γ, IL-2, and TNF-α on CD4+ T lymphocytes in peripheral blood of cattle can effectively distinguish tuberculosis-positive and tuberculosis-negative samples. This method may facilitate evaluating the level of cellular immune response after bovine pathogen infection and vaccine injection.

Comparative analysis between cytomorphology and flow cytometry methods in central nervous system infiltration assessment in oncohematological patients

ABSTRACT Introduction Oncohematological patients require the evaluation for possible infiltration of the central nervous system (CNS) by neoplastic cells at diagnosis and/or during the monitoring of the chemotherapeutic treatment. Morphological analysis using conventional microscopy is considered the method of choice to evaluate the cerebrospinal fluid (CSF) samples, despite technical limitations. Objective This study aimed to compare the performance of the cytomorphology and flow cytometric immunophenotyping (FC) in the detection of CNS infiltration. Method We evaluated 520 CSF samples collected from 287 oncohematological patients for whom the detection of neoplastic cells was simultaneously requested by cytomorphology and FC. Results Laboratory analyses revealed 435/520 (83.7%) conclusive results by the two methods evaluated, among which 385 (88.5%) were concordant. Discordance between the methods was observed in 50/435 (11.5%) samples, 45 (90%) being positive by FC. Furthermore, the FC defined the results in 69/72 (95.8%) inconclusive samples by cytomorphology. The positivity of FC was particularly higher among hypocellular samples. Among 431 samples with a cell count of < 5/μL, the FC identified neoplastic cells in 75 (17.4%), while the cytomorphology reported positive results in 26 (6%). Among the samples that presented adequate cell recovery for evaluation by both methods (506/520), the comparative analysis between FC and cytomorphology revealed a Kappa coefficient of 0.45 (CI: 0.37-0.52), interpreted as a moderate agreement. Conclusion The data showed that the CSF analysis by FC helps in the definition of CNS infiltration by neoplastic cells, particularly in the cases with dubious morphological analysis or in the evaluation of samples with low cellularity.

A novel differential diagnosis algorithm for chronic lymphocytic leukemia using immunophenotyping with flow cytometry

Abstract Introduction The availability of a clinical decision algorithm for diagnosis of chronic lymphocytic leukemia (CLL) may greatly contribute to the diagnosis of CLL, particularly in cases with ambiguous immunophenotypes. Herein we propose a novel differential diagnosis algorithm for the CLL diagnosis using immunophenotyping with flow cytometry. Methods The hierarchical logistic regression model (Backward LR) was used to build a predictive algorithm for the diagnosis of CLL, differentiated from other lymphoproliferative disorders (LPDs). Results A total of 302 patients, of whom 220 (72.8%) had CLL and 82 (27.2%), B-cell lymphoproliferative disorders other than CLL, were included in the study. The Backward LR model comprised the variables CD5, CD43, CD81, ROR1, CD23, CD79b, FMC7, sIg and CD200 in the model development process. The weak expression of CD81 and increased intensity of expression in markers CD5, CD23 and CD200 increased the probability of CLL diagnosis, (p < 0.05). The odd ratio for CD5, C23, CD200 and CD81 was 1.088 (1.050 - 1.126), 1.044 (1.012 - 1.077), 1.039 (1.007 - 1.072) and 0.946 (0.921 - 0.970) [95% C.I.], respectively. Our model provided a novel diagnostic algorithm with 95.27% of sensitivity and 91.46% of specificity. The model prediction for 97.3% (214) of 220 patients diagnosed with CLL, was CLL and for 91.5% (75) of 82 patients diagnosed with an LPD other than CLL, was others. The cases were correctly classified as CLL and others with a 95.7% correctness rate. Conclusions Our model highlighting 4 markers (CD81, CD5, CD23 and CD200) provided high sensitivity and specificity in the CLL diagnosis and in distinguishing of CLL among other LPDs.

Immunophenotypic characterization of acute leukemias in Bahia, Brazil

ABSTRACT Objective To characterize the immunophenotypic profile of acute leukemias in the population of the state of Bahia, Brazil. Methods This is a descriptive, retrospective study. From 2014 to 2018, 796 new cases of acute leukemia were evaluated. The data were obtained from analysis of reports and records of tests performed by flow cytometry immunophenotyping. All individuals of all age groups diagnosed as acute lymphoblastic leukemia or acute myeloid leukemia were included in the study. Demographic variables and expression of leukemia antigens were evaluated. Results Most cases were diagnosed as acute myeloid leukemia and 42.7% as acute lymphoblastic leukemia. Significant differences were found in expression of markers in acute leukemias when age groups were compared, as well as in demographic characteristics. B-cell acute lymphoblastic leukemia was more prevalent than cases of T-cell origin. Assessing the aberrant markers in acute myeloid leukemias, the non-acute promyelocytic leukemia group presented expression of CD7 and CD56 as the most frequent ones. In B-cell acute lymphoblastic leukemia, the most frequent aberrant markers were CD66c, CD13 and CD33. Conclusion Significant differences were found as to several antigens when comparing adults and children, and these findings may contribute to future studies correlating the phenotypic profile to genetic characteristics and therapeutic response, including specific antigen therapies, which may be better targeted.

The dynamics of genome size and GC contents evolution in genus Nicotiana / A dinâmica do tamanho do genoma e a evolução dos conteúdos de GC no gênero Nicotiana

Hybridization and Polyploidization are most common of the phenomenon observed in plants, especially in the genus Nicotiana leading to the duplication of genome. Although genomic changes associated with these events has been studied at various levels but the genome size and GC content variation is less understood because of absence of sufficient genomic data. In this study the flow cytometry technique was used to uncover the genome size and GC contents of 46 Nicotiana species and we compared the genomic changes associated with the hybridization events along evolutionary time scale. The genome size among Nicotiana species varied between 3.28 pg and 11.88 pg whereas GC contents varied between 37.22% and 51.25%. The tetraploid species in genus Nicotiana including section Polydiclae, Repandae, Nicotiana, Rustica and Sauveolentes revealed both up and downsizing in their genome sizes when compared to the sum of genomes of their ancestral species. The genome sizes of three homoploid hybrids were found near their ancestral species. Loss of large genome sequence was observed in the evolutionary more aged species (>10 Myr) as compared to the recently evolved one’s (<0.2 Myr). The GC contents were found homogenous with a mean difference of 2.46% among the Nicotiana species. It is concluded that genome size change appeared in either direction whereas the GC contents were found more homogenous in genus Nicotiana.

A hibridização e a poliploidização são os fenômenos mais comuns observados em plantas, principalmente no gênero Nicotiana, levando à duplicação do genoma. Embora as mudanças genômicas associadas a esses eventos tenham sido estudadas em vários níveis, o tamanho do genoma e a variação do conteúdo de GC são menos compreendidos devido à ausência de dados genômicos suficientes. Neste estudo, a técnica de citometria de fluxo foi usada para descobrir o tamanho do genoma e o conteúdo de GC de 46 espécies de Nicotiana, e comparamos as mudanças genômicas associadas aos eventos de hibridização ao longo da escala de tempo evolutiva. O tamanho do genoma entre as espécies de Nicotiana variou entre 3,28 pg e 11,88 pg, enquanto os conteúdos de GC variaramentre 37,22% e 51,25%. As espécies tetraploides do gênero Nicotiana, incluindo as seções Polydiclae, Repandae, Nicotiana, Rustica e Sauveolentes, revelaram aumento e redução do tamanho do genoma quando comparados à soma dos genomas de suas espécies ancestrais. Os tamanhos do genoma de três híbridos homoploides foram encontrados perto de suas espécies ancestrais. A perda da grande sequência do genoma foi observada nas espécies evolutivas mais velhas (> 10 Myr) em comparação com as que evoluíram recentemente (< 0,2 Myr). Os teores de GC foram homogêneos com diferença média de 2,46% entre as espécies de Nicotiana. Conclui-se que a mudança no tamanho do genoma apareceu em ambas as direções, enquanto os conteúdos de GC foram encontrados mais homogêneos no gênero Nicotiana.

The dynamics of genome size and GC contents evolution in genus Nicotiana

Abstract Hybridization and Polyploidization are most common of the phenomenon observed in plants, especially in the genus Nicotiana leading to the duplication of genome. Although genomic changes associated with these events has been studied at various levels but the genome size and GC content variation is less understood because of absence of sufficient genomic data. In this study the flow cytometry technique was used to uncover the genome size and GC contents of 46 Nicotiana species and we compared the genomic changes associated with the hybridization events along evolutionary time scale. The genome size among Nicotiana species varied between 3.28 pg and 11.88 pg whereas GC contents varied between 37.22% and 51.25%. The tetraploid species in genus Nicotiana including section Polydiclae, Repandae, Nicotiana, Rustica and Sauveolentes revealed both up and downsizing in their genome sizes when compared to the sum of genomes of their ancestral species. The genome sizes of three homoploid hybrids were found near their ancestral species. Loss of large genome sequence was observed in the evolutionary more aged species (>10 Myr) as compared to the recently evolved ones ( 0.2 Myr). The GC contents were found homogenous with a mean difference of 2.46% among the Nicotiana species. It is concluded that genome size change appeared in either direction whereas the GC contents were found more homogenous in genus Nicotiana.

Resumo A hibridização e a poliploidização são os fenômenos mais comuns observados em plantas, principalmente no gênero Nicotiana, levando à duplicação do genoma. Embora as mudanças genômicas associadas a esses eventos tenham sido estudadas em vários níveis, o tamanho do genoma e a variação do conteúdo de GC são menos compreendidos devido à ausência de dados genômicos suficientes. Neste estudo, a técnica de citometria de fluxo foi usada para descobrir o tamanho do genoma e o conteúdo de GC de 46 espécies de Nicotiana, e comparamos as mudanças genômicas associadas aos eventos de hibridização ao longo da escala de tempo evolutiva. O tamanho do genoma entre as espécies de Nicotiana variou entre 3,28 pg e 11,88 pg, enquanto os conteúdos de GC variaram entre 37,22% e 51,25%. As espécies tetraploides do gênero Nicotiana, incluindo as seções Polydiclae, Repandae, Nicotiana, Rustica e Sauveolentes, revelaram aumento e redução do tamanho do genoma quando comparados à soma dos genomas de suas espécies ancestrais. Os tamanhos do genoma de três híbridos homoploides foram encontrados perto de suas espécies ancestrais. A perda da grande sequência do genoma foi observada nas espécies evolutivas mais velhas (> 10 Myr) em comparação com as que evoluíram recentemente ( 0,2 Myr). Os teores de GC foram homogêneos com diferença média de 2,46% entre as espécies de Nicotiana. Conclui-se que a mudança no tamanho do genoma apareceu em ambas as direções, enquanto os conteúdos de GC foram encontrados mais homogêneos no gênero Nicotiana.

Analytical performance verification protocols and specifications of CD34 +cell enumeration by flow cytometry / 中华检验医学杂志

Objective:To investigate the analytical performance verification protocols and performance specifications of CD34+cell enumeration by flow cytometry for clinical laboratories.Methods:According to international guidelines and National Health Standard of China, we designed the performance verification protocols of CD34 +cell enumeration (including percent count and absolute count) by flow cytometry. Four quality assessment materials, three leukapheresis products and three samples of peripheral blood were selected to verify the precision, linearity, carryover, trueness and accuracy of FACSCanto Ⅱ measurement system, and the assessment criterion was set according to the detection technologies of clinical laboratories. Results:The CVs of intra-run precision of percent count and absolute count were 2.5% to 8.9% and 3.0% to 9.0%; the CVs of inter-run precision were 2.8% to 10.5% and 3.8% to 9.9%, respectively. The slopes of linearity regression equation of low range (3.6/μl to 123.6/μl) and high range (113.2/μl to 1196.3/μl) were 0.993 2 and 0.965 2, and R2 were 0.999 6 and 0.993 9, and the biases were -8.67% to 0.22%. The carryover of percent and absolute count were 0.07% and 0.00%. When percent count≤0.2% or absolute count≤20/μl, the absolute biases of trueness were in the range of ±0.006% or ±0.5/μl, and the absolute biases of accuracy were in the range of ±0.02% or ±0.9/μl; when percent count>0.2% or absolute count>20/μl, the relative biases of trueness were in the range of ±5.65%, and the relative biases of accuracy were in the range of ±8.19%. The verification results met the assessment criterion set in this study. Conclusions:The performance verification protocols and assessment criterion formulated in this study not only conform to the recommendations of domestic and foreign guidelines, but also conform to state of the detection technologies of native clinical laboratories, which can be taken as a reference of performance verification for clinical laboratories.