imaging flow cytometry
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Cancers ◽  
2021 ◽  
Vol 13 (24) ◽  
pp. 6386
Author(s):  
Antonio J. Ruiz-Rodríguez ◽  
Maria P. Molina-Vallejo ◽  
Inés Aznar-Peralta ◽  
Cristina González Puga ◽  
Inés Cañas García ◽  
...  

The isolation of circulating tumour cells (CTCs) in colorectal cancer (CRC) mostly relies on the expression of epithelial markers such as EpCAM, and phenotypic characterisation is usually performed under fluorescence microscopy with only one or two additional markers. This limits the ability to detect different CTC subpopulations based on multiple markers. The aim of this work was to develop a novel protocol combining two platforms (IsoFluxTM and ImageStream®X) to improve CTC evaluation. Cancer cell lines and peripheral blood from healthy donors were used to evaluate the efficiency of each platform independently and in combination. Peripheral blood was extracted from 16 early CRC patients (before loco-regional surgery) to demonstrate the suitability of the protocol for CTC assessment. Additionally, peripheral blood was extracted from nine patients one month after surgery to validate the utility of our protocol for identifying CTC subpopulation changes over time. Results: Our protocol had a mean recovery efficiency of 69.5% and a limit of detection of at least four cells per millilitre. We developed an analysis method to reduce noise from magnetic beads used for CTC isolation. CTCs were isolated from CRC patients with a median of 37 CTCs (IQ 13.0–85.5) at baseline. CTCs from CRC patients were significantly (p < 0.0001) larger than cytokeratin (CK)-negative cells, and patients were stratified into two groups based on BRAFV600E and PD-L1 expression on CK-positive cells. The changes observed over time included not only the number of CTCs but also their distribution into four different subpopulations defined according to BRAFV600E and PD-L1 positivity. We developed a novel protocol for semi-automatic CTC isolation and phenotypic characterisation by combining two platforms. Assessment of CTCs from early CRC patients using our protocol allowed the identification of two clusters of patients with changing phenotypes over time.


2021 ◽  
Vol 65 (4) ◽  
Author(s):  
Anett Kristin Larsen ◽  
Jaione Simón-Santamaría ◽  
Kjetil Elvevold ◽  
Bo Göran Ericzon ◽  
Kim Erlend Mortensen ◽  
...  

Autofluorescent granules of various sizes were observed in primary human liver endothelial cells (LSECs) upon laser irradiation using a wide range of wavelengths. Autofluorescence was detected in LAMP-1 positive vesicles, suggesting lysosomal location. Confocal imaging of freshly prepared cultures and imaging flow cytometry of non-cultured cells revealed fluorescence in all channels used. Treatment with a lipofuscin autofluorescence quencher reduced autofluorescence, most efficiently in the near UV-area. These results, combined with the knowledge of the very active blood clearance function of LSECs support the notion that lysosomally located autofluorescent material reflected accumulation of lipofuscin in the intact liver. These results illustrate the importance of careful selection of fluorophores, especially when labelling of live cells where the quencher is not compatible.


Molecules ◽  
2021 ◽  
Vol 26 (23) ◽  
pp. 7096
Author(s):  
Michał Konieczny ◽  
Peter Rhein ◽  
Katarzyna Czaczyk ◽  
Wojciech Białas ◽  
Wojciech Juzwa

The aim of the research was to design an advanced analytical tool for the precise characterization of microbial aggregates from biofilms formed on food-processing surfaces. The approach combined imaging flow cytometry with a machine learning-based interpretation protocol. Biofilm samples were collected from three diagnostic points of the food-processing lines at two independent time points. The samples were investigated for the complexity of microbial aggregates and cellular metabolic activity. Thus, aggregates and singlets of biofilm-associated microbes were simultaneously examined for the percentages of active, mid-active, and nonactive (dead) cells to evaluate the physiology of the microbial cells forming the biofilm structures. The tested diagnostic points demonstrated significant differences in the complexity of microbial aggregates. The significant percentages of the bacterial aggregates were associated with the dominance of active microbial cells, e.g., 75.3% revealed for a mushroom crate. This confirmed the protective role of cellular aggregates for the survival of active microbial cells. Moreover, the approach enabled discriminating small and large aggregates of microbial cells. The developed tool provided more detailed characteristics of bacterial aggregates within a biofilm structure combined with high-throughput screening potential. The designed methodology showed the prospect of facilitating the detection of invasive biofilm forms in the food industry environment.


Biology ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 1213
Author(s):  
Patricia Peris-Frau ◽  
Irene Sánchez-Ajofrín ◽  
Alicia Martín Maestro ◽  
Carolina Maside ◽  
Daniela Alejandra Medina-Chávez ◽  
...  

The heterogeneous nature of ejaculates highlights the relevance of studying the behavior of different sperm subpopulations. Changes in sperm motility and the increase in tyrosine phosphorylation are key events that usually occur during capacitation and can be modified by the cryopreservation process. However, the relationship between both events remains poorly defined throughout capacitation in the different sperm subpopulations. Fresh and frozen-thawed spermatozoa were incubated in capacitating (CAP) and non-capacitating (NC) media up to 240 min. Sperm kinematics, tyrosine phosphorylation and mitochondrial activity were measured by the CASA system and imaging flow cytometry. Four motile sperm subpopulations (SP) were identified in fresh and frozen-thawed ram semen after the cluster analysis. Incubation under CAP conditions over time led to greater changes in the percentage of spermatozoa included in each subpopulation compared to NC conditions, being different between fresh and frozen-thawed spermatozoa. The SP1, characterized by slow spermatozoa, progressively increased after 15 min in frozen-thawed samples incubated in both media but not in fresh ones. The SP4, characterized by fast and non-linear spermatozoa, showed a marked increase during CAP, but not under NC conditions, occurring more rapidly in frozen-thawed spermatozoa. This subpopulation (SP4) was also the only one positively and strongly correlated with mitochondrial activity and all phosphorylated sperm regions during capacitation, either in fresh or frozen-thawed samples. Our results indicated that in vitro capacitation induced significant changes in the distribution of motile sperm subpopulations, being affected by cryopreservation. Notwithstanding, the subpopulation which probably represents hyperactivated-like spermatozoa (SP4) also increased in frozen-thawed samples, occurring faster and simultaneously to the increment of mitochondrial activity and tyrosine phosphorylation of different sperm regions.


2021 ◽  
Vol 22 (22) ◽  
pp. 12436
Author(s):  
Marvin Droste ◽  
Tobias Tertel ◽  
Stefanie Jeruschke ◽  
Robin Dittrich ◽  
Evangelia Kontopoulou ◽  
...  

Small extracellular vesicles isolated from urine (uEVs) are increasingly recognized as potential biomarkers. Meanwhile, different uEV preparation strategies exist. Conventionally, the performance of EV preparation methods is evaluated by single particle quantification, Western blot, and electron microscopy. Recently, we introduced imaging flow cytometry (IFCM) as a next-generation single EV analysis technology. Here, we analyzed uEV samples obtained with different preparation procedures using nanoparticle tracking analysis (NTA), semiquantitative Western blot, and IFCM. IFCM analyses demonstrated that urine contains a predominant CD9+ sEV population, which exceeds CD63+ and CD81+ sEV populations. Furthermore, we demonstrated that the storage temperature of urine samples negatively affects the recovery of CD9+ sEVs. Although overall reduced, the highest CD9+ sEV recovery was obtained from urine samples stored at −80 °C and the lowest from those stored at −20 °C. Upon comparing the yield of the different uEV preparations, incongruencies between NTA and IFCM data became apparent. Results obtained by both NTA and IFCM were consistent with Western blot analyses for EV marker proteins; however, NTA results correlated with the amount of the impurity marker uromodulin. Despite demonstrating that the combination of ultrafiltration and size exclusion chromatography appears as a reliable uEV preparation technique, our data challenge the soundness of traditional NTA for the evaluation of different EV preparation methods.


Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2619-2619
Author(s):  
Kathy Fuller ◽  
Henry Hui ◽  
Jason Stanley ◽  
Wendy N. Erber

Abstract Chronic lymphocytic leukaemia is a genetically heterogeneous disease with treatment and prognosis varying based on chromosomal abnormalities. These are detectable in up to 80% of cases when tested on the nuclei of interphase cells by fluorescence in situ hybridisation (FISH). Despite the clinical importance of FISH in management, as only up to 200 nuclei are generally assessed, it is not suitable for minimal residual disease (MRD) assessment. Since clinical decisions are based on detection thresholds of 10 -4, MRD assays are restricted to flow cytometry and molecular based assessment. Here we have explored the utility of a cutting-edge automated imaging flow cytometry method that incorporates cell immunophenotype and FISH ("immuno-flowFISH") to detect chromosomal abnormalities in CLL. Aims: Our aim was to determine the capability of immuno-flowFISH using imaging flow cytometry to detect del(17p) and +12 in CLL, and, the lowest limit of detection. We hypothesized that this integrated automated immuno-flowFISH method would be suitable for MRD assessment of CLL. Methods: Blood from 75 patients with CLL, at diagnosis or on therapy, was analysed. For MRD studies, cells from the CI cell line were spiked into normal blood at concentrations of 0.001 - 10%. After red cell lysis, samples were incubated with CD3, CD5 and CD19 fluorophore-conjugated antibodies (fluorophores: BV480, BV605, AF647). Following fixation and membrane permeabilization, DNA was denatured at 78 oC for 5 mins. FISH probes to 17p12 and centromeres of chromosomes 12 and 17 were added and hybridized for 24 hours at 42 oC. Nuclei were then stained with SYTOX AADvanced and up to 600,000 cells acquired on the Amnis ® ImageStream ®XMk II imaging flow cytometer. Digital images (x60 objective) and quantitative data derived from computational algorithms (IDEAS software) were used to assess FISH signals overlying cell nuclei. IDEAS was then used to assess the number and percent CD19/CD5-positive CLL cells with FISH abnormalities. Results: Between 10,000 and 600,000 cells (mean 60,000) were acquired. CLL (CD19/CD5-positive) and T- (CD3/CD5-positive) cells could be clearly identified by their immunophenotype and assessed individually for probe signals. FISH signals were identifiable on the digital images as specific "spots" overlying the SYTOX AADvanced nuclear stain. The IDEAS software could enumerate the number of FISH spots per cell and this was confirmed by quantitative mean channel fluorescence intensity for each probe. A chromosome 12 or 17 abnormality was detected in 23 of the 75 CLL cases. Of these, 10 cases had only one 17p signal (but 2 for the centromere of chromosome 17), indicative of del(17p). Del(17p) was detected in 2-35% of CD19/CD5-positive cells (i.e. 0.4-23% or 270-35,441 of all cells), the lowest seen in a patient on cytoreductive therapy. In 13/75 cases, there were 3 FISH signals for CEP12, consistent with trisomy 12 (+12) in 0.1-46% of all cells analysed; the lowest number of 0.1% was when 26 out of 26,000 cells analysed were CD19/CD5-positive and had +12. We also performed multi-FISH, incorporating CEP12, CEP17 and 17p probes together with the CD3, CD5 and CD19 antibodies. This required 7-fluorophores (antibodies, probes and nucleus) and confirmed the ability to detect del(17p) and chromosome 12 copy number simultaneously in a single analysis. Spiking of CI CLL cells into normal blood demonstrated that +12 could be detected to a lowest limit of 10 -5. In all analyses, CLL cells had normal diploid spots for the control CEP17 probe, and the CD3/CD5-positive T cells had dual signals for CEP12, CEP17 and 17p12 probes on numerical analysis and on digital imagery. Conclusion: This study of confirms that high-throughput automated imaging flow cytometry, integrating FISH and immunophenotyping, can detect chromosomal defects in CLL. The lowest limit of detection for a FISH-detectable abnormality was 10 -5. This high sensitivity and specificity exceeds current clinical practice (10 -4), and was achieved through the analysis of many thousands of cells and positive identification of CLL cells based on their phenotype. This immuno-flowFISH method does not require any prior cell separation and is automated. It adds a new dimension to chromosomal analysis in CLL, both at diagnosis and for MRD monitoring. The high precision and specificity of immuno-flowFISH illustrates that this has a real place as a new MRD assessment tool for CLL. Disclosures No relevant conflicts of interest to declare.


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