Single-Cell Cytokine Gene Expression in Peripheral Blood Cells Correlates with Latent Tuberculosis Status
Pooja VirRiccardo ArrigucciKarim LakehalAmy L. DavidowRichard PineSanjay TyagiYuri BushkinAlfred LardizabalMaria Laura Gennaro
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Abstract:
RNA flow cytometry (FISH-Flow) achieves high-throughput measurement of single-cell gene expression by combining in-situ nucleic acid hybridization with flow cytometry. We tested whether antigen-specific T-cell responses detected by FISH-Flow correlated with latent tuberculosis infection (LTBI), a condition affecting one-third of the world population. Peripheral-blood mononuclear cells from donors, identified as positive or negative for LTBI by current medical practice, were stimulated ex vivo with mycobacterial antigen. IFNG and IL2 mRNA production was assayed by FISH-Flow. Concurrently, immunophenotypes of the cytokine mRNA-positive cells were characterized by conventional, antibody-based staining of cell-surface markers. An association was found between donor LTBI status and antigen-specific induction of IFNG and IL2 transcripts. Induction of these cytokine genes, which was detected by FISH-Flow in a quarter the time required to see release of the corresponding proteins by ELISA, occurred primarily in activated CD4+ T cells via T-cell receptor engagement. Moreover, NK cells contributed to IFNG gene induction. These results show that antigen-driven induction of T-cell cytokine mRNA is a measurable single-cell parameter of the host responses associated with latent tuberculosis. FISH-Flow read-outs contribute a multi-scale dimension to the immunophenotyping afforded by antibody-based flow cytometry. Multi-scale, single-cell analyses may satisfy the need to determine disease stage and therapy response for tuberculosis and other infectious pathologies.Keywords:
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This article provides an overview of the role of flow cytometry in the diagnosis, prognosis, and follow‐up of T and NK‐cell lymphoproliferative disorders. For each category, we will briefly discuss the immunophenotypic features of normal T and NK cells, and address technical issues in flow cytometry, the approach to diagnosis in various contexts, pitfalls in interpretation, and its use in follow‐up and post‐therapy management. In addition to reviewing the diagnostic, prognostic, and therapeutic utility of flow cytometric immunophenotyping in several of specific T and NK cell entities, we will also cover some of the new immunophenotypic markers. Furthermore, we will touch upon incorporation of flow cytometry in the final diagnosis, including correlation with other ancillary tests. © 2019 International Clinical Cytometry Society
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Over the last 10 years, immunophenotyping of haematologic malignancies has become an indispensable diagnostic supplement to the classical morphological approach. Immunophenotyping of haematopoietic cells is performed with the use of a number of monoclonal antibodies (MOABs), which are directed specifically against structures of blood cells that become expressed at the different stages of differentiation and maturation. Cells to which the fluorescently labelled MOABs are directed can be recognised and measured using fluorescence microscopy or fluorescence flow cytometry. Many MOABs, fluorochromes and user-friendly flow cytometers have become available in the last 15 years, as a result of which immunophenotyping is now routinely applied in clinical practice. Immunophenotyping has the potential to classify leukaemias and other malignant lymphomas according to cell type and stage of maturation. This information is important for the establishment of the right diagnosis and prognosis, and for the optimal treatment choice. In a number of cases immunophenotyping provides information which cannot be obtained by simple morphological investigation. The immunophenotyping of blood and bone-marrow cells is also a sensitive method for detecting minimal residual disease after an apparent complete remission has been achieved.
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Background: Acute leukemia area a group of neoplastic disorders characterized by proliferation and accumulation of immature hematopoeitic cells in bone marrow, blood, and other tissues. The present study was conducted to have a detailed understanding of immunophenotyping profi le, the morphologic and immunophenotypic discrepancy and importance of immunophenotyping in diagnosis of acute leukemia. Objectives: To study immunophenotyping profi le in acute leukemia (acute myeloid leukemia [AML], acute lymphoid leukemia, and mixed lineage leukemia) and to study its importance in diagnosis. Materials and Methods: This study was performed in Medical College, Jabalpur. 160 patients diagnosed morphologically with AML, acute lymphoblastic leukemia and mixed lineage leukemia seen were included in the study. Results: Only in 73% cases of acute leukemia did fi nd similarity in morphological appearance and immunophenotyping. In remaining 27% cases morphological fi ndings did not correlate with immunophenotyping expression. Diagnosis in these 27% patients changed after immunophenotyping. Conclusions: It is imperative and absolutely essential to ascertain the lineage of leukemia by immunophenotyping before starting on treatment as more than 25% of patients would not respond or later relapse if treatment is initiated on morphological diagnosis.
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(1) Background: Physical stimuli may activate peripheral blood mononuclear cells (PBMCs) to secrete cytokines, which may favor pro-inflammatory responses or trigger reparative phenomena. The purpose of this study is to evaluate the action of Polarized Polychromatic Incoherent Low Energy Radiation (PILER) on human in vitro PBMCs, by detection of the possible effects on cytokine production; (2) Methods: isolated PBMCs were irradiated with a PILER lamp at different exposure times, at a distance of 10 cm, before incubation. The supernatants were collected after 24 h and 48 h and cytokines evaluated by ELISA; (3) Results: Our results showed a decrease in the levels of pro-inflammatory IL-12p70, IL-17A, IFN-γ, and TNF-α cytokines, whereas IL-10 and TGF-β1 with regulatory activity increased; (4) Conclusions: PILER irradiation affected the cytokine production by isolated PBMCs driving the immune response toward an anti-inflammatory/reparative profile.
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Objective It is to study the method and clinical significance of acute leukemia immunophenotyping by multi-parameter flow cytometry.Methods Immunophenotyping analysis was done by three color flow cytometry with the method of CD_(45)/SSC double parameter and scatter spot picture.Results In the 79 cases of leukemia,42 cases expressed medullary system antigen in which CD_(33) expression was the highest,12 cases(28%) with CD7 expression,2 cases(5%) with CD_2 expression,2 cases(5%) with CD_3 expression,2 cases(5%) with CD_(19) expression.39 cases expressed lymphocyte system antigen in which CD_(10) expression was the highest,6 cases(6%) with CD_(33) expression,4?cases(10%) with CD_(64) expression,2 cases was double expression leukemia.Conclusion Multi-parameter flow cytometry can accurately type acute leukemia,so it has an important value for the treatment choice and prognosis diagnosis of the patients with leukemia.
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Objective To study the relationship of immunophenotype and FAB phenotype and guide clinical analysis and treatment.Methods Cell morphology checking and immunophenotyping were performed at the same time for the 505 patients with leukemia,flow cytometry had used for immunophenotype,on the end the results had been analyzed to understand the relationship between the two phenotypes.Results ①Among the 505 cases of leukemia,there were AML(248)、ALL(200)、CML(18)、CLL(15)、HAL(14)、UAL(10) in FAB phenotype and AML(163)、Ly+AML(83)、ALL(108)、My+ALL(106)、HAL(32)、UAL(13) in immunophenotype.② In AML the accordance rate and part accordance rate of the two phenotypes was 61.3% and 30.2%.In ALL the accordance rate was and part accordance rate was 53% and 44.5%.In HAL the accordance rate was 42.9%.Conclusion Immunophenotype plays an important role to distinguish AML、ALL subtype、variants leukemia and HAL.Immunophenotyping was shaper and more accurate than FAB phenotype,and is a supplementary and correction,but it can not replace the FAB phenotype.Combination of both can improve the diagnostic accuracy.
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