Keerthana Prasad

Image Analysis Tools for Evaluation of Microscopic Views of Immunohistochemically Stained Specimen in Medical Research---a Review

The aim of this study is to review the methods being used for image analysis of microscopic views of immunohistochemically stained specimen in medical research. The solutions available range from general purpose software to commercial packages. Many studies have developed their own custom written programs based on some general purpose software available. Many groups have reported development of computer aided image analysis programs aiming at obtaining faster, simpler and cheaper solutions. Image analysis tools namely Aperio, Lucia, Metaview, Metamorph, ImageJ, Scion, Adobe Photoshop, Image Pro Plus are also used for evaluation of expressions using immunohistochemical staining. An overview of such methods used for image analysis is provided in this paper. This study concludes that there is good scope for development of freely available software for staining intensity quantification, which a medical researcher could easily use without requiring high level computer skills.

Evidence-based assessment of antiosteoporotic activity of petroleum-ether extract of Cissus quadrangularis Linn. on ovariectomy-induced osteoporosis

The increasing incidence of postmenopausal osteoporosis and its related fractures have become global health issues in the recent days. Postmenopausal osteoporosis is the most frequent metabolic bone disease; it is characterized by a rapid loss of mineralized bone tissue. Hormone replacement therapy has proven efficacious in preventing bone loss but not desirable to many women due to its side-effects. Therefore we are in need to search the natural compounds for a treatment of postmenopausal symptoms in women with no toxic effects. In the present study, we have evaluated the effect of petroleum-ether extract of Cissus quadrangularis Linn. (CQ), a plant used in folk medicine, on an osteoporotic rat model developed by ovariectomy. In this experiment, healthy female Wistar rats were divided into four groups of six animals each. Group 1 was sham operated. All the remaining groups were ovariectomized. Group 2 was fed with an equivolume of saline and served as ovariectomized control (OVX). Groups 3 and 4 were orally treated with raloxifene (5.4 mg/kg) and petroleum-ether extract of CQ (500 mg/kg), respectively, for 3 months. The findings were assessed on the basis of animal weight, morphology of femur, and histochemical localization of alkaline phosphatase (ALP) (an osteoblastic marker) and tartrate-resistant acid phosphatase (TRAP) (an osteoclastic marker) in upper end of femur. The study revealed for the first time that the petroleum-ether extract of CQ reduced bone loss, as evidenced by the weight gain in femur, and also reduced the osteoclastic activity there by facilitating bone formation when compared to the OVX group. The osteoclastic activity was confirmed by TRAP staining, and the bone formation was assessed by ALP staining in the femur sections. The color intensity of TRAP and ALP enzymes from the images were evaluated by image analysis software developed locally. The effect of CQ was found to be effective on both enzymes, and it might be a potential candidate for prevention and treatment of postmenopausal osteoporosis. The biological activity of CQ on bone may be attributed to the phytogenic steroids present in it.

Image Analysis Approach for Development of a Decision Support System for Detection of Malaria Parasites in Thin Blood Smear Images

This paper describes development of a decision support system for diagnosis of malaria using color image analysis. A hematologist has to study around 100 to 300 microscopic views of Giemsa-stained thin blood smear images to detect malaria parasites, evaluate the extent of infection and to identify the species of the parasite. The proposed algorithm picks up the suspicious regions and detects the parasites in images of all the views. The subimages representing all these parasites are put together to form a composite image which can be sent over a communication channel to obtain the opinion of a remote expert for accurate diagnosis and treatment. We demonstrate the use of the proposed technique for use as a decision support system by developing an android application which facilitates the communication with a remote expert for the final confirmation on the decision for treatment of malaria. Our algorithm detects around 96% of the parasites with a false positive rate of 20%. The Spearman correlation r was 0.88 with a confidence interval of 0.838 to 0.923, p < 0.0001.

Microanatomical and immunohistochemical study of the human radial nerve at the antecubital fossa

BACKGROUND Poor prognosis of radial nerve repair in elderly patients may be due to changes in intraneural anatomy with age. Also, chances of Complex Regional Pain Syndrome-Type I (CRPS-I) following radial nerve injury are comparatively high. The present study is to find the fascicular pattern of radial nerve (at antecubital fossa), microanatomic morphometric characteristics of its connective tissue components and changes with age and study of intraneural sympathetic fiber content. METHODS Twenty human (21-87 years) cadaveric radial nerves have been collected from antecubital fossa and the study has been performed at magnifications (10x, 20x and 40x objective) after routine histological (hematoxylin & eosin stain) processing was done for morphometric analysis (total cross-sectional, fascicular and non-fascicular area) and immunohistochemical (tyrosine hydroxylase) processing for sympathetic fibers. RESULTS The radial nerve is of a polyfascicular type with a grouped pattern of nerve fascicular distribution. The number of fascicles range from 9 to 17, whereas the number of fascicles per square millimeter of a cross-sectional area is 1.95. In elderly cases, there is significant increase in total radial nerve cross-sectional area due to an increase in its non-fascicular connective tissue area and excessive adipose tissue deposition in interfascicular domains. The average sympathetic fiber area is 0.046 mm(2) without definite relationship to age. CONCLUSION There is an increase in total nerve cross-sectional area of the radial nerve in elderly cases. There is no relationship of sympathetic content to age. Our study makes an attempt to build a normal data base for radial nerve which might be helpful during the application of diagnostic procedures.

Histomorphometric and sympathetic innervation of the human internal thoracic artery

INTRODUCTION: Internal thoracic artery (ITA) is an established arterial graft for the coronary artery by‐pass surgery. Special micro‐anatomical features of the ITA wall may protect it from age related pathological changes. One of the complications seen after coronary artery bypass grafting is vasospasm. Sympathetic nerves may be involved in vasospasm. OBJECTIVE: To ascertain the sympathetic innervation of the internal thoracic artery and to assess the effect of aging on this artery by histomorphometry. METHOD: Fifty‐four human internal thoracic artery samples were collected from 27 cadavers (19 male and 8 female) with ages of 19 to 83 years. Samples were divided into three age groups: G1, 19–40 years; G2, 41–60 years; G3, ≥61 years. Sections (thickness 5 µm) of each sample were taken and stained with hematoxylin–eosin and Verhoeff–Van Gieson stains. Five of fifty‐four samples were processed for tyrosine hydroxylase immunostaining. RESULTS: The thickness of the tunica intima was found to be constant in all age groups, whereas the thickness of the tunica media decreased in proportion to age. Verhoeff–Van Gieson staining showed numerous elastic laminae in the tunica media. Significant differences (p<0.0001) in the number of elastic laminae were found between G1 with G2 cadavers, between G2 and G3 cadavers and between G3 and G1 cadavers. Tyrosine hydroxylase immunostaining demonstrated sympathetic fibers, located mainly in the tunica adventitia and the adventitia–media border. The sympathetic nerve fiber area and sympathetic index were found to be 0.0016 mm2 and 0.012, respectively. DISCUSSION: Histology of the ITA showed features of the elastic artery. This may be associated with lower incidence of Atherosclerosis or intimal hyperplasia in ITA samples even in elderly cases. Low sympathetic index (0.012) of ITA may be associated with fewer incidences of sympathetic nervous systems problems (vasospasm) of the ITA. CONCLUSION: Sympathetic nerve fibers are present in the adventitia of the internal thoracic artery. This is an elastic artery, although anatomically it is considered to be medium‐sized. The sympathetic index may be used for analysis of sympathetic nerve fiber‐related problems of the internal thoracic artery.

Applications of 'TissueQuant'- A color intensity quantification tool for medical research

This paper demonstrates the use of TissueQuant - an image analysis tool for quantification of color intensities which was developed for use in medical research where the stained biological specimen such as tissue or antigen needs to be quantified. TissueQuant provides facilities for user interaction to choose and quantify the color of interest and its shades. Gaussian weighting functions are used to provide a color score which quantifies how close the shade is to the user specified reference color. We describe two studies in medical research which use TissueQuant for quantification. The first study evaluated the effect of petroleum-ether extract of Cissus quadrangularis (CQ) on osteoporotic rats. It was found that the analysis results correlated well with the manual evaluation, p < 0.001. The second study evaluated the nerve morphometry and it was found that the adipose and non adipose tissue content was maximum in radial nerve among the five nerves studied.

Asymmetry analysis of breast thermograms using automated segmentation and texture features

In this article, we present a new approach for breast thermogram image analysis by developing a fully automatic segmentation of right and left breast for asymmetry analysis, using shape features of the breast and Polynomial curve fitting. Segmentation results are validated with their respective Ground Truths. Histogram and grey level co-occurrence matrix-based texture features are extracted from the segmented images. Statistical test shows that features are highly significant in detection of breast cancer. We have obtained an accuracy of 90%, sensitivity of 87.5% and specificity of 92.5% for a set of eighty images with forty normal and forty abnormal using SVM RBF classifier.

Quantitative Fraction Evaluation of Dermal Collagen and Elastic Fibres in the Skin Samples Obtained in Two Orientations from the Trunk Region

Background. Histomorphic evaluation of dermal collagen and elastic fibres was analysed by image analysis technique. The quantification of dermal elements was performed in skin tissues, collected in horizontal and vertical directions from trunk region and discussed under the perspective of consequences of scar related complications. Materials and Method. Total number of 200 skin samples collected from 5 areas of trunk region were processed histologically and subjected to tissue-quant image analysis. Statistical analysis involving mean with SEM and paired t test by SPSS were employed to the percentage values obtained from image analysis. Result. Among the chosen 5 areas of trunk region, abdomen showed the statistically significant difference for both collagen and elastic content between horizontal and vertical orientations (P< 0.05), whereas upper back, presternal, and lateral chest areas showed significant difference (P< 0.05) only for collagen and groin only for elastic content. Conclusion. The differences in the distribution of dermal collagen and elastic fibres in 2 directions of the samples from the same areas might be attributed to final outcome of wound healing process by influencing the appearance and behaviour of scar related complications in the region of trunk.

Histomorphometric and sympathetic innervation of the human renal artery: A cadaveric study

Background and Aim: Renal artery stenosis (RAS) and acute renal failure may be due to the intimal hyperplasia and sympathetic fibers of the renal artery (RA), respectively. The purpose of this study was to characterize arterial wall and sympathetic innervation of the human RA. Materials and Methods: Fifty-two fresh human RA samples (proximal part) were collected from 26 cadavers (19 males and 7 females), between the ages of 19 and 83 years, during autopsy. Samples were divided into three age groups: Group 1, 19-40 years; Group 2, 41-60 years; Group 3, over 61 years. 5-μm thick sections of each sample were taken and stained with hematoxylin-eosin and Verhoeff-Van Gieson. Five out of 52 samples were processed for tyrosine hydroxylase (TH) immunostaining. Results: Our histological studies revealed that tunica media of RA showed smooth muscle cells and fine irregularly arranged elastic fibers. Intimal hyperplasia was the most common finding. The present study showed that thickness of tunica intima and media were found to increase with age. Sympathetic nerves were present in the tunica adventitia and outer media of the RA. The mean adventitial and sympathetic nerve fiber areas were found to be 0.595 and 0.071 mm2, respectively. Sympathetic index (SI) to RA was calculated by dividing the sympathetic fiber area by the adventitial area of the RA. SI of RA was found to be 0.140. Conclusion: We conclude that RA showed the structure of musculo-elastic artery. SI may be used for the analysis of sympathetic fiber related problems of the human RA or kidneys.

Microanatomical and immunohistochemical study of the human lateral antebrachial cutaneous nerve of forearm at the antecubital fossa and its clinical implications

Changes in the intraneural anatomy with age can cause poor prognosis of nerve repair in patients after nerve injury. The occurrence of Complex Regional Pain Syndrome‐Type II, secondary to peripheral nerve injury, is common. The purpose of this study is to asses changes in cross‐sectional anatomy of the lateral antebrachial cutaneous nerve of forearm (LCNF) at the antecubital fossa in the fascicular, nonfascicular components (adipose and nonadipose tissue), and sympathetic fibers area with respect to age. For the purpose of the study, 32 human (37–88 years) fresh cadaveric LCNF were collected from left‐antecubital fossae and processed for histological, morphometric analysis [total cross‐sectional (Asc), fascicular (Af), and nonfascicular area (Anonf)], and immunohistochemical method (tyrosine hydroxylase) for sympathetic fibers. The LCNFs average total cross‐sectional area was 3.024 mm2, and fascicular area was 0.582 mm2. The average number of fascicles per mm2 was 3.09. The cross‐sectional area in the nerve was mainly occupied by nonfascicular connective tissue (80.75%). There was increased adipose tissue deposition (48.48% of Asc) and decreased collagen fibers (32.24% of Asc) in interfascicular domains without any definite relationship with age. The average sympathetic fiber area was 0.026 mm2 within the nerve fascicular area without any correlation with age. In LCNF, there was more adipose tissue and less collagen fibers deposition in the interfascicular domains of all age cases, and this may act as an obstacle for nerve fiber regeneration on using LCNF as an interpositional nerve graft. Clin. Anat. 23:693–701, 2010.