Flow cytometry inmunophenotyping can identify and characterize solid tumour cells in clinical samples: A preliminary study

2019 ◽  
Vol 493 ◽  
pp. S148-S149
Author(s):  
C. Quirós ◽  
L. Fernández ◽  
H. Torres ◽  
F. Domínguez ◽  
F.V. Álvarez ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Solid Tumour ◽  
Tumour Cells ◽  
Clinical Samples ◽  
Preliminary Study

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.

Approaching stability challenges for flow cytometry in a regulated bioanalytical environment

Bioanalysis ◽  
2019 ◽  
Vol 11 (20) ◽  
pp. 1845-1858 ◽  
Author(s):  
Anamica Muruganandham ◽  
Carolyne Dumont ◽  
Kuiyi Xing
Keyword(s):  
Flow Cytometry ◽  
Critical Parameter ◽  
Storage Period ◽  
Reliable Data ◽  
Clinical Samples ◽  
Sample Collection ◽  
Testing Laboratory ◽  
Stability Parameters ◽  
Stability Issues ◽  
Limited Period

Stability of samples for flow cytometry is a critical parameter since storage period of samples is restricted to only a limited period after collection. For most studies, clinical samples have to be shipped to a testing laboratory, in contrast to preclinical samples, which can be analyzed on-site or off-site. Therefore, evaluating stability is critical to provide flexibility on testing of samples to obtain reliable data. A wide variety of factors contributes to establishing stability from sample collection through acquisition. We provided suggestions for experimental and stability parameters to be taken into consideration when designing a flow cytometry method. The case studies presented represent how certain stability issues were overcome to perform flow cytometry assays in a regulated bioanalytical environment.

Preliminary Study on In Situ Realtime Quantitation of Target Bacteria on the Principle of Flow Cytometry

2017 ◽  
Vol 262 ◽  
pp. 224-227
Author(s):  
Gen Murakami ◽  
Yuichi Sugai ◽  
Kyuro Sasaki
Keyword(s):  
Flow Cytometry ◽  
Bacterial Culture ◽  
Scattered Light ◽  
Bacterial Suspension ◽  
Culture Solution ◽  
Bacterial Cells ◽  
Monitoring Wells ◽  
Measurement Principle ◽  
Preliminary Study

In-situ realtime method that can monitor the target bacteria should be used to determine the real situation of the bacteria in deep parts of heaps in heap bioleaching plants. This study suggest to apply flow cytometry technology to in-situ realtime monitoring of target bacteria. Flow cytometry is a method that can rapidly quantify the bacterial cells in bacterial suspension based on the detection of lights that are emitted from bacterial cells. In this study, we estimated the possibility of the application of flow cytometry to the selective detection of target bacteria. The bacterial culture solution that had been diluted by water including other bacteria was provided for fluorescence spectral analysis and scattered light analysis that were functions of flow cytometry. Our target bacteria could be selectively detected by those analyses in this study, therefore, it was shown that the flow cytometry could be useful for detecting target bacteria selectively. Because the measurement principle of flow cytometry is quite simple, it can be expected to be installed into deep heaps through the monitoring wells and determine the dominance of target bacteria in-situ and realtime in the future.

scholarly journals Flow cytometry of human semen: a preliminary study of a non-invasive method for the detection of spermatogenetic defects

2005 ◽  
Vol 20 (12) ◽  
pp. 3469-3475 ◽  
Author(s):  
N. Levek-Motola ◽  
Y. Soffer ◽  
L. Shochat ◽  
A. Raziel ◽  
L.M. Lewin ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Human Semen ◽  
Non Invasive ◽  
Invasive Method ◽  
Preliminary Study

scholarly journals Artifacts associated with mithramycin fluorescence in the clinical detection and quantitation of aneuploidy by flow cytometry.

1982 ◽  
Vol 30 (4) ◽  
pp. 317-322 ◽  
Author(s):  
R E Cunningham ◽  
K S Skramstad ◽  
A E Newburger ◽  
S E Shackney
Keyword(s):  
Flow Cytometry ◽  
Room Temperature ◽  
Human Melanoma ◽  
Time Dependent ◽  
Clinical Samples ◽  
Fixation Time ◽  
C Cells ◽  
Temperature Dependent ◽  
Clinical Detection

Ethanol-fixed cells stored at 4 degrees C exhibit fixation time-dependent hyperchromatism in comparison with freshly fixed cells when stained with mithramycin and examined by flow cytometry. This hyperchromatism has been found to be temperature-dependent, developing fully within 72 hr at room temperature, and within 2 hr at 37 degrees C. Cells from normal donors that are stained with mithramycin exhibit spurious aneuploid peaks. These spurious aneuploid peaks can be eliminated by incubating ethanol-fixed cells at 37 degrees C for 2 hr prior to staining; true aneuploidy is not affected by this procedure. In rare instances, cytoplasmic fluorescence can be observed in mithramycin-stained cells. In addition, unexplained hypochromatism and hyperchromatism can be observed in some clinical samples, particularly in human melanoma. The effects of these unexplained staining artifacts can be minimized or eliminated by adopting strict criteria for the clinical detection of aneuploidy by flow cytometry.

scholarly journals Applications of Flow Cytometry to Clinical Microbiology

2000 ◽  
Vol 13 (2) ◽  
pp. 167-195 ◽  
Author(s):  
Alberto Álvarez-Barrientos ◽  
Javier Arroyo ◽  
Rafael Cantón ◽  
César Nombela ◽  
Miguel Sánchez-Pérez
Keyword(s):  
Flow Cytometry ◽  
Clinical Microbiology ◽  
Ex Vivo ◽  
Analytical Techniques ◽  
Clinical Samples ◽  
Clinical Microbiology Laboratory ◽  
Commercial Kits ◽  
Antimicrobial Treatments ◽  
Near Future

SUMMARY Classical microbiology techniques are relatively slow in comparison to other analytical techniques, in many cases due to the need to culture the microorganisms. Furthermore, classical approaches are difficult with unculturable microorganisms. More recently, the emergence of molecular biology techniques, particularly those on antibodies and nucleic acid probes combined with amplification techniques, has provided speediness and specificity to microbiological diagnosis. Flow cytometry (FCM) allows single- or multiple-microbe detection in clinical samples in an easy, reliable, and fast way. Microbes can be identified on the basis of their peculiar cytometric parameters or by means of certain fluorochromes that can be used either independently or bound to specific antibodies or oligonucleotides. FCM has permitted the development of quantitative procedures to assess antimicrobial susceptibility and drug cytotoxicity in a rapid, accurate, and highly reproducible way. Furthermore, this technique allows the monitoring of in vitro antimicrobial activity and of antimicrobial treatments ex vivo. The most outstanding contribution of FCM is the possibility of detecting the presence of heterogeneous populations with different responses to antimicrobial treatments. Despite these advantages, the application of FCM in clinical microbiology is not yet widespread, probably due to the lack of access to flow cytometers or the lack of knowledge about the potential of this technique. One of the goals of this review is to attempt to mitigate this latter circumstance. We are convinced that in the near future, the availability of commercial kits should increase the use of this technique in the clinical microbiology laboratory.

scholarly journals Pre-metastatic niche triggers SDF-1/CXCR4 axis and promotes organ colonisation by hepatocellular circulating tumour cells via downregulation of Prrx1

2019 ◽  
Vol 38 (1) ◽  
Author(s):  
Yujun Tang ◽  
Yishi Lu ◽  
Yuan Chen ◽  
Lei Luo ◽  
Lei Cai ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Cell Line ◽  
Lentiviral Vector ◽  
Tumour Cells ◽  
Clinical Samples ◽  
Circulating Tumour Cells ◽  
Metastatic Niche ◽  
Metastatic Sites

Abstract Background Circulating tumour cells (CTCs), especially mesenchymal CTCs, are important determinants of metastasis, which leads to most recurrence and mortality in hepatocellular carcinoma (HCC). However, little is known about the underlying mechanisms of CTC colonisation in pre-metastatic niches. Methods Detection and classification of CTCs in patients were performed using the CanPatrol™ system. A lentiviral vector expressing Prrx1-targeting shRNA was constructed to generate a stable HCC cell line with low expression of Prrx1. The effect of Prrx1 knockdown on stemness, migration, and drug resistance of the cell line was assessed, including involvement of SDF-1/CXCR4 signalling. Promising clinical applications of an inhibitor of STAT3 tyrosine phosphorylation, C188–9, and specific blockade with CXCR4 antibody were explored. Results The number of mesenchymal CTCs in blood was closely associated with tumour recurrence or metastasis. Pre-metastatic niche-derived SDF-1 could downregulate Prrx1, which induced the stemness, drug resistance, and increased expression of CXCR4 in HCC cells through the STAT3 pathway in vitro. In vivo, mice bearing tumours of Prrx1 low-expressing cells had significantly shorter survival. In xenograft tumours and clinical samples, loss of Prrx1 was negatively correlated with increased expression of CXCR4 in lung metastatic sites compared with that in the primary foci. Conclusions These findings demonstrate that decreased expression of Prrx1 stimulates SDF-1/CXCR4 signalling and contributes to organ colonisation with blood CTCs in HCC. STAT3 inhibition and specific blockade of CXCR4 have clinical potential as therapeutics for eliminating organ metastasis in advanced HCC.

scholarly journals A Cell-Based Capture Assay for Rapid Virus Detection

Viruses ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 1165
Author(s):  
Elad Milrot ◽  
Efi Makdasi ◽  
Boaz Politi ◽  
Tomer Israely ◽  
Orly Laskar
Keyword(s):  
Flow Cytometry ◽  
Fluorescence Microscopy ◽  
Virus Detection ◽  
Vero Cells ◽  
Detection Sensitivity ◽  
Clinical Samples ◽  
Response Analysis ◽  
Proof Of Concept ◽  
Virus Identification ◽  
Capture Assay

Routine methods for virus detection in clinical specimens rely on a variety of sensitive methods, such as genetic, cell culture and immuno-based assays. It is imperative that the detection assays would be reliable, reproducible, sensitive and rapid. Isolation of viruses from clinical samples is crucial for deeper virus identification and analysis. Here we introduce a rapid cell-based assay for isolation and detection of viruses. As a proof of concept several model viruses including West Nile Virus (WNV), Modified Vaccinia Ankara (MVA) and Adenovirus were chosen. Suspended Vero cells were employed to capture the viruses following specific antibody labeling which enables their detection by flow cytometry and immuno-fluorescence microscopy assays. Using flow cytometry, a dose response analysis was performed in which 3.6e4 pfu/mL and 1e6 pfu/mL of MVA and WNV could be detected within two hours, respectively. When spiked to commercial pooled human serum, detection sensitivity was slightly reduced to 3e6 pfu/mL for WNV, but remained essentially the same for MVA. In conclusion, the study demonstrates a robust and rapid methodology for virus detection using flow cytometry and fluorescence microscopy. We propose that this proof of concept may prove useful in identifying future pathogens.

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