How to measure image quality in tissue-based diagnosis (diagnostic surgical pathology)

Background

Automated image analysis, measurements of virtual slides, and open access electronic measurement user systems require standardized image quality assessment in tissue-based diagnosis.

Aims

To describe the theoretical background and the practical experiences in automated image quality estimation of colour images acquired from histological slides.

Theory, material and measurements

Digital images acquired from histological slides should present with textures and objects that permit automated image information analysis. The quality of digitized images can be estimated by spatial independent and local filter operations that investigate in homogenous brightness, low peak to noise ratio (full range of available grey values), maximum gradients, equalized grey value distribution, and existence of grey value thresholds. Transformation of the red-green-blue (RGB) space into the hue-saturation-intensity (HSI) space permits the detection of colour and intensity maxima/minima. The feature distance of the original image to its standardized counterpart is an appropriate measure to quantify the actual image quality. These measures have been applied to a series of H&;E stained, fluorescent (DAPI, Texas Red, FITC), and immunohistochemically stained (PAP, DAB) slides. More than 5,000 slides have been measured and partly analyzed in a time series.

Results

Analysis of H&;E stained slides revealed low shading corrections (10%) and moderate grey value standardization (10 – 20%) in the majority of cases. Immunohistochemically stained slides displayed greater shading and grey value correction. Fluorescent stained slides are often revealed to high brightness. Images requiring only low standardization corrections possess at least 5 different statistically significant thresholds, which are useful for object segmentation. Fluorescent images of good quality only posses one singular intensity maximum in contrast to good images obtained from H&;E stained slides that present with 2 – 3 intensity maxima.

Conclusion

Evaluation of image quality and creation of formally standardized images should be performed prior to automatic analysis of digital images acquired from histological slides. Spatial dependent and local filter operations as well as analysis of the RGB and HSI spaces are appropriate methods to reproduce evaluated formal image quality.

     

First arrival dates of the Nightingale (<Emphasis Type="Italic">Luscinia megarhynchos</Emphasis>) to Central Croatia in the early 20th century and at the turn of the 21<Superscript>st</Superscript> century

Global climate change has important impacts on animal life-cycles. One of the responses to global warming is an earlier arrival time of many migratory bird species. The first arrival date of the Nightingale (Luscinia megarhynchos) in Central Croatia was studied for the periods of 1901–1917 and 1991–2005. Data were derived from the first spring observations and first capture data. A statistically significant advance of 11 ± 1.4 days was recorded. The difference in the mean April temperature between two study periods was significant, while changes in NAO winter index were not significant. Adequate data from the beginning of the 20th century exist; however, recent studies were done on a relatively short-term basis. Therefore, the analysis of two discrete datasets may help to fill the gaps in the knowledge about the climate change response.     

Molecular determinants of sphingomyelin specificity of a eukaryotic pore-forming toxin

Sphingomyelin (SM) is abundant in the outer leaflet of the cell plasma membrane, with the ability to concentrate in so-called lipid rafts. These specialized cholesterol-rich microdomains not only are associated with many physiological processes but also are exploited as cell entry points by pathogens and protein toxins. SM binding is thus a widespread and important biochemical function, and here we reveal the molecular basis of SM recognition by the membrane-binding eukaryotic cytolysin equinatoxin II (EqtII). The presence of SM in membranes drastically improves the binding and permeabilizing activity of EqtII. Direct binding assays showed that EqtII specifically binds SM, but not other lipids and, curiously, not even phosphatidylcholine, which presents the same phosphorylcholine headgroup. Analysis of the EqtII interfacial binding site predicts that electrostatic interactions do not play an important role in the membrane interaction and that the two most important residues for sphingomyelin recognition are Trp(112) and Tyr(113) exposed on a large loop. Experiments using site-directed mutagenesis, surface plasmon resonance, lipid monolayer, and liposome permeabilization assays clearly showed that the discrimination between sphingomyelin and phosphatidylcholine occurs in the region directly below the phosphorylcholine headgroup. Because the characteristic features of SM chemistry lie in this subinterfacial region, the recognition mechanism may be generic for all SM-specific proteins.     

Quantitative analysis of basal dendritic tree of layer III pyramidal neurons in different areas of adult human frontal cortex

Large long projecting (cortico-cortical) layer IIIc pyramidal neurons were recently disclosed to be in the basis of cognitive processing in primates. Therefore, we quantitatively examined the basal dendritic morphology of these neurons by using rapid Golgi and Golgi Cox impregnation methods among three distinct Brodmann areas (BA) of an adult human frontal cortex: the primary motor BA4 and the associative magnopyramidal BA9 from left hemisphere and the Broca's speech BA45 from both hemispheres. There was no statistically significant difference in basal dendritic length or complexity, as dendritic spine number or their density between analyzed BA's. In addition, we analyzed each of these BA's immunocytochemically for distribution of SMI-32, a marker of largest long distance projecting neurons. Within layer IIIc, the highest density of SMI-32 immunopositive pyramidal neurons was observed in associative BA9, while in primary BA4 they were sparse. Taken together, these data suggest that an increase in the complexity of cortico-cortical network within human frontal areas of different functional order may be principally based on the increase in density of large, SMI-32 immunopositive layer IIIc neurons, rather than by further increase in complexity of their dendritic tree and synaptic network.     

Acute appendicitis and ileal perforation with a toothpick treated by laparoscopy

A 69-year-old man underwent an emergency laparoscopic procedure after the acute appendicitis diagnosis has been established. Laparoscopic exploration showed inflamed appendix and perforation of terminal ileum with a swallowed part of the wooden toothpick. The treatment consisted of typical laparoscopic appendectomy and laparoscopic removal of the foreign body, followed by laparoscopic closure of the perforation site and lavage of the abdominal cavity. The postoperative course was uneventful and the patient was discharged from the hospital on day 3 after the operation.     

Quantitative trait, genetic, environmental, and geographical distances among populations of the C4 grass Trachypogon plumosus in Neotropical savannas

Venezuelan savannas are exposed to land‐use changes and biological invasions which compromise their persistence and function. The native C₄ grass Trachypogon plumosus is the most important component of the savannas under diverse combinations of climate and soils, suggesting substantial interpopulation variation. We examined quantitative traits and isozyme variation of nine populations of this grass and related these estimates to geographical and environmental features of sampled locations. Isozyme diversity estimates were based on 10 polymorphic enzyme systems whereas 21 quantitative traits, from field and controlled growth conditions, were evaluated. Distance matrices for quantitative traits, isozyme, geographical and environmental data were subjected to clustering analysis. Correspondence between quantitative trait distance and genetic distance, and their association to geographical and environmental distances were analysed with Mantel tests. All quantitative traits differed significantly among populations. The average Q_ST calculated for eight quantitative traits measured in the greenhouse was 0.157. Isozyme diversity differed significantly among populations. About 28% of total isozyme variation occurred among populations. Significant positive associations were detected between environmental, quantitative field traits, and geographical distance as well as between the later and genetic distances. Genetic distances did not correspond significantly with quantitative traits nor did environmental distances. Ecologically meaningful associations were detected between field quantitative traits, environmental, and geographical data using cluster analysis. Our results support the hypothesis that processes of the neutral type are mainly responsible for the variation patterns observed in T. plumosus populations in Venezuelan savannas. Variation observed for quantitative traits among populations seems to be due to the effect of environmental conditions on phenotypically plastic traits, and not the result of directional selection favouring different phenotypes in different populations.     

Amplification of bacitracin transporter genes in the bacitracin producing Bacillus licheniformis

We have amplified the previously cloned and sequenced genes of the bacitracin exporter (bcr), a member of the ATP-binding transport protein family, within the chromosome of the bacitracin producing Bacillus licheniformis. Amplification of the transporter genes was followed by greatly increased bacitracin resistance. Antibiotic production was enhanced at a low level of bcr genes amplification. An enlarged increase in the copy number of the bcr genes negatively affects the overall growth of bacteria.     

A quantitative analysis of kinase inhibitor selectivity

Kinase inhibitors are a new class of therapeutics with a propensity to inhibit multiple targets. The biological consequences of multi-kinase activity are poorly defined, and an important step toward understanding the relationship between selectivity, efficacy and safety is the exploration of how inhibitors interact with the human kinome. We present interaction maps for 38 kinase inhibitors across a panel of 317 kinases representing >50% of the predicted human protein kinome. The data constitute the most comprehensive study of kinase inhibitor selectivity to date and reveal a wide diversity of interaction patterns. To enable a global analysis of the results, we introduce the concept of a selectivity score as a general tool to quantify and differentiate the observed interaction patterns. We further investigate the impact of panel size and find that small assay panels do not provide a robust measure of selectivity.     

Soil seed bank and the effect of needle litter layer on seedling emergence in a tropical pine plantation

The soil seed bank is the basis for community establishment and permanence and plays a primary role in natural restoration of degraded or altered ecosystems. As part of a restoration project, this study aimed to quantify the soil seed bank and to evaluate the effect of the needle litter layer on seedling emergence. Soil samples from a pine plantation were collected at random in the field and set to germinate in a greenhouse. Half of them were covered by a 6cm layer of dead pine needles simulating field conditions. In the field, 20 x 20cm plots were established, half were left intact and half were cleaned from the litter needles. All four treatments had 15 replicates and seedling emergence was recorded during six months. Soil seed bank density was 1 222/m2 from 17 morphotypes. In the field, the number of morphotypes and seedlings was only 9% and 6% respectively, of those emerged in the greenhouse, possibly due to watering and lack of predation in the latter. In both cases, herbs and graminoids were the dominant emerging seedlings, making up to 70-90% of the total. The needle layer didn't prevent seeds from reaching the soil but strongly reduced (> 50%) seedling emergence, although high variability within treatments resulted in no statistically significant differences. These results show that the needle layer hinders germination and/or emergence of seedlings from the seed bank. Its removal may be a recommended technique to accelerate natural restoration in pine plantations.     

Preserved postischemic heart function in sucrose-fed type 2 diabetic OLETF rats

Cardiovascular disease is one of the most important causes of morbidity and mortality in diabetes mellitus, but there has been controversy over functional impairment of diabetic hearts and their tolerance to ischemia. We studied ischemic heart function in type 2 diabetic rats with different degrees of hyperglycemia and its relationship with cardiac norepinephrine release. Otsuka Long-Evans Tokushima Fatty rats (OLETF) and age-matched Long-Evans Tokushima Otsuka normal rats (LETO) were used. One group of OLETF rats was given 30% sucrose in drinking water (OLETF-S). Hearts were isolated and perfused in a working heart preparation and subjected to 30 min ischemia followed by 40 min reperfusion at age of 12 months. Hemodynamics and coronary norepinephrine overflow were examined. Fasting plasma glucose in OLETF increased markedly at 12 months and sucrose administration exacerbated hyperglycemia in diabetic rats (LETO 6.6 +/- 0.5, OLETF 8.3 +/- 0.7, OLETF-S 15.0 +/- 1.7 mmol/L, P < 0.01). Basic cardiac output in OLETF was decreased as compared with LETO and OLETF-S (LETO 29.4 +/- 2.5, OLETF 24.0 +/- 2.4, OLETF-S 27.0 +/- 0.9 ml/min/g, P < 0.05) and remained very low after ischemia, while in OLETF-S it was well preserved (OLETF 4.2 +/- 2.1, OLETF-S 13.7 +/- 2.6 ml/min/g, P < 0.01). Correspondently, cardiac norepinephrine released during ischemia and reperfusion was lower in OLETF-S (OLETF 2.3 +/- 1.0, OLETF-S 0.7 +/- 0.1 pmol/ml, P < 0.01). Thus, OLETF hearts were more vulnerable to ischemia but sucrose feeding rendered their hearts resistant to ischemia. Less norepinephrine release may play a role in preventing postischemic functional deterioration in sucrose-fed diabetic hearts.