scholarly journals Quantitative and qualitative studies of micronucleus induction in mouse erythrocytes using flow cytometry. I. Measurement of micronucleus induction in peripheral blood polychromatic erythrocytes by chemicals with known and suspected genotoxicity

Mutagenesis ◽  
1997 ◽  
Vol 12 (1) ◽  
pp. 1-8 ◽  
Author(s):  
Jan Grawé ◽  
Michael Nüsse ◽  
Ilse-Dore Adler
Keyword(s):  
Flow Cytometry ◽  
Peripheral Blood ◽  
Qualitative Studies ◽  
Micronucleus Induction ◽  
Polychromatic Erythrocytes

scholarly journals Flow cytometry of reticulocytes applied to clinical hematology

Blood ◽  
1983 ◽  
Vol 61 (6) ◽  
pp. 1091-1097 ◽  
Author(s):  
HJ Tanke ◽  
PH Rothbarth ◽  
JM Vossen ◽  
GJ Koper ◽  
JS Ploem
Keyword(s):  
Flow Cytometry ◽  
Peripheral Blood ◽  
Blood Bank ◽  
Control Group ◽  
Allogeneic Bone ◽  
Blood Samples ◽  
Frequency Distributions ◽  
Hematologic Disorders ◽  
Rna Content ◽  
Polychromatic Erythrocytes

Abstract Reticulocytes in fixed human blood samples were stained for RNA with the fluorescent dye pyronin Y and measured by flow cytometry. The resulting relative frequency distributions of the RNA fluorescence intensities concurred with the different stages in maturation from early reticulocytes to mature red cells. A computer program was written to calculate from these frequency distributions the relative number of reticulocytes, their relative RNA content, and the median of the reticulocyte population (RNA index). This method was applied to 30 healthy blood bank donors (control group), as well as to patients with various hematologic disorders showing abnormal erythropoietic activity. The measured percentage of reticulocytes, RNA content, and RNA index were found to correlate well with the various hematologic disorders. Changes in erythropoiesis could be clearly followed, as was demonstrated by analyzing blood samples from children with aplastic anemia or acute myeloid leukemia, who were treated with allogeneic bone marrow transplantation. Measurements on blood samples from healthy blood bank donors showed that with this method, small changes in the reticulocyte population, such as the appearance of polychromatic erythrocytes in the peripheral blood 5–8 hr after donation, can be detected. The statistical reliability and the information provided on the maturation stage of the entire reticulocyte population make flow cytometry of peripheral blood reticulocytes a more informative method for the study of hematologic abnormalities than conventional methods for reticulocyte counting and classification.

scholarly journals Flow cytometry of reticulocytes applied to clinical hematology

Blood ◽  
1983 ◽  
Vol 61 (6) ◽  
pp. 1091-1097
Author(s):  
HJ Tanke ◽  
PH Rothbarth ◽  
JM Vossen ◽  
GJ Koper ◽  
JS Ploem
Keyword(s):  
Flow Cytometry ◽  
Peripheral Blood ◽  
Blood Bank ◽  
Control Group ◽  
Allogeneic Bone ◽  
Blood Samples ◽  
Frequency Distributions ◽  
Hematologic Disorders ◽  
Rna Content ◽  
Polychromatic Erythrocytes

Reticulocytes in fixed human blood samples were stained for RNA with the fluorescent dye pyronin Y and measured by flow cytometry. The resulting relative frequency distributions of the RNA fluorescence intensities concurred with the different stages in maturation from early reticulocytes to mature red cells. A computer program was written to calculate from these frequency distributions the relative number of reticulocytes, their relative RNA content, and the median of the reticulocyte population (RNA index). This method was applied to 30 healthy blood bank donors (control group), as well as to patients with various hematologic disorders showing abnormal erythropoietic activity. The measured percentage of reticulocytes, RNA content, and RNA index were found to correlate well with the various hematologic disorders. Changes in erythropoiesis could be clearly followed, as was demonstrated by analyzing blood samples from children with aplastic anemia or acute myeloid leukemia, who were treated with allogeneic bone marrow transplantation. Measurements on blood samples from healthy blood bank donors showed that with this method, small changes in the reticulocyte population, such as the appearance of polychromatic erythrocytes in the peripheral blood 5–8 hr after donation, can be detected. The statistical reliability and the information provided on the maturation stage of the entire reticulocyte population make flow cytometry of peripheral blood reticulocytes a more informative method for the study of hematologic abnormalities than conventional methods for reticulocyte counting and classification.

A P-Selectin/CD62P Monoclonal Antibody (LYP-20), in Tandem with Flow Cytometry, Detects in vivo Activated Circulating Rat Platelets in Severe Vascular Trauma

1994 ◽  
Vol 72 (05) ◽  
pp. 745-749 ◽  
Author(s):  
Elza Chignier ◽  
Maud Parise ◽  
Lilian McGregor ◽  
Caroline Delabre ◽  
Sylvie Faucompret ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
Platelet Activation ◽  
Peripheral Blood ◽  
Vascular Trauma ◽  
Activated Platelets ◽  
Group I ◽  
Group Ii ◽  
Rat Platelets

SummaryP-selectin, also known as CD62P, GMP140 or PADGEM, is present in platelet a-granules and endothelial cell Weibel-Palade bodies and is very rapidly expressed on the surface of these cells on activation. In this study, an anti P-selectin monoclonal antibody (LYP20) was used, in tandem with flow cytometry, to identify activated platelets at the site of induced vascular trauma or in peripheral blood. Moreover, electron microscopy was performed to characterize sites of vascular trauma and quantify the number of adhering platelets. The same induced vascular trauma was observed to result into animals responding in 2 different ways (Group I, Group II) following the degree of platelet activation. Five rats, out of 14 with induced vascular trauma, had more than half of their circulating platelets expressing P-selectin when drawn at the site of the trauma (67.4% ± 3.44) or in peripheral blood (78.5% ± 2.5) (Group I). In the remaining 9 animals a much smaller proportion of circulating platelets expressed P-selectin when assayed from trauma sites (18% ± 3.34) or in peripheral blood (18.0% ± 4.30) (Group II). Enhanced P-selectin expression by circulating platelets in Group I, compared to Group II, appears to be linked to the degree of activated platelets adhering at sites of trauma (171 ± 15 × 103 platelets versus 48 ± 31 × 103 platelets per mm2). In the 5 control animals, that were not operated on, platelets expressing P-selectin when drawn at the site of a mock trauma (7.0% ± 1.84) or in the peripheral blood (11.2% ± 3.30) showed little activation. In addition, no platelet adhesion was seen on the vascular bed of these animals. Results from this study show that analysis of P-selectin (CD62P) expression, in circulating platelets, is a valuable and rapid marker of platelet activation following severe vascular trauma induced in rats. However, activated platelets were not detected to the same extent in the peripheral blood of all animals having undergone vascular trauma. It is conceivable that platelets, depending on the degree of activation, may be actively sequestered in organs and prevented from circulating. Alternatively, P-selectin may be rapidly endocytosed, or not expressed, by activated circulating platelets depending on the type of agonists implicated in vivo activation.

scholarly journals FRI0015 PHENOTYPE AND FUNCTION OF THE PERIPHERAL BLOOD DENDRITIC CELLS OF PSORIASIS PATIENTS WITH AND WITHOUT ARTHRITIS

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 578.1-579
Author(s):  
S. Schnitte ◽  
A. Fuchs ◽  
T. Funk ◽  
A. C. Pecher ◽  
D. Dörfel ◽  
...  
Keyword(s):  
Risk Factors ◽  
Flow Cytometry ◽  
Dendritic Cells ◽  
Psoriatic Arthritis ◽  
T Lymphocytes ◽  
Peripheral Blood ◽  
Surface Proteins ◽  
Research Support ◽  
Cross Sectional ◽  
Cell Counts

Background:Psoriasis is a frequent skin disease that can appear with an arthritic manifestation in approximately 30% of the cases [1]. The underlying excessive immune reaction caused by pro-inflammatory cytokines can be triggered by several risk factors [2]. Various subgroups of Dendritic cells (DCs) in the skin play a crucial role in the induction of the dermal inflammatory response [3].Objectives:As the role of peripheral blood DCs remains unknown and the cause of an arthritic manifestation is still not completely understood [4], this project aimed to detect differences in phenotype or function of peripheral blood DCs in psoriatic patients with or without arthritis.Methods:We analyzed peripheral blood cells of 60 psoriasis patients with and without arthritis. Different DC subpopulations were detected by flow cytometry. Monocyte-derived DCs were cultured with or without Lipopolysaccharides to gain immature (iDC) and mature (mDC) cells. The DC phenotype was determined by staining with CD80, CD83, CD86, CD206, CCR7, CD1a, HLA-DR, CD40, GPN-MB, DC209 and CD14. Their T-cell stimulatory capability was analyzed by co-incubation with Carboxyfluorescein succinimidyl ester stained lymphocytes and the quantification of CD4+ T-lymphocytes afterwards. To measure the migration capacity DCs were seated into transwell chambers with a semipermeable membrane and partly supplemented with Macrophage Inflammatory Protein 3 Beta (Mip3b). Migrated cells were detected by flow cytometry. Measured cell counts were normalized to cell counts without Mip3b stimulation.Results:Comparing the factor of increase of migrated mDC counts due to mip3b stimulation, we detected a significant lower rate in samples of patients with arthritis (PsA) compared to those of patients without (Ps). Assays of mDCs without mip3b stimulation showed a significant higher count of migrated cells in the samples of the arthritic group [Figure 1]. Cell counts with Mip3b stimulation did vary slightly in the groups. The DC subpopulations and the expression of analyzed cell surface proteins did not show significant differences. The amounts of stimulated T-Lymphocytes did not differ significantly.Figure 1.Migration essay showing mDCs following Mip3b (+miß3b) as multiples of mDCs without stimulation (-mip3b). The factor of increase is significantly lower in patients with arthritis (PsA) compared to patients without (Ps). Absolute counts of migrated mDCs without Mip3b are significantly higher in the arthritic group. Cell counts with stimulation do not differ significantly (data not shown). N=24, p<0.05Conclusion:CCL19 (Mip3b) is a potent ligand to the CCR7 receptor inducing migration of DCs towards the lymphatic node [5]. The CCR7 amounts on the DC surface did not differ significantly in the groups. The mDCs without CCL19 stimulation migrated in higher amounts in samples of arthritic patients. Cell counts of stimulated DCs showed only slight differences. These results could be generated by a different appearance of the DCs of arthritic patients that might facilitate migration. Further experiments focusing on this aspect should be performed. A possible effect of disruptive factors (age, sex, medication…) needs to be clarified.References:[1]Henes, J.C., et al.,High prevalence of psoriatic arthritis in dermatological patients with psoriasis: a cross-sectional study.Rheumatol Int, 2014.34(2): p. 227-34.[2]Lee, E.B., et al.,Psoriasis risk factors and triggers.Cutis, 2018.102(5s): p. 18-20.[3]Kim, T.G., S.H. Kim, and M.G. Lee,The Origin of Skin Dendritic Cell Network and Its Role in Psoriasis.Int J Mol Sci, 2017.19(1).[4]Veale, D.J. and U. Fearon,The pathogenesis of psoriatic arthritis.Lancet, 2018.391(10136): p. 2273-2284.[5]Ricart, B.G., et al.,Dendritic cells distinguish individual chemokine signals through CCR7 and CXCR4.J Immunol, 2011.186(1): p. 53-61.Acknowledgments:This project was financially supported by Novartis Pharma GmbH.Disclosure of Interests:Sarah Schnitte Grant/research support from: Reaserch grant by Novartis, Alexander Fuchs: None declared, Tanja Funk: None declared, Ann-Christin Pecher: None declared, Daniela Dörfel: None declared, Jörg Henes Grant/research support from: Novartis, Roche-Chugai, Consultant of: Novartis, Roche, Celgene, Pfizer, Abbvie, Sanofi, Boehringer-Ingelheim,

542 Expansion of cytotoxic NK Cells from PBMCs using individualized cytokine combination

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A578-A578
Author(s):  
Andreia Maia ◽  
Joana Lerias ◽  
Markus Maeurer ◽  
Mireia Castillo-Martin
Keyword(s):  
Flow Cytometry ◽  
Nk Cells ◽  
Natural Killer ◽  
Peripheral Blood ◽  
Blood Donors ◽  
Nk Cell ◽  
Tumour Cells ◽  
List Type ◽  
Mhc I ◽  
Cytokine Combination

BackgroundAdoptive immunotherapy relies on the use of T-cells to target tumour cells, through Major Histocompatibility Complex (MHC) Class I recognition(1). However, many tumours display alterations in the MHC-I pathway, a well-described immune evasion mechanism(2). Natural Killer (NK) cells recognize transformed cells independently from the presence of MHC-I and may be a reliable therapeutic option for patients with altered tumour MHC-I expression. The source of NK cells may be autologous or allogeneic and NK cells are also clinically relevant recipients of transgenic receptors (TCRs or antibodies) targeting tumour cells. NK cells have been categorized according to their CD56 and CD16 surface expression into different subpopulations: cytotoxic (CD56+CD16+) and regulatory (CD56brightCD16-)(3). Expanding cytotoxic NK cells is challenging, since the frequency of NK cells is low in peripheral blood(4) and there is also – at this point – not an optimal expansion protocol available.The goal of this project is to determine the best cytokine combination that facilitates expansion of cytotoxic NK cells that either target tumor cells directly or serve as recipients for transgenic receptors.MethodsPeripheral Blood Mononuclear Cells (PBMCs) were extracted using Ficoll methodology from blood donors and cultured in T25 flasks with Cell Genix Medium supplemented with 10% human serum and antibiotics. NK cells were expanded supplemented with feeder cells (ratio 1:1) and different cytokine combinations (1000 U/mL of IL-2, 10 U/ml of IL-12, 180 U/mL of IL-15 and/or 1 U/mL of IL-21) during 20 days. The immunophenotype of expanded NK cells was analyzed at days 0, 5, 10, 15 and 20 by flow cytometry. The cytotoxicity of NK cells was measured by a CD107a Assay or by a Total Cytotoxicity and Apoptosis Assay at days 10 and 20. Thirteen different cytokine combinations were tested.Results4/13 cytokine combinations produced a statistically significant increase of the absolute number of NK cells with a higher percentage of cytotoxic NK cells (figure 1). However, induction of cytotoxicity was not associated with a strong NK cell expansion. The regulatory NK cells subset (CD56brightCD16-) showed the highest percentage of CD107a-expressing cells, more than the CD56+CD16+, the most cytotoxic subpopulation of NK cells.Abstract 542 Figure 1Representative percentage of NK cells in total lymphocytes (A), CD56+CD16+ subpopulation in total NK cells (B), and CD56brightCD16- subpopulation amongst total NK cells (C) at different time points (5, 10, 15 and 20 days) expanded from PBMCs* p-value < 0.05ConclusionsThis work shows that we are able to grow and efficiently expand NK cells from PBMCs with different cytokine combinations leading to clinically relevant NK cell numbers as well as cytotoxic functions. This enables to produce NK cell products for therapy and as recipients for transgenic tumor antigen-specific receptors.AcknowledgementsThe authors would like to thank the Champalimaud Foundation Biobank, the Vivarium Facility and the Flow Cytometry Platform of the Champalimaud Centre for the Unknown.Ethics ApprovalThis study was approved by the Champalimaud Foundation Ethics Committee and by the Ethics Research Committee of NOVA Medical School of NOVA University of Lisbon.ConsentWritten informed consent was obtained from the blood donors to use their samples for research purposes.ReferencesRosenberg SA, Restifo NP, Yang JC, Morgan RA, Mark E. Adoptive cell transfer: a clinical path to effective cancer immunotherapy. Nat Rev Cancer 2008;8(4):299–308.Aptsiauri N, Ruiz-Cabello F, Garrido F. The transition from HLA-I positive to HLA-I negative primary tumors: the road to escape from T-cell responses. Curr Opin Immunol 2018;51:123–32.Di Vito C, Mikulak J, Mavilio D. On the way to become a natural killer cell. Front Immunol. 2019;10(August):1–15.Zotto G Del, Antonini F, Pesce S, Moretta F, Moretta L. Comprehensive phenotyping of human PB NK Cells by Flow Cytometry. 2020;1–9.

Micronucleus test by flow cytometry: application to mouse peripheral blood

1988 ◽  
Vol 203 (5) ◽  
pp. 383
Author(s):  
H. Norppa ◽  
M. Hayashi ◽  
T. Sofuni ◽  
Y. Kodama ◽  
M. Ishidate
Keyword(s):  
Flow Cytometry ◽  
Peripheral Blood ◽  
Micronucleus Test
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