Multicolour and lineage-specific interphase chromosome Flow-FISH: method development and clinical validation.

Flow cytometry can be applied in the detection of fluorescence in situ hybridisation (FISH) signals to efficiently analyse chromosomal aberrations. However, such interphase chromosome (IC) Flow-FISH protocols are currently limited to detecting a single colour. Furthermore, combining IC Flow-FISH with conventional multicolour flow cytometry is difficult because the DNA-denaturation step in FISH assay also disrupts cellular integrity and protein structures, precluding subsequent antigen-antibody binding and hindering concurrent labeling of surface antigens and FISH signals. We developed a working protocol for concurrent multicolour flow cytometry detection of nuclear IC FISH signals and cell surface markers. The protocol was validated by assaying sex chromosome content of blood cells, which was indicative of chimerism status in patients who had received sex-mismatched allogeneic haematopoietic stem cell transplants (allo-HSCT). The method was also adapted to detect trisomy 12 in chronic lymphocytic leukaemia (CLL) subjects. We first demonstrated the feasibility of this protocol in detecting multiple colours and concurrent nuclear and surface signals with high agreement. In clinical validation experiments, chimerism status was identified in clinical samples (n=56) using the optimised IC Flow-FISH method; the results tightly corresponded to those of conventional slide-based FISH (R2=0.9649 for XX cells and 0.9786 for XY cells). In samples from patients who received sex-mismatched allo-HSCT, individual chimeric statuses in different lineages could be clearly distinguished with high flexibility in gating strategies. Furthermore, in CLL samples with trisomy 12, this method could demonstrate that enriched trisomy 12 FISH signal was present in B cells rather than in T cells. Finally, by performing combined labelling of chromosome 12, X chromosome, and surface markers, we could detect rare residual recipient CLL cells with trisomy 12 after allo-HSCT. This adaptable protocol for multicolour and lineage-specific IC Flow-FISH advances the technique to allow for its potential application in various clinical contexts where conventional FISH assays are currently being utilised.

Specific cultivation-independent enumeration of viable cells in probiotic products using a combination of fluorescence in situ hybridization and flow cytometry.

This study introduces an optimized integration of flow cytometry and fluorescence in situ hybridization (Flow-FISH) as an approach for the specific enumeration of gram-positive bacteria in probiotic products, overcoming the limitations of conventional methods. The enhanced Flow-FISH technique synergizes the rapid and automated capabilities of flow cytometry with the high specificity of FISH, facilitating the differentiation of viable cells at the species level within probiotic blends. By analyzing lyophilized samples of Lacticaseibacillus rhamnosus, Lactiplantibacillus plantarum, and Bifidobacterium animalis subsp. lactis, and a commercial product, the study highlights the optimized Flow-FISH protocol's advantages, including reduced hybridization times to 1.5 h and elimination of centrifugation steps. Comparative evaluations with the widely accepted enumeration methods plate count and Live/Dead (L/D) staining were conducted. The study revealed that Flow-FISH produces higher viable cell counts than plate count, thereby challenging the traditional "gold standard" by highlighting its predisposition to underestimate actual viable cell numbers. Against L/D staining, Flow-FISH achieved comparable results, which, despite the different foundational premises of each technique, confirms the accuracy and reliability of our method. In conclusion, the optimized Flow-FISH protocol represents a significant leap forward in probiotic research and quality control. This method provides a rapid, robust, and highly specific alternative for the enumeration of probiotic bacteria, surpassing traditional methodologies. Its ability to enable a more detailed and reliable analysis of probiotic products paves the way for precise quality control and research insights, underscoring its potential to improve the field significantly.

Association between leukocyte telomere length and sex by quantile regression analysis

ABSTRACT Introduction: Telomere length (TL) is a biomarker of cellular proliferative history. In healthy individuals, leukocyte TL shortens with age and associates with the lifespan of men and women. However, most of studies had used linear regression models to address the association of the TL attrition, aging and sex. Methods: We evaluated the association between the TL, aging and sex in a cohort of 180 healthy subjects by quantile regression. The TL of nucleated blood cells was measured by fluorescent in situ hypridization (flow-FISH) in a cohort of 89 men, 81 women, and 10 umbilical cord samples. The results were validated by quantitative polymerase chain reaction (qPCR) and compared to a linear regression analysis. Results: By quantile regression, telomere dynamics slightly differed between sexes with aging: women had longer telomeres at birth and slower attrition rate than men until the sixth decade of life; after that, TL eroded faster and became shorter than that in men. These differences were not observed by linear regression analysis, as the overall telomere attrition rates in women and men were similar (42 pb per year, p < 0.0001 vs. 45 pb kb per year, p < 0.0001). Also, qPCR did not recapitulate flow-FISH findings, as the telomere dynamics by qPCR followed a linear model. Conclusion: The quantile regression analysis accurately reproduced a third-orderpolynomial TL attrition rate in both women and men, but it depended on the technique applied to measure TL. The Flow-FISH reproduced the expected telomere dynamics through life and, differently from the qPCR, was able to detect the subtle TL variations associated with sex and aging.

[Application of Flow Cytometry Combined Fluorescence in Situ Hybridization to Indentify the Lymphocyte Subtypies with Epstein-Barr Virus Infection].

OBJECTIVE: To establish the technique that take the advantages of flow cytometry combined fluorescence in situ hybridization (Flow-FISH) to identify the Epstein-Barr virus(EBV) infected lymphocyte subtypies in patients' peripheral blood sample. METHODS: Peripheral Blood monocyte from 9 patients with EBV infection enrolled at Children's Hospital in Chongqing Medical University were isolated by Ficoll-paque centrifugal separation. The expressions of EBER1, EBER2 in cell were detected by qRT-PCR. The surface markers of cell were detected by Flow cytometry after staining with their antibodies. The cell was treated Fix-Permeabilization Buffer before hybridization with fluorescent labeled probe at 37 ℃ overnight. The cell status, surface markers and targeted mRNA are detected by flow cytometry and fluorescence microscope. RESULTS: It was optimized that the Fix-Permeabilization Buffer and recipe with 0.2% Tween-20 were picked out as providing a good cell integrity and high resolution of surface markers. Hybridization with 20% formamide and 7% dextran sulfate at 37 ℃ overnight is the optimal hybridization condition as a good hybridization effect, a detectable cell integrity and a high resolution of cell markers under flow cytometry detection. Finally, upon the established Flow-FISH method, lymphocyte subpopulations of the EBV+ cells from cell lines and blood samples of patients were identified successfully. CONCLUSION: A Flow-FISH technology is established, which can be applied in the identification of EBV infected cell subtypes. This research provides a foundmental for its application in clinical test in EBV+ related proliferative diseases.

Dissecting Molecular Phenotypes Through FACS-Based Pooled CRISPR Screens.

Pooled CRISPR screens are emerging as a powerful tool to dissect regulatory networks, by assessing how a protein responds to genetic perturbations in a highly multiplexed manner. A large number of genes are perturbed in a cell population through genomic integration of one single-guide RNA (sgRNA) per cell. A subset of cells with the phenotype of interest can then be enriched through fluorescence-activated cell sorting (FACS). SgRNAs with altered abundance after phenotypic enrichment allow identification of genes that either promote or attenuate the investigated phenotype. Here we provide detailed guidelines on how to design and execute a pooled CRISPR screen to investigate molecular phenotypes. We describe how to generate a custom sgRNA library and how to perform a FACS-based screen using readouts such as intracellular antibody staining or Flow-FISH to assess phosphorylation levels or RNA abundance. Through the variety of available perturbation systems and readout options many different molecular and cellular phenotypes can now be tackled with pooled CRISPR screens.

Association between leukocyte telomere length and sex by quantile regression analysis.

INTRODUCTION: Telomere length (TL) is a biomarker of cellular proliferative history. In healthy individuals, leukocyte TL shortens with age and associates with the lifespan of men and women. However, most of studies had used linear regression models to address the association of the TL attrition, aging and sex. METHODS: We evaluated the association between the TL, aging and sex in a cohort of 180 healthy subjects by quantile regression. The TL of nucleated blood cells was measured by fluorescent in situ hypridization (flow-FISH) in a cohort of 89 men, 81 women, and 10 umbilical cord samples. The results were validated by quantitative polymerase chain reaction (qPCR) and compared to a linear regression analysis. RESULTS: By quantile regression, telomere dynamics slightly differed between sexes with aging: women had longer telomeres at birth and slower attrition rate than men until the sixth decade of life; after that, TL eroded faster and became shorter than that in men. These differences were not observed by linear regression analysis, as the overall telomere attrition rates in women and men were similar (42 pb per year, p < 0.0001 vs. 45 pb kb per year, p < 0.0001). Also, qPCR did not recapitulate flow-FISH findings, as the telomere dynamics by qPCR followed a linear model. CONCLUSION: The quantile regression analysis accurately reproduced a third-order polynomial TL attrition rate in both women and men, but it depended on the technique applied to measure TL. The Flow-FISH reproduced the expected telomere dynamics through life and, differently from the qPCR, was able to detect the subtle TL variations associated with sex and aging.

Application of Flow Cytometry Combined Fluorescence in Situ Hybridization to Indentify the Lymphocyte Subtypies with Epstein-Barr Virus Infection / 中国实验血液学杂志

OBJECTIVE@#To establish the technique that take the advantages of flow cytometry combined fluorescence in situ hybridization (Flow-FISH) to identify the Epstein-Barr virus(EBV) infected lymphocyte subtypies in patients' peripheral blood sample.@*METHODS@#Peripheral Blood monocyte from 9 patients with EBV infection enrolled at Children's Hospital in Chongqing Medical University were isolated by Ficoll-paque centrifugal separation. The expressions of EBER1, EBER2 in cell were detected by qRT-PCR. The surface markers of cell were detected by Flow cytometry after staining with their antibodies. The cell was treated Fix-Permeabilization Buffer before hybridization with fluorescent labeled probe at 37 ℃ overnight. The cell status, surface markers and targeted mRNA are detected by flow cytometry and fluorescence microscope.@*RESULTS@#It was optimized that the Fix-Permeabilization Buffer and recipe with 0.2% Tween-20 were picked out as providing a good cell integrity and high resolution of surface markers. Hybridization with 20% formamide and 7% dextran sulfate at 37 ℃ overnight is the optimal hybridization condition as a good hybridization effect, a detectable cell integrity and a high resolution of cell markers under flow cytometry detection. Finally, upon the established Flow-FISH method, lymphocyte subpopulations of the EBV+ cells from cell lines and blood samples of patients were identified successfully.@*CONCLUSION@#A Flow-FISH technology is established, which can be applied in the identification of EBV infected cell subtypes. This research provides a foundmental for its application in clinical test in EBV+ related proliferative diseases.

DNA-methylation-based telomere length estimator: comparisons with measurements from flow FISH and qPCR.

Telomere length (TL) is a marker of biological aging associated with several health outcomes. High throughput reproducible TL measurements are needed for large epidemiological studies. We compared the novel DNA methylation-based estimator (DNAmTL) with the high-throughput quantitative PCR (qPCR) and the highly accurate flow cytometry with fluorescent in situ hybridization (flow FISH) methods using blood samples from healthy adults. We used Pearson's correlation coefficient, Bland Altman plots and linear regression models for statistical analysis. Shorter DNAmTL was associated with older age, male sex, white race, and cytomegalovirus seropositivity (p<0.01 for all). DNAmTL was moderately correlated with qPCR TL (N=635, r=0.41, p < 0.0001) and flow FISH total lymphocyte TL (N=144, r=0.56, p < 0.0001). The agreements between flow FISH TL and DNAmTL or qPCR were acceptable but with wide limits of agreement. DNAmTL correctly classified >70% of TL categorized above or below the median, but the accuracy dropped with increasing TL categories. The ability of DNAmTL to detect associations with age and other TL-related factors in the absence of strong correlation with measured TL may indicate its capture of aspects of telomere maintenance mechanisms and not necessarily TL. The inaccuracy of DNAmTL prediction should be considered during data interpretation and across-study comparisons.

FISH Variants.

FISH has gained an irreplaceable place in microbiology because of its ability to detect and locate a microorganism, or a group of organisms, within complex samples. However, FISH role has evolved drastically in the last few decades and its value has been boosted by several advances in signal intensity, imaging acquisitions, automation, method robustness, and, thus, versatility. This has resulted in a range of FISH variants that gave researchers the ability to access a variety of other valuable information such as complex population composition, metabolic activity, gene detection/quantification, or subcellular location of genetic elements. In this chapter, we will review the more relevant FISH variants, their intended use, and how they address particular challenges of classical FISH.

Flow-FISH as a Tool for Studying Bacteria, Fungi and Viruses.

Many techniques are currently in use to study microbes. These can be aimed at detecting, identifying, and characterizing bacterial, fungal, and viral species. One technique that is suitable for high-throughput analysis is flow cytometry-based fluorescence in situ hybridization, or Flow-FISH. This technique employs (fluorescently labeled) probes directed against DNA or (m)RNA, for instance targeting a gene or microorganism of interest and provides information on a single-cell level. Furthermore, by combining Flow-FISH with antibody-based protein detection, proteins of interest can be measured simultaneously with genetic material. Additionally, depending on the type of Flow-FISH assay, Flow-FISH can also be multiplexed, allowing for the simultaneous measurement of multiple gene targets and/or microorganisms. Together, this allows for, e.g., single-cell gene expression analysis or identification of (sub)strains in mixed cultures. Flow-FISH has been used in mammalian cells but has also been extensively employed to study diverse microbial species. Here, the use of Flow-FISH for studying microorganisms is reviewed. Specifically, the detection of (intracellular) pathogens, studying microorganism biology and disease pathogenesis, and identification of bacterial, fungal, and viral strains in mixed cultures is discussed, with a particular focus on the viruses EBV, HIV-1, and SARS-CoV-2.

[A case report of familial dyskeratosis congenital. Case report].

Dyskeratosis congenita (DC) is a hereditary syndrome of bone marrow failure, which develops because of telomeres defects and combines with cancer predisposition. Its classical clinical features are skin pigmentation, nail dystrophy, oral leukoplakia (skin-mucosa triad). The goal is to describe the algorithm of diagnosis, clinical specificities of DC and specific treatment for cases of DC in one family. The present report includes descriptions of diagnosis and treatment of family members diagnosed for the first time as having a DC. The report shows an importance of all diagnostic stages: from a medical history and clinical picture to an application of modern high-tech diagnostic methods (flow-FISH, NGS). The report underlines an importance of diagnosis of all family members for excluding an asymptomatic form after a case of DC has been already detected in that family. A high frequency of a toxicity and secondary neoplasia makes it necessary to realize an individual approach at treatment of each patient with DC (the earliest start of androgen treatment, prompt decision of implementation of allogenic hematopoietic stem cell transplantation). The knowledge of pathogenesis, clinical features and principles of diagnosis and therapy of this disease is relevant to pediatricians and hematologists.

Combined Single-Cell Measurement of Cytokine mRNA and Protein in Immune Cells.

A key feature of immune cells, such as T cells, is their rapid responsiveness to activation. The response rate of T cells depends on the signal strength, and the type of signals they receive. Studying the underlying mechanisms that define responsiveness, however, is confounded by the fact that immune cells do not uniformly respond to activation. Tools that measure gene products on a single-cell level therefore provide additional insights in T cell biology. Here we describe flow cytometry-based fluorescence in situ hybridization (Flow-FISH), a high-throughput assay that allows for the simultaneous measurement of cytokine mRNA and protein levels of the gene(s) of interest by flow cytometry. We present several possible applications of Flow-FISH in human and murine T cells that-with minor adjustments-should also be applicable for other cell types.

Comparison of flow-FISH and MM-qPCR telomere length assessment techniques for the screening of telomeropathies.

Assessment of telomere length (TL) in peripheral blood leukocytes is part of the diagnostic algorithm applied to patients with acquired bone marrow failure syndromes (BMFSs) and dyskeratosis congenita (DKC). Monochrome multiplex-quantitative polymerase chain reaction (MM-qPCR) and fluorescence in situ hybridization (flow-FISH) are methodologies available for TL screening. Dependent on TL expressed in relation to percentiles of healthy controls, further genetic testing for inherited mutations in telomere maintenance genes is recommended. However, the correct threshold to trigger this genetic workup is still under debate. Here, we prospectively compared MM-qPCR and flow-FISH regarding their capacity for accurate identification of DKC patients. All patients (n = 105) underwent genetic testing by next-generation sequencing and in 16 patients, mutations in DKC-relevant genes were identified. Whole leukocyte TL of patients measured by MM-qPCR was found to be moderately correlated with lymphocyte TL measured by flow-FISH (r² = 0.34; P < 0.0001). The sensitivity of both methods was high, but the specificity of MM-qPCR (29%) was significantly lower compared with flow-FISH (58%). These results suggest that MM-qPCR of peripheral blood cells is inferior to flow-FISH for clinical routine screening for suspected DKC in adult patients with BMFS due to lower specificity and a higher rate of false-positive results.

A simple procedure for detecting Dekkera bruxellensis in wine environment by RNA-FISH using a novel probe.

Dekkera bruxellensis, considered the major microbial contaminant in wine production, produces 4-ethylphenol, a cause of unpleasant odors. Thus, identification of this yeast before wine spoilage is crucial. Although challenging, it could be achieved using a simple technique: RNA-FISH. To reach it is necessary to design probes that allow specific detection/identification of D. bruxellensis among the wine microorganisms and in the wine environment and, if possible, using low formamide concentrations. Therefore, this study was focused on: a) designing a DNA-FISH probe to identify D. bruxellensis that matches these requirements and b) determining the applicability of the RNA-FISH procedure after the end of the alcoholic fermentation and in wine. A novel DNA-FISH D. bruxellensis probe with good performance and specificity was designed. The application of this probe using an in-suspension RNA-FISH protocol (applying only 5% of formamide) allowed the early detection/identification of D. bruxellensis at low cell densities (5 × 102 cell/mL). This was possible by flow cytometry independently of the growth stage of the target cells, both at the end of the alcoholic fermentation and in wine even in the presence of high S. cerevisiae cell densities. Thus, this study aims to contribute to facilitate the identification of D. bruxellensis before wine spoilage occurs, preventing economic losses to the wine industry.

Telomere Length Calibration from qPCR Measurement: Limitations of Current Method.

Telomere length (TL) comparisons from different methods are challenging due to differences in laboratory techniques and data configuration. This study aimed to assess the validity of converting the quantitative polymerase chain reaction (qPCR) telomere/single copy gene (T/S) ratio to TL in kilobases (kb). We developed a linear regression equation to predict TL from qPCR T/S using flow cytometry with fluorescence in situ hybridization (flow FISH) TL data from 181 healthy donors (age range = 19⁻53) from the National Marrow Donor Program (NMDP) biorepository. TL measurements by qPCR and flow FISH were modestly correlated (R² = 0.56, p < 0.0001). In Bland-Altman analyses, individuals with the shortest (≤10th percentile) or longest (≥90th) flow FISH TL had an over- or under-estimated qPCR TL (bias = 0.89 and -0.77 kb, respectively). Comparisons of calculated TL from the NMDP samples and 1810 age- and sex-matched individuals from the National Health and Nutrition Examination Survey showed significant differences (median = 7.1 versus 5.8 kb, respectively, p < 0.0001). Differences in annual TL attrition were also noted (31 versus 13 bp/year, respectively, p = 0.02). Our results demonstrate that TL calculated in kb from qPCR T/S may yield biased estimates for individuals with the shortest or longest TL, those often of high clinical interest. We also showed that calculated TL in kb from qPCR data are not comparable across populations and therefore are not necessarily useful.

Long-term effects of superoxide and DNA repair on lizard telomeres.

Telomeres are the non-coding protein-nucleotide "caps" at chromosome ends that contribute to chromosomal stability by protecting the coding parts of the linear DNA from shortening at cell division, and from erosion by reactive molecules. Recently, there has been some controversy between molecular and cell biologists, on the one hand, and evolutionary ecologists on the other, regarding whether reactive molecules erode telomeres during oxidative stress. Many studies of biochemistry and medicine have verified these relationships in cell culture, but other researchers have failed to find such effects in free-living vertebrates. Here, we use a novel approach to measure free radicals (superoxide), mitochondrial "content" (a combined measure of mitochondrial number and size in cells), telomere length and DNA damage at two primary time points during the mating season of an annual lizard species (Ctenophorus pictus). Superoxide levels early in the mating season vary widely and elevated levels predict shorter telomeres both at that time as well as several months later. These effects are likely driven by mitochondrial content, which significantly impacts late season superoxide (cells with more mitochondria have more superoxide), but superoxide effects on telomeres are counteracted by DNA repair as revealed by 8-hydroxy-2'-deoxyguanosine assays. We conclude that reactive oxygen species and DNA repair are fundamental for both short- and long-term regulation of lizard telomere length with pronounced effects of early season cellular stress detectable on telomere length near lizard death.

Correlation of Leukocyte Telomere Length Measurement Methods in Patients with Dyskeratosis Congenita and in Their Unaffected Relatives.

Several methods have been employed to measure telomere length (TL) in human studies. It has been difficult to directly compare the results from these studies because of differences in the laboratory techniques and output parameters. We compared TL measurements (TLMs) by the three most commonly used methods, quantitative polymerase chain reaction (qPCR), flow cytometry with fluorescence in situ hybridization (flow FISH) and Southern blot, in a cohort of patients with the telomere biology disorder dyskeratosis congenita (DC) and in their unaffected relatives (controls). We observed a strong correlation between the Southern blot average TL and the flow FISH total lymphocyte TL in both the DC patients and their unaffected relatives (R² of 0.68 and 0.73, respectively). The correlation between the qPCR average TL and that of the Southern blot method was modest (R² of 0.54 in DC patients and of 0.43 in unaffected relatives). Similar results were noted when comparing the qPCR average TL and the flow FISH total lymphocyte TL (R² of 0.49 in DC patients and of 0.42 in unaffected relatives). In conclusion, the strengths of the correlations between the three widely used TL assays (qPCR, flow FISH, and Southern blot) were significantly different. Careful consideration is warranted when selecting the method of TL measurement for research and for clinical studies.

Assessment of Telomere Length, Phenotype, and DNA Content.

Telomere sequences at the end of chromosomes control somatic cell division; therefore, telomere length in a given cell population provides information about its replication potential. This unit describes a method for flow cytometric measurement of telomere length in subpopulations using fluorescence in situ hybridization of fluorescently-labeled probes (Flow-FISH) without prior cell separation. After cells are stained for surface immunofluorescence, antigen-antibody complexes are covalently cross-linked onto cell membranes before FISH with a telomere-specific probe. Cells with long telomeres are included as internal standards. Addition of a DNA dye permits exclusion of proliferating cells during data analysis. DNA ploidy measurements of cells of interest and internal standard are performed on separate aliquots in parallel to Flow-FISH. Telomere fluorescence of G0/1 cells of subpopulations and internal standards obtained from Flow-FISH are normalized for DNA ploidy, and telomere length in subsets of interest is expressed as a fraction of the internal standard telomere length. © 2017 by John Wiley & Sons, Inc.