Your browser doesn't support javascript.
Show: 20 | 50 | 100
Results 1 - 6 de 6
Filter
1.
Methods Mol Biol ; 2386: 43-60, 2022.
Article in English | MEDLINE | ID: covidwho-1513947

ABSTRACT

A comprehensive study of the cellular components of the immune system demands both deep and broad immunophenotyping of numerous cell subsets in an effective and practical way. Novel full-spectrum technology reveals the complete emission spectrum of each dye maximizing the amount of information that can be obtained on a single sample regarding conventional flow cytometry and provide an expanded knowledge of biological processes. In this chapter, we describe a 37-color protocol that allows to identify more than 45 different cell populations on whole blood samples of SARS-CoV-2-infected patients.


Subject(s)
COVID-19 , Flow Cytometry , Immunophenotyping/methods , COVID-19/blood , Color , Humans , Immune System
2.
Adv Sci (Weinh) ; 8(18): e2100323, 2021 09.
Article in English | MEDLINE | ID: covidwho-1316190

ABSTRACT

Blood cell analysis is a major pillar of biomedical research and healthcare. These analyses are performed in central laboratories. Rapid shipment from collection site to the central laboratories is currently needed because cells and biomarkers degrade rapidly. The dried blood spot from a fingerstick allows the preservation of cellular molecules for months but entire cells are never recovered. Here leucocyte elution is optimized from dried blood spots. Flow cytometry and mRNA expression profiling are used to analyze the recovered cells. 50-70% of the leucocytes that are dried on a polyester solid support via elution after shaking the support with buffer are recovered. While red blood cells lyse upon drying, it is found that the majority of leucocytes are preserved. Leucocytes have an altered structure that is improved by adding fixative in the elution buffer. Leucocytes are permeabilized, allowing an easy staining of all cellular compartments. Common immunophenotyping and mRNAs are preserved. The ability of a new biomarker (CD169) to discriminate between patients with and without Severe Acute Respiratory Syndrome induced by Coronavirus 2 (SARS-CoV-2) infections is also preserved. Leucocytes from blood can be dried, shipped, and/or stored for at least 1 month, then recovered for a wide variety of analyses, potentially facilitating biomedical applications worldwide.


Subject(s)
Communicable Diseases/diagnosis , Diagnostic Tests, Routine/methods , Dried Blood Spot Testing/methods , Hematology/methods , Immunophenotyping/methods , Antibodies, Viral/blood , Biomarkers/blood , Blood Specimen Collection/methods , COVID-19/diagnosis , Cell Separation/methods , Communicable Diseases/virology , Erythrocytes/virology , Flow Cytometry/methods , Humans , Leukocytes/virology , RNA, Messenger/blood , SARS-CoV-2/genetics
3.
J Immunol ; 207(2): 720-734, 2021 07 15.
Article in English | MEDLINE | ID: covidwho-1311404

ABSTRACT

Most shared resource flow cytometry facilities do not permit analysis of radioactive samples. We are investigating low-dose molecular targeted radionuclide therapy (MTRT) as an immunomodulator in combination with in situ tumor vaccines and need to analyze radioactive samples from MTRT-treated mice using flow cytometry. Further, the sudden shutdown of core facilities in response to the COVID-19 pandemic has created an unprecedented work stoppage. In these and other research settings, a robust and reliable means of cryopreservation of immune samples is required. We evaluated different fixation and cryopreservation protocols of disaggregated tumor cells with the aim of identifying a protocol for subsequent flow cytometry of the thawed sample, which most accurately reflects the flow cytometric analysis of the tumor immune microenvironment of a freshly disaggregated and analyzed sample. Cohorts of C57BL/6 mice bearing B78 melanoma tumors were evaluated using dual lymphoid and myeloid immunophenotyping panels involving fixation and cryopreservation at three distinct points during the workflow. Results demonstrate that freezing samples after all staining and fixation are completed most accurately matches the results from noncryopreserved equivalent samples. We observed that cryopreservation of living, unfixed cells introduces a nonuniform alteration to PD1 expression. We confirm the utility of our cryopreservation protocol by comparing tumors treated with in situ tumor vaccines, analyzing both fresh and cryopreserved tumor samples with similar results. Last, we use this cryopreservation protocol with radioactive specimens to demonstrate potentially beneficial effector cell changes to the tumor immune microenvironment following administration of a novel MTRT in a dose- and time-dependent manner.


Subject(s)
Cryopreservation/methods , Flow Cytometry/methods , Leukocytes, Mononuclear/immunology , Melanoma, Experimental/pathology , Myeloid Cells/immunology , Animals , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Cell Line, Tumor , Immunophenotyping/methods , Mice , Mice, Inbred C57BL , Natural Killer T-Cells/immunology , Pandemics , Signal Transduction/immunology , Tumor Microenvironment/immunology
4.
J Med Virol ; 93(3): 1589-1598, 2021 03.
Article in English | MEDLINE | ID: covidwho-1196482

ABSTRACT

A novel member of human coronavirus, named severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has been recently recognized in China and rapidly spread worldwide. Studies showed the decreasing of peripheral blood lymphocytes in a majority of patients. In this study, we have reported the clinical features, laboratory characteristics, the frequency of peripheral blood lymphocyte subpopulations, and their apoptosis pattern in Iranian coronavirus infectious disease (COVID-19) patients. Demographic and clinical data of 61 hospitalized confirmed cases with COVID-19 at Imam Khomeini Hospital were collected and analyzed. Peripheral blood mononuclear cells were isolated from all samples and the apoptosis pattern was evaluated using Annexin V/propidium iodide method. The frequency of lymphocyte subsets, including T-CD4+ , T-CD8+ , NK, B cells, and monocytes, was measured in all patients and 31 controls by flow cytometry. Our findings demonstrated that the percentage of lymphocytes, CD4+ , and CD8+ T cells were decreased in COVID-19 patients compared with the control group. Regarding the clinical severity, the number of lymphocytes, CD4+ , CD8+ T cells, and NK cells were also decreased in severe cases when compared with mild cases. Finally, our data have also indicated the increase in apoptosis of mononuclear cells from COVID-19 patients which was more remarkable in severe clinical cases. The frequency of immune cells is a useful indicator for prediction of severity and prognosis of COVID-19 patients. These results could help to explain the immunopathogenesis of SARS-CoV-2 and introducing novel biomarkers, therapeutic strategies, and vaccine candidates.


Subject(s)
B-Lymphocytes/cytology , CD4-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/cytology , Immunophenotyping/methods , Killer Cells, Natural/cytology , SARS-CoV-2/immunology , Adult , Aged , Apoptosis/immunology , Biomarkers/blood , COVID-19/immunology , Female , Flow Cytometry , Humans , Iran , Lymphocyte Count , Lymphopenia/immunology , Male , Middle Aged
5.
PLoS One ; 16(1): e0244855, 2021.
Article in English | MEDLINE | ID: covidwho-1052436

ABSTRACT

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative agent of the pandemic human respiratory illness COVID-19, is a global health emergency. While severe acute disease has been linked to an expansion of antibody-secreting plasmablasts, we sought to identify B cell responses that correlated with positive clinical outcomes in convalescent patients. We characterized the peripheral blood B cell immunophenotype and plasma antibody responses in 40 recovered non-hospitalized COVID-19 subjects that were enrolled as donors in a convalescent plasma treatment study. We observed a significant negative correlation between the frequency of peripheral blood memory B cells and the duration of symptoms for convalescent subjects. Memory B cell subsets in convalescent subjects were composed of classical CD24+ class-switched memory B cells, but also activated CD24-negative and natural unswitched CD27+ IgD+ IgM+ subsets. Memory B cell frequency was significantly correlated with both IgG1 and IgM responses to the SARS-CoV-2 spike protein receptor binding domain (RBD) in most seropositive subjects. IgM+ memory, but not switched memory, directly correlated with virus-specific antibody responses, and remained stable over 3 months. Our findings suggest that the frequency of memory B cells is a critical indicator of disease resolution, and that IgM+ memory B cells may play an important role in SARS-CoV-2 immunity.


Subject(s)
B-Lymphocyte Subsets/immunology , COVID-19/immunology , Adult , Aged , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Antibody Formation , B-Lymphocytes/immunology , Convalescence , Disease Progression , Female , Humans , Immunity/immunology , Immunoglobulin G/immunology , Immunoglobulin M/metabolism , Immunophenotyping/methods , Male , Middle Aged , Recovery of Function/immunology , SARS-CoV-2/immunology , SARS-CoV-2/isolation & purification
6.
Nat Commun ; 11(1): 5243, 2020 10 16.
Article in English | MEDLINE | ID: covidwho-872700

ABSTRACT

SARS-CoV-2 is the novel coronavirus responsible for the current COVID-19 pandemic. Severe complications are observed only in a small proportion of infected patients but the cellular mechanisms underlying this progression are still unknown. Comprehensive flow cytometry of whole blood samples from 54 COVID-19 patients reveals a dramatic increase in the number of immature neutrophils. This increase strongly correlates with disease severity and is associated with elevated IL-6 and IP-10 levels, two key players in the cytokine storm. The most pronounced decrease in cell counts is observed for CD8 T-cells and VD2 γδ T-cells, which both exhibit increased differentiation and activation. ROC analysis reveals that the count ratio of immature neutrophils to VD2 (or CD8) T-cells predicts pneumonia onset (0.9071) as well as hypoxia onset (0.8908) with high sensitivity and specificity. It would thus be a useful prognostic marker for preventive patient management and improved healthcare resource management.


Subject(s)
Betacoronavirus/immunology , CD8-Positive T-Lymphocytes/immunology , Neutrophils/immunology , Receptors, Antigen, T-Cell, gamma-delta/immunology , Biomarkers/blood , COVID-19 , Coronavirus Infections/immunology , Coronavirus Infections/pathology , Cytokine Release Syndrome/immunology , Cytokine Release Syndrome/pathology , Flow Cytometry , Humans , Immunophenotyping/methods , Interleukin-10/blood , Interleukin-6/blood , Lymphocyte Count , Pandemics , Pneumonia, Viral/immunology , Pneumonia, Viral/pathology , SARS-CoV-2 , Severity of Illness Index
SELECTION OF CITATIONS
SEARCH DETAIL
...