Advances In Flow Cytometry Research Articles

Extracellular Histones Profiles of Pediatric H3K27-Altered Diffuse Midline Glioma.

Diffuse midline glioma, H3 K27-altered (DMG) is a fatal tumour that arises in the midline structures of the brain. When located in the pons, it is more commonly referred to as diffuse intrinsic pontine glioma (DIPG). DMG/DIPG is usually diagnosed when children are < 10 years, and it has a median overall survival of < 12 months after diagnosis. Radiological imaging is still the gold standard for DIPG diagnosis while the use of biopsy procedures led to our knowledge on its biology, such as with the identification of the canonical histone H3K27M mutation. However, the need to improve survival encourages the development of non-invasive, fast and inexpensive assays on biofluids for optimizing molecular diagnoses in DMG/DIPG. Here, we propose a rapid, new, imaging and epigenetics-based approach to diagnose DMG/DIPG in the plasma of paediatric patients. A total of 20 healthy children (mean age: 10.5 years) and 24 children diagnosed with DMG/DIPG (mean age: 8.5 years) were recruited. Individual histones (H2A, H2B, H3, H4, macroH2A1.1 and macroH2A1.2), histone dimers and nucleosomes were assayed in biofluids by means of a new advanced flow cytometry ImageStream(X)-adapted method. We report a significant increase in circulating histone dimers and tetramers (macroH2A1.1/H2B versus control: p value < 0.0001; macroH2A1.2/H2B versus control: p value < 0.0001; H2A/H2B versus control: p value < 0.0001; H3/H4 versus control: p value = 0.008; H2A/H2B/H3/H4 versus control: p value < 0.0001) and a significant downregulation of individual histones (H2B versus control: p value < 0.0001; H3 versus control: p value < 0.0001; H4 versus control: p value < 0.0001). Moreover, histones were also detectable in the cerebrospinal fluid (CSF) of patients with DMG/DIPG and in the supernatant of SF8628, OPBG-DIPG002 and OPBG-DIPG004 DMG/DIPG cell lines, with patterns mostly similar to each other, but distinct compared to blood plasma. In summary, we identified circulating histone signatures able to detect the presence of DMG/DIPG in biofluids of children, using a rapid and non-invasive ImageStream(X)-based imaging technology, which may improve diagnosis and benefit the patients.

TRBP2, a Major Component of the RNAi Machinery, Is Subjected to Cell Cycle-Dependent Regulation in Human Cancer Cells of Diverse Tissue Origin.

Transactivation Response Element RNA-binding Protein (TRBP2) is a double-stranded RNA-binding protein widely known for its critical contribution to RNA interference (RNAi), a conserved mechanism of gene-expression regulation mediated through small non-coding RNA moieties (ncRNAs). Nevertheless, TRBP2 has also proved to be involved in other molecular pathways and biological processes, such as cell growth, organism development, spermatogenesis, and stress response. Mutations or aberrant expression of TRBP2 have been previously associated with diverse human pathologies, including Alzheimer's disease, cardiomyopathy, and cancer, with TRBP2 playing an essential role(s) in proliferation, invasion, and metastasis of tumor cells. Hence, the present study aims to investigate, via employment of advanced flow cytometry, immunofluorescence, cell transgenesis and bioinformatics technologies, new, still elusive, functions and properties of TRBP2, particularly regarding its cell cycle-specific control during cancer cell division. We have identified a novel, mitosis-dependent regulation of TRBP2 protein expression, as clearly evidenced by the lack of its immunofluorescence-facilitated detection during mitotic phases, in several human cancer cell lines of different tissue origin. Notably, the obtained TRBP2-downregulation patterns seem to derive from molecular mechanisms that act independently of oncogenic activities (e.g., malignancy grade), metastatic capacities (e.g., low versus high), and mutational signatures (e.g., p53-/- or p53ΔΥ126) of cancer cells. Taken together, we herein propose that TRBP2 serves as a novel cell cycle-dependent regulator, likely exerting mitosis-suppression functions, and, thus, its mitosis-specific downregulation can hold strong promise to be exploited for the efficient and successful prognosis, diagnosis, and (radio-/chemo-)therapy of diverse human malignancies, in the clinic.

Abstract 2068: Multi-pass flow cytometry for high-marker panels with minimal spillover spread

Abstract Multiparametric flow cytometry on immune cells is essential in cancer research, disease diagnosis, and treatment monitoring. Collecting data on &amp;gt;10 markers at a time is ideal due to deeper cell characterization and assay sensitivity, however, high-marker panels are subject to significant spillover spread from spectrally overlapping fluorophores that compromise data quality, standardization, and interpretation. Clinical flow cytometry still relies on the use of multi-tube low-marker (&amp;lt;10) panels that minimize spillover spread and ease analysis and interpretation of results. To improve and expand the clinical utility of flow cytometry, there is a need for higher marker measurements without use of highly overlapping fluorophores, which would also serve to streamline the use of samples and reagents. Here, we present the development of a novel barcoding method using micron-sized laser particles (LPs) that confer an optical signature to individual cells at high-throughput, enabling tracking of cells through multiple passes in a flow cytometer. This process is possible using a custom cytometer equipped to detect LPs and capture samples. Sequential staining of samples with releasing or photobleaching antibodies/fluors between cycles enables merging of multiple smaller panels that each have minimal spillover, resulting in a high-marker dataset with drastic reduction in spillover compared to conventional single-acquisition flow cytometry. We demonstrate the utility of this technology through three different assays. Human PBMCs were barcoded with LPs and stained with 1) a panel of releasable antibodies (Miltenyi Biotec, 10 markers, 2 passes), 2) a laboratory-designed panel that utilized photobleaching (32 markers, 3 passes), and 3) a panel of dried and validated DuraClone antibodies that utilized photobleaching (Beckman Coulter, 18 markers, 2 passes). For each assay, cells were stained, acquired, photobleached/released, captured, re-stained, re-acquired and steps were repeated either once (for 2 passes) or twice (for 3 passes). In all cases, we found drastically reduced spillover, better data quality, and identified the same cell subtypes detected by conventional flow cytometry. Reproducibility was high across multiple (3+) replicates with CVs &amp;lt;30% among all populations with &amp;gt;1% frequency of total. New cell populations were identified that would not be possible with separate acquisitions of each pass. Recent advances in flow cytometry now allow more markers to be analyzed together, but at the cost of increasing spectral overlap and spillover spread. Multi-pass flow cytometry confines spillover to individual panels, reducing overall interference and simplifying analysis. This enhances data quality and usability while streamlining resources, facilitating the clinical uptake of complex flow cytometry in cancer research, diagnosis, and treatment. Citation Format: Trevor Brown, Sarah Forward, Emane Rose Assita, Geoffrey Abbott, Marissa Fahlberg, Sheldon J. Kwok. Multi-pass flow cytometry for high-marker panels with minimal spillover spread [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2068.

Abstract 91: Comprehensive 27-marker standard panel for immune monitoring of pre-clinical tumor mouse models using spectral analyzer technology

Abstract Within the last decade, major technological advances in flow cytometry and (single cell) RNA-sequencing have deepened our understanding of complex anti-tumoral immune responses. This allows a comprehensive immune monitoring of novel therapies in pre-clinical models. Conventional flow cytometry is reaching its technical limitation. The emitted fluorescence signal of the target population stained with antibodies is evaluated with simple bandpass filters, which leads to spectral overlap and thus the number of parameters that can be analyzed simultaneously is limited. The spectral analyzer technology combines both methods, providing great flexibility in evaluating different immune cell populations. This provides the opportunity to obtain target information by measuring the entire fluorescence spectrum measuring each wavelength individually and thus evaluating the true signal of each fluorochrome unaffected by autofluorescence or spillover. Our standard all-in-one flow cytometry panel uses the full capacity of a conventional BD Fortessa flow cytometer and enables the differentiation of important immune cell populations in tumors, such as T cells (CD4+, CD8+, regulatory T cells), B and NK cells as well as macrophages (M1/M2), MDSCs (granulocytes and monocytes) and dendritic cells. However, it would be of great advantage to gain further insights into activation and effector functions of the immune cells using markers like CD44 &amp; CD62L, CD69, PD-1, Lag-3, Tim-3 and PDL-1, CD80, CD86, CD40 and Ki-67. The Sony ID7000 spectral analyzer enables the simultaneous assessment of 27 markers to evaluate immune phenotypes, with the ability to include up to 4 individual target antibodies. Flow cytometry data from various tumor models are presented, demonstrating the enormous utility and potential of the spectral analyzer, especially with limited and precious tumor sample material, by evaluating different immune cell populations simultaneously without reaching its technical limits. The spectral analyzer technology enables us to mechanistically investigate differences in tumor therapies such as PD-1 treatment or other immune cell modulators not only phenotypically but also with regard to activation and differentiation of immune cell populations. Citation Format: Jonas F. Hummel, Philipp Metzger, Roland Sonntag, Dennis Borrero-Wolff, Cynthia Obodozie, Holger Weber. Comprehensive 27-marker standard panel for immune monitoring of pre-clinical tumor mouse models using spectral analyzer technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 91.

Abstract 6788: Comparative analysis of anti-HER2 antibody-drug conjugates in tumor spheroid models

Abstract Robust techniques are needed to screen the anti-tumor activity of antibody-drug conjugates (ADCs) in vitro. Traditional monolayer models provide a cost-effective and scalable option but lack many features of the tumor microenvironment, such as complex cell-cell and cell-extracellular matrix interactions. Tumor spheroids afford a more translational model for quantification of ADCs. Here, spheroids are used to compare the mechanism of action (MoA) of anti-HER2 ADCs. Single spheroids were formed from mono- or co-cultures of HER2 over-expressing cancer cells and HER2 negative control cells. Incucyte® Nuclight Green Lentivirus was used to label HER2 positive BT474 cells, whilst HER2 negative MDA-MB-231 cells were left unlabeled. Antibodies were added after 72 hours of spheroid formation in 96-well ultra-low attachment plates. Brightfield and fluorescence images were captured every 3 hours using the Incucyte® Live-Cell Analysis System and quantified, for metrics such as spheroid size and fluorescence intensity, to give an indication of ADC cytotoxicity. Co-culture spheroids were dissociated at assay endpoint and the remaining proportion of green and unlabeled cells was measured using the iQue® Advanced Flow Cytometry Platform. The area of the BT474 mono-culture spheroids decreased in a concentration-dependent manner in the presence of both ADCs tested (trastuzumab emtansine and trastuzumab deruxtecan), indicating increased cytotoxicity. In contrast, only the highest concentration of trastuzumab, which is the monoclonal antibody backbone on which they are based, resulted in a reduction in spheroid size. This displays the increased anti-tumor activity due to the addition of the ADC payloads. In the co-culture model, only the trastuzumab deruxtecan induced a reduction in spheroid area, with the size of the spheroids in the presence of trastuzumab emtansine and trastuzumab remaining comparable to control levels. Analysis of the spheroid composition using flow cytometry showed that for the BT474 cells, again, percentage cell death compared to the IgG control was greater for the two ADCs, at 96.4% and 97.2%, respectively. This death was greater than with trastuzumab, which induced 46.4% cell death. Unlike in the mono-culture assays, a high level of death of the HER2 low expressing cells was also seen in the co-culture in the presence of trastuzumab deruxtecan (94.1% death compared to control). This displays the unique bystander activity of trastuzumab deruxtecan, which has led to its approval as a treatment for both HER2 high and HER2 low breast cancers. These data display how the Incucyte® Live-Cell Analysis System combined with iQue® Advanced Flow Cytometry can be leveraged to provide comprehensive assessment of the in vitro function of ADCs and how it can enable differences in their MoAs to be distinguished in 3D spheroid models. Citation Format: Kirsty McBain, Kalpana Barnes, Nicola Bevan. Comparative analysis of anti-HER2 antibody-drug conjugates in tumor spheroid models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6788.

The future of equine semen analysis.

We are currently experiencing a period of rapid advancement in various areas of science and technology. The integration of high throughput 'omics' techniques with advanced biostatistics, and the help of artificial intelligence, is significantly impacting our understanding of sperm biology. These advances will have an appreciable impact on the practice of reproductive medicine in horses. This article provides a brief overview of recent advances in the field of spermatology and how they are changing assessment of sperm quality. This article is written from the authors' perspective, using the stallion as a model. We aim to portray a brief overview of the changes occurring in the assessment of sperm motility and kinematics, advances in flow cytometry, implementation of 'omics' technologies, and the use of artificial intelligence/self-learning in data analysis. We also briefly discuss how some of the advances can be readily available to the practitioner, through the implementation of 'on-farm' devices and telemedicine.

Dissociation protocols influence the phenotypes of lymphocyte and myeloid cell populations isolated from the neonatal lymph node.

Frequencies and phenotypes of immune cells differ between neonates and adults in association with age-specific immune responses. Lymph nodes (LN) are critical tissue sites to quantify and define these differences. Advances in flow cytometry have enabled more multifaceted measurements of complex immune responses. Tissue processing can affect the immune cells under investigation that influence key findings. To understand the impact on immune cells in the LN after processing for single-cell suspension, we compared three dissociation protocols: enzymatic digestion, mechanical dissociation with DNase I treatment, and mechanical dissociation with density gradient separation. We analyzed cell yields, viability, phenotypic and maturation markers of immune cells from the lung-draining LN of neonatal and adult mice two days after intranasal respiratory syncytial virus (RSV) infection. While viability was consistent across age groups, the protocols influenced the yield of subsets defined by important phenotypic and activation markers. Moreover, enzymatic digestion did not show higher overall yields of conventional dendritic cells and macrophages from the LN. Together, our findings show that the three dissociation protocols have similar impacts on the number and viability of cells isolated from the neonatal and adult LN. However, enzymatic digestion impacts the mean fluorescence intensity of key lineage and activation markers that may influence experimental findings.

Cellular Dynamics Following CAR T Cell Therapy Are Associated with Response, Resistance and Cytokine Release Syndrome in Relapsed/Refractory Multiple Myeloma

Introduction The introduction of B cell maturation antigen (BCMA)-targeting chimeric antigen receptor (CAR) T cells revolutionized the treatment of relapsed/refractory multiple myeloma (RRMM). While long-term follow-up of CD19 CAR T cells demonstrated a connection between CAR T cell expansion and persistence with outcome, very little is known about persistence and effects on bystander cells of BCMA-targeting CAR T cells. Methods We analyzed 27 RRMM patients (pts) treated with Idecabtagene vicleucel (Ide-cel) at our center. Pts were grouped based on their remission evaluated 100 days after infusion into responders (partial response or better, n=16) or non-responders (progressive disease, n=11). Peripheral blood (pb) was drawn at day of leukapheresis, at day of infusion/after lymphodepletion (LD, d0) and at several time points following Ide-cel infusion (d7, d14, d30 and d100). In order to reflect a comprehensive picture of the longitudinal dynamics of CAR and non-CAR T cells, we subjected pb mononuclear cells to advanced flow cytometry. Additionally, we assessed the impact of cellular dynamics on the occurrence of cytokine release syndrome (CRS), tocilizumab application and the incidence of cytopenia. Results The median number of infused CAR T cells was 430x10 6 (range: 248.8 - 489.1x10 6) with one exception (25.7x10 6). There was no correlation between the median number of infused cells and in vivo expansion of CAR T cells after infusion as well as response or the development of CRS or cytopenia. Significant differences between responders and non-responders were already detected on day of leukapheresis: While non-responders showed similar numbers of CD4+ and CD8+ T cells, responders had significantly higher CD8+ T cell counts as compared to CD4+ T cells (p&amp;lt;0.05, Figure 1). Additionally, significantly reduced percentages of effector memory CD8+ T cells were detected in responders as compared to non-responders. The latter group showed significantly increased numbers of CD3+CD8+ T cells at the day of infusion indicating a less effective LD in non-responders. Following CAR T cell infusion, peak expansion occurred two weeks after infusion and was significantly higher in responders as compared to non-responders (p&amp;lt;0.001, Figure 2). CD3+ CAR T cell compartment predominantly comprised of CD8+ T cells regardless of response (median: 85%). Analysis of the distribution of CAR and non-CAR T cells after infusion revealed that the majority of CD4+ T cells in responders and non-responders were untransfected. Similar results were detected for CD8+ T cells in non-responders. However, in responders CD8+ CAR T cells approximated CD8+ non-CAR T cells with no significant differences on d7 and d14. With regard to T cell differentiation, almost the entire CD4+ T cells exhibited either a central memory or effector memory phenotype after CAR T cell infusion. Shortly after CAR T cell infusion, effector memory CD8+ T cells outbalanced all other CD8+ T cell subtypes. Most pts had no detectable CAR T cells 100 days after infusion, regardless of remission. Overall, 22 pts (81%) developed CRS shortly after CAR T cell infusion and 7 of these (32%) required tocilizumab treatment. Whereas percentages of CD3+ CAR T cells were significantly elevated in both CRS positive groups (p&amp;lt;0.05) shortly after infusion, absolute numbers were significantly increased only in CRS positive pts without tocilizumab. Analysis of blood counts following CAR T cell infusion revealed that cytopenias persisted longer in CRS pts treated with tocilizumab, which was also reflected by significantly increased numbers of days below thresholds for all cell lines (anemia, thrombocytopenia and leukopenia, p&amp;lt;0.01). Even though the occurrence of CRS and especially the treatment with tocilizumab affected blood cell composition, it did not correlate to response. Conclusion We demonstrate that BCMA-targeting CAR T cells only transiently persist in pb after infusion. Still, initial CAR T cell expansion, predominantly caused by effector memory CD8+ CAR T cells, was linked to response and CRS in RRMM pts. A correlation between CRS and tocilizumab administration with prolonged cytopenias following CAR T cell infusion was also observed. Our data provide first evidence that responders and non-responders can early be distinguished by differential cellular composition in CAR and non-CAR T cell compartments with distinct features already present at day of apheresis.

Rapid purification and multiparametric characterization of circulating small extracellular vesicles utilizing a label-free lab-on-a-chip device

Nano-scale extracellular vesicles are lipid-bilayer delimited particles that are naturally secreted by all cells and have emerged as valuable biomarkers for a wide range of diseases. Efficient isolation of small extracellular vesicles while maintaining yield and purity is crucial to harvest their potential in diagnostic, prognostic, and therapeutic applications. Most conventional methods of isolation suffer from significant shortcomings, including low purity or yield, long duration, need for large sample volumes, specialized equipment, trained personnel, and high costs. To address some of these challenges, our group has reported a novel insulator-based dielectrophoretic device for rapid isolation of small extracellular vesicles from biofluids and cell culture media based on their size and dielectric properties. In this study, we report a comprehensive characterization of small extracellular vesicles isolated from cancer-patients’ biofluids at a twofold enrichment using the device. The three-fold characterization that was performed using conventional flow cytometry, advanced imaging flow cytometry, and microRNA sequencing indicated high yield and purity of the isolated small extracellular vesicles. The device thus offers an efficient platform for rapid isolation while maintaining biomolecular integrity.

Platelet Function and Maturity and Related microRNA Expression in Whole Blood in Patients with ST-Segment Elevation Myocardial Infarction.

Reduced effect of antiplatelet therapy has been reported in patients with ST-segment elevation myocardial infarction (STEMI). MicroRNAs (miRs) may influence platelet function and maturity, and subsequently the effect of antiplatelet therapy. We aimed to explore the association between miR expression and platelet function and maturity in patients with acute STEMI and healthy individuals. We performed an observational study of STEMI patients admitted directly to primary percutaneous coronary intervention. Patients were treated with antiplatelet therapy according to guidelines. Within 24 hours after admission, blood samples were obtained to measure: the expression of 10 candidate miRs, platelet function markers using advanced flow cytometry, platelet aggregation, serum thromboxane B2, and platelet maturity markers. Furthermore, blood samples from healthy individuals were obtained to determine the normal variation. In total, 61 STEMI patients and 50 healthy individuals were included. STEMI patients had higher expression of miR-21-5p, miR-26b-5p, and miR-223-3p and lower expression of miR-150-5p, miR423-5p, and miR-1180-3p than healthy individuals. In STEMI patients, the expression of miR-26b-5p showed the most consistent association with platelet function (all p-values <0.05, Spearman's rho ranging from 0.27 to 0.41), while the expression of miR-150-5p and miR-223-3p showed negative associations with platelet function. No association between miR expression and platelet maturity markers was observed. In patients with STEMI, the expression of six miRs was significantly different from healthy individuals. The expression of miR-26b-5p may affect platelet function in acute STEMI patients and potentially influence the effect of antiplatelet therapy.

Sperm Quality Assessment in Stallions: How to Choose Relevant Assays to Answer Clinical Questions.

Stallion sperm analysis is indicated for infertility diagnosis, pre-sale expertise, production of fresh or frozen doses, and frozen straw quality control. Various collection methods are described, and numerous assays can be performed on semen. Determining an approach for each of these cases is challenging. This review aims to discuss how to obtain relevant clinical results, answering stallion owners' concerns. Semen can be collected with an artificial vagina on a phantom or a mare, by electro-ejaculation under anesthesia, or after pharmacological induction. The collection method influences the semen volume and concentration, while the total sperm number depends on the testicular production and collection frequency. In the seminal plasma, acidity, pro-oxidant activity, and some enzymes have repercussions for the semen quality and its conservation. Moreover, non-sperm cells of seminal plasma may impact semen conservation. Motility analysis remains a core parameter, as it is associated with fresh or frozen dose fertility. Computer-assisted motility analyzers have improved repeatability, but the reproducibility between laboratories depends on the settings that are used. Morphology analysis showing spermatozoa defects is useful to understand production and maturation abnormalities. Staining of the spermatozoa is used to evaluate viability, but recent advances in flow cytometry and in fluorochromes enable an evaluation of multiple intracellular parameters. Spermatozoa protein expression already has clinical applications, for example, as a fertility and freezing ability predictor. At present, stallion semen analysis ranges from macroscopic evaluation to assessing spermatozoa proteins. However, clinically, all these data may not be relevant, and the lack of standardization may complicate their interpretation.

The antimicrobial substance's content influences the membrane blocking mechanism in cross-flow filtration – Flow cytometric assessment of the membrane's activity

The study aims to investigate the antibacterial properties of carvacrol-loaded cellulose acetate membranes in cross-flow filtration using an advanced microbiological flow cytometry method and analyze the influence of carvacrol's presence on the pore-blocking mechanisms. The commercial cellulose acetate microfiltration membranes were impregnated with carvacrol using supercritical carbon dioxide as a solvent and transport medium. This technique enables active substance distribution throughout the whole polymer volume on the molecular level. Carvacrol loadings of up to 30 % were obtainable, whereby the membranes preserved their microstructure and functionality for loadings of up to 25 %. The protocol for flow cytometric assessment of the cell deposition on the membrane surface during the cross-flow filtration was developed. The results showed that the presence of carvacrol considerably decreased Staphylococcus aureus deposition on the membrane (from the order of magnitude 107/(cm2membrane surface·mLpermeate) to 105/(cm2membrane surface·mLpermeate)). The membranes containing 20 % and 25 % carvacrol showed the largest permeate flux and the smallest number of deposited bacteria. The largest fraction of dead bacteria (42 %) was detected in the membrane containing 25 % carvacrol. Mathematical modeling showed that the pore-blocking mechanism changed with the increase in carvacrol content, from the cake filtration phenomenon via the intermediate blocking towards standard and complete blocking mechanisms.

Molecular techniques drive cutting edge advancements in management of cutaneous T cell lymphoma.

Cutaneous 5T cell lymphoma (CTCL), characterized by malignant T cells infiltrating the skin with potential for dissemination, remains a challenging disease to diagnose and treat due to disease heterogeneity, treatment resistance, and lack of effective and standardized diagnostic and prognostic clinical tools. Currently, diagnosis of CTCL practically relies on clinical presentation, histopathology, and immunohistochemistry. These methods are collectively fraught with limitations in sensitivity and specificity. Fortunately, recent advances in flow cytometry, polymerase chain reaction, high throughput sequencing, and other molecular techniques have shown promise in improving diagnosis and treatment of CTCL. Examples of these advances include T cell receptor clonotyping via sequencing to detect CTCL earlier in the disease course and single-cell RNA sequencing to identify gene expression patterns that commonly drive CTCL pathogenesis. Experience with these techniques has afforded novel insights which may translate into enhanced diagnostic and therapeutic approaches for CTCL.

Phenotypic profiling of CD34+ cells by advanced flow cytometry improves diagnosis of juvenile myelomonocytic leukemia.

Diagnostic criteria for juvenile myelomonocytic leukemia (JMML) are currently well defined, however in some patients diagnosis still remains a challenge. Flow cytometry is a well established tool for diagnosis and follow-up of hematological malignancies, nevertheless it is not routinely used for JMML diagnosis. Herewith, we characterized the CD34+ hematopoietic precursor cells collected from 31 children with JMML using a combination of standardized EuroFlow antibody panels to assess the ability to discriminate JMML cells from normal/reactive bone marrow cell as controls (n=29) or from cells of children with other hematological diseases mimicking JMML (n=9). CD34+ precursors in JMML showed markedly reduced B-cell and erythroid-committed precursors compared to controls, whereas monocytic and CD7+ lymphoid precursors were significantly expanded. Moreover, aberrant immunophenotypes were consistently present in CD34+ precursors in JMML, while they were virtually absent in controls. Multivariate logistic regression analysis showed that combined assessment of the number of CD34+CD7+ lymphoid precursors and CD34+ aberrant precursors or erythroid precursors had a great potential in discriminating JMMLs versus controls. Importantly our scoring model allowed highly efficient discrimination of truly JMML versus patients with JMML-like diseases. In conclusion, we show for the first time that CD34+ precursors from JMML patients display a unique immunophenotypic profile which might contribute to a fast and accurate diagnosis of JMML worldwide by applying an easy to standardize single eight-color antibody combination.

Advanced flow cytometry for biomedical applications.

Flow cytometry (FC) is a versatile tool with excellent capabilities to detect and measure multiple characteristics of a population of cells or particles. Notable advancements in in vivo photoacoustic FC, coherent Raman FC, microfluidic FC, and so on, have been achieved in the last two decades, which endows FC with new functions and expands its applications in basic research and clinical practice. Advanced FC broadens the tools available to researchers to conduct research involving cancer detection, microbiology (COVID-19, HIV, bacteria, etc.), and nucleic acid analysis. This review presents an overall picture of advanced flow cytometers and provides not only a clear understanding of their mechanisms but also new insights into their practical applications. We identify the latest trends in this area and aim to raise awareness of advanced techniques of FC. We hope this review expands the applications of FC and accelerates its clinical translation.

Decoding of exosome heterogeneity for cancer theranostics.

Extracellular Vesicles (EVs) discovery establishes a new milestone in cancer research. It explains cancer biology in a new way. EVs classified some significant subclasses such as macrovesicle (originated from plasma membrane), apoptotic bodies (originated from the plasma membrane and endoplasmic reticulum), and exosomes (originated from endosomes).1 The theranostic signature of exosomes in cancer healing is impressive, but its heterogeneity leads most complicated domain of research. Therefore we comment on decoding exosome heterogeneity for future efficiency and effective cancer theranostics development. EVs are the cell-secreted one of the most important entities. The exosome attracted much attention due to its versatile uses. Overall, its participation in intercellular communication and its inner cargos (DNA, RNA, proteins, lipids and glycan)2 can reprogram the recipient cells. In addition, it also shows the healthy or pathologic complication status of the parent cells. In cancer, tumor and exosomes interlink a complex chapter of cancer biology. Exosomes play an important role in accelerated tumor growth, immune cell reprogramming,2 angiogenesis,3 extracellular matrix remodelling,4, 5 metastasis,4, 5 epithelial-mesenchymal transition (EMT),4, 5 organ-specific metastasis,5, 6 drug resistance and cancer stem cell development.3, 4 Clinically aspect, the exosome is the source of the cancer biomarkers.7-9 Exosome heterogeneity1 is evolving into a major conflict in cancer biomarker and therapeutic research.10 The exosome heterogeneity (Figure 1) is based on several facts such as origin, size, quantity, and internal molecular diversity.1 Exosome size distribution is the most complicated side of tumor-derived exosomes (TEXs). Scientific evidence shows that the same cells release differently-sized exosomes. This size-oriented biodiversity is reflected in using different isolation methods.9, 10 The tumor cells release more exosomes compared to healthy cells. The higher release of exosomes from tumor cells depends on several factors, such as drug and therapeutic exposures, hypoxic conditions, and senescence. The functional diversity of exosomes is related to the surface molecular expression and inner cargos of exosomes. All these complicated/complex parameters lead to a challenging ecosystem development of exosome-based cancer theragnostics (combination of biomarkers and therapeutics research). This scenario requires a solution to solve these challenges. Based on several innovative nano platform-based approaches transform these challenges into an opportunity for exosome researcher in a detailed exploration of exosome.11 Exosomes isolation, size and quantity related complication solved via microfluidic device, fluorescence antibody based exosome tracking, Nanoparticle tracking analysis (NTA) (above 60 nm exosome size challenging for its application),12, 13 magnetic and surface plasma resonance principle based single exosome screening,1 advanced sensitive flow cytometry (it is capable of the separation of exosome subpopulation) and electrophoresis chip. Molecular profiling of exosomes is one of the most critical tasks revealing the functional diversity of bioactive cargo molecules of exosomes. Droplet digital polymerase chain reaction (it is sensitive to the detection of rear mutations in tumor-derived exosomes),1 microfluidic technology combined with a chip system, and an electrochemical principle-based micro RNA profiling of exosomes explain the cancer complication of a new dimension.1 The downstream process of exosome profiling needs a muti-omics approach for proper molecular diversity to understand.1 The single cells exosome profiling approach combines with machine learning for more precision cancer marker development.14 All these technological advancements support constructing the next-generation promising cancer theranostic era based on exosomes.15, 16 Finally, decoding exosome heterogeneity opens a new door for exosome-based precision medicine17 and vaccines18 for cancer. We hope this article encourages large-scale EV research to explore a single exosome profiling approach and future exosome-based cancer precision medicine development. Not applicable. The authors declare no conflict of interest. There is no funding for this study.

Quantifying in vitrocomplement-dependent cytotoxicity (CDC) using advanced flow cytometry

Abstract Complement dependent cytotoxicity (CDC) is a key Fc mediated function of monoclonal antibody (mAb) therapeutics. mAb binding to antigen triggers a complex molecular cascade with sequential recruitment of serum proteins, eventuating in target cell lysis. Here we demonstrate CDC quantification in a streamlined, in vitro, advanced flow cytometry assay. Anti-CD20 mAbs were incubated with target cell lines in 96- or 384-well plates. Human serum (15%) was then added to induce CDC. Cells were labeled with iQue® Cell Membrane Integrity (R/Red) Dye to enable assessment of cell death using the iQue® Advanced Flow Cytometry Platform. Induction of CDC by anti-CD20 mAbs correlated with target cell CD20 expression, with most cell death observed with high-CD20 expressing Ramos cells. Maximal Ramos cell death induced by Rituximab and Truxima® was 68% and 70%, respectively. Comparatively, cell death of mid-CD20 expressing Raji cells was lower at 34% and 24%. No CDC was induced with CD20 negative Jurkat cells. Another anti-CD20-IgG1 mAb was compared against two isotype mutants: IgG1fut (non-fucosylated) and IgG1NQ (non-glycosylated). Their activity was profiled towards three previously described effector functions: antibody-dependent cellular cytotoxicity (ADCC); antibody-dependent cellular phagocytosis (ADCP) and CDC. CDC induction was comparable between the three mAbs, however their ADCC and ADCP activity differed. The IgG1NQ did not exert ADCP or ADCC activity, whilst the IgG1fut showed more ADCC and less ADCP than the native. These data exemplify the use of advanced flow cytometry to quantify Fc function of mAbs towards a range of effector mechanisms, highlighting the potential to profile libraries of novel therapeutics in minimal time.

A novel isolation method for spontaneously released extracellular vesicles from brain tissue and its implications for stress-driven brain pathology

BackgroundExtracellular vesicles (EVs), including small EVs (sEVs) such as exosomes, exhibit great potential for the diagnosis and treatment of brain disorders, representing a valuable tool for precision medicine. The latter demands high-quality human biospecimens, especially in complex disorders in which pathological and specimen heterogeneity, as well as diverse individual clinical profile, often complicate the development of precision therapeutic schemes and patient-tailored treatments. Thus, the collection and characterization of physiologically relevant sEVs are of the utmost importance. However, standard brain EV isolation approaches rely on tissue dissociation, which can contaminate EV fractions with intracellular vesicles.MethodsBased on multiscale analytical platforms such as cryo-EM, label-free proteomics, advanced flow cytometry, and ExoView analyses, we compared and characterized the EV fraction isolated with this novel method with a classical digestion-based EV isolation procedure. Moreover, EV biogenesis was pharmacologically manipulated with either GW4869 or picrotoxin to assess the validity of the spontaneous-release method, while the injection of labelled-EVs into the mouse brain further supported the integrity of the isolated vesicles.ResultsWe hereby present an efficient purification method that captures a sEV-enriched population spontaneously released by mouse and human brain tissue. In addition, we tested the significance of the release method under conditions where biogenesis/secretion of sEVs was pharmacologically manipulated, as well as under animals’ exposure to chronic stress, a clinically relevant precipitant of brain pathologies, such as depression and Alzheimer’s disease. Our findings show that the released method monitors the drug-evoked inhibition or enhancement of sEVs secretion while chronic stress induces the secretion of brain exosomes accompanied by memory loss and mood deficits suggesting a potential role of sEVs in the brain response to stress and related stress-driven brain pathology.ConclusionsOverall, the spontaneous release method of sEV yield may contribute to the characterization and biomarker profile of physiologically relevant brain-derived sEVs in brain function and pathology.7SKHktGsByupRjRZYNwNfMVideo

Advanced Flow Cytometry Using the SYTO-13 Dye for the Assessment of Platelet Reactivity and Maturity in Whole Blood

Newly produced immature platelets are larger, contain higher amounts of residual RNA, and are more reactive than mature platelets. Flow cytometry using the SYTO-13 dye is a method for the subdivision of immature platelets from mature platelets based on the labelling of intracellular platelet RNA, enabling the simultaneous investigation of the reactivity of each platelet population. This method provides detailed information on several aspects of platelet physiology using a combination of platelet surface markers and agonists. Currently, no standardized protocol exists across laboratories. Here, we describe a flow cytometry protocol in detail to investigate platelet reactivity and its relation to platelet maturity. We analyzed 20 healthy individuals with the protocol and compared the platelet subpopulation with the highest SYTO-13 labelling (in the first quintile, "SYTO-high") corresponding to the most immature platelets (highest RNA content) with the platelet subpopulation with the lowest SYTO-13 labelling (in the fifth quintile, "SYTO-low") corresponding to the mature platelets with the lowest RNA content. SYTO-high platelets had overall significantly increased platelet reactivity compared with that of SYTO-low platelets. The presented method may be a valuable research tool for the analysis of platelet reactivity and its relation to platelet maturity.

Assaying Mitochondrial Function by Multiparametric Flow Cytometry.

Flow cytometry has been a vital tool in cell biology for decades based on its versatile ability to detect and quantifiably measure both physical and chemical attributes of individual cells within a larger population. More recently, advances in flow cytometry have enabled nanoparticle detection. This is particularly applicable to mitochondria, which, as intracellular organelles have distinct subpopulations that can be evaluated based on differences in functional, physical, and chemical attributes, in a manner analogous to cells. This includes distinctions based on size, mitochondrial membrane potential (ΔΨm), chemical properties, and protein expression on the outer mitochondrial membrane in intact, functional organelles and internally in fixed samples. This method allows for multiparametric analysis of subpopulations of mitochondria, as well as collection for downstream analysis down to the level of a single organelle. The present protocol describes a framework for analysis and sorting mitochondria by flow cytometry, termed fluorescence activated mitochondrial sorting (FAMS), based on the separation of individual mitochondria belonging to subpopulations of interest using fluorescent dyes and antibody labeling.