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1.
Sci Rep ; 13(1): 20978, 2023 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-38017254

RESUMO

Immunobiography describes the life-long effects of exogenous or endogenous stimuli on remodeling of immune cell biology, including the development of memory T and B-cells. The present study aimed at investigating the rhythms of changes in phenotypic features of memory T and B-cells along childhood and adolescence. A descriptive-observational investigation was conducted including 812 healthy volunteers, clustered into six consecutive age groups (9Mths-1Yr; 2Yrs; 3-4Yrs; 5-7Yrs; 8-10Yrs; 11-18Yrs). Immunophenotypic analysis of memory T-cell (CD4+ and CD8+) and B-cell subsets were performed by flow cytometry. The results pointed out that memory-related biomarkers of T and B-cells displayed a bimodal profile along healthy childhood and adolescence, regardless of sex. The first stage of changes occurs around 2Yrs, with predominance of naive cells, while the second and more prominent wave occurs around the age 8-10Yrs, with the prevalence of memory phenotypes. The neighborhood connectivity profile analysis demonstrated that the number of correlations reaches a peak at 11-18Yrs and lower values along the childhood. Males presented higher and conserved number of correlations when compared to females. Altogether, our results provide new insights into immunobiography and a better understanding of interactions among the cellular subsets studied here during childhood and adolescence.


Assuntos
Linfócitos T CD4-Positivos , Linfócitos T CD8-Positivos , Masculino , Feminino , Humanos , Adolescente , Criança , Linfócitos B , Imunofenotipagem , Citometria de Fluxo , Memória Imunológica , Subpopulações de Linfócitos T
2.
Front Immunol ; 14: 1268686, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37915569

RESUMO

Background: Multiparameter flow cytometry (FC) immunophenotyping is a key tool for detailed identification and characterization of human blood leucocytes, including B-lymphocytes and plasma cells (PC). However, currently used conventional data analysis strategies require extensive expertise, are time consuming, and show limited reproducibility. Objective: Here, we designed, constructed and validated an automated database-guided gating and identification (AGI) approach for fast and standardized in-depth dissection of B-lymphocyte and PC populations in human blood. Methods: For this purpose, 213 FC standard (FCS) datafiles corresponding to umbilical cord and peripheral blood samples from healthy and patient volunteers, stained with the 14-color 18-antibody EuroFlow BIgH-IMM panel, were used. Results: The BIgH-IMM antibody panel allowed identification of 117 different B-lymphocyte and PC subsets. Samples from 36 healthy donors were stained and 14 of the datafiles that fulfilled strict inclusion criteria were analysed by an expert flow cytometrist to build the EuroFlow BIgH-IMM database. Data contained in the datafiles was then merged into a reference database that was uploaded in the Infinicyt software (Cytognos, Salamanca, Spain). Subsequently, we compared the results of manual gating (MG) with the performance of two classification algorithms -hierarchical algorithm vs two-step algorithm- for AGI of the cell populations present in 5 randomly selected FCS datafiles. The hierarchical AGI algorithm showed higher correlation values vs conventional MG (r2 of 0.94 vs. 0.88 for the two-step AGI algorithm) and was further validated in a set of 177 FCS datafiles against conventional expert-based MG. For virtually all identifiable cell populations a highly significant correlation was observed between the two approaches (r2>0.81 for 79% of all B-cell populations identified), with a significantly lower median time of analysis per sample (6 vs. 40 min, p=0.001) for the AGI tool vs. MG, respectively and both intra-sample (median CV of 1.7% vs. 10.4% by MG, p<0.001) and inter-expert (median CV of 3.9% vs. 17.3% by MG by 2 experts, p<0.001) variability. Conclusion: Our results show that compared to conventional FC data analysis strategies, the here proposed AGI tool is a faster, more robust, reproducible, and standardized approach for in-depth analysis of B-lymphocyte and PC subsets circulating in human blood.


Assuntos
Linfócitos B , Plasmócitos , Humanos , Reprodutibilidade dos Testes , Imunofenotipagem , Leucócitos
3.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 31(5): 1327-1332, 2023 Oct.
Artigo em Chinês | MEDLINE | ID: mdl-37846680

RESUMO

OBJECTIVE: To analyze the immunological phenotype of chronic myeloid leukemia (CML), and explore its characteristics and significance. METHODS: The immunophenotypes of 40 CML children and 40 controls were analyzed by multicolor flow cytometry. CD45/SSC, as the basic gate, was used to delineate neutrophils. Then, the distribution of cluster differentiation (CD) molecules on the surface of granulocytes was analyzed in three ranges (≥1%, ≥5%, and ≥20%), and the expression rates of CD molecules (≥1% included in the statistical analysis) and the mean fluorescence intensity (MFI) were compared between the two groups. RESULTS: The proportion of granulocytes in the CML group was (82.1±6.4)%, which was significantly higher than (57.8±11.8)% in the control group (P <0.001). The expression rates of CD15/CD11b/CD33/CD13 in CML and control groups were high, and both distributed in the range of ≥20%. The differentiation trajectory of CD33/CD13 was normal and there were no significant differences in the expression rate and MFI between the two groups. However, both the expression rate of CD11b and CD15 MFI in the CML group were significantly lower than those in the control group (P <0.001). There were no significant differences in the expression rate and MFI of CD10 between the two groups, and the expression levels of CD10 between the two groups were consistent in different distributions. In the CML group, there was a large number of cases with abnormal high expression of CD56, 52.5% of the cases had a CD56 expression rate of ≥5%, and 42.5% had a CD56 expression rate of ≥20%, while the control group did not express CD56 (<1%). The expression distribution of CD117 was different between the two groups. In the range of expression rate ≥5%, there were 35.0% cases in the CML group, while only 2.5% in the control group. The expression rate of CD117 in the CML group was higher than that in the control group (P <0.001), though there was no significant difference in MFI. CONCLUSION: The immunophenotyping of CML is characterized by increased proportion of mature neutrophils, decreased CD15 MFI, decreased proportion of CD11b and abnormal high expression of CD56 and CD117. Flow cytometric analysis of immunophenotype can effectively distinguish normal granulocytes from chronic granulocytes, and help in the diagnosis of CML.


Assuntos
Leucemia Mielogênica Crônica BCR-ABL Positiva , Leucemia Mieloide , Criança , Humanos , Citometria de Fluxo , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Granulócitos , Neutrófilos , Imunofenotipagem
4.
Front Immunol ; 14: 1227905, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37799722

RESUMO

Background: Nirmatrelvir has been authorized for the treatment of both hospitalized and non-hospitalized COVID-19 patients. However, the association between T lymphocyte subsets and the outcome of hospitalized COVID-19 patients treated with oral Nirmatrelvir has not been investigated. The objective of this study was to examine whether lymphocyte subsets could serve as biomarkers to assess the risk of mortality in COVID-19 patients undergoing Nirmatrelvir treatment, with the aim of enhancing medication management for COVID-19 patients. Methods: We conducted a retrospective cohort study at the Xiangya Hospital of Central South University in China between December 5, 2022 and January 31, 2023. The study reported demographic, clinical, T lymphocyte subsets, and inflammatory cytokine data of COVID-19 patients. We evaluated the associations of T lymphocyte subsets on admission with the composite outcome or death of patients using univariate and multivariable Cox regression analyses with hazards ratios (HRs) and 95% confidence intervals (CIs). Results: We identified 2118 hospitalized COVID-19 patients during the study period, and conducted a follow-up of up to 38 days. Of these, 131 patients received Nirmatrelvir, with 56 (42.7%) in the composite outcome group, and 75 (57.3%) in the non-composite outcome group. Additionally, 101 (77.1%) patients were discharged, while 30 (22.9%) died. Our results showed a significant decrease in the CD3+, CD4+, and CD8+ T cell counts of patients in the composite outcome group and mortality group compared to the non-composite outcome group and discharged group, respectively. Multivariate Cox regression analysis showed that the significant decrease in CD8+ T cell count in peripheral blood was independently associated with the composite outcome in COVID-19 patients treated with Nirmatrelvir, with an HR of 1.96 (95%CI: 1.01-3.80). The significant decrease in CD4+ and CD8+ T cell counts in peripheral blood increased the hazard of developing mortality, with HRs of 6.48 (95%CI: 1.47-28.63) and 3.75 (95%CI: 1.27-11.11), respectively. Conclusion: Our study revealed a significant positive correlation between a decrease in CD8+ T cell counts and progression and mortality of hospitalized COVID-19 patients treated with Nirmatrelvir. Lower counts (/µL) of CD8+ T cell (<201) were associated with a higher risk of in-hospital severity and death. Our findings may provide valuable references for physicians in optimizing the use of Nirmatrelvir.


Assuntos
COVID-19 , Humanos , Imunofenotipagem , Prognóstico , Estudos Retrospectivos , Linfócitos T CD8-Positivos/química , Biomarcadores
5.
Br J Biomed Sci ; 80: 11573, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37822353

RESUMO

Background: B-Cell Lymphoproliferative Disorders (B-LPDs) are a group of heterogenous disorders characterised by the accumulation of B-cells in peripheral blood, bone marrow, lymph nodes and spleen. They have a variable disease course and outcome and many share similar features making differential diagnosis challenging. Therefore, accurate diagnosis is fundamental in particular for determining treatment options. Immunophenotyping by flow cytometry plays a crucial role in the diagnosis of B-LPDs. However, overlapping immunophenotyping patterns exist and the use of novel monoclonal antibodies has become increasingly important in immunophenotyping analysis. More recently differential expression of CD200 has been reported in various B-LPDs and that CD200 may improve the differentiation between chronic lymphocytic leukaemia (CLL) and mantle cell lymphoma (MCL). In this study CD200 expression is evaluated in different B-LPDs. Methods: A total of 100 samples were collected and analysed by immunophenotyping flow cytometry over a period of 1 year (2017-2018), by a panel of monoclonal antibodies including CD200. The percentage of CD200 and its expression intensity was evaluated and compared between different groups of B-LPDs. Results: All of the 50 cases of CLL expressed CD200 with moderate to bright intensity, 6 MCL cases lacked the expression of CD200. Furthermore, all 5 cases of hairy cell leukaemia (HCL) expressed CD200. Out of all B-LPDs evaluated, CD200 expression in HCL cases was noted to be the brightest. The other 39 cases were not found to be B-LPDs. Conclusion: CD200 has an important role in differentiating CLL from MCL, HCL has a consistent bright expression of CD200. By adding CD200 to the combinations of markers in routine testing panel, Immunophenotyping by flow cytometry can be an effective tool in the diagnosis of B-LPDs especially in cases with atypical immunophenotyping pattern. Our result support that CD200 can be added to routine testing panel as it is useful in differentiating them.


Assuntos
Leucemia Linfocítica Crônica de Células B , Linfoma de Célula do Manto , Humanos , Anticorpos Monoclonais/metabolismo , Linfócitos B/metabolismo , Linfócitos B/patologia , Diagnóstico Diferencial , Citometria de Fluxo , Imunofenotipagem , Leucemia Linfocítica Crônica de Células B/diagnóstico , Leucemia Linfocítica Crônica de Células B/metabolismo , Linfoma de Célula do Manto/diagnóstico , Linfoma de Célula do Manto/patologia
6.
J Cancer Res Ther ; 19(5): 1335-1339, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37787304

RESUMO

Background: Leukemic cells express a characteristic set of "cluster of differentiation" (CD) markers, which forms the basis of the current WHO classification. Leukemia-associated aberrant immunophenotype (LAIP) refers to expression of unusual CD markers by leukemic cells, which are not normally expressed by their respective lineage. The incidence of LAIP varies considerably, and its clinical implications, prognostic relevance, and sensitivity to therapy are still debatable. This study was conducted to identify the immunophenotypic aberrancies in newly diagnosed leukemias in our Institute. Method: This was an observational study, which included newly diagnosed leukemias on flow cytometry. Aberrant immunophenotypic expressions were recorded whenever present and were correlated with prognostic factors like age, gender, and total leucocyte count (TLC). Results: The study included 110 newly diagnosed cases of leukemias (85 acute and 25 chronic) over 1.5 years. Immunophenotypic aberrancies were detected in 40.4% of the cases. The highest incidence of aberrations was detected in acute myeloid leukemia (60.7%). LAIPs were detected in 50% of T-acute lymphoblastic leukemia and 25% cases of in B-cell acute lymphoblastic leukemia (B-ALL). Aberrant CD33 and CD56 expression in B-ALL correlated with poor prognostic factors like higher age and higher TLC, respectively. Immunophenotypic aberrancies were present in 28% cases of chronic lymphocytic leukemia. Conclusion: The results of this study have generated valuable baseline data on the incidence of LAIPs in this region. This information is vital because establishing LAIPs at the time of diagnosis is crucial for disease monitoring. Some LAIPs are associated with underlying cytogenetic abnormalities and hence impact the management and prognosis.


Assuntos
Leucemia Linfocítica Crônica de Células B , Leucemia Mieloide Aguda , Leucemia-Linfoma Linfoblástico de Células Precursoras , Humanos , Citometria de Fluxo/métodos , Centros de Atenção Terciária , Leucemia Mieloide Aguda/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/epidemiologia , Imunofenotipagem
7.
Cell Mol Biol (Noisy-le-grand) ; 69(9): 31-36, 2023 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-37807337

RESUMO

Acute myeloid leukemia (AML) is a heterogeneous malignancy characterized by the clonal expansion of myeloid precursor cells in the bone marrow. In this study, we investigated the interplay of hematological parameters, CD markers, genetic polymorphisms, and database mutations in the interleukin 15 (IL15) gene in AML patients. We enrolled 59 newly diagnosed AML patients and analyzed their bone marrow specimens using flow cytometry and molecular techniques. The hematological parameters of the AML patients revealed a significant increase in platelet count and RBC, Hb, and HCT levels compared to healthy individuals. CD marker expression analysis revealed upregulation of CD33, CD45, CD13, CD117, CD38, HLA-DR, CD15, CD64, MPO, CD34, and CD11c in AML patients. Molecular analysis showed 15 mutations in different positions of exon 8 of the IL15 gene, with the most frequent mutation being a homozygous mutation resulting from a nucleotide substitution. Additionally, 10 novel heterozygous mutations were identified in different locations of chromosome 4, with a low variant rate. Finally, database analysis of gnomAD and Mutagene revealed a high number of potential driver mutations in the IL15 gene in leukemia patients. These results provide valuable insights into the genetic and immunophenotypic characteristics of AML patients and highlight the potential role of IL15 in AML pathogenesis.


Assuntos
Interleucina-15 , Leucemia Mieloide Aguda , Humanos , Interleucina-15/genética , Antígenos CD/metabolismo , Leucemia Mieloide Aguda/genética , Mutação/genética , Imunofenotipagem , Citometria de Fluxo/métodos , Polimorfismo Genético
8.
Clin Exp Metastasis ; 40(6): 505-515, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37812366

RESUMO

Diagnosing malignant pleural effusions (MPE) is challenging when patients lack a history of cancer and cytopathology does not detect malignant cells in pleural effusions (PE). We investigated whether a systematic analysis of PE by flow cytometry immunophenotyping (FCI) had any impact on the diagnostic yield of MPE. Over 7 years, 570 samples from patients with clinical suspicion of MPE were submitted for the FCI study. To screen for epithelial malignancies, a 3-color FCI high sensitivity assay was used. The FCI results, qualified as "malignant" (FCI+) or "non-malignant" (FCI-), were compared to integrated definitive diagnosis established by clinicians based on all available information. MPE was finally diagnosed in 182 samples and FCI detected 141/182 (77.5%). Morphology further confirmed FCI findings by cytopathology detection of malignant cells in PE (n = 91) or histopathology (n = 29). Imaging tests and clinical history supported the diagnosis in the remaining samples. The median percentage of malignant cells was 6.5% for lymphoma and 0.23% for MPE secondary to epithelial cell malignancies. FCI identified a significantly lower percentage of EpCAM+ cells in cytopathology-negative MPE than in cytopathology-positive cases (0.02% vs. 1%; p < 0.0001). Interestingly, 29/52 MPE (55.8%) where FCI alerted of the presence of malignant cells were new diagnosis of cancer. Overall, FCI correctly diagnosed 456/522 samples (87.4%) suitable for comparison with cytopathology. These findings show that high sensitivity FCI significantly increases the diagnostic yield of MPE. Early detection of FCI + cases accelerates the diagnostic pathway of unsuspected MPE, thus supporting its implementation in clinical diagnostic work-up as a diagnostic tool.


Assuntos
Derrame Pleural Maligno , Humanos , Derrame Pleural Maligno/diagnóstico , Citometria de Fluxo/métodos , Imunofenotipagem
9.
Front Immunol ; 14: 1151748, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37795090

RESUMO

Background: Immune cell expression profiling from patient samples is critical for the successful development of immuno-oncology agents and is useful to understand mechanism-of-action, to identify exploratory biomarkers predictive of response, and to guide treatment selection and combination therapy strategies. LAG-3 is an inhibitory immune checkpoint that can suppress antitumor T-cell responses and targeting LAG-3, in combination with PD-1, is a rational approach to enhance antitumor immunity that has recently demonstrated clinical success. Here, we sought to identify human immune cell subsets that express LAG-3 and its ligands, to characterize the marker expression profile of these subsets, and to investigate the potential relationship between LAG-3 expressing subsets and clinical outcomes to immuno-oncology therapies. Methods: Comprehensive high-parameter immunophenotyping was performed using mass and flow cytometry of tumor-infiltrating lymphocytes (TILs) and peripheral blood mononuclear cells (PBMCs) from two independent cohorts of samples from patients with various solid tumor types. Profiling of circulating immune cells by single cell RNA-seq was conducted on samples from a clinical trial cohort of melanoma patients treated with immunotherapy. Results: LAG-3 was most highly expressed by subsets of tumor-infiltrating CD8 T central memory (TCM) and effector memory (TEM) cells and was frequently co-expressed with PD-1. We determined that these PD-1+ LAG-3+ CD8 memory T cells exhibited a unique marker profile, with greater expression of activation (CD69, HLA-DR), inhibitory (TIM-3, TIGIT, CTLA-4) and stimulatory (4-1BB, ICOS) markers compared to cells that expressed only PD-1 or LAG-3, or that were negative for both checkpoints. In contrast to tumors, LAG-3 expression was more limited in circulating immune cells from healthy donors and solid tumor patients. Additionally, we found abundant expression of the LAG-3 ligands MHC-II and galectin-3 in diverse immune cell types, whereas FGL1 and LSECtin were minimally expressed by immune cells in the tumor microenvironment (TME). Lastly, we found an inverse relationship between baseline and on-treatment levels of circulating LAG3 transcript-expressing CD8 memory T cells and response to combination PD-1 and CTLA-4 blockade in a clinical trial cohort of melanoma patients profiled by scRNAseq. Conclusions: These results provide insights into the nature of LAG-3- and ligand-expressing immune cells within the TME, and suggest a biological basis for informing mechanistic hypotheses, treatment selection strategies, and combination immunotherapy approaches to support continued development of dual PD-1 and LAG-3 blockade.


Assuntos
Melanoma , Receptor de Morte Celular Programada 1 , Humanos , Antígeno CTLA-4 , Receptor de Morte Celular Programada 1/metabolismo , Leucócitos Mononucleares , Imunofenotipagem , Ligantes , Microambiente Tumoral , Fibrinogênio/uso terapêutico
10.
Crit Rev Oncol Hematol ; 192: 104186, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37863402

RESUMO

Plasmacytoid dendritic cells (pDCs) are a specific dendritic cell type stemming from the myeloid lineage. Clinically and pathologically, neoplasms associated with pDCs are classified as blastic plasmacytoid dendritic cell neoplasm (BPDCN), mature plasmacytoid dendritic myeloid neoplasm (MPDMN) and pDC expansion in myeloid neoplasms (MNs). BPDCN was considered a rare and aggressive neoplasm in the 2016 World Health Organization (WHO) classification. MPDMN, known as mature pDC-derived neoplasm, is closely related to MNs and was first recognized in the latest 2022 WHO classification, proposing a new concept that acute myeloid leukemia cases could show clonally expanded pDCs (pDC-AML). With the advances in detection techniques, an increasing number of pDC expansion in MNs have been reported, but whether the pathogenesis is similar to that of MPDMN remains unclear. This review focuses on patient characteristics, diagnosis and treatment of pDC expansion in MNs to gain further insight into this novel and unique provisional subtype.


Assuntos
Neoplasias Hematológicas , Leucemia Mieloide Aguda , Neoplasias Cutâneas , Humanos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/terapia , Leucemia Mieloide Aguda/metabolismo , Imunofenotipagem , Células Dendríticas/patologia , Neoplasias Hematológicas/diagnóstico , Neoplasias Hematológicas/terapia , Neoplasias Hematológicas/patologia , Neoplasias Cutâneas/patologia
11.
Indian J Med Res ; 158(2): 161-174, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37787259

RESUMO

Background & objectives: Accurate diagnosis of immunodeficiencies requires a critical comparison of values with age-matched controls. In India, the existing reference values for rare lymphocyte subsets are currently not available and we rely on the data originating from other countries for the interpretation of the results. Furthermore, there is limited information on normal variation for these rare-subset parameters in Indian children. So, this study aimed to establish normative values for clinically important lymphocyte subsets in Indian children at different age groups. Methods: 148 children aged ≥16 yr were enrolled in this study. The study population included 61 per cent males and 39 per cent females and was divided into the following groups: cord blood (n=18), 0-6 months (n=9), 6-12 months (n=13), 1-2 yr (n=19), 2-5 yr (n=27), 5-10 yr (n=25) and 10-16 yr (n=37). The absolute and relative percentage of lymphocytes, T, B, natural killer cell, along with activated, naïve and memory subsets, was determined by flow cytometry. Results: Median values and the 10th and 90th percentiles were obtained for 34 lymphocyte sub-populations. The T and B naïve compartments showed a decreasing trend, whereas memory cells showed an increase with age. The activated T cell subset shows an increasing pattern up to one year and then declines gradually. Double negative T cells are relatively stable. TCRgd+T cell percentage increases with age. Interpretation & conclusions: This single-centre pilot study provides preliminary data that justifies the need for future large-scale multi centric studies to generate a reference range for interpreting extended immunophenotyping profiles in the paediatric age group, making it possible for clinicians to assess the immunological status in inborn errors of immunity, infectious and autoimmune diseases.


Assuntos
Subpopulações de Linfócitos , Subpopulações de Linfócitos T , Masculino , Feminino , Criança , Humanos , Projetos Piloto , Contagem de Linfócitos , Imunofenotipagem , Citometria de Fluxo , Índia/epidemiologia , Valores de Referência
12.
Cell Rep Methods ; 3(10): 100612, 2023 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-37883923

RESUMO

Immunophenotyping is a powerful approach for deciphering responses of the immune system to drug screening and immunotherapy. In this issue of Cell Report Methods, Liechti et al. have advanced this approach by developing a pipeline, which allows high-throughput but still accurate single-cell immunophenotyping in time.


Assuntos
Sistema Imunitário , Imunoterapia , Imunofenotipagem
13.
Cell Rep Methods ; 3(10): 100619, 2023 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-37883924

RESUMO

High-dimensional flow cytometry is the gold standard to study the human immune system in large cohorts. However, large sample sizes increase inter-experimental variation because of technical and experimental inaccuracies introduced by batch variability. Our high-throughput sample processing pipeline in combination with 28-color flow cytometry focuses on increased throughput (192 samples/experiment) and high reproducibility. We implemented quality control checkpoints to reduce technical and experimental variation. Finally, we integrated FlowSOM clustering to facilitate automated data analysis and demonstrate the reproducibility of our pipeline in a study with 3,357 samples. We reveal age-associated immune dynamics in 2,300 individuals, signified by decreasing T and B cell subsets with age. In addition, by combining genetic analyses, our approach revealed unique immune signatures associated with a single nucleotide polymorphism (SNP) that abrogates CD45 isoform splicing. In summary, we provide a versatile and reliable high-throughput, flow cytometry-based pipeline for immune discovery and exploration in large cohorts.


Assuntos
Subpopulações de Linfócitos B , Leucócitos , Humanos , Imunofenotipagem , Reprodutibilidade dos Testes , Citometria de Fluxo/métodos
14.
Cells ; 12(19)2023 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-37830568

RESUMO

Mesenchymal stem cells (MSCs) can differentiate into multiple connective tissue lineages, including osteoblasts, chondrocytes, and adipocytes. MSCs secrete paracrine molecules that are associated with immunomodulation, anti-fibrotic effects, and angiogenesis. Due to their orchestrative potential, MSCs have been therapeutically applied for several diseases. An important aspect of this process is the delivery of high-quality MSCs to patients at the right time, and cryo-biology and cryo-preservation facilitate the advancement of the logistics thereof. This study aimed to compare the biological signatures between freshly preserved and cryo-preserved MSCs by using big data sourced from the Pharmicell database. From 2011 to 2022, data on approximately 2300 stem cell manufacturing cases were collected. The dataset included approximately 60 variables, including viability, population doubling time (PDT), immunophenotype, and soluble paracrine molecules. In the dataset, 671 cases with no missing data were able to receive approval from an Institutional Review Board and were analyzed. Among the 60 features included in the final dataset, 20 were selected by experts and abstracted into two features by using a principal component analysis. Circular clustering did not introduce any differences between the two MSC preservation methods. This pattern was also observed when using viability, cluster of differentiation (CD) markers, and paracrine molecular indices as inputs for unsupervised analysis. The individual average PDT and cell viability at most passages did not differ according to the preservation method. Most immunophenotypes (except for the CD14 marker) and paracrine molecules did not exhibit different mean levels or concentrations between the frozen and unfrozen MSC groups. Collectively, the biochemical signatures of the cryo-preserved and unfrozen bone marrow MSCs were comparable.


Assuntos
Antígenos CD , Células-Tronco Mesenquimais , Humanos , Proliferação de Células , Antígenos CD/genética , Adipócitos , Imunofenotipagem
15.
Clin Lab Med ; 43(4): 521-547, 2023 12.
Artigo em Inglês | MEDLINE | ID: mdl-37865501

RESUMO

Myelodysplastic syndromes/neoplasms (MDS) are a heterogeneous class of hematopoietic stem cell neoplasms characterized by ineffective hematopoiesis leading to peripheral cytopenias. This group of diseases is typically diagnosed using a combination of clinical, morphologic, and genetic criteria. Many studies have described the value of multiparametric flow cytometry (MFC) in the diagnosis, classification, and prognostication of MDS. This review summarizes the approach to MDS diagnosis and immunophenotypic characterization using MFC and describes the current state while highlighting future opportunities and potential pitfalls.


Assuntos
Síndromes Mielodisplásicas , Neoplasias , Humanos , Citometria de Fluxo , Síndromes Mielodisplásicas/diagnóstico , Imunofenotipagem
16.
Int J Mol Sci ; 24(17)2023 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-37686408

RESUMO

Hitherto, calcified aortic valves (AVs) and failing bioprosthetic heart valves (BHVs) have been investigated by similar approaches, mostly limited to various immunostaining techniques. Having employed multiple immunostaining combinations, we demonstrated that AVs retain a well-defined cellular hierarchy even at severe stenosis, whilst BHVs were notable for the stochastic degradation of the extracellular matrix (ECM) and aggressive infiltration by ECM-digesting macrophages. Leukocytes (CD45+) comprised ≤10% cells in the AVs but were the predominant cell lineage in BHVs (≥80% cells). Albeit cells with uncertain immunophenotype were rarely encountered in the AVs (≤5% cells), they were commonly found in BHVs (≥80% cells). Whilst cell conversions in the AVs were limited to the endothelial-to-mesenchymal transition (represented by CD31+α-SMA+ cells) and the formation of endothelial-like (CD31+CD68+) cells at the AV surface, BHVs harboured numerous macrophages with a transitional phenotype, mostly CD45+CD31+, CD45+α-SMA+, and CD68+α-SMA+. In contrast to immunostaining, which was unable to predict cell function in the BHVs, our whole-specimen, nondestructive electron microscopy approach (EM-BSEM) was able to distinguish between quiescent and matrix-degrading macrophages, foam cells, and multinucleated giant cells to conduct the ultrastructural analysis of organelles and the ECM, and to preserve tissue integrity. Hence, we suggest EM-BSEM as a technique of choice for studying the cellular landscape of BHVs.


Assuntos
Agressão , Valvas Cardíacas , Microscopia Eletrônica de Varredura , Imunofenotipagem , Divisão Celular
17.
PLoS One ; 18(9): e0291662, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37729123

RESUMO

OBJECTIVES: In myelodysplastic syndromes (MDS), neoplastic myeloblast (CD34+CD13+CD33+ cells) numbers often increase over time, leading to secondary acute myeloid leukemia (AML). In recent studies, blasts in some MDS patients have been found to express a megakaryocyte-lineage molecule, CD41, and such patients show extremely poor prognosis. This is the first study to evaluate whether myeloblasts transition to CD41+ blasts over time and to investigate the detailed immunophenotypic features of CD41+ blasts in MDS. METHODS: We performed a retrospective cohort study, in which time-dependent changes in blast immunophenotypes were analyzed using multidimensional flow cytometry (MDF) in 74 patients with MDS and AML (which progressed from MDS). RESULTS: CD41+ blasts (at least 20% of CD34+ blasts expressing CD41) were detected in 12 patients. In five of these 12 patients, blasts were CD41+ from the first MDF analysis. In the other seven patients, myeloblasts (CD34+CD33+CD41- cells) transitioned to megakaryoblasts (CD34+CD41+ cells) over time, which was often accompanied by disease progression (including leukemic transformation). These CD41+ patients were more frequently observed among patients with monosomal and complex karyotypes. CD41+ blasts were negative for the erythroid antigen, CD235a, and positive for CD33 in all cases, but CD33 expression levels were lower in three cases when compared with CD34+CD41- blasts. Among the five CD41+ patients who underwent extensive immunophenotyping, CD41+ blasts all expressed CD61, but two cases had reduced CD42b expression, three had reduced/absent CD13 expression, and three also expressed CD7. CONCLUSIONS: Myeloblasts become megakaryoblastic over time in some MDS patients, and examining the megakaryocyte lineage (not only as a diagnostic work-up but also as follow-up) is needed to detect CD41+ MDS. The immunophenotypic features revealed in this study may have diagnostic relevance for CD41+ MDS patients.


Assuntos
Células Precursoras de Granulócitos , Síndromes Mielodisplásicas , Humanos , Imunofenotipagem , Células Progenitoras de Megacariócitos , Estudos Retrospectivos , Antígenos CD34
18.
Clin Lab ; 69(9)2023 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-37702695

RESUMO

BACKGROUND: Acute lymphoblastic leukemia (ALL) encompasses a group of lymphoid neoplasms that morphologically and immunophenotypically resemble B-lineage and T-lineage precursor cells. Our objective is to describe the immunophenotypic aspects of acute lymphoblastic leukemia (All) diagnosed by flow cytometry at the hematology laboratory of IBN ROCHD University Hospital Center and to compare them with those reported in other series. METHODS: This is a descriptive study over a period from August 2016 to October 2021, during a 5 year-and-2-month period. Immunophenotyping was performed at the flow cytometry unit on a Beckman-Coulter with 6 colors and 2 lasers in the hematology laboratory of the same hospital and data was collected retrospectively from the patients' files, their medical prescription files, kalisil software, and a data collection form we have established. RESULTS: The 440 patients had ages ranging from 1 month to 76 years, with a median of 9.5 years and the overall male-to-female ratio was 1.44. The immunological subtyping revealed that 82.5% of cases were B-ALL and 17.5% were T-ALL; of these B-ALL cases 230 (63.36%) were children (range: 0.1 - 15 years) and 134 (36.91%) were adults (range: 16 - 76 years); T-ALL were distributed in both age groups, 49 cases (37.7%) were children (range: 2 - 15 years) and 28 (21.56%) were adults (range: 18 - 63 years). All patients presented at least one abnormal blood count; thrombocytopenia has been observed in 89.4% of cases, anemia in 86.5% of cases, hyperleukocytosis in 79.8% of cases, leukopenia in 10.6% of cases, and pancytopenia in 4.8% of cases. The frequency of B-cell markers in B-ALL was found to be 363 (100%) for CD19, 323 (88.94%) for CD10, 290 (80%) for CD79a, and 73 (20%) for CD20. CD34 expression was found in 73 (20%) cases of B-ALL. HLA-DR was found in 54 (15%) cases, while TdT was found in 43 (13%) cases. Aberrant expression of myeloid antigens was found in 94 (26%) cases of B-ALL. Among T-ALL, the positivity of CD3 and CD7 was 100% (77 cases), while CD5 was positive in 58 (75%) cases. CD34 expression was found in only 19 cases of T-ALL. CD4 and CD8 expression was checked in only 9 adult patients and 4 pediatric cases. Out of them, 77.82% of cases were negative for both CD4 and CD8. CD4 and CD8 double positivity was seen in only 11.1% of cases, and 22.4% of cases showed either CD4 or CD8 positivity. CONCLUSIONS: We concluded that our study was similar to reports in the Americas and Europe, and it was the first large one that describes the immunophenotypic profile of ALL in the Moroccan population.


Assuntos
Leucemia-Linfoma Linfoblástico de Células Precursoras , Leucemia-Linfoma Linfoblástico de Células T Precursoras , Adulto , Humanos , Feminino , Masculino , Criança , Pré-Escolar , Adolescente , Leucemia-Linfoma Linfoblástico de Células T Precursoras/diagnóstico , Imunofenotipagem , Marrocos , Estudos Retrospectivos , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Hospitais Universitários , Antígenos CD34
19.
EBioMedicine ; 95: 104769, 2023 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-37672979

RESUMO

BACKGROUND: Efficient biomarker discovery and clinical translation depend on the fast and accurate analytical output from crucial technologies such as multiplex imaging. However, reliable cell classification often requires extensive annotations. Label-efficient strategies are urgently needed to reveal diverse cell distribution and spatial interactions in large-scale multiplex datasets. METHODS: This study proposed Self-supervised Learning for Antigen Detection (SANDI) for accurate cell phenotyping while mitigating the annotation burden. The model first learns intrinsic pairwise similarities in unlabelled cell images, followed by a classification step to map learnt features to cell labels using a small set of annotated references. We acquired four multiplex immunohistochemistry datasets and one imaging mass cytometry dataset, comprising 2825 to 15,258 single-cell images to train and test the model. FINDINGS: With 1% annotations (18-114 cells), SANDI achieved weighted F1-scores ranging from 0.82 to 0.98 across the five datasets, which was comparable to the fully supervised classifier trained on 1828-11,459 annotated cells (-0.002 to -0.053 of averaged weighted F1-score, Wilcoxon rank-sum test, P = 0.31). Leveraging the immune checkpoint markers stained in ovarian cancer slides, SANDI-based cell identification reveals spatial expulsion between PD1-expressing T helper cells and T regulatory cells, suggesting an interplay between PD1 expression and T regulatory cell-mediated immunosuppression. INTERPRETATION: By striking a fine balance between minimal expert guidance and the power of deep learning to learn similarity within abundant data, SANDI presents new opportunities for efficient, large-scale learning for histology multiplex imaging data. FUNDING: This study was funded by the Royal Marsden/ICR National Institute of Health Research Biomedical Research Centre.


Assuntos
Pesquisa Biomédica , Aprendizado Profundo , Neoplasias Ovarianas , Humanos , Feminino , Imunofenotipagem , Terapia de Imunossupressão
20.
Cytometry A ; 103(11): 839-850, 2023 11.
Artigo em Inglês | MEDLINE | ID: mdl-37768325

RESUMO

High-dimensional immunoprofiling is essential for studying host response to immunotherapy, infection, and disease in murine model systems. However, the difficulty of multiparameter panel design combined with a lack of existing murine tools has prevented the comprehensive study of all major leukocyte phenotypes in a single assay. Herein, we present a 40-color flow cytometry panel for deep immunophenotyping of murine lymphoid tissues, including the spleen, blood, Peyer's patches, inguinal lymph nodes, bone marrow, and thymus. This panel uses a robust set of surface markers capable of differentiating leukocyte subsets without the use of intracellular staining, thus allowing for the use of cells in downstream functional experiments or multiomic analyses. Our panel classifies T cells, B cells, natural killer cells, innate lymphoid cells, monocytes, macrophages, dendritic cells, basophils, neutrophils, eosinophils, progenitors, and their functional subsets by using a series of co-stimulatory, checkpoint, activation, migration, and maturation markers. This tool has a multitude of systems immunology applications ranging from serial monitoring of circulating blood signatures to complex endpoint analysis, especially in pre-clinical settings where treatments can modulate leukocyte abundance and/or function. Ultimately, this 40-color panel resolves a diverse array of immune cells on the axes of time, tissue, and treatment, filling the niche for a modern tool dedicated to murine immunophenotyping.


Assuntos
Imunidade Inata , Tecido Linfoide , Camundongos , Animais , Citometria de Fluxo/métodos , Linfócitos T , Células Matadoras Naturais , Imunofenotipagem
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