Phenotyping Multiple Subsets of Immune Cells In Situ in Formalin-Fixed, Paraffin-Embedded Tissue Sections.

Some somatic illnesses such as peripheral tumours can present with psychiatric symptoms. Many of these are characterized by changes in biomarkers related to the inflammation or immune response. Here, we describe a multispectral imaging protocol that can be used to phenotype immune and other cell types through simultaneous imaging of multiple proteins in sections of peripheral solid tumours and other tissues. This approach can also be used to assess the spatial organization of these cells within the tissue.

Clinical Case of the Month:A 54 Year-Old Woman With Fever and Chills of Four-Days Duration.

CASE REPORT: A 54 year-old woman with diabetes mellitus type two and end-stage renal disease on hemodialysis presented to the emergency department with a four day history of generalized malaise, fever, and chills. Her symptoms were also associated with occasional dyspnea without a cough. She reported intermittent chronic diarrhea with hemodialysis which was currently unchanged. On the day of admission, she could not tolerate hemodialysis due to her symptoms. Over the past year she admitted to night sweats and a 40 pound weight loss. She denied having palpitations, chest pain, hemoptysis, lymph node swelling, sick contacts, or recent travel. The remainder of the review of systems was negative.

Automated prognostic pattern detection shows favourable diffuse pattern of FOXP3(+) Tregs in follicular lymphoma.

BACKGROUND: Histopathological prognostication relies on morphological pattern recognition, but as numbers of biomarkers increase, human prognostic pattern-recognition ability decreases. Follicular lymphoma (FL) has a variable outcome, partly determined by FOXP3 Tregs. We have developed an automated method, hypothesised interaction distribution (HID) analysis, to analyse spatial patterns of multiple biomarkers which we have applied to tumour-infiltrating lymphocytes in FL. METHODS: A tissue microarray of 40 patient samples was used in triplex immunohistochemistry for FOXP3, CD3 and CD69, and multispectral imaging used to determine the numbers and locations of CD3(+), FOXP3/CD3(+) and CD69/CD3(+) T cells. HID analysis was used to identify associations between cellular pattern and outcome. RESULTS: Higher numbers of CD3(+) (P=0.0001), FOXP3/CD3(+) (P=0.0031) and CD69/CD3(+) (P=0.0006) cells were favourable. Cross-validated HID analysis of cell pattern identified patient subgroups with statistically significantly different survival (35.5 vs 142 months, P=0.00255), a more diffuse pattern associated with favourable outcome and an aggregated pattern with unfavourable outcome. CONCLUSIONS: A diffuse pattern of FOXP3 and CD69 positivity was favourable, demonstrating ability of HID analysis to automatically identify prognostic cellular patterns. It is applicable to large numbers of biomarkers, representing an unsupervised, automated method for identification of undiscovered prognostic cellular patterns in cancer tissue samples.

Using a quantitative risk register to promote learning from a patient safety reporting system.

BACKGROUND: Patient safety reporting systems are now used in most health care delivery organizations. These systems, such as the one in use at Virginia Mason (Seattle) since 2002, can provide valuable reports of risk and harm from the front lines of patient care. In response to the challenge of how to quantify and prioritize safety opportunities, a risk register system was developed and implemented. METHODS: Basic risk register concepts were refined to provide a systematic way to understand risks reported by staff. The risk register uses a comprehensive taxonomy of patient risk and algorithmically assigns each patient safety report to 1 of 27 risk categories in three major domains (Evaluation, Treatment, and Critical Interactions). For each category, a composite score was calculated on the basis of event rate, harm, and cost. The composite scores were used to identify the "top five" risk categories, and patient safety reports in these categories were analyzed in greater depth to find recurrent patterns of risk and associated opportunities for improvement. RESULTS: The top five categories of risk were easy to identify and had distinctive "profiles" of rate, harm, and cost. The ability to categorize and rank risks across multiple dimensions yielded insights not previously available. These results were shared with leadership and served as input for planning quality and safety initiatives. This approach provided actionable input for the strategic planning process, while at the same time strengthening the Virginia Mason culture of safety. CONCLUSIONS: The quantitative patient safety risk register serves as one solution to the challenge of extracting valuable safety lessons from large numbers of incident reports and could profitably be adopted by other organizations.

Practical Guidance for Developing Small-Molecule Optical Probes for In Vivo Imaging.

The WMIS Education Committee (2019-2022) reached a consensus that white papers on molecular imaging could be beneficial for practitioners of molecular imaging at their early career stages and other scientists who are interested in molecular imaging. With this consensus, the committee plans to publish a series of white papers on topics related to the daily practice of molecular imaging. In this white paper, we aim to provide practical guidance that could be helpful for optical molecular imaging, particularly for small molecule probe development and validation in vitro and in vivo. The focus of this paper is preclinical animal studies with small-molecule optical probes. Near-infrared fluorescence imaging, bioluminescence imaging, chemiluminescence imaging, image-guided surgery, and Cerenkov luminescence imaging are discussed in this white paper.

Doped Ferrite Nanoparticles Exhibiting Self-Regulating Temperature as Magnetic Fluid Hyperthermia Antitumoral Agents, with Diagnostic Capability in Magnetic Resonance Imaging and Magnetic Particle Imaging.

This paper reports a comprehensive investigation of a magnetic nanoparticle (MNP), named M55, which belongs to a class of innovative doped ferrite nanomaterials, characterized by a self-limiting temperature. M55 is obtained from M48, an MNP previously described by our group, by implementing an additional purification step in the synthesis. M55, after citrate and glucose coating, is named G-M55. The present study aimed to demonstrate the properties of G-M55 as a diagnostic contrast agent for MRI and magnetic particle imaging (MPI), and as an antitumoral agent in magnetic fluid hyperthermia (MFH). Similar specific absorption rate values were obtained by standard MFH and by an MPI apparatus. This result is of interest in relation to the application of localized MFH by MPI apparatus. We demonstrated the biocompatibility of G-M55 in a triple-negative human breast cancer line (MDA-MB-231), and its efficacy as an MFH agent in the same cell line. We also demonstrated the efficacy of MFH treatment with G-M55 in an experimental model of breast cancer. Overall, our results pave the way for the clinical application of G-M55 as an MFH agent in breast cancer therapy, allowing not only efficient treatment by both standard MFH apparatus and MPI but also temperature monitoring.

Cellular context in epigenetics: quantitative multicolor imaging and automated per-cell analysis of miRNAs and their putative targets.

Epigenetics in general and microRNA (miRNA) in particular are an important and growing field of research, and while significant advances in the role of miRNA in a variety of diseases including cancer have been made, the majority of the information on the relationship between miRNA and its putative target proteins have been made on homogenized tissue, which, while useful, provides no information on tissue- or cell-specific signatures or relationships. Developments in spectral imaging have made it possible to image and quantitate samples labeled for both a miR and its putative target, using either brightfield or fluorescence labeling. Advances in automated morphometric and cellular quantitation enable the extraction of quantitative, tissue-specific measures of marker in each cell of a tissue section. This per-cell quantitative data can then be displayed as scatter plots, in a manner analogous to flow cytometry data, and multimarker phenotypes can be determined from threshold-based quadrant analysis. This paper describes the utility of the Nuance™ multispectral imaging system and the inForm™ image analysis software for assessing morphological distributions of miRNAs and their putative targets in thin tissue sections, and provides methodologies for the per-cell quantitative analysis of both markers.

Mind Over Magnets - How Magnetic Particle Imaging is Changing the Way We Think About the Future of Neuroscience.

Magnetic particle imaging (MPI) is an emerging imaging technique, which has the potential to provide the sensitivity, specificity and temporal resolution necessary for novel imaging advances in neurological applications. MPI relies on the detection of superparamagnetic iron-oxide nanoparticles, which allows for visualization and quantification of iron or iron-labeled cells throughout a subject. The combination of these qualities can be used to image many neurological conditions including cancer, inflammatory processes, vascular-related issues and could even focus on cell therapies and theranostics to treat these problems. This review will provide a basic introduction to MPI, discuss the current use of this technology to image neurological conditions, and touch on future applications including the potential for clinical translation.

Whither Magnetic Hyperthermia? A Tentative Roadmap.

The scientific community has made great efforts in advancing magnetic hyperthermia for the last two decades after going through a sizeable research lapse from its establishment. All the progress made in various topics ranging from nanoparticle synthesis to biocompatibilization and in vivo testing have been seeking to push the forefront towards some new clinical trials. As many, they did not go at the expected pace. Today, fruitful international cooperation and the wisdom gain after a careful analysis of the lessons learned from seminal clinical trials allow us to have a future with better guarantees for a more definitive takeoff of this genuine nanotherapy against cancer. Deliberately giving prominence to a number of critical aspects, this opinion review offers a blend of state-of-the-art hints and glimpses into the future of the therapy, considering the expected evolution of science and technology behind magnetic hyperthermia.

Utilizing 18F-FDG PET/CT Imaging and Quantitative Histology to Measure Dynamic Changes in the Glucose Metabolism in Mouse Models of Lung Cancer.

A hallmark of advanced tumors is a switch to aerobic glycolysis that is readily measured by [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography (18F-FDG PET) imaging. Co-mutations in the KRAS proto-oncogene and the LKB1 tumor suppressor gene are frequent events in lung cancer that drive hypermetabolic, glycolytic tumor growth. A critical pathway regulating the growth and metabolism of these tumors is the mechanistic target of the rapamycin (mTOR) pathway, which can be effectively targeted using selective catalytic mTOR kinase inhibitors. The mTOR inhibitor MLN0128 suppresses glycolysis in mice bearing tumors with Kras and Lkb1 co-mutations, referred to as KL mice. The therapy response in KL mice is first measured by 18F-FDG PET and computed tomography (CT) imaging before and after the delivery of MLN0128. By utilizing 18F-FDG PET/CT, researchers are able to measure dynamic changes in the glucose metabolism in genetically engineered mouse models (GEMMs) of lung cancer following a therapeutic intervention with targeted therapies. This is followed by ex vivo autoradiography and a quantitative immunohistochemical (qIHC) analysis using morphometric software. The use of qIHC enables the detection and quantification of distinct changes in the biomarker profiles following treatment as well as the characterization of distinct tumor pathologies. The coupling of PET imaging to quantitative histology is an effective strategy to identify metabolic and therapeutic responses in vivo in mouse models of disease.

The SAGE Guideline Model: achievements and overview.

The SAGE (Standards-Based Active Guideline Environment) project was formed to create a methodology and infrastructure required to demonstrate integration of decision-support technology for guideline-based care in commercial clinical information systems. This paper describes the development and innovative features of the SAGE Guideline Model and reports our experience encoding four guidelines. Innovations include methods for integrating guideline-based decision support with clinical workflow and employment of enterprise order sets. Using SAGE, a clinician informatician can encode computable guideline content as recommendation sets using only standard terminologies and standards-based patient information models. The SAGE Model supports encoding large portions of guideline knowledge as re-usable declarative evidence statements and supports querying external knowledge sources.

Technical considerations in longitudinal multispectral small animal molecular imaging.

In a previous study, we investigated physical methods to reduce whole-body, diet-related autofluorescence interference in several mouse strains through changes in animal diet. Measurements of mice with an in vivo multispectral imaging system over a 21-day period allowed for the quantification of concentration changes in multiple in vivo fluorophores. To be an effective instrument, a multispectral imaging system requires a priori spectral knowledge, the form and importance of which is not necessarily intuitive, particularly when noninvasive in vivo longitudinal imaging studies are performed. Using an optimized spectral library from a previous autofluorescence-reduction study as a model, we investigated two additional spectral definition techniques to illustrate the results of poor spectral definition in a longitudinal fluorescence imaging study. Here we systematically evaluate these results and show how poor spectral definition can lead to physiologically irrelevant results. This study concludes that the proper selection of robust spectra corresponding to each specific fluorescent molecular label of interest is of integral importance to enable effective use of multispectral imaging techniques in longitudinal fluorescence studies.

Phenotyping Multiple Subsets of Immune Cells In Situ in FFPE Tissue Sections: An Overview of Methodologies.

The recent clinical success of new cancer immunotherapy agents and methods is driving the need to understand the role of immune cells in solid tissues, especially tumors. Immune cell phenotyping via flow cytometry, while a cornerstone of immunology, is not spatially resolved and cannot analyze immune cell subsets in situ in clinical biopsy sections or to determine their interrelationships. To address this problem, a number of methodologies have been developed in attempts to phenotype immune and other cells in images acquired from tissue sections and to assess their organization in the tumor and its microenvironment. This chapter review the staining and multiplex image analysis methods that have been developed for phenotyping immune and other cells in formalin-fixed, paraffin-embedded (FFPE) tissue sections.

Autofluorescence removal, multiplexing, and automated analysis methods for in-vivo fluorescence imaging.

The ability to image and quantitate fluorescently labeled markers in vivo has generally been limited by autofluorescence of the tissue. Skin, in particular, has a strong autofluorescence signal, particularly when excited in the blue or green wavelengths. Fluorescence labels with emission wavelengths in the near-infrared are more amenable to deep-tissue imaging, because both scattering and autofluorescence are reduced as wavelengths are increased, but even in these spectral regions, autofluorescence can still limit sensitivity. Multispectral imaging (MSI), however, can remove the signal degradation caused by autofluorescence while adding enhanced multiplexing capabilities. While the availability of spectral "libraries" makes multispectral analysis routine for well-characterized samples, new software tools have been developed that greatly simplify the application of MSI to novel specimens.

Systemic effects of shock and resuscitation monitored by visible hyperspectral imaging.

Hyperspectral imaging (HSI) has been useful in monitoring several medical conditions, which to date have generally involved local changes in skin oxygenation of isolated regions of interest such as skin flaps or small burns. Here, by contrast, we present a study in which HSI was used to assess the local cutaneous manifestations of significant systemic events. HSI of the ventral surface of the lower jaw was used to monitor changes in skin oxygenation during hypovolemic shock induced by hemorrhage with additional pulmonary contusion injury in a porcine model, and to monitor the subsequent recovery of oxygenation with resuscitation. Quantitative and qualitative changes were observed in the level of skin oxygenation during shock and recovery. Quantitative values were obtained by fitting reference spectra of oxyhemoglobin and deoxyhemoglobin to sample spectra. Qualitative changes included changes in the observed spatial distribution or pattern of skin oxygenation. A mottled pattern of oxygen saturation was observed during hemorrhagic shock, but not observed during hypovolemic shock or following resuscitation. Historically, the assessment of skin color and mottling has been an important, albeit inexact, component of resuscitation algorithms. Now, it is possible to analyze these variables during shock and resuscitation in an objective manner. The clinical utility of these advances needs to be determined.

Dynamic PET and Optical Imaging and Compartment Modeling using a Dual-labeled Cyclic RGD Peptide Probe.

PURPOSE: The aim of this study is to determine if dynamic optical imaging could provide comparable kinetic parameters to that of dynamic PET imaging by a near-infrared dye/(64)Cu dual-labeled cyclic RGD peptide. METHODS: The integrin α(v)ß(3) binding RGD peptide was conjugated with a macrocyclic chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) for copper labeling and PET imaging and a near-infrared dye ZW-1 for optical imaging. The in vitro biological activity of RGD-C(DOTA)-ZW-1 was characterized by cell staining and receptor binding assay. Sixty-min dynamic PET and optical imaging were acquired on a MDA-MB-435 tumor model. Singular value decomposition (SVD) method was applied to compute the dynamic optical signal from the two-dimensional optical projection images. Compartment models were used to quantitatively analyze and compare the dynamic optical and PET data. RESULTS: The dual-labeled probe (64)Cu-RGD-C(DOTA)-ZW-1 showed integrin specific binding in vitro and in vivo. The binding potential (Bp) derived from dynamic optical imaging (1.762 ± 0.020) is comparable to that from dynamic PET (1.752 ± 0.026). CONCLUSION: The signal un-mixing process using SVD improved the accuracy of kinetic modeling of 2D dynamic optical data. Our results demonstrate that 2D dynamic optical imaging with SVD analysis could achieve comparable quantitative results as dynamic PET imaging in preclinical xenograft models.

In vivo optical imaging and dynamic contrast methods for biomedical research.

This paper provides an overview of optical imaging methods commonly applied to basic research applications. Optical imaging is well suited for non-clinical use, since it can exploit an enormous range of endogenous and exogenous forms of contrast that provide information about the structure and function of tissues ranging from single cells to entire organisms. An additional benefit of optical imaging that is often under-exploited is its ability to acquire data at high speeds; a feature that enables it to not only observe static distributions of contrast, but to probe and characterize dynamic events related to physiology, disease progression and acute interventions in real time. The benefits and limitations of in vivo optical imaging for biomedical research applications are described, followed by a perspective on future applications of optical imaging for basic research centred on a recently introduced real-time imaging technique called dynamic contrast-enhanced small animal molecular imaging (DyCE).

Distinguished photons: a review of in vivo spectral fluorescence imaging in small animals.

Fluorescence-based molecular imaging in small animals is having a major impact on drug development and disease research and the ability to detect multiple molecular species at once is becoming increasingly important. Unlike bioluminescence, in fluorescence, ubiquitous autofluorescent signals from the skin need to be separated from that of labeled fluorophores to ensure proper quantitative data. Since its introduction in 2004, spectral imaging methods have become an important part of in vivo fluorescence imaging of small animals by enabling easy multiplexed imaging methods and through the quantitative removal of interfering skin autofluorescence signals. This article is a review of the literature on spectral imaging methods and applications in fluorescence imaging of small animals.