Development and Validation of an Image Analysis System for the Measurement of Cell Proliferation in Mammary Glands of Rats.

Reliable detection and measurement of cell proliferation are essential in the preclinical assessment of carcinogenic risk of therapeutics. In this context, the assessment of mitogenic potential on mammary glands is crucial in the preclinical safety evaluation of novel insulins. The existing manual counting is time-consuming and subject to operator bias. To standardize the processes, make it faster, and resistant to errors, we developed a semiautomated image analysis system (CEPA software, which is open-source) for counting of proliferating cells in photomicrographs of mammary gland sections of rats labeled with Ki-67. We validated the software and met the predefined targets for specificity, accuracy, and reproducibility. In comparison to manual counting, the respective mean differences in absolute labeling indices (LIs) for CEPA software were 3.12% for user 1 and 3.05% for user 2. The respective regression analysis revealed a good correlation between the CEPA software user and manual counting. Moreover, the CEPA software showed enhanced reproducibility between independent users. The interuser variability is centered around 0 and the absolute difference was about 0.53% LI. Based on validation data, our software has superiority to the manual counting and is a valid and reliable tool for the routine analysis of cell proliferation in mammary glands from rats exposed to insulin analogs.

Testing an Alternative Method for Estimating the Length of Fungal Hyphae Using Photomicrography and Image Processing.

This study aimed to develop and test an unbiased and rapid methodology to estimate the length of external arbuscular mycorrhizal fungal (AMF) hyphae in soil. The traditional visual gridline intersection (VGI) method, which consists in a direct visual examination of the intersections of hyphae with gridlines on a microscope eyepiece after aqueous extraction, membrane-filtration, and staining (e.g., with trypan blue), was refined. For this, (i) images of the stained hyphae were taken by using a digital photomicrography technique to avoid the use of the microscope and the method was referred to as "digital gridline intersection" (DGI) method; and (ii), the images taken in (i) were processed and the hyphal length was measured by using ImageJ software, referred to as the "photomicrography-ImageJ processing" (PIP) method. The DGI and PIP methods were tested using known grade lengths of possum fur. Then they were applied to measure the hyphal lengths in soils with contrasting phosphorus (P) fertility status. Linear regressions were obtained between the known lengths (Lknown) of possum fur and the values determined by using either the DGI (LDGI) (LDGI = 0.37 + 0.97 × Lknown, r2 = 0.86) or PIP (LPIP) methods (LPIP = 0.33 + 1.01 × Lknown, r2 = 0.98). There were no significant (P > 0.05) differences between the LDGI and LPIP values. While both methods provided accurate estimation (slope of regression being 1.0), the PIP method was more precise, as reflected by a higher value of r2 and lower coefficients of variation. The average hyphal lengths (6.5-19.4 m g-1) obtained by the use of these methods were in the range of those typically reported in the literature (3-30 m g-1). Roots growing in P-deficient soil developed 2.5 times as many hyphae as roots growing in P-rich soil (17.4 vs 7.2 m g-1). These tests confirmed that the use of digital photomicrography in conjunction with either the grid-line intersection principle or image processing is a suitable method for the measurement of AMF hyphal lengths in soils for comparative investigations.

The use of guideline images to improve histological estimation of hepatic steatosis.

BACKGROUND & AIMS: Guideline images of specific fat proportionate area (FPA) percentages have recently been published to aid the histological assessment of liver steatosis as subjective estimates of FPA are usually overestimated. To assess, (i) the effect of guideline images on accuracy and concordance of estimated FPA (eFPA), (ii) experience of steatosis grading systems on eFPA, (iii) the effect of magnification on assessment of FPA (iv) and produce a range of guideline images at x4 objective magnification (OM). METHODS: Two circulations of sample images (C1 and C2) were circulated to UK liver external quality assessment histopathology scheme members who were asked to independently evaluate steatosis. Each circulation consisted of 15 images taken at both x20 and x4OM representing the full range of steatosis. C1 was distributed first, then C2 with guideline images of FPA 6 weeks later. RESULTS: Participants overestimated FPA in C1. In C2, there was significant improvement in accuracy (P < 0.001) of eFPA for sample images with mFPA >5%. Concordance of x4OM eFPA was substantial in both circulations (C1 K = 0.878, C2 K = 0.724). CONCLUSION: The tendency to overestimate eFPA has been corroborated and can be largely corrected with the use of guideline images (without needing digital image analysis). There is a need to redefine steatosis grades that are clinically significant and validated using an accurate quantification of steatosis.

The dark side of photomicrographs in pathology reports: liability and practical concerns hidden from view.

The rapid evolution of digital imaging has facilitated the ability to include photomicrographs in pathology reports. Although these pictures may seem to be an informative accompaniment to the written report, there are many problems raised by the images, which are not generally recognized. These include lack of quality standards, selection of representative images, and liability implications, which are addressed by well-established legal precedent. For dermatologists there is no such thing as a casual interest in a photomicrograph on a report, for it acts to distribute a share of liability by obligating the clinician to interpret the image properly. The risk management ramifications of these unintended consequences should be strongly considered by clinicians who favor the receipt of photomicrographic images in their pathology reports.

Differentiation of bone and soft tissues in formalin-fixed, paraffin-embedded tissue by using methylene blue/acid fuchsin stain.

OBJECTIVE: To find a staining method for formalin-fixed, paraffin-embedded tissue that would distinguish bone from surrounding soft tissues, including muscle, periosteal tissue and bone marrow. STUDY DESIGN: A variety of stains were tested and compared with hematoxylin-eosin. The potential value of any given stain was evaluated based on its ability to stain bone and soft tissues different colors or shades that could be readily identified in photomicrographs. Stains were evaluated using both endochondral (tibia) and intramembranous bone (calvaria) samples. RESULTS: In contrast to standard hematoxylin-eosin stain, which stains both bone and soft tissues pink, the methylene blue/acid fuchsin stain demonstrates remarkable contrast between bone and other tissues. Methylene blue/acid fuchsin stained bone bright pink and the surrounding soft tissues blue-purple. CONCLUSION: In addition to the superior staining properties of methylene blue/acid fuchsin, other benefits of this stain include its stability, ease of use and low cost. This stain has many potential applications in the study of erosive bone disease in humans and also in animal models for research.

Is retinal photography useful in the measurement of stroke risk?

BACKGROUND: The retinal microcirculation can be viewed non-invasively to give a unique perspective of the cerebral microcirculation in vivo. Studying pathological changes of retinal blood vessels (microaneurysms, retinal haemorrhages, and retinal arteriolar narrowing) may help to understand the causes of various cerebrovascular disorders. Retinal photography provides such an opportunity. RECENT DEVELOPMENTS: Several recent studies have shown that retinal microvascular changes are reliably documented by retinal photographs. These retinopathy changes seem to be fairly common in the general population, even in people without hypertension or diabetes. Retinopathy is related to incident clinical stroke and stroke mortality and to MRI-defined subclinical cerebral white-matter lesions and cerebral atrophy, independent of blood pressure, diabetes, and other cerebrovascular risk factors. WHERE NEXT?: Retinal microvascular abnormalities seem to be markers of concomitant cerebral microangiopathy, and retinal photography may be useful for the investigation of microvascular disorders of the brain in clinical and epidemiological settings. Future research should be aimed at the development of standardised photographic methods for the assessment of retinal microvascular changes, the replication of these findings in other populations and in people with other cerebrovascular disorders, and the examination of the increased accuracy of stoke-risk stratification given by retinal photography

Digital imaging guidelines for pathology: a proposal for general and academic use.

Digital imaging is an inexpensive and widely available tool that is used by most pathologists in patient reports, education, publication, diagnosis, and data archival. Its popularity is due, in part, to the ease of modifying, storing, enhancing, and annotating images. Since digital manipulation is essentially undetectable in the final product, it poses the potential risk for fraudulent manipulation and heightens the possibility of unintentional misrepresentation. In an attempt to ensure a high degree of uniformity and quality, and to create a professional standardization amongst pathologists, digital imaging guidelines are proposed for use in general and academic practice.

Transfer of biological traces in cases of hanging and ligature strangulation.

In hanging and ligature strangulation, the noose mostly causes a mark or groove which is formed partly by compression of the skin and partly by abrasion with loss of the upper epidermal layers. The horny scales abraded from the neck may be transferred to the strangulation device or to the interposed textiles where they are sometimes visible at stereomicroscopic examination or even to the naked eye as silver-grey particles. The morphologic features of the epidermal transfer due to hanging and ligature strangulation is demonstrated by 14 case examples. The biological traces may be sufficient for comparative DNA typing by means of PCR-based methods. In 9 out of the 14 cases, genomic DNA typing was successful. Analysis of mtDNA succeeded in another two cases, although genomic DNA could not be detected. Beside the accumulation of solid epidermic particles the paper describes deposition of serous and fatty tissue fluid at the ligature (mainly adjacent to skin ridges).

Evaluating the accuracy of different sperm counting chambers by performing strict counts of photographed beads.

PURPOSE: To suggest a method for evaluating the accuracy of different kinds of sperm counting chambers by eliminating errors concerned with human skills or semen properties. In this method, various concentrations were prepared from stocks of commercially available latex beads (Accu-beads, Hamilton-Thorn Research, Beverly, MA). Samples from identical preparations were loaded into different types of chambers, namely, hemocytometer (Neubauer, improved double, Superior Ltd, Germany), Makler (Sefi Medical Instruments, Haifa, Israel) and Horwell (Horwell Ltd, London, UK). Beads were counted by both direct microscopic observations and by strict scanning of their photographed images. RESULTS: In all cases, counts by direct observation were about 5% higher than strict counts of the same photographed beads. Counting photographed beads showed high reproducibility (average CV of 5.1%) between samples in the two wells of the hemocytometer. Counts of photographed beads, sampled from identical stocks, were on average slightly lower in the Makler chamber (20.7 x 10(6)/ml) and much higher in the Horwell chamber (47.4 x 10(6)/ml) than counts in the hemocytometer (21.5 x 10(6)/ml). Samples from three different batches of Accubeads revealed slight variation in counts between the batches and an average concentration of 11% above the number indicated on the commercial product. CONCLUSIONS; A technique that combined loading latex beads from identical stock into various chambers, proper covering of the tested samples and strict counting of photographed beads provided precise and reproducible results. By eliminating most errors related to human skills and semen properties, this method is suitable for evaluating the accuracy of counting chambers.

Improving pictures in dermatopathology.

We have found that a remarkable improvement in the resolution of the details and in the richness of halftones can be obtained using 4" x 5" black-and-white professional films specifically prepared for scientific and documentary photography. Through modification of the developing time and developer dilution, a close control of the contrast and gray tones is also possible. Using this large-format film, a contact print can be made and top-quality pictures obtained even for a subject as difficult as skin pathology.

Age-related changes of the human skin surface microrelief.

In the present study we describe an original semi-automated image analysis method to quantitate the changes of the geometric properties of cutaneous microrelief with age. The skin surface is composed of very fine lines intersecting each other and forming polygons. The number, average size, perimeter and equivalent diameter of these polygons were quantified on replicas of 190 persons, 101 females and 89 males, from 6 months to 95 years of age. A good correlation was found between the age of the subjects and these four parameters. The evolution of these parameters was not linear but could be best described by a polynomial equation of fourth order for the number of polygons per field, and by an exponential equation for the three other parameters: average surface, perimeter and equivalent diameter of the polygons. A few details of the evolution with age of the skin surface patterns were different for males and females.

Technique for preparing high-quality microphotographs by fluorescence microscopy.

Simple techniques resulting in high-quality microphotographs by fluorescence microscopy are described for the preparation of Salmonella cultures and slides.