Use of colloidal gold in diagnostic surgical pathology

Colloidal gold immuno-electron microscopy is a powerful tool for defining antigenicity at the subcellular level. Such studies permit correlation with cell fractionation studies. They also allow one to assess the specificity of a particular antibody. The most useful reagent for immuno-electron microscopy is colloidal gold stabilized by a binding protein, either staphylococcal protein A or immunoglobulin. This method permits highly discrete labeling, and the system is useful for most antibodies used in diagnostic pathology.     

Application of alternative fixatives to formalin in diagnostic pathology

Fixation is a critical step in the preparation of tissues for histopathology. The objective of this study was to investigate the effects of different fixatives vs formalin on proteins and DNA, and to evaluate alternative fixation for morphological diagnosis and nucleic acid preservation for molecular methods. Forty tissues were fixed for 24 h with six different fixatives: the gold standard fixative formalin, the historical fixatives Bouin and Hollande, and the alternative fixatives Greenfix, UPM and CyMol. Tissues were stained (Haematoxylin-Eosin, Periodic Acid Schiff, Trichromic, Alcian-blue, High Iron Diamine), and their antigenicity was determined by immunohistochemistry (performed with PAN-CK, CD31, Ki-67, S100, CD68, AML antibodies). DNA extraction, KRAS sequencing, FISH for CEP-17, and flow cytometry analysis of nuclear DNA content were applied. For cell morphology the alternative fixatives (Greenfix, UPM, CyMol) were equivalent to formalin. As expected, Hollande proved the best fixative for morphology. The morphology obtained with Bouin was comparable to that with formalin. Hollande was the best fixative for histochemistry. Bouin proved equivalent to formalin. The alternative fixatives were equivalent to formalin, although with greater variability in haematoxylin-eosin staining. It proved possible to obtain immunohistochemical staining largely equivalent to that following formalin-fixation with the following fixatives: Greenfix, Hollande, UPM and CyMol. The tissues fixed in Bouin did not provide results comparable to those obtained with formalin. The DNA extracted from samples fixed with alternative fixatives was found to be suitable for molecular analysis.     

Histology,imaging and new diagnostic work-flows in pathology

Introduction

Since their introduction in 1999, fully automated, high speed, high-resolution whole slide imaging devices have become increasing more reliable, fast and capable. While by no means perfect, these devices have evolved to a point where one can consider placing them in a pre-diagnostic role in a clinical histology lab.

Methods

At the Massachusetts General Hospital, we are running a pilot study placing high end WSI devices in our main clinical histology lab (after the cover slipper and before slides are sent to the pathologist) to examine the requirement for both the machine and the laboratory.

Results

Placing WSI systems in the clinical lab stresses the system in terms of reliability and throughput. Significantly however, success requires significant modification to the lab workflow. It is likely laboratories need to move from manual, large batch processes to increasingly automated, continuous flow (or mini-batch) processes orchestrated by the LIS using bar coding to track and direct slides, and incorporating the decision to image into the specimen type and the histology orders. Furthermore, image quality, capture speed and reliability are functions of the quality of the histology presented to the WSI devices.

Conclusion

Imaging in pathology does not begin in a WSI robot but in the grossing room and in the histology lab. As more and more imaging devices are placed in histology lab, the inter-relationships histology and pathology imaging will become increasing understood.

     

Histochemistry: historical development and current use in pathology

We describe the history of the histochemical stains that contributed most to the development of modern pathology during the last two centuries. Histochemical stains are presented in a list, which provides the essential information about year, country and main use of each to enable the reader to follow the chronological and geographical history of histochemistry. In addition to the historical evaluation of histochemistry development, we investigate how many classical histochemical stains survive in a modern laboratory of pathology and how often they are used for diagnostic practice compared to immunohistochemical (IHC) techniques. A ratio of about one histochemical reaction to 13 IHC reactions was tabulated. Finally, our data make it possible to define different cultural approaches to the terminology of histochemical and IHC stains: the former were based on eponyms, which link the stain with the name of its inventor, while the latter use a more impersonal biological terminology.     

The biological effects of terahertz laser radiation as a fundamental premise for designing diagnostic and treatment methods

This review considers experimental research of the biological effects of terahertz radiation and their results, which provide a basis for medical application of laser terahertz radiation. Therapeutic and diagnostic uses of laser terahertz radiation are described with reference to preliminary basic investigations at the organismic, cell, and molecular levels. It is emphasized that preliminary basic studies of this kind are of importance as another avenue of research to design diagnostic and therapeutic techniques in addition to terahertz imaging and spectroscopy.

     

Cause and effect considerations in diagnostic pathology and pathology phenotyping of genetically engineered mice (GEM)

Over the next several decades, biology is embarking on its most ambitious project yet: to annotate the human genome functionally, prioritizing and focusing on those genes relevant to development and disease. Model systems are fundamental prerequisites for this task, and genetically engineered mice (GEM) are by far the most accessible mammalian system because of their anatomical, physiological, and genetic similarity to humans. The scientific utility of GEM has become commonplace since the technology to produce them was established in the early 1980s. Conceptually, however, an efficiently coordinated high-throughput approach that permits correlation between newly discovered genes, functional properties of their protein products, and biological relevance of these products as drug targets has yet to be established. The discipline of veterinary anatomical pathology (hereafter referred to as pathology) is not immune to this requirement for evolution and adaptation, and to address relationships and tissue consequences between tens of thousands of genes and their cognate proteins, novel interdisciplinary technologies and approaches must emerge. Although many of the techniques of pathology are well established, in the context of pathology's contribution to functional annotation of the genome, several conceptually important and unresolved issues remain to be addressed. While an ever-increasing arsenal of genetic and molecular tool-sets are available to evaluate and understand the function of genes and their pathophysiological mechanisms, pathology will continue to play an essential role in confirming cause and effect relationships of gene function in development and disease. This role will continue to be dependent on keen observation, a systematic but disciplined approach, expert knowledge of strain-dependent anatomical differences and incidental lesions, and relevant tissue-based evidence. Miniaturization and high-throughput adaptation of these methods must also continue so that they can complement parallel phenotyping efforts, provide pathology-based data in pace with concurrent phenotyping efforts, and continue to find new utility in the collective effort of functional annotation.     

Toxoplasmosis in pregnancy: evaluation of diagnostic methods

Toxoplasmosis in pregnancy is usually subclinic or associated with non specific symptoms. Diagnosis and timing of infection are usually based on serological tests. In this short review we tried to summarize the serological patterns we can encounter and to discuss the interpretation of test results.     

Sequence of diagnostic methods in destructive lung diseases

Of 240 examinees with different pulmonary pathology 100 verified cases of destructive diseases were selected. Nine diagnostic methods were applied to them. The effectiveness of each method was evaluated, and 3 strategies of the diagnosis of pulmonary destructive diseases were proposed. The cybernetic mathematical apparatus was used.     

Deficiency of current methods in assaying endochitinase activity

Since chitin is degraded by a combination of both endo- and exochitinases, it is likely that both enzymes will be present in a crude extract. Currently used substrates for detecting endochitinase activity suffer from the fact that they could easily be digested by the contaminating exochitinase, thus giving either a false-positive or an inaccurate reading of the endochitinase activity. Using Photorhabdus luminescens, a bacterium symbiotically associated with insect-parasitic nematode Heterorhabditis bacteriophora as an exemplary system, we have identified these two chitinases by a simple "fluorimetric zymography" procedure. The exochitinase is a metalloenzyme and its activity is inhibited by 1,10-phenanthroline. Once the exochitinase activity is detected in a crude extract, its contribution must be eliminated before accurate determination of the endochitinase activity can be carried out. Specific properties of these enzymes including the pH activity profile, the requirement of metal ions for activity, and the molecular weight of the enzymes are among the factors to be considered in developing assaying procedures for endochitinase.     

Medical diagnostic radiation exposures and risk of gliomas

High-dose ionizing radiation is an established risk factor for glioma, but it remains unknown whether moderate- and low-dose radiation increase glioma risk. In this analysis, we assessed the evidence that self-reported exposures to diagnostic ionizing radiation, including computerized tomography (CT) scans, is associated with increased risk of adult glioma. While no independent association was observed for CT scans alone (3+ scans compared to none P = 0.08 and 1-2 scans compared to none P = 0.68), our findings suggest an increased risk of adult gliomas with cumulative exposure to three or more CT scans to the head and neck region (OR = 1.97, 95% CI: 0.92-4.23) limited to those who reported a family history of cancer: the P value for the interaction between having three or more CT scans and family history of cancer was 0.08. The stratum-specific adjusted OR for those with family history of cancer was more than three times that for the sub-group without family history of cancer. While there is some potential for symptom-related bias, one might expect this to be present for all diagnostic procedures rather than specific to one procedure. The interaction between CT scans and glioma with family history of cancer supports the biological plausibility of our findings, because similar results have been found for breast cancer and radiation. This observational data will increase awareness about potential risks associated with CT scans and the need to minimize the use of unnecessary examinations.     

PCR diagnostic methods for Ascosphaera infections in bees

Fungi in the genus Ascosphaera are the causative agents of chalkbrood, a major disease affecting bee larval viability. Identification of individual Ascosphaera species based on morphological features has been difficult due to a lack of distinguishing characteristics. Most identifications are based on the size and shape of the ascomata, spore balls and conidia. Unfortunately, much overlap occurs in the size of these structures, and some Ascosphaera species will not produce sexual structures in vitro. We report a quick and reliable diagnostic method for identifying Ascosphaera infections in Megachile bees (leafcutting bees) using PCR markers that employ genus-specific primers for Ascosphaera, and species-specific primers for species known to be associated with Megachile spp. Using these methods, species identifications can be performed directly on bees, including asymptomatic individuals. Furthermore, the PCR markers can detect co-infections of multiple Ascosphaera species in a single host. We also identified a marker for Ascosphaera apis, the predominant cause of chalkbrood in Apis mellifera, the honey bee. Our diagnostic methods eliminate the need for culturing samples, and could be used to process a large number of field collected bee larvae.     

Comparison of the diagnostic methods used in maxillofacial trauma

A prospective study was performed to evaluate the diagnostic methods used in acute maxillofacial trauma. Clinical examination, routine facial x-rays with linear tomography, and computer tomography were compared in 49 patients. Computer tomography was found to be the most accurate test in the diagnosis of facial bone injury, especially complex fractures. Computer tomography also provided valuable information regarding soft-tissue injury of the face. The radiation doses of linear tomography and computer tomography were calculated to be below the level known to cause cataract formation. Computer tomography is a safe, reliable adjunct in the diagnosis of acute maxillofacial trauma and should be strongly considered after initial screening measures are completed.