Diagnostic Validation of a Whole-Slide Imaging Scanner in Cytological Samples: Diagnostic Accuracy and Comparison With Light Microscopy.

Digital slides created by whole-slide imaging scanners can be evaluated by pathologists located in remote sites, but the process must be validated before this technology can be applied to routine cytological diagnosis. The aim of this study was to validate a whole-slide imaging scanner for cytological samples. Sixty cytological samples, whose diagnoses were confirmed by gold-standard examinations (histology or flow cytometry), were digitalized using a whole-slide imaging scanner. Digital slides and glass slides were examined by 3 observers with different levels of cytopathological expertise. No significant differences were noted between digital and glass slides in regard to the number of cases correctly diagnosed, or the sensitivity, specificity, or diagnostic accuracy, irrespective of the observers' expertise. The agreements between the digital slides and the gold-standard examinations were moderate to substantial, while the agreements between the glass slides and the gold-standard examinations were substantial for all 3 observers. The intraobserver agreements between digital and glass slides were substantial to almost perfect. The interobserver agreements when evaluating digital slides were moderate between observers 1 and 2 and between observers 1 and 3 while they were substantial between observers 2 and 3. In conclusion, our study demonstrated that the digital slides produced by the whole-slide imaging scanner are adequate to diagnose cytological samples and are similar among clinical pathologists with differing levels of expertise.

Qualitative identification of imidacloprid in postmortem animal tissue by gas chromatography-tandem mass spectrometry.

During an avian mass mortality event investigation at the National Fish and Wildlife Forensic Laboratory in Ashland, OR, imidacloprid became an insecticide of concern. A qualitative analytical toxicology screen of seeds, plucks (tongue, esophagus, and trachea), and ventricular contents was requested. A method for the extraction and qualitative analysis of the insecticide in animal tissues was therefore developed. The procedure relies on a combined Food Emergency Response Network (FERN) and QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) approach to sample extraction followed by qualitative analysis by gas chromatography-tandem mass spectrometry. Since imidacloprid is not amenable to the conditions of gas chromatography, a trimethylsilyl derivative was created and characterized. Proposed mechanisms for the creation of this derivative and its mass spectrum are described. The imidacloprid-trimethylsilyl (TMS) derivative was detected in all samples submitted.

Observational Study Design in Veterinary Pathology, Part 1: Study Design.

Observational studies are the basis for much of our knowledge of veterinary pathology and are highly relevant to the daily practice of pathology. However, recommendations for conducting pathology-based observational studies are not readily available. In part 1 of this series, we offer advice on planning and conducting an observational study with examples from the veterinary pathology literature. Investigators should recognize the importance of creativity, insight, and innovation in devising studies that solve problems and fill important gaps in knowledge. Studies should focus on specific and testable hypotheses, questions, or objectives. The methodology is developed to support these goals. We consider the merits and limitations of different types of analytic and descriptive studies, as well as of prospective vs retrospective enrollment. Investigators should define clear inclusion and exclusion criteria and select adequate numbers of study subjects, including careful selection of the most appropriate controls. Studies of causality must consider the temporal relationships between variables and the advantages of measuring incident cases rather than prevalent cases. Investigators must consider unique aspects of studies based on archived laboratory case material and take particular care to consider and mitigate the potential for selection bias and information bias. We close by discussing approaches to adding value and impact to observational studies. Part 2 of the series focuses on methodology and validation of methods.

Observational Study Design in Veterinary Pathology, Part 2: Methodology.

Observational studies are a basis for much of our knowledge of veterinary pathology, yet considerations for conducting pathology-based observational studies are not readily available. In part 1 of this series, we offered advice on planning and carrying out an observational study. Part 2 of the series focuses on methodology. Our general recommendations are to consider using already-validated methods, published guidelines, data from primary sources, and quantitative analyses. We discuss 3 common methods in pathology research-histopathologic scoring, immunohistochemistry, and polymerase chain reaction-to illustrate principles of method validation. Some aspects of quality control include use of clear objective grading criteria, validation of key reagents, assessing sample quality, determining specificity and sensitivity, use of technical and biologic negative and positive controls, blinding of investigators, approaches to minimizing operator-dependent variation, measuring technical variation, and consistency in analysis of the different study groups. We close by discussing approaches to increasing the rigor of observational studies by corroborating results with complementary methods, using sufficiently large numbers of study subjects, consideration of the data in light of similar published studies, replicating the results in a second study population, and critical analysis of the study findings.

Microarray-Based Gene Expression Analysis for Veterinary Pathologists: A Review.

High-throughput, genome-wide transcriptome analysis is now commonly used in all fields of life science research and is on the cusp of medical and veterinary diagnostic application. Transcriptomic methods such as microarrays and next-generation sequencing generate enormous amounts of data. The pathogenetic expertise acquired from understanding of general pathology provides veterinary pathologists with a profound background, which is essential in translating transcriptomic data into meaningful biological knowledge, thereby leading to a better understanding of underlying disease mechanisms. The scientific literature concerning high-throughput data-mining techniques usually addresses mathematicians or computer scientists as the target audience. In contrast, the present review provides the reader with a clear and systematic basis from a veterinary pathologist's perspective. Therefore, the aims are (1) to introduce the reader to the necessary methodological background; (2) to introduce the sequential steps commonly performed in a microarray analysis including quality control, annotation, normalization, selection of differentially expressed genes, clustering, gene ontology and pathway analysis, analysis of manually selected genes, and biomarker discovery; and (3) to provide references to publically available and user-friendly software suites. In summary, the data analysis methods presented within this review will enable veterinary pathologists to analyze high-throughput transcriptome data obtained from their own experiments, supplemental data that accompany scientific publications, or public repositories in order to obtain a more in-depth insight into underlying disease mechanisms.

Practical Stereology Applications for the Pathologist.

Qualitative histopathology is the gold standard for routine examination of morphological tissue changes in the regulatory or academic environment. The human eye is exceptional for pattern recognition but often cannot detect small changes in quantity. In cases where detection of subtle quantitative changes is critical, more sensitive methods are required. Two-dimensional histomorphometry can provide additional quantitative information and is quite useful in many cases. However, the provided data may not be referent to the entire tissue and, as such, it makes several assumptions, which are sources of bias. In contrast, stereology is design based rather than assumption based and uses stringent sampling methods to obtain accurate and precise 3-dimensional information using geometrical and statistical principles. Recent advances in technology have made stereology more approachable and practical for the pathologist in both regulatory and academic environments. This review introduces pathologists to the basic principles of stereology and walks the reader through some real-world examples for the application of these principles in the workplace.

The Pathologist 2.0: An Update on Digital Pathology in Veterinary Medicine.

Using light microscopy to describe the microarchitecture of normal and diseased tissues has changed very little since the middle of the 19th century. While the premise of histologic analysis remains intact, our relationship with the microscope is changing dramatically. Digital pathology offers new forms of visualization, and delivery of images is facilitated in unprecedented ways. This new technology can untether us entirely from our light microscopes, with many pathologists already performing their jobs using virtual microscopy. Several veterinary colleges have integrated virtual microscopy in their curriculum, and some diagnostic histopathology labs are switching to virtual microscopy as their main tool for the assessment of histologic specimens. Considering recent technical advancements of slide scanner and viewing software, digital pathology should now be considered a serious alternative to traditional light microscopy. This review therefore intends to give an overview of the current digital pathology technologies and their potential in all fields of veterinary pathology (ie, research, diagnostic service, and education). A future integration of digital pathology in the veterinary pathologist's workflow seems to be inevitable, and therefore it is proposed that trainees should be taught in digital pathology to keep up with the unavoidable digitization of the profession.

The Veterinary Forensic Necropsy: A Review of Procedures and Protocols.

Investigation of animal-related crime, and therefore submission of forensic cases to veterinary pathology facilities, is increasing, yet many veterinary pathologists are unfamiliar and often uncomfortable with involvement in the forensic necropsy. This article discusses various aspects of the forensic necropsy without specific attention to any particular species group or crime. General advice is given on procedures, documentation, and recording of the examination, and the article indicates how these features may differ from those used in investigation of natural disease. It also discusses evidence management, including recordkeeping, identification of evidence, labeling of photographs, and use of standard operating procedures and protocols. Various written and visual methods for documentation of the forensic necropsy are covered, and adjunctive topics such as sample collection, assessment, and description of wounds and taphonomy are included. Cause, mechanism, and manner of death are defined, and guidance to the use of these terms is given. The aim of this article is to offer guidance on procedural aspects of the forensic necropsy that will help those developing their forensic services, contribute to standardization of the provision of forensic veterinary pathology, and build the confidence of the "uncomfortable" forensic veterinary pathologist.

Identification, Collection, and Preservation of Veterinary Forensic Evidence: On Scene and During the Postmortem Examination.

Although it is the obligation of the veterinary forensic pathologist to be competent in identifying, collecting, and preserving evidence from the body, it is also necessary for them to understand the relevance of conditions on the crime scene. The body is just one piece of the puzzle that needs to be considered when determining the cause of death. The information required for a complete postmortem analysis should also include details of the animal's environment and items of evidence present on the crime scene. These factors will assist the veterinary forensic pathologist in the interpretation of necropsy findings. Therefore, the veterinary forensic pathologist needs to have a basic understanding of how the crime scene is processed, as well as the role of the forensic veterinarian on scene. In addition, the veterinary forensic pathologist must remain unbiased, necessitating an understanding of evidence maintenance and authentication.

Postmortem Changes in Animal Carcasses and Estimation of the Postmortem Interval.

A thorough understanding of the physical and chemical changes that occur in the body after death is critical for accurate interpretation of gross and microscopic pathology at autopsy. Furthermore, knowledge of the postmortem processes and the factors that affect them will aid in the estimation of the postmortem interval (PMI). The estimation of the PMI is important in many human and animal death investigations. Despite many decades of research, accuracy in estimation of the time of death has not significantly improved, and no single method can be reliably used to accurately estimate the time of death. Great care should be taken when formulating such an estimate, for it is dependent on multiple circumstantial and environmental factors, and the accuracy and precision of the estimate decrease as the PMI increases. The majority of the research in the field has been conducted on human bodies, but many relevant conclusions may be drawn regarding the expected postmortem changes in animals and the estimation of the PMI. The veterinary pathologist must use great caution when attempting to extrapolate data and apply formulas designed for use in humans. Methods reviewed include gross changes, microscopic changes, temperature-based methods, postmortem chemistry, molecular methods, microbial assay, ocular changes, radiography, entomology, and others. Although only several of these methods are currently practical for use in the workup of cases, it is expected that future research will result in improved techniques with enhanced accuracy in the estimation of the PMI, which will benefit both human and veterinary forensic investigations.

Forensic Veterinary Pathology: Sharp Injuries in Animals.

Sharp-force injuries are injuries caused by a mechanical force using sharp objects against the skin. Sharp-force injuries are mainly classified as stab, incised, chop, and therapeutic wounds and are less frequent than blunt-force injuries in animals. The analysis of the edges of the wound is crucial, especially if more than one type of lesion is involved. It may be difficult to differentiate between sharp trauma and blunt trauma, because lacerations can resemble incised wounds. The accurate documentation and examination of these injuries may indicate the instrument involved, the relationship between the animal and the perpetrator, and the force of the stab. Situations in which this type of trauma occurs may involve social violence, accidents, hunting, veterinary medical management, and religious rituals. The causes of death related to this type of trauma include hypovolemic shock, pneumothorax, or asphyxiation due to aspiration of blood. Necropsy findings should provide objective and unbiased information about the cause and manner of death to aid the investigation and further judgment of a possible crime.

Veterinary Forensics: Firearms and Investigation of Projectile Injury.

Projectile injury represents an estimated 14% of reported animal cruelty cases in the United States. Cases involving projectiles are complicated by gross similarities to other common types of injury, including bite wounds and motor vehicle injuries, by weapons and ammunition not commonly recognized or understood by veterinary medical professionals, and by required expertise beyond that employed in routine postmortem examination. This review describes the common types of projectile injuries encountered within the United States, as well as firearms and ammunition associated with this form of injury. The 3 stages of ballistics-internal, external, and terminal-and wounding capacity are discussed. A general understanding of firearms, ammunition, and ballistics is necessary before pursuing forensic projectile cases. The forensic necropsy is described, including gunshot wound examination, projectile trajectories, different imaging procedures, collection and storage of projectile evidence, and potential advanced techniques for gunpowder analysis. This review presents aspects of projectile injury investigation that must be considered in tandem with standard postmortem practices and procedures to ensure reliable conclusions are reached for medicolegal as well as diagnostic purposes.

Integrating the Forensic Sciences in Wildlife Case Investigations: A Case Report of Pentobarbital and Phenytoin Toxicosis in a Bald Eagle (Haliaeetus leucocephalus).

The application of medical knowledge to the purpose of law is the foundation of forensic pathology. A forensic postmortem examination often involves the expertise of multiple scientific disciplines to reconstruct the full story surrounding the death of an animal. Wildlife poses additional challenges in forensic investigations due to little or no associated history, and the disruptive effects of decomposition. To illustrate the multidisciplinary nature of wildlife forensic medicine, the authors outline a case of secondary pentobarbital/phenytoin toxicosis in a bald eagle (Haliaeetus leucocephalus). The eagle was the single fatality in a group of 8 birds that fed on euthanized domestic cat remains that had been improperly disposed of in a landfill. Cooperation between responding law enforcement officers, pathologists, and other forensic scientists led to the successful diagnosis and resolution of the case.

Veterinary Forensic Pathology: Drowning and Bodies Recovered From Water.

Determining the cause of death in animals recovered from bodies of water, swimming pools, or other water-containing vessels is challenging. Animals recovered from water may or may not have drowned. The diagnosis of drowning is usually one of exclusion, requiring information from the crime scene, recovery scene, the medical history or reliable witness accounts. While there are characteristic macroscopic and microscopic lesions of drowning, none are specific and are dependent on the volume and tonicity of the drowning medium. Beyond interpreting the postmortem findings, the court may ask pathologists to comment on the behavioral and welfare implications of drowning. This requires an understanding of the drowning process, which is a complex series of sequential, concurrent, and overlapping cardiorespiratory reflexes, electrolyte and blood gas abnormalities, aspiration, physical exhaustion, and breathlessness eventually culminating in death. This review addresses the mechanisms, lesions, and diagnostic issues associated with drowning in nonaquatic companion animals.

Veterinary Forensic Toxicology.

Veterinary pathologists working in diagnostic laboratories are sometimes presented with cases involving animal poisonings that become the object of criminal or civil litigation. Forensic veterinary toxicology cases can include cases involving animal cruelty (malicious poisoning), regulatory issues (eg, contamination of the food supply), insurance litigation, or poisoning of wildlife. An understanding of the appropriate approach to these types of cases, including proper sample collection, handling, and transport, is essential so that chain of custody rules are followed and proper samples are obtained for toxicological analysis. Consultation with veterinary toxicologists at the diagnostic laboratory that will be processing the samples before, during, and after the forensic necropsy can help to ensure that the analytical tests performed are appropriate for the circumstances and findings surrounding the individual case.

Electrocution of Raptors on Power Lines: A Review of Necropsy Methods and Findings.

Decades after the problem was first identified, power line electrocution continues to be a cause of avian mortality. Currently, several federal laws protect eagles and other migratory birds, meaning that utility companies may be liable for electrocution-related deaths. Veterinarians and veterinary pathologists called upon to diagnose and treat electrocuted birds should keep this in mind when conducting clinical and postmortem examinations. This review details necropsy findings and methods used to diagnose electrocution. A combination of gross, subgross, and radiographic examinations can aid in identification of subtle injury. Diagnosis is made based on the presence of skin and/or feather burns. Other necropsy findings may include skin lacerations, subcutaneous burns, bruising, limb avulsion, hemopericardium, and vascular rupture. At the US Fish and Wildlife Service's National Forensics Laboratory, from 2000 to 2015, 417 raptor deaths were determined to have been caused by electrocution. Bald eagles and golden eagles were the most commonly submitted species. In a retrospective review of 377 cases, for which whole bodies were submitted, 18% of the electrocuted birds had only a single, small (less than 3 cm in diameter) external burn. Small, isolated burns tended to occur on the undersides of the wings at and distal to the elbow and on the lower legs and feet. These areas should be most carefully examined in cases where electrocution injury is not immediately apparent.

Forensic Entomology in Animal Cruelty Cases.

Forensic entomology can be useful to the veterinary professional in cases of animal cruelty. A main application of forensic entomology is to determine the minimum postmortem interval by estimating the time of insect colonization, based on knowledge of the rate of development of pioneer colonizers and on insect species succession during decomposition of animal remains. Since insect development is temperature dependent, these estimates require documentation of the environmental conditions, including ambient temperature. It can also aid in the detection and recognition of wounds, as well as estimate the timing of periods of neglect. Knowledge of the geographic distribution of insects that colonize animal remains may suggest that there has been movement or concealment of the carcass or can create associations between a suspect, a victim, and a crime scene. In some instances, it can aid in the detection of drugs or toxins within decomposed or skeletonized remains. During animal cruelty investigations, it may become the responsibility of the veterinary professional to document and collect entomological evidence from live animals or during the necropsy. The applications of forensic entomology are discussed. A protocol is described for documenting and collecting entomological evidence at the scene and during the necropsy, with additional emphasis on recording geographic location, meteorological data, and collection and preservation of insect specimens.

Forensic Pathology of Traumatic Brain Injury.

Traumatic brain injury constitutes a significant proportion of cases requiring forensic examination, and it encompasses (1) blunt, nonmissile head injury, especially involving motor vehicle accidents, and (2) penetrating, missile injury produced by a range of high- and lower-velocity projectiles. This review examines the complex pathophysiology and biomechanics of both types of neurotrauma and assesses the macroscopic and histologic features of component lesions, which may be used to determine the cause and manner of death resulting from an intentional assault or accident. Estimation of the survival time postinjury by pathologic examination is also important where malicious head injury is suspected, in an attempt to ascertain a time at which the traumatic event might have been committed, thereby evaluating the authenticity of statements made by the alleged perpetrator.

Nondrowning Asphyxia in Veterinary Forensic Pathology: Suffocation, Strangulation, and Mechanical Asphyxia.

Asphyxia in a forensic context refers to death by rapid cerebral anoxia or hypoxia due to accidental or nonaccidental injury. Death due to nondrowning asphyxia can occur with strangulation, suffocation, and mechanical asphyxia, each of which is categorized based on the mechanism of injury. Individuals dying due to various types of asphyxia may or may not have lesions, and even those lesions that are present may be due to other causes. The interpretation or opinion that death was due to asphyxia requires definitive and compelling evidence from the postmortem examination, death scene, and/or history. Beyond the postmortem examination, pathologists may be faced with questions of forensic importance that revolve around the behavioral and physiological responses in animals subjected to strangulation, suffocation, or mechanical asphyxia to determine if the animal suffered. While there is no prescriptive answer to these questions, it is apparent that, because of physiological and anatomical differences between humans and animals, for some mechanisms of asphyxia, consciousness is maintained for longer periods and the onset of death is later in animals than that described for people. Veterinary pathologists must be cognizant that direct extrapolation from the medical forensic literature to animals may be incorrect. This article reviews the terminology, classification, mechanisms, and lesions associated with asphyxial deaths in companion animals and highlights significant comparative differences of the response to various types of asphyxia in animals and people.

Circumstances, Descriptive Characteristics, and Pathologic Findings in Dogs Suspected of Starving.

The authors reviewed the case circumstances, population characteristics, gross, and histopathologic findings in 40 cases of emaciated dogs with a suspected diagnosis of starvation. The dogs' estimated age ranged from 3 months to geriatric. Nineteen breeds were represented, including small-breed (n = 11), large-breed (n = 13), and pit bull-type (n = 16) dogs. The median body condition score was 1 out of 9 (Purina scale). Various diseases were identified as the cause of death in 7 dogs, while the cause of death in the other 33 dogs was starvation due to exogenous causes (SEC). Circumstances associated exclusively with SEC included being found in a vacated residence and death during temperature extremes or severe weather. Dogs with SEC did not differ significantly from diseased dogs in body condition score, sex, neuter status, or breed category (small, large, or pit bull type). Gross findings associated exclusively with SEC included severe hair matting and traumatic injuries. Diseased dogs had an empty stomach significantly more often than SEC dogs, which frequently had food and/or foreign material in the stomach. In 5 of the 7 cases where disease was the cause of death, disease involved the gastrointestinal tract. Gross and histopathologic changes commonly found in SEC and diseased dogs included the following: gross loss of muscle mass and absence of subcuticular fat; serous atrophy of omental, perirenal, epicardial, and bone marrow fat; atrophy of the liver, skin, thyroid gland, and testicle; gastric mucosal petechiae and ecchymoses; melena; and splenic hemosiderophages.