Imaging Flow Cytometry Method Research Articles

“Double Hit” Myeloma: Detection By FISH and Flow Cytometry in Circulating Myeloma Plasma Cells

“Double hit” myeloma is defined as the presence of 2 of the known high-risk genetic abnormalities in myeloma. This includes amp(1q), del(17p) and IGH translocations involving chromosomes 4, 16 and 20. Given the prognostic significance of these abnormalities, with poor overall survival, identification is crucial. Testing is traditionally by fluorescence in situ hybridization (FISH) on a single-site bone marrow sample at baseline or at specific disease timepoints. We explored whether amp(1q) and del(17p) “double hit” abnormalities could be detected in circulating myeloma plasma cells (CMPC). Aims: We assessed our novel imaging flow cytometry method that simultaneously integrates immunophenotyping and FISH. Called “immuno-flowFISH” this single cell method combines antigenic detection of neoplastic plasma cells, FISH for DNA loci of interest and imaging flow cytometry for automated assessment. Methods: Blood from 16 myeloma patients, at diagnosis or on therapy was studied. Following density gradient centrifugation, cells were incubated with CD38/CD138/CD319-AF647-conjugated antibodies to identify plasma cells, CD19-BV480 (to differentiate normal from neoplastic plasma cells) and CD3-BV510 (T-lymphocyte control). After fixation and cell membrane permeabilization, DNA was denatured (78 oC for 5 minutes). The cells were then incubated with FISH probes to 1q21 ( CKS1B) and 17p13 loci, and to the centromere of chromosome 17 (C17), and hybridized (24 hours for 37 oC). The nuclei were stained with SYTOX AADvanced and 50,000 to 500,000 cells acquired on the Amnis ® ImageStream ®X Mk II imaging flow cytometer (Cytek Biosciences). Digital images and quantitative data derived from computational algorithms (IDEAS software) were used to assess FISH signals overlying the counterstained nuclei. Results: A mean of 264,937 cells were acquired and 110,696 analysed. Cells with a neoplastic plasma cell phenotype (CD38/CD138/CD319-positive, CD19-negative) were present in all samples. The number of these CMPC ranged from 7 to 797, amounting to 0.011- 0.546% of all cells. FISH abnormalities were present in CMPC of 10 of the 16 cases (see Table). Isolated amp(1q) (three CKS1B FISH signals) was present in 4 cases, and isolated del(17p), (one 17p13 and two C17 FISH spots) in CMPC in 3 cases. “Double hit” abnormalities, with both amp(1q) and del(17p), were present in another 3 cases, with the following cytogenetic patterns (illustrated in the Figure): 1. amp(1q) and del(17p) in different cells (Case 8); 2. amp(1q) and del(17p) in the same cell (35.7% of CMPC), in addition to isolated amp(1q) (28.6% of CMPC) (Case 9); 3. amp(1q) and del(17p) in the same cells only (Case 10). All circulating myeloma plasma cells had diploid signals for the chromosome C17 centromeric probe and the CD3-positive T-cells diploid signals for all probes. Conclusion: Incorporating antigenic identification and FISH, with imaging flow cytometric analysis, can detect “double hit” amp(1q) and del(17p) in circulating myeloma plasma cells at high sensitivity (10 -5). This novel approach allowed sub-clonal interrogation, with amp(1q) and del(17p) “double hits” detected in the same (Cases 9, 10) and separate CMPC (Case 8). It could also identify sub-clonal acquisition of secondary del(17p) (Case 9), which may be a consequence of disease evolution or intra-tumoral clonal heterogeneity. FISH analysis of circulating plasma cells using a flow cytometric platform is achievable to low levels of disease through assessment of thousands of cells. It holds significant promise for sensitive holistic real-time blood surveillance to detect high-risk and sub-clonal “double hit” alterations at the single cell level. Blood monitoring for these genetic aberrations overcomes the limitations of single site marrow analysis and may provide insights into the disease trajectory, clonal evolution and prognosis.

Label‐Free Imaging Flow Cytometry for Cell Classification Based on Multiple Interferometric Projections Using Deep Learning

A new label‐free imaging flow cytometry method for noninvasive and automated biological cell classification is presented. Each cell is rolled during flow, and its off‐axis holograms from multiple viewpoints are acquired. Using the reconstructed quantitative phase profiles of the cell projections, highly discriminating features, enabling cell detection, classification, and differentiation, are extracted via a modified ResNet‐18 deep convolutional neural network architecture. The model is first validated by classifying metastatic breast carcinoma cells (MCF‐7) and normal human mammary epithelial cells (MCF‐10A). An increase in classification accuracy by 1% is achieved when processing five interferometric projections versus processing a single interferometric projection. This model is further tested on four types of white blood cells and exhibits an accuracy increase of 5% when processing 12 interferometric projections versus processing a single interferometric projection. This approach is shown to be superior to that of using conventional 2D‐rotation augmentation, and can be used to decrease substantially the number of cell examples needed for training the classification model without impairing the results. This novel concept has great potential to be incorporated into label‐free imaging flow cytometry and improve cell classification, and be used to detect various types of medical conditions and diseases.

Development of an Imaging Flow Cytometry Method for Fungal Cytological Profiling and Its Potential Application in Antifungal Drug Development.

Automated imaging techniques have been in increasing demand for the more advanced analysis and efficient characterization of cellular phenotypes. The success of the image-based profiling method hinges on assays that can rapidly and simultaneously capture a wide range of phenotypic features. We have developed an automated image acquisition method for fungal cytological profiling (FCP) using an imaging flow cytometer that can objectively measure over 250 features of a single fungal cell. Fungal cells were labeled with calcofluor white and FM4-64FX, which bind to the cell wall and lipophilic membrane, respectively. Images of single cells were analyzed using IDEAS® software. We first acquired FCPs of fungal cells treated with fluconazole, amphotericin B, and caspofungin, each with a distinct mode of action, to establish FCP databases of profiles associated with specific antifungal treatment. Once fully established, we investigated the potential application of this technique as a screening methodology to identify compounds with novel antifungal activity against Candida albicans and Cryptococcus neoformans. Altogether, we have developed a rapid, powerful, and novel image-profiling method for the phenotypic characterization of fungal cells, also with potential applications in antifungal drug development.

An Imaging Flow Cytometry Method for Assessment of Human Natural Killer Cells

An Imaging Flow Cytometry Method for Assessment of Human Natural Killer Cells

FISH By Imaging Flow Cytometry in CLL for Diagnosis and MRD Assessment

▪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. DisclosuresNo relevant conflicts of interest to declare.

Multi-probe FISH Analysis of Immunophenotyped Chronic Lymphocytic Leukemia by Imaging Flow Cytometry.

Imaging flow cytometry is an automated method that enables cells and fluorescent signals to be visualized and quantified. Here, we describe a new imaging flow cytometry method whereby fluorescence in situ hybridization (FISH) is integrated with cell phenotyping. The method, called "immuno-flowFISH," provides an exciting new dimension for the analysis of genomic changes in cytological samples (e.g., blood, bone marrow). Cells are analyzed in suspension without any requirement for prior cell isolation or separation. Multiple antibodies and FISH probes, each with a unique fluorophore, can be added and many thousands of cells analyzed. Specific cell populations are identified by their antigenic profile and then analyzed for the presence of chromosomal defects. Immuno-flowFISH was applied to the assessment of chronic lymphocytic leukemia (CLL), a mature B-cell neoplasm where chromosomal abnormalities predict prognosis and treatment requirements. This integrated immunophenotyping and multi-probe FISH strategy could detect both structural and numerical chromosomal changes involving chromosomes 12 and 17 in CLL cells. Given that many thousands of cells were analyzed and the leukemic cells were positively identified by their immunophenotype, this multi-probe method adds precision to the cytogenomic analysis of CLL. © 2021 Wiley Periodicals LLC.

An Imaging Flow Cytometry Method to Measure Citrullination of H4 Histone as a Read-out for Neutrophil Extracellular Traps Formation.

The formation of neutrophil extracellular traps (NETs) is thought to play a critical role in infections and propagating sterile inflammation. Histone citrullination is an essential and early step in NETs formation, detectable prior to the formation of the hallmark extracellular DNA-scaffolded strands. In addition to the classical microscopy method, new technologies are being developed for studies of NETs and their detection, both for research and clinical purposes. Classical microscopy studies of NETs are subjective, low throughput and semi-quantitative, and limited in their ability to capture the early steps. We have developed this novel Imaging Flow Cytometry (IFC) method that specifically identifies and quantifies citrullination of histone H4 as a NETs marker and its relationship with other alterations at nuclear and cellular level. These include nuclear decondensation and super-condensation, multi-lobulated nuclei versus 1-lobe nuclei and cell membrane damage. NETs markers can be quantified following variable periods of treatment with NETs inducers, prior to the formation of the specific extracellular DNA-scaffolded strands. Because these high throughput image-based cell analysis features can be performed with statistical rigor, this protocol is suited for both experimental and clinical applications as well as clinical evaluations of NETosis as a biomarker.

Detection of Del(17p) in Hematological Malignancies By Imaging Flow Cytometry

Del(17p) in chronic lymphocytic leukemia (CLL) and plasma cell myeloma has a unique genomic profile leading to refractoriness to conventional therapies and poor overall survival. Detection is generally by fluorescence in situ hybridization (FISH) on a slide with analysis of up to 200 nuclei, not necessarily all of being neoplastic cells. The small cell sample analyzed, and high threshold for a positive result (>5% positive cells) makes FISH a low precision test. This means false negative results are inevitable, especially if del(17p) is only in a minor clone, potentially leading to suboptimal treatment. To address this, we developed an automated FISH method with analysis on an imaging flow cytometer, an instrument with the functionality of a standard flow cytometer which generates high-resolution digital images of each cell. By combining immunophenotyping with FISH, on whole cells in a single test, we can detect chromosomal defects in cells with a specific phenotype. Aims: Our aim was to determine the capability of this automated integrated immunophenotyping-FISH imaging flow cytometry method to detect del(17p) in CLL and myeloma, the two commonest hematological malignancies, and in which this genetic defect encodes poor prognosis. We hypothesized that it would be more sensitive and specific than standard FISH. Methods: Bone marrow and/or blood samples in EDTA anticoagulant from 19 cases of CLL or myeloma, at diagnosis or on therapy, were studied. After red cell lysis, cells were incubated with fluorochrome-conjugated antibodies to CD3, CD5, CD19, CD38, and CD138 antigens (fluorochromes: BV480, BV605, AF647). Following fixation, cell membranes were permeabilized and double-stranded DNA denatured (78oC for 5 mins). FISH probes to the centromere of chromosome 17 (CEP17, Spectrum Green) and 17p12 locus (Orange-Red) were added and hybridized for 24 hours at 37oC. Nuclei were stained with SYTOX AADvanced. Data for up to 200,000 cells was collected on the Amnis® ImageStream®XMk II imaging flow cytometer. Digital images (x60) and quantitative data derived from computational algorithms (IDEAS software) were used to assess FISH signals overlying the nuclei of CD5/CD19-positive CLL cells or CD38/CD138-positive plasma cells for each probe. Digital images and quantitative data were assessed for FISH signals within immunophenotyped cells. Results: Between 10,000 and 200,000 (mean 60,000) cells were analyzed per sample. The FISH signals were seen on the digital images and confirmed by quantitative mean channel fluorescence intensity of the probes. There were 12 CLL cases with one 17p FISH signal in the CD5/CD19-positive population, with the number of del(17p) CLL cells ranging from 2 - 35% (or 0.4 - 23% of all cells analyzed) (Fig 1). This amounted to between 270 and 35,441 cells in the analysed sample with del(17p). The lowest del(17p) burden was in a patient on cytoreductive therapy. All CLL cells had normal diploid spots for the control CEP17 probe, and the CD3/CD5-positive T cells had dual signals for both CEP17 and 17p12 probes. There were 5 myeloma cases with 1 FISH signal for 17p overlying the nucleus of the CD38/CD138-positive plasma cells (Fig 2). In these cases, 52-90% of cells (15,600-18,000 cells) had a plasma cell phenotype and 13-19% of these (or 2,340-2,964 CD38/CD138-positive cells) showed del(17p). This represented 1-5% of all cells in the sample. All gated plasma cells had 2 FISH spots for CEP17. Conclusion: This imaging flow cytometry method that integrates FISH with immunophenotyping could detect del(17p) in CLL and myeloma with a lowest limit of detection of 0.4% and 1% respectively. The high sensitivity was achieved as many thousands of cells were analyzed, and 17p was only assessed in gated cells with the phenotype of interest (i.e. CD5/CD19 or CD38/CD138). This method, that includes positive cell identification by phenotype, precludes the need for prior cell sorting or purification. Imaging flow cytometry for del(17p) by "immuno-flowFISH" brings a new dimension to FISH analysis at diagnosis and for disease monitoring. Its high precision and specificity will enable detection of del(17p), even when only present in minor sub-clones, for therapeutic decision making and prognostic stratification. This technique has a real place in clinical assessment of del(17p) in CLL and myeloma and could be applied for other significant chromosomal defects of therapeutic and prognostic significance. Figure 1 Disclosures Augustson: Roche: Other: Support of parent study and funding of editorial support. Cheah:Celgene, F. Hoffmann-La Roche, Abbvie, MSD: Research Funding; Celgene, F. Hoffmann-La Roche, MSD, Janssen, Gilead, Ascentage Pharma, Acerta, Loxo Oncology, TG therapeutics: Honoraria.

A novel imaging flow cytometry method for the detection of histone H4 acetylation in myeloid cells

The histone deacetylase inhibitor (HDACI) valproic acid has been shown to inhibit the growth of multiple paediatric tumour types and is well tolerated in a childhood cancer setting. The current study was designed to develop a novel imaging flow cytometry method for the detection of histone H4 acetylation in white blood cells obtained from childhood cancer patients treated with valproic acid. HL-60 cells and whole blood samples from healthy volunteers were incubated with valproic acid (0-8mM) for 0-24hours, with additional blood samples collected from ependymoma patients receiving valproic acid on the SIOP Ependymoma II clinical trial. An imaging flow cytometry method was developed using an ImageStream®χ flow cytometer, collecting 100000 images per sample following excitation of PE tagged acH4 antibody and DAPI. The mean percentage of acH4-positive cells increased to a greater extent than increases in mean and median fluorescence intensity following incubation with valproic acid. Comparable results were observed for in vitro and ex vivo experiments, and the assay was shown to be appropriate for clinical sample analysis. Myeloid cells exhibited a smaller proportion of acH4-positive cells than the lymphoid population, but a greater fold increase above basal levels. The percentage of acH4-positive myeloid cells has the potential to be used as a robust pharmacodynamic biomarker for the measurement of acH4 for HDACIs. The developed assay is now being utilised in a clinical trial involving the treatment of childhood ependymoma patients with valproic acid.

Abstract 2583: Imaging flow cytometry assay development and validation for the detection of histone H4 acetylation in white blood cells

Abstract Introduction: Histone deacetylases have been identified as oncogenes in several cancer types, providing an attractive target for anticancer treatment. In this respect, the histone deacetylase inhibitor valproic acid has been shown to inhibit the growth of multiple paediatric tumor types and is well tolerated in children with refractory solid or CNS tumors. Objective: The aim of the current study was to develop and validate a novel imaging flow cytometry method for the detection of histone H4 acetylation in lymphoid and myeloid cell populations, and to assess the applicability of the method to a clinical trial setting. Method: HL-60 cells and whole blood samples from healthy volunteers were incubated with valproic acid (0.5-8 mM) for 0.5-24 hours, followed by RBC lysis for the whole blood samples and fixed with cold methanol. Additional blood samples were collected from patients with ependymoma who were receiving valproic acid as part of the SIOP Ependymoma II clinical trial. An imaging flow cytometry method was developed using an ImageStreamχ flow cytometer, collecting WBCs with excitation of PE conjugated acH4 antibody and DAPI. Data were collected using Inspire™ software and further analysed by Ideas™ software 6.2. Both in vitro and ex vivo experiments were repeated on at least 3 occasions. Result: In the HL-60 cell line the mean percentage of acH4 positive cells was 1.98% in the vehicle control sample, increasing to 10.9-77.9% when treated with 0.5-8 mM valproic acid for 6 hours, with percentages of 8.7-49.0% observed following incubation with 4 mM valproic acid for 0.5-24 hours. Comparable data were generated in lymphoid and myeloid WBC populations following ex vivo incubation of whole blood samples with valproic acid. Increases in the percentage of acH4 positive cells were observed in samples collected at 4 hours post-administration in patients receiving valproic acid as compared to pre-treatment samples. Myeloid cells appeared to have a smaller proportion of acH4 positive cells than observed in the lymphoid population but a greater fold increase above basal levels. Conclusion: A new assay for detection of histone H4 acetylation in WBCs by imaging flow cytometry has been developed and optimised. The assay showed increases in acH4 positivity in both in vitro and ex vivo experiments following exposure to valproic acid. The method can be used for the measurement of acH4 as a pharmacodynamic biomarker for histone deacetylase inhibitors in drug development and monitoring of drug efficacy in clinical trials. Citation Format: Suriyon Uitrakul, Gareth James Veal, Claire Hutton, David Jamieson. Imaging flow cytometry assay development and validation for the detection of histone H4 acetylation in white blood cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2583.

An imaging flow cytometry method to assess ricin trafficking in A549 human lung epithelial cells.

The endocytosis and trafficking of ricin in mammalian cells is an important area of research for those producing ricin anti-toxins and other ricin therapeutics. Ricin trafficking is usually observed by fluorescence microscopy techniques. This gives good resolution and leads to a detailed understanding of the internal movement of ricin within cells. However, microscopy techniques are often hampered by complex analysis and quantification techniques, and the inability to look at ricin trafficking in large populations of cells. In these studies we have directly labelled ricin and assessed if its trafficking can be observed using Imaging Flow Cytometry (IFC) both to the cytoplasmic region of cells and specifically to the Golgi apparatus. Using IDEAS® data analysis software the specific fluorescence location of the ricin within the cells was analysed. Then, using cytoplasmic masking techniques to quantify the number of cells with endocytosed cytoplasmic ricin or cells with Golgi-associated ricin, kinetic endocytosis curves were generated. Here we present, to the authors' knowledge, the first example of using imaging flow cytometry for evaluating the subcellular transport of protein cargo, using the trafficking of ricin toxin in lung cells as a model.

Foundations of identifying individual chromosomes by imaging flow cytometry with applications in radiation biodosimetry

Biodosimetry is an important tool for triage in the case of large-scale radiological or nuclear emergencies, but traditional microscope-based methods can be tedious and prone to scorer fatigue. While the dicentric chromosome assay (DCA) has been adapted for use in triage situations, it is still time-consuming to create and score slides. Recent adaptations of traditional biodosimetry assays to imaging flow cytometry (IFC) methods have dramatically increased throughput. Additionally, recent improvements in image analysis algorithms in the IFC software have resulted in improved specificity for spot counting of small events. In the IFC method for the dicentric chromosome analysis (FDCA), lymphocytes isolated from whole blood samples are cultured with PHA and Colcemid. After incubation, lymphocytes are treated with a hypotonic solution and chromosomes are isolated in suspension, labelled with a centromere marker and stained for DNA content with DRAQ5. Stained individual chromosomes are analyzed on the ImageStream®X (EMD-Millipore, Billerica, MA) and mono- and dicentric chromosome populations are identified and enumerated using advanced image processing techniques. Both the preparation of the isolated chromosome suspensions as well as the image analysis methods were fine-tuned in order to optimize the FDCA. In this paper we describe the method to identify and score centromeres in individual chromosomes by IFC and show that the FDCA method may further improve throughput for triage biodosimetry in the case of large-scale radiological or nuclear emergencies.

Imagestream detection and characterisation of circulating tumour cells – A liquid biopsy for hepatocellular carcinoma?

The lack of progress in developing and delivering new therapies for hepatocellular carcinoma (HCC) is in part attributed to the risk related avoidance of tumour biopsy at diagnosis. Circulating tumour cells (CTCs) are a potential source of tumour tissue that could aid biological or biomarker research, treatment stratification and monitoring. An imaging flow cytometry method, using immunofluorescence of cytokeratin, EpCAM, AFP, glypican-3 and DNA-PK together with analysis of size, morphology and DNA content, for detection of HCC CTCs was developed and applied to 69 patient and 31 control samples. The presence of CTCs as a prognostic indicator was assessed in multivariate analyses encompassing recognised prognostic parameters. Between 1 and 1642 CTCs were detected in blood samples from 45/69 HCC patients compared to 0/31 controls. CTCs positive for the epithelial markers cytokeratin and EpCAM were detected in 29% and 18% of patients respectively, while an additional 28% of patients had CTCs negative for all markers other than size and evidence of hyperploidy. CTC number correlated significantly with tumour size and portal vein thrombosis (PVT). The median survival of patients with >1 CTC was 7.5months versus >34months for patients with <1 CTC (p<0.001, log-rank), with significance retained in a multivariate analysis (HR 2.34, 95% CI 1.005-5.425, p=0.049) including tumour size and PVT. The use of multiple parameters enhanced HCC CTC detection sensitivity, revealing biological associations and predictive biomarker potential that may be able to guide stratified medicine decisions and future research. Characteristics of tumour tissues can be used to predict outcomes for individual patients with cancer, as well as help to choose their best treatment. Biopsy of liver cancers carries risks, however, and is usually avoided. Some cancer cells enter the blood, and although they are very rare, we have developed a method of finding and characterising them in patients with liver cancer, which we hope will provide a low risk means of guiding treatment.

Validation of the Cytokinesis-block Micronucleus Assay Using Imaging Flow Cytometry for High Throughput Radiation Biodosimetry.

The cytokinesis-block micronucleus assay can be employed in triage radiation biodosimetry to determine the dose of radiation to an exposed individual by quantifying the frequency of micronuclei in binucleated lymphocyte cells. Partially automated analysis of the assay has been applied to traditional microscope-based methods, and most recently, the assay has been adapted to an automated imaging flow cytometry method. This method is able to automatically score a larger number of binucleated cells than are typically scored by microscopy. Whole blood samples were irradiated, divided into 2 mL and 200 μL aliquots, cultured for 48 h and 72 h, and processed to generate calibration curves from 0-4 Gy. To validate the method for use in radiation biodosimetry, nine separate whole blood samples were then irradiated to known doses, blinded, and processed. Results indicate that dose estimations can be determined to within ±0.5 Gy of the delivered dose after only 48 h of culture time with an initial blood volume of 200 μL. By performing the cytokinesis-block micronucleus assay using imaging flow cytometry, a significant reduction in the culture time and volume requirements is possible, which greatly increases the applicability of the assay in high throughput triage radiation biodosimetry.

Quantifying autophagosomes and autolysosomes in cells using imaging flow cytometry.

Autophagy dysregulation has been implicated in numerous diseases and many therapeutic agents are known to modulate this pathway. Therefore, the ability to accurately monitor autophagy is critical to understanding its role in the pathogenesis and treatment of many diseases. Recently an imaging flow cytometry method measuring colocalization of microtubule associated protein 1B light chain 3 (LC3) and lysosomal signals via Bright Detail Similarity (BDS) was proposed which enabled evaluation of autophagic processing. However, since BDS only evaluates colocalization of LC3 and lysosomal signals, the number of autophagy organelles was not taken into account. We found that in cells classified as having Low BDS, there was a large degree of variability in accumulation of autophagosomes. Therefore, we developed a new approach wherein BDS was combined with number of LC3+ puncta, which enabled us to distinguish between cells having very few autophagy organelles versus cells with accumulation of autophagosomes or autolysosomes. Using this method, we were able to distinguish and quantify autophagosomes and autolysosomes in breast cancer cells cultured under basal conditions, with inhibition of autophagy using chloroquine, and with induction of autophagy using amino acid starvation. This technique yields additional insight into autophagy processing making it a useful supplement to current techniques.

Automated analysis of the cytokinesis-block micronucleus assay for radiation biodosimetry using imaging flow cytometry

The cytokinesis-block micronucleus (CBMN) assay is employed in biological dosimetry to determine the dose of radiation to an exposed individual from the frequency of micronuclei (MN) in binucleated lymphocyte cells. The method has been partially automated for the use in mass casualty events, but it would be advantageous to further automate the method for increased throughput. Recently, automated image analysis has been successfully applied to the traditional, slide-scoring-based method of the CBMN assay. However, with the development of new technologies such as the imaging flow cytometer, it is now possible to adapt this microscope-based assay to an automated imaging flow cytometry method. The ImageStream(X) is an imaging flow cytometer that has adequate sensitivity to quantify radiation doses larger than 1 Gy while adding the increased throughput of traditional flow cytometry. The protocol and analysis presented in this work adapts the CBMN assay for the use on the ImageStream(X). Ex vivo-irradiated whole blood samples cultured for CBMN were analyzed on the ImageStream(X), and preliminary results indicate that binucleated cells and MN can be identified, imaged and enumerated automatically by imaging flow cytometry. Details of the method development, gating strategy and the dose response curve generated are presented and indicate that adaptation of the CBMN assay for the use with imaging flow cytometry has potential for high-throughput analysis following a mass casualty radiological event.