Optimising multiplex immunofluorescence staining for characterising the tumour immune micro-environment.

Exploring the tumour microenvironment provides insight into the unique interaction between the host and tumour. Ultimately, its study improves understanding of how an individual mounts and achieves an anti-tumour immune response. In the context of colorectal cancer, immune biomarkers within the tumour microenvironment outperform traditional histopathological staging in predicting disease recurrence. Multiplex immunofluorescence enables simultaneous assessment of multiple markers to provide a highly accurate classification of immune cells and their spatial characterisation relative to tumour tissue. Further, automated slide staining provides staining consistency and reduces labour costs. Image acquisition using a non-spectral scanner allows more researchers to utilise multiplexed immunofluorescence for translational research. Herein we describe the optimisation process of conducting automated staining using a five-colour, tyramide signal amplification-based multiplex immunofluorescence panel. Using antibodies against CD3, CD8, CD103 and cytokeratin, the panel characterises T cell populations within human colorectal adenocarcinoma tissue. We provide an overview of primary antibody titration and the development of tyramide signal amplification immunofluorescence monoplex assays. We detail the processes of antibody stripping and the role of exogenous horseradish peroxidase inhibition to facilitate multiplexing. An account of determining the staining sequence and fluorophore assignment is provided. We describe image acquisition using a standard fluorescence microscope slide scanner and the management of spectral crosstalk using this system. Finally, we briefly document the digital image analysis required to characterise cells and determine their spatial distribution within the colorectal tumour microenvironment.

PlateletSeq: A novel method for discovery of blood-based biomarkers.

Platelets are small circulating fragments of cells that play important roles in thrombosis, haemostasis, immune response, inflammation and cancer growth. Although anucleate, they contain a rich RNA repertoire which offers an opportunity to characterise changes in platelet gene expression in health and disease. Whilst this can be achieved with conventional RNA sequencing, a large input of high-quality RNA, and hence blood volume, is required (unless a pre-amplification step is added), along with specialist bioinformatic skills for data analysis and interpretation. We have developed a transcriptomics next-generation sequencing-based approach that overcomes these limitations. Termed PlateletSeq, this method requires very low levels of RNA input and does not require specialist bioinformatic analytical skills. Here we describe the methodology, from sample collection to processing and data analysis. Specifically, blood samples can be stored for up to 8 days at 4 °C prior to analysis. Platelets are isolated using multi-step centrifugation and a purity of ≤ 1 leucocyte per 0.26x106 platelets is optimal for gene expression analysis. We have applied PlateletSeq to normal adult blood samples and show there are no age-associated variations and only minor gender-associated differences. In contrast, platelets from patients with myeloproliferative neoplasms show differences in platelet transcript profiles from normal and between disease subtypes. This illustrates the potential applicability of PlateletSeq for biomarker discovery and studying platelet biology in patient samples. It also opens avenues for assessing platelet quality in other fields such as transfusion research.

Imaging flow cytometry in the assessment of leukocyte-platelet aggregates.

Platelets are subcellular blood elements with a well-established role in haemostasis. Upon activation platelets undergo granule exocytosis, resulting in α-granule P-Selectin being expressed on the cell membrane. This allows binding of activated platelets to P-Selectin glycoprotein ligand 1 (PSGL-1) expressing leukocytes, forming leukocyte-platelet aggregates (LPAs). Whole blood flow cytometry (FCM) has demonstrated that elevated circulating LPAs (especially monocyte LPAs) are linked to atherothrombosis in high risk patients, and that activated platelet binding influences monocytes towards a pro-adhesive and pro-atherogenic phenotype. However, a limitation of conventional FCM is the potential for coincident events to resemble LPAs despite no tethering. Imaging cytometry can be used to characterize LPA formation and distinguish circulating MPAs from coincidental events. Platelets and leukocyte subsets are identified by expression of surface markers (e.g. the lipopolysachharide receptor CD14 on monocytes, glycoprotein Ib CD42b on platelets). In conventional FCM, all events with both leukocyte and platelet characteristics are designated as LPAs. However, by using an 'internal' mask based on the brightfield image and the fluorescent platelet identifier, imaging flow cytometry is able to distinguish leukocytes with tethered platelets (genuine LPAs) from leukocyte with coincidental, untethered platelets nearby. Mechanisms (e.g. adhesion molecules) or consequences (e.g. signal transduction) can then be separately analysed in platelet tethered and untethered leukocytes. Imaging flow cytometry therefore provides a more accurate approach for both enumeration and analysis of LPAs than conventional FCM.

Delayed Stream Segregation in Older Adults: More Than Just Informational Masking.

OBJECTIVE: To determine whether the time course for the buildup of auditory stream segregation differs between younger and older adults. DESIGN: Word recognition thresholds were determined for the first and last keywords in semantically anomalous but syntactically correct sentences (e.g., "A rose could paint a fish") when the target sentences were masked by speech-spectrum noise, 3-band vocoded speech, 16-band vocoded speech, intact and colocated speech, and intact and spatially separated speech. A significant reduction in thresholds from the first to the last keyword was interpreted as indicating that stream segregation improved with time. RESULTS: The buildup of stream segregation is slowed for both age groups when the masker is intact, colocated speech. CONCLUSIONS: Older adults are more disadvantaged; for them, stream segregation is also slowed even when a speech masker is spatially separated, conveys little meaning (3-band vocoding), and vocal fine structure cues are impoverished but envelope cues remain available (16-band vocoding).

Non-severe thermal burn injuries induce long-lasting downregulation of gene expression in cortical excitatory neurons and microglia.

Burn injuries are devastating traumas, often leading to life-long consequences that extend beyond the observable burn scar. In the context of the nervous system, burn injury patients commonly develop chronic neurological disorders and have been suggested to have impaired motor cortex function, but the long-lasting impact on neurons and glia in the brain is unknown. Using a mouse model of non-severe burn injury, excitatory and inhibitory neurons in the primary motor cortex were labelled with fluorescent proteins using adeno-associated viruses (AAVs). A total of 5 weeks following the burn injury, virus labelled excitatory and inhibitory neurons were isolated using fluorescence-activated cell sorting (FACS). In addition, microglia and astrocytes from the remaining cortical tissue caudal to the motor cortex were immunolabelled and isolated with FACS. Whole transcriptome RNA-sequencing was used to identify any long-lasting changes to gene expression in the different cell types. RNA-seq analysis showed changes to the expression of a small number of genes with known functions in excitatory neurons and microglia, but not in inhibitory neurons or astrocytes. Specifically, genes related to GABA-A receptors in excitatory neurons and several cellular functions in microglia were found to be downregulated in burn injured mice. These findings suggest that non-severe burn injuries lead to long lasting transcriptomic changes in the brain, but only in specific cell types. Our findings provide a broad overview of the long-lasting impact of burn injuries on the central nervous system which may help identify potential therapeutic targets to prevent neurological dysfunction in burn patients.

Imaging flow cytometric detection of del(17p) in bone marrow and circulating plasma cells in multiple myeloma.

BACKGROUND: Detection of del(17p) in myeloma is generally performed by fluorescence in situ hybridization (FISH) on a slide with analysis of up to 200 nuclei. The small cell sample analyzed makes this a low precision test. We report the utility of an automated FISH method, called "immuno-flowFISH", to detect plasma cells with adverse prognostic risk del(17p) in bone marrow and blood samples of patients with myeloma. METHODS: Bone marrow (n = 31) and blood (n = 19) samples from 35 patients with myeloma were analyzed using immuno-flowFISH. Plasma cells were identified by CD38/CD138-immunophenotypic gating and assessed for the 17p locus and centromere of chromosome 17. Cells were acquired on an AMNIS ImageStreamX MkII imaging flow cytometer using INSPIRE software. RESULTS: Chromosome 17 abnormalities were identified in CD38/CD138-positive cells in bone marrow (6/31) and blood (4/19) samples when the percent plasma cell burden ranged from 0.03% to 100% of cells. Abnormalities could be identified in 14.5%-100% of plasma cells. CONCLUSIONS: The "immuno-flowFISH" imaging flow cytometric method could detect del(17p) in plasma cells in both bone marrow and blood samples of myeloma patients. This method was also able to detect gains and losses of chromosome 17, which are also of prognostic significance. The lowest levels of 0.009% (bone marrow) and 0.001% (blood) for chromosome 17 abnormalities was below the detection limit of current FISH method. This method offers potential as a new means of identifying these prognostically important chromosomal defects, even when only rare cells are present and for serial disease monitoring.

"Immuno-FlowFISH": Applications for Chronic Lymphocytic Leukemia.

Imaging flow cytometry has the capacity to bridge the gap that currently exists between the diagnostic tests that detect important phenotypic and genetic changes in the clinical assessment of leukemia and other hematological malignancies or blood-based disorders. We have developed an "Immuno-flowFISH" method that leverages the quantitative and multi-parametric power of imaging flow cytometry to push the limits of single-cell analysis. Immuno-flowFISH has been fully optimized to detect clinically significant numerical and structural chromosomal abnormalities (i.e., trisomy 12 and del(17p)) within clonal CD19/CD5+ CD3- Chronic Lymphocytic Leukemia (CLL) cells in a single test. This integrated methodology has greater accuracy and precision than standard fluorescence in situ hybridization (FISH). We have detailed this immuno-flowFISH application with a carefully catalogued workflow, technical instructions, and a repertoire of quality control considerations to supplement the analysis of CLL. This next-generation imaging flow cytometry protocol may provide unique advancements and opportunities in the holistic cellular assessment of disease for both research and clinical laboratory settings.

Chimeric antigen receptor T-cells: Properties, production, and quality control.

Chimeric antigen receptor (CAR) T-cell therapy is a novel adoptive T-cell immunotherapy for haematological malignancies. First introduced into clinical practice in 2017, CAR T-cell therapy is now finding its place in the management of lymphoid malignancies, primarily of B-cell lineage, including lymphoblastic leukaemia, non-Hodgkin lymphoma and plasma cell myeloma, with remarkable therapeutic outcomes. CAR T-cells are a customised therapeutic product for each patient. Manufacture commences with collection of autologous T-cells, which are then genetically engineered ex vivo to express transmembrane CARs. These chimeric proteins consist of an antibody-like extracellular antigen-binding domain, to recognise specific antigens on the surface of tumour cells (e.g. CD19), linked to the intracellular co-stimulatory signalling domains of a T-cell receptor (e.g. CD137). The latter is required for in vivo CAR T-cell proliferation, survival, and durable efficacy. Following reinfusion, CAR T-cells harness the cytotoxic capacity of a patient's immune system. They overcome major mechanisms of tumour immuno-evasion and have potential to generate robust cytotoxic anti-tumour responses. This review discusses the background to CAR T-cell therapies, including their molecular design, mechanisms of action, methods of production, clinical applications and established and emerging technologies for CAR T-cell evaluation. It highlights the need for standardisation, quality control and monitoring of CAR T-cell therapies, to ensure their safety and efficacy in clinical management.

Locally performed postoperative circulating tumour DNA testing performed during routine clinical care to predict recurrence of colorectal cancer.

BACKGROUND: Identifying patients at high risk for colorectal cancer recurrence is essential for improving prognosis. In the postoperative period, circulating tumour DNA (ctDNA) has been demonstrated as a significant prognostic indicator of recurrence. These results have been obtained under the strict rigours of clinical trials, but not validated in a real-world setting using in-house testing. We report the outcomes of locally performed postoperative ctDNA testing conducted during routine clinical care and the association with the recurrence of colorectal cancer. METHODS: We recruited 36 consecutive patients with newly diagnosed colorectal cancer between 2018 and 2020. Postoperative plasma samples were collected at the first outpatient review following resection. Tumour-informed ctDNA analysis was performed using droplet digital polymerase chain reaction or targeted next-generation sequencing. RESULTS: At the time of surgery, there were 24 patients (66.7%) with localized cancer, nine (25%) with nodal spread, and three (8.3%) with metastatic disease. The median time from surgery to plasma sample donation was 22 days (IQR 20-28 days). At least one somatic mutation was identified in primary tumour tissue for 28 (77.8%) patients. Postoperative ctDNA was detected in five patients (13.9%). The median duration of follow-up was 32.0 months (IQR 27.2-38.1 months). Two patients (5.56%) developed metastatic recurrence. However, neither had detectable postoperative ctDNA. There were no instances of loco-regional recurrence. CONCLUSION: Analysis of postoperative ctDNA testing can be performed locally, however this study did not reproduce the adverse association between detectable postoperative ctDNA and the development of colorectal cancer recurrence seen in clinical trials.

IGH cytogenetic abnormalities can be detected in multiple myeloma by imaging flow cytometry.

AIMS: Cytogenetic abnormalities involving the IGH gene are seen in up to 55% of patients with multiple myeloma. Current testing is performed manually by fluorescence in situ hybridisation (FISH) on purified plasma cells. We aimed to assess whether an automated imaging flow cytometric method that uses immunophenotypic cell identification, and does not require cell isolation, can identify IGH abnormalities. METHODS: Aspirated bone marrow from 10 patients with multiple myeloma were studied. Plasma cells were identified by CD38 and CD138 coexpression and assessed with FISH probes for numerical or structural abnormalities of IGH. Thousands of cells were acquired on an imaging flow cytometer and numerical data and digital images were analysed. RESULTS: Up to 30 000 cells were acquired and IGH chromosomal abnormalities were detected in 5 of the 10 marrow samples. FISH signal patterns seen included fused IGH signals for IGH/FGFR3 and IGH/MYEOV, indicating t(4;14) and t(11;14), respectively. In addition, three IGH signals were identified, indicating trisomy 14 or translocation with an alternate chromosome. The lowest limit of detection of an IGH abnormality was in 0.05% of all cells. CONCLUSIONS: This automated high-throughput immuno-flowFISH method was able to identify translocations and trisomy involving the IGH gene in plasma cells in multiple myeloma. Thousands of cells were analysed and without prior cell isolation. The inclusion of positive plasma cell identification based on immunophenotype led to a lowest detection level of 0.05% marrow cells. This imaging flow cytometric FISH method offers the prospect of increased precision of detection of critical genetic lesions involving IGH and other chromosomal defects in multiple myeloma.

The effect of priming on release from informational masking is equivalent for younger and older adults.

OBJECTIVE: Previous studies have shown that presenting younger listeners with all but the last word of a target anomalous sentence immediately before presenting the full sentence in a noisy background produces a greater release from masking when the masker is two-talker anomalous speech than when it is speech-spectrum noise, thereby demonstrating that an auditory prime can produce a release from informational masking. The purpose of this study was to investigate whether older adults could gain the same benefit from auditory primes as younger adults and what bottom-up auditory factors contribute to the advantage provided by auditory primes in releasing speech from informational masking. DESIGN: A total of 76 younger adults (university students) and 76 older adults (volunteers from the local community) participated in this study. All participants spoke English as a first language and had normal hearing below 4 kHz. RESULTS: In experiment 1, younger adults performed better in the presence of the speech masker, whereas older adults performed equivalently under both types of masking, but auditory priming produced an equivalent amount of release from informational masking in both younger and older adults. To examine the degree to which familiarity with the target talker's voice contributed to the priming effects observed in the first experiment, in experiment 2, we primed individuals with sentences that were spoken by the target talker but with lexical content that was unrelated to the target sentences. There was no release from informational masking for either age group. Next, to investigate the extent to which the release from informational masking in the first experiment was due to the amplitude envelope cues provided by the prime, in experiment 3, we noise vocoded the prime (using 3 bands) to remove semantic content while retaining some cues about the prime's amplitude envelope. When the primes were noise vocoded, there was no release from informational masking for either younger or older adults. Finally, to examine whether older adults' performance in the presence of the speech masker in the first experiment was due to an age-related decline in the ability to take advantage of dips in the amplitude envelope of the speech masker, in experiment 4, we noise vocoded the speech masker. We found a significant improvement in performance, but the amount of improvement was equivalent for both age groups. CONCLUSIONS: Auditory priming resulted in equivalent amounts of release from informational masking in both younger and older adults. The benefit provided by auditory primes was not due to cues provided in the prime about the target talker's voice or cues provided in the prime about fluctuations in the amplitude envelope of the target sentences. Importantly, there was an age-related decline in performance in the presence of a two-talker masker relative to a continuous speech-spectrum noise masker; however, this age-related decline in performance cannot be attributed to age-related differences in the ability to take advantage of fluctuations in the amplitude envelope of the speech masker.

Analysis of human chromosomes by imaging flow cytometry.

Chromosomal analysis is traditionally performed by karyotyping on metaphase spreads, or by fluorescent in situ hybridization (FISH) on interphase cells or metaphase spreads. Flow cytometry was introduced as a new method to analyze chromosomes number (ploidy) and structure (telomere length) in the 1970s with data interpretation largely based on fluorescence intensity. This technology has had little uptake for human cytogenetic applications primarily due to analytical challenges. The introduction of imaging flow cytometry, with the addition of digital images to standard multi-parametric flow cytometry quantitative tools, has added a new dimension. The ability to visualize the chromosomes and FISH signals overcomes the inherent difficulties when the data is restricted to fluorescence intensity. This field is now moving forward with methods being developed to assess chromosome number and structure in whole cells (normal and malignant) in suspension. A recent advance has been the inclusion of immunophenotyping such that antigen expression can be used to identify specific cells of interest for specific chromosomes and their abnormalities. This capability has been illustrated in blood cancers, such as chronic lymphocytic leukemia and plasma cell myeloma. The high sensitivity and specificity achievable highlights the potential imaging flow cytometry has for cytogenomic applications (i.e., diagnosis and disease monitoring). This review introduces and describes the development, current status, and applications of imaging flow cytometry for chromosomal analysis of human chromosomes.

Simultaneous flow cytometric characterization of multiple cell types and metabolic states in the rat brain after repeated mild traumatic brain injury.

BACKGROUND: Cellular responses at the sub-acute phase of mild traumatic brain injury (mTBI), and their contribution to ongoing damage, are unclear, complex and require simultaneous assessment of multiple cells to elucidate. NEW METHOD: An 11-colour flow cytometry method for analysing brain cells was evaluated in a weight-drop rat model of repeated mTBI. Animals received sham, one, two or three mTBI delivered at 24 h intervals (n = 6/group). Cerebrum homogenates were prepared 11 days after first mTBI, in two cohorts of n = 3/group to enable same-day staining of fresh tissue. Percentages of neurons, astrocytes, microglia, mature oligodendrocytes and NeuN + CC1+ cells, neutrophils, macrophages and non-myeloid leukocytes, and their immunoreactivity for cell damage indicators (inducible nitric oxide synthase; iNOS, proliferating cell nuclear antigen; PCNA, 8-Oxo-2'-deoxyguanosine; 8OHDG and 4-hydroxynonenal; HNE), were assessed. RESULTS: Median fluorescence intensity (MFI) of iNOS in activated microglia increased following two, but not one or three, mTBI (p = 0.04). However, there were differences between processing cohorts in terms of percentages and MFI of some PCNA+, iNOS+, 8OHDG + and HNE + cell populations. COMPARISON WITH EXISTING METHODS: Previous applications of flow cytometry for rat brain analysis were typically limited to three or four markers. This method uses 11 markers to identify nine cell populations and evaluate their immunoreactivity to four metabolic indicators of cell damage. CONCLUSIONS: Flow cytometry can be useful for discerning injury-related changes in multiple rat brain cells. However, markers sensitive to subtle changes in experimental conditions must be identified in pilot experiments and subsequently analysed in the same tissue-processing cohort.

Immunomodulatory effects of statins and autoimmune rheumatic diseases: novel intracellular mechanism involved.

Inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, known as statins, are the most commonly prescribed agents for the treatment of hypercholesterolemia. However, the effects of statins may extend beyond their influences on serum cholesterol levels resulting in cholesterol-independent or pleiotropic effects. Clinical, animal and in vitro studies suggest that statins have additional clinical uses because of their anti-inflammatory and immunomodulatory effects, in part due to their capacity to interfere with the mevalonate pathway and inhibit prenylation of Rho family GTPases. This review focuses on the molecular mechanisms of the anti-inflammatory and immunomodulatory effects of statins. In base to all these information, we suggest that statins could have similar inhibitory effects on MAPKs pathways in cells from RA patients, including osteoclasts and fibroblasts.

TGFα expression in myeloid malignancies.

BACKGROUND: Transforming growth factor α (TGFα) is a peptide growth factor known to be expressed in normal haemopoiesis. It is also expressed in a range of epithelial neoplasms but has not been assessed in haemopoietic malignancies. We have performed an immunohistochemical evaluation of TGFα in acute and chronic myeloid malignancies. METHODS: TGFα expression was semiquantitatively assessed in 69 normal bone marrow trephines and 157 cases of myeloid malignancy using an immunohistochemical approach. RESULTS: Blast cells of myeloid origin in acute myeloid leukaemia (AML), myelodysplasia and accelerated and blast phases of chronic myeloid leukaemia (CML) were TGFα positive. In acute promyelocytic leukaemia the neoplastic cells had significantly weaker TGFα expression than seen in other forms of AML. The blast cells in CML-accelerated and blast phases were positive with similar expression to AML. CONCLUSIONS: TGFα is expressed in neoplastic myeloblasts and could, therefore, be used as blast cell biomarker in diagnostic haematopathology. In addition, TGFα immunohistochemistry may be of use in identifying a therapeutic target.