Automated Tissue Processor Research Articles

Histopathological and Biochemical Effects of Methanol and Aqueous Extracts of the Leaf of Irvingia Gabonensis (African wild mango) on the liver of Albino Wistar Rat

Background: The use of Irvingia gabonensis locally for different purposes from eating down to drinking its extracts and concoctions for treating various ailments has been a common practice over the ages. The liver is a very vital metabolic organ and the central site for food and drug metabolism and subsequent excretion. In that case, the understanding of the effects of these foods and drugs both orthodox and herbal is ever invaluable both to the clinicians and the patients. This research work aims to investigate the effects of its leaf extract on the liver using histological and biochemical parameters. Methods: The experimental rats (n=20) used were divided into five (5) groups of four (4) each. Group one served as control and received only water and standard pellets. Groups two and three were administered with the methanolic extract of 150 mg/ kg and 300 mg/kg respectively. Groups four and five were administered with the aqueous extract of 250 mg/kg and 500 mg/kg respectively. The liver samples were harvested, fixed and processed in an automated tissue processor. The blood sample obtained through retro-orbital puncture was made to clot. The serum was then used to analyze the ALT, AST, ALP and Bilirubin content of the liver. Results were obtained and analyzed statistically, showing a significant (p < 0.05) increase in the AST and ALP levels in groups three (3) and five (5). The Serum bilirubin and ALT levels showed insignificant (p > 0.05) increases in all the groups. The photomicrograph indicates mild fibrosis and sinusoidal dilations and cellular infiltrations in groups three and five with no visual changes in the rest of the groups. Conclusion: The findings suggest that at high doses of both methanolic and aqueous extracts (300 and 500mg/kg respectively), the leaf extract is mildly toxic to the liver.

HISTO-PATHOLOGICAL SPECTRUM OF SKIN LESIONS IN TERTIARY CARE CENTRE: A THREE YEAR STUDY

Introduction: The largest sensory organ of human body is the skin, which accounts for about 15% of total body weight. It is a complex organ having multiple functions. Primarily it acts as a barrier against various harmful environmental agents (1).Skin diseases affect all age groups and are much common in developing countries. In the field of dermatology, 2000 different skin diseases are well known(2) Objectives: The objectives of the study were as follows: (1) To estimate the prevalence of various non-neoplastic and neoplastic skin lesions in our tertiary care hospital. (2) To study the age and sex-wise distribution of non-neoplastic and neoplastic skin lesions. Methods: The present study comprised a total of 102 skin biopsies of non-neoplastic lesions and neoplastic skin lesions, relevant data like age, sex, and sites of skin lesion were obtained from request forms provided to histopathology section. Specimens received were fixed in 10% formalin. Biopsy bits were submitted as whole and processed in automated tissue processor for routine paraffin embedding. Results: Out of 102 biopsies that were analyzed, most belonged to 21- 60years with slight male predominance with a total of 85 cases being Non-Neoplastic skin lesions, of which Chronic dermatitis 18 (28.5%) was the most common histopathological diagnosis, followed by Hansens disease 21(24.7%) among granulomatous skin disease, and a total of 17 cases of neoplastic skin lesions were identified of which dermatofibroma 2(11.8%) and eccrine spiradenoma 2(11.8%) were the benign variants , and Squamous cell carcinoma 3(17.6%) was the most common malignant variant. Conclusion: Histopathological evaluation of skin biopsies holds gold standard technique for diagnosing various skin lesions and their microscopic evaluation helps in assessing variegated morphological features of different types of skin lesions.

Clinico-histopathological evaluation of spindle cell soft tissue tumours

Introduction: Spindle cells are of mesenchymal origin and part of connective tissue. Soft tissues defined as non-epithelial, extra skeletal tissues of the body that may be voluntary muscles, fat and fibrous tissue and vessels. Soft tissue tumours are defined as mesenchymal proliferation which occur in the extraskeletal nonepithelial tissues of the body, excluding the viscera, coverings of brain and lymphoreticular system. Aims: The aims of the study is to classify according to WHO, evaluate the incidence rate and age, sex, site wise distribution and histopathological findings of the various types of spindle cell soft tissue tumours and comparing it with national and international study.Materials and Methods: The present study was done on surgical specimens received in Department of Pathology from August 2019 to August 2021. The Autolysed, poorly preserved specimens were excluded from the study. The specimens were fixed, grossly examined, representative tissue bits taken, processed in automated tissue processor for routine paraffin embedding, Tissue sectioning by microtomy and staining with the routine hematoxylin and eosin method done. Special stains were used if necessary. The microscopic examination was done and the results were analysed. Results: Out of 110 cases of spindle cell soft tissue tumors, most common tumors were Peripheral nerve sheath tumours (34.5%) followed by Fibroblastic and myofibroblastic tumours (22.7%). Most of the cases in present study occur between 21-30 years of age and more common in males. Higher rates of tumours located in head and neck region (36.4%). Conclusion: Spindle cell soft tissue tumours are classified according to morphological and histogenetic basis. Definite diagnosis especially for difficult cases should be made by performing immunohistochemistry and molecular biology. Present study was unique on the basis of that it includes spindle cell soft tissue tumours.

HISTOPATHOLOGICAL STUDY OF NON-NEOPLASTIC LESIONS OF SKIN

Objectives: The objectives of the study were as follows: (1) To find the prevalence of various non-neoplastic skin lesions in our tertiary care hospital. (2) To study the age- and sex-wise distribution of non-neoplastic skin lesions. (3) To compare the results of the present study with other studies. Methods: The present study comprised total 200 punch biopsy specimens of diagnosed cases of non-neoplastic lesions of skin, detailed clinical data, age, sex, and sites of skin lesion of patients which were included in the study. Specimens received were fixed in 10% formalin. Biopsy bits were submitted as whole and processed in automated tissue processor for routine paraffin embedding. Results: A total of 200 lesions were analyzed. Maximum cases were in the age group of 21–30 years followed by 31–40 years age. Fifty-five (27.5%) cases were of non-infectious erythematous, papular, and squamous diseases followed by 54 (27%) cases of vesiculobullous and vesiculopustular diseases, 37 (18.5%) cases of bacterial diseases, 20 (10%) cases of fungal diseases, and 34 (17%) cases of vascular lesions, connective tissue disorders, non-infectious granulomas, perforating disorders, etc. Conclusion: Skin lesions were common in males than in females with male: female ratio of 1.38:1. Younger age group was more commonly affected. Non-infectious erythematous, papular, and squamous lesions were most common with psoriasis being the most common followed by non-infectious vesiculobullous and vesiculopustular lesions with pemphigus vulgaris being the most common, followed by infective lesions consisting of bacterial, fungal, and viral lesions. Other lesions consisted of vascular skin lesions, connective tissue disorders non-infectious granulomas, perforating disorders, etc.

Principles of Manual Tissue Processing in an Oral and Maxillofacial Pathology Laboratory in Lagos, Nigeria


 
 
 
 
 
 Introduction: Tissue processing describes the steps involved in infiltrating a well fixed tissue with wax for section cutting on a microtome. Paraffin tissue processing can be performed either manually or by using an automated tissue processor and errors in any of the steps involved would significantly affect histopathology. The aim of this article was to describe to principles of manual tissue processing as performed in the Oral and Maxillofacial Pathology Laboratory of the College of Medicine, University of Lagos, Nigeria (OMFP-CMUL Lab).
 Methods: We report the principles of manual tissue processing as performed in the OMFP-CMUL lab using the index case of an 11-year-old girl with a histopathologic diagnosis of unicystic ameloblastoma.
 Results: Steps of tissue processing included fixation, specimen accessioning, gross examination, decalcification (for tissue specimens containing calcium deposits), dehydration, clearing, infiltration, embedding, sectioning, labeling of tissue blocks, tissue floatation bathing, making the slide and numbering, staining, drying, cover-slipping, and microscopic examination.
 Discussion: Common errors seen in histopathology laboratories were avoided by the quality control practices of the residents, consultants, laboratory scientists and technicians within the department.
 Authors
 U P Egbunah 1, A O Akinshipo 2, O F Ajayi 2 3
 

Tissue Processing.

Tissue processing is the technique by which fixed tissues are made suitable for embedding within a supportive medium such as paraffin, and consists of three sequential steps: dehydration, clearing, and infiltration. In most clinical and research settings, tissue processing is accomplished using an automated tissue processor, with or without microwave-assistance. To ensure high-quality results, processing protocols should be tailored to tissue size and composition by modifying variables such as reagents used and the timing of the various steps. Herein, we provide an overview of tissue processing theory and outline a basic tissue processing method for use with a conventional automated fluid transfer/enclosed processor. The principles described will assist readers in optimizing tissue processing for their own projects.

Abnormal Uterine Bleeding – A Clinico-pathological Study

Background:
 Abnormal Uterine Bleeding (AUB) can occur at any age in a woman`s reproductive period and needs to be assessed very carefully and immediately. When it occurs in the older age group, a more meticulous screening for malignancy is imperative so that treatment can be more radical. Dilatation and curettage is a simple, cost effective, safe and a reliable investigation and it gives us a direct access to the target organ.
 Methods:
 Study was conducted prospectively on 162 patients presenting with AUB in reproductive, perimenopausal and postmenopausal age group. All the endometrial samples procured from the endometrial curettage were fixed in 10% buffered formalin for 12-24 hours, processed in the automated tissue processor, cut and stained with Hematoxylin & Eosin (H & E) stain and were finally studied in detail for the morphological findings under light microscopy.
 Result:
 In our study Secretory endometrium was most common type, which was followed by Proliferative endometrium. Disordered proliferative endometrium and Endometrial hyperplasia were the commonest histopathological patterns seen in AUB of organic type. Endometrial carcinoma was seen more commonly in postmenopausal age group. Further, in our study Mc Cluggage criteria was applied to all the samples to categorize endometrial samples which were unassessable and inadequate.
 Conclusion: 
 Evaluation of Endometrial samples is important in all patients with Abnormal Uterine Bleeding (AUB) to find out the Organic Pathology. Histopathological typing of endometrium is crucial for appropriate therapy. Its interpretation is quite challenging and also may show considerable interobserver variability. In AUB, the endometrial samples should be taken during the bleeding episode itself. Dilatation and curettage is a simple, cost effective, safe and reliable investigation and gives us a direct access to the target organ.

Prognostic Significance of Ki-67 and p53 Immunoexpression in Breast Carcinoma Patients with Positive Axillary Lymph Nodes

Introduction: Breast carcinoma is a heterogenous disorder in which the treatment modality should be based on a risk assessment approach and only patients with higher relative risk of recurrence and distant metastasis should be the appropriate candidates for adjuvant therapy. This is where role of prognostic variables like Ki-67 and p53 immunoexpression as biomarkers of proliferative activity of tumor cells becomes important. Aims and Objectives: The aim of this study was to evaluate the significance of Ki-67 and p53 immunoexpression as prognostic biomarkers in breast carcinoma patients with positive axillary lymph nodes. Material and Methods: A total of 182 breast cancer patients were included in the study, assessed for various clinicopathological characteristics and after receiving the mastectomy/lobectomy specimen, tissue sections about 1cm thick were taken from representative areas, processed in an automated tissue processor (Histokinette) and simultaneously stained for routine hematoxylin and eosin stain along with Ki-67 and p53 immunostains and patients followed up. Results: High Ki-67 and p53 immunoexpression was significantly associated with larger tumor size, higher tumor grade, axillary lymph node metastasis, higher disease stage at presentation, lymphovascular invasion, early recurrence, shortened disease free survival and worse overall survival. Conclusions: Ki-67 and p53 immunoexpression can be used as a surrogate biomarker along with other clinicopathological characteristics in resource constrained facilities for better prediction of prognosis of breast cancer patients with positive axillary lymph nodes.

Celiac disease and its histopathology

Introduction: Celiac disease, also known as celiac sprue, non-tropical sprue, gluten-induced enteropathy, or gluten-sensitive enteropathy (GSE), is a chronic immune-mediated disorder of the small intestine characterized by malabsorption after ingestion of wheat gluten or related proteins in rye (secalins) and barley (hordeins) in individuals with a certain genetic background. Materials and Method: The study was conducted in Mahatma Gandhi Medical College and hospital, Jaipur. 53 Duodenal biopsies were taken who have symptoms of diarrhea, iron deficiency anemia, amenorrhea, recurrent mouth ulceration. All biopsies taken from duodenal sitewere brought in 10% buffered formalin. After overnight fixation in formalin, the tissues were processed in automated tissue processor for dehydration, clearing, and paraffin embedding. Sections were cut in rotary microtome 4 micrometer thickness. The section was stained for Hematoxylin and Eosin stain. Results: The present study included 53 Duodenal biopsies. Out of 53 duodenal biopsies, 12 biopsies have shown presence of Celiac disease. Rest 41 biopsies have shown chronic duodenitis. Conclusion: Celiac disease is a common autoimmune condition with mainly intestinal, but also extra-intestinal manifestations. The histologic hallmark of GlutenSensitive Enteropathy is both increased inflammation and architectural derangement in the small intestinal villi.The small intestinal mucosa of untreated persons with celiac disease usually display villous atrophy (Marsh III), or, less commonly, isolated crypt hyperplasia (Marsh II), together with intraepithelial lymphocytic proliferation.

Histopathological patterns of ovarian lesions: A study of 161 cases

Ovarian lesions are commonly encountered pathologies that cannot be categorized clinicoradiologically. Definite diagnosis is of great importance for therapeutic and prognostic purposes. Histopathology gives accurate diagnosis in most cases. Few cases need supportive tests like immunohistochemistry. Objective: to study the histomorphological diversity of ovarian lesions, their age and location in North of Iraq (Mosul and Duhok). Patients and methods: In the period extended from January 2008 to December 2011, 161 cases of ovarian lesions were collected from pathology departments in Azadi General Hospital “Duhok” and Al-Jamhori Teaching Hospital “Mosul”. Automated tissue processor was used for histologic study and Streptavidin-biotin method on paraffin sections was applied for immunohistochemistry. Result: There was a wide age range, most being in the third decade. The right ovaries were more common involved than the left. Histologically, 58 (36%) cases were non-neoplastic and 103 were neoplastic including 90 (55.9%) benign and 9 (5.6%) malignant tumors. The remaining 4 (2.4%) cases comprised borderline serous cystadenoma. Conclusion: Most ovarian lesions were functional non-neoplastic followed by benign neoplastic. Apart from few cases, diagnosis was merely histological without any ancillary test.

Timeliness of surgical pathology results: a departmental audit of histopathological services

Aim To determine the turnaround time (TAT) for surgical pathology specimens (SPS) seen in our department and to identify the various determinants (variables) that affect it. Materials and method This is a cross-sectional descriptive study (prospective) of all SPS that were sent to the histopathology laboratory of University of Uyo Teaching Hospital (UUTH). A special proforma/questionnaire was designed for this study taking into account the various stages of converting a histopathological request to a result. Results Following specimen accessioning, the mean number of days before grossing was 1.65 ± 0.97 days. Tissue processing took a mean of 2.30 ± 2.05 days. Many of the processed SPS (histopathological slides) were reviewed and reported by histopathology resident doctors and consultant histopathologists within second and third days after processing in the laboratory (24.1% and 23.2% respectively), with a mean of 5.18 ± 13.07 days. Typing of result and verification of result took a mean of 2.99 ± 13.16 days. The shortest time interval to transform a histological request to a standard result was four days while the mean TAT was 8.47 ± 3.34 days. There was a significant correlation between the histological diagnosis and TAT. Conclusion Overnight fixation should be limited to large operating room specimens and not generalised. Provision of automated tissue processor, multi-headed and projecting microscopes will improve the TAT. All laboratory staff should be made to know that each stained glass slide represents a living patient and that the results are awaited.

Mullerian-inhibiting substance augments ovarian response to superovulation in mice with diminished ovarian reserve

To determine whether pretreatment with Mullerian-inhibiting substance (MIS) affects the oocyte yield of superovulation in a mouse model of diminished ovarian reserve (DOR). Preclinical placebo-controlled animal study. The DOR mouse model was created by giving intraperitoneal (IP) injections of 4-vinylcyclohexene diepoxide (VCD) to 6-week old nu/nu mice (n=3) at 160 mg/kg daily for 5 days. Control mice (n=3) were injected IP with vehicle control (corn oil). Mice were euthanized 14 days later. Dissected ovaries were fixed in 4% paraformaldehyde, embedded in paraffin blocks in an automated tissue processor, cut into 8-um sections, and mounted onto glass slides, which were stained with hematoxylin and eosin. Under light microscopy, ovarian follicles were classified into five groups - primordial, primary, secondary, antral, and atretic - and counted. A cohort of DOR mice was subsequently treated with subcutaneous recombinant human MIS protein (750 ug/kg, n=3) or saline (n=4) twice daily for 40 days. After 30 days of observation, superovulation was performed in standard fashion with pregnant mare serum gonadotropin 7.5 IU IP, followed by human chorionic gonadotropin (hCG) 7.5 IU IP 49 hours later to trigger ovulation. 16 hours after hCG, oocytes were dissected from the oviducts of euthanized mice and were counted. Morphologic assessment of collected oocytes was performed under a stereo microscope, and the percentage of degenerated oocytes collected from each mouse was noted. Oocyte yields and percentage of degenerated oocytes of MIS-treated and saline control mice were compared using student’s t-test. A p-value of < 0.05 was considered significant. VCD administration as described above creates a DOR phenotype in mice, by depleting primordial follicle reserve by 73%, while maintaining ovulatory function. DOR mice treated with MIS produced, on average, 29 oocytes through superovulation compared to saline control mice who produced only 11 oocytes (p = 0.038). Furthermore, more oocytes were morphologically abnormal in controls compared to MIS-treated mice (52.2% versus 30.1%, p = 0.026). In the setting of DOR, treatment with MIS protein significantly enhances oocyte yield following superovulation and leads to a lower proportion of degenerated oocytes. These findings suggest a future clinical application of MIS in IVF protocols, particularly for patients with DOR.

Incidence of Helicobacter Pylori in upper gastrointestinal lesion

Introduction: Helicobacter pylori are gram-negative bacillus. It is noninvasive and live in gastric mucus, with the small proportion of bacteria adherent to the mucosa. Its spiral shape and flagella render Helicobacter pylori motile in mucus environment. Helicobacter pylori is an important pathogen in human causing chronic gastritis and playing a major role in the development of peptic ulcers and gastric cancer. Material and Methods: Study was conducted in Mahatma Gandhi Medical College and hospital, Jaipur (from Jan 2014 to March 2015). 57 Gastrointestinal biopsies were taken who have symptom of dyspepsia. All biopsies taken from different sites were brought in 10%buffered formalin. After overnight fixation in formalin, the tissues were processed in automated tissue processor for dehydration, clearing, and paraffin embedding. Section were cut in rotary microtome at 4 micrometer thickness. The section was stained for Hematoxylin and Eosin stain and Geimsa overnight stain. Result: The present studyincluded 57 cases of upperGIT biopsies (gastro esophageal duodenal biopsies). The incidence of Helicobacter Pylori was seen with chronic gastritis in our study. Conclusion: H.pyloriareetiologically associated with chronic active gas triti s, duodenal ulcer, gastric ulcer, primarygastric B-cell lymphoma or mucosalassociated lymphoid type lymphoma (MALT lymphoma) and gastric adenocarcinoma. Helicobacter pylori induced chronic gas trit is were encountered in 16(47.22%) casesofthe presentstudy. Overnight Giemsa stain was done on all the patients and a histopathological diagnosis was made along with the study of the incidence of Helicobacterpylori positivity in all these patients.

Pattern of occurrence of primary head and neck cancers presenting in Benin City, Southern Nigeria

Context: Head and neck cancers (HNCs) are the 10th most common cancer globally. About half a million new cases are diagnosed yearly with an increasing incidence in developing countries. Aim: The main objective of this study is to evaluate the epidemiological and histopathological patterns of all HNCs in Benin City, Southern Nigeria, and to compare these findings with other similar research works globally. Materials and Methods: This is a retrospective review of histopathology records of HNCs diagnosed over a period of 5 years (January 2008 and December 2012) at the University of Benin Teaching Hospital. All specimens were fixed in 10% formalin solution, processed with Histokinette automated tissue processor, paraffin embedded, and sectioned at 3–5 microns with microtome machine before staining with hematoxylin and eosin. The histopathological diagnoses were analyzed with respect to age, sex, tumor types, and the International Classification of Disease Oncology Ninth Revision. Results: A total of 259 HNC cases were encountered. One hundred and forty males (54.1%) and 119 females (45.9%) were seen giving a ratio of 1.2:1. The mean age for all malignancies was 41.8 years ± 19.8 standard deviation of the 259 patients, 83.4% had carcinomas, 7.7% had lymphomas, 5.4% had blastomas, and 3.5% had sarcomas. Squamous cell carcinoma (SCC) constituted 66.9% of all carcinomas. Non-Hodgkins lymphoma accounted for 85% of all lymphomas while retinoblastoma constituted 78.6% of primitive cells. Conclusion: The larynx was the most common topographic tumor site of primary HNCs in our study and the most common HNC were of epithelial origin (carcinoma) with SCC as the most prevalent malignancy.

Histopathological Findings in Uterus and Cervix of Hysterectomy Specimens

&lt;strong&gt;Background:&lt;/strong&gt; For centuries, the female reproductive system has been affected by various abnormalities and diseases and hence has been the subject of interest and the basis for the gynaecological practice. The uterus being a vital reproductive and hormone-responsive organ, is subjected to a variety of physiological changes and benign and malignant disorders. &lt;strong&gt;Aims and Objectives:&lt;/strong&gt; To study the indications of hysterectomy, to study gross and histopathological features of uterus and cervix in hysterectomy specimens and correlate with clinical findings. &lt;strong&gt;Material and Methods:&lt;/strong&gt; This was a descriptive study of the gross and histopathological findings of uterus and cervix in 150 hysterectomy specimens received in the department. The hysterectomy specimens received were fixed in 10% formalin for 24 hours, were examined grossly and necessary sections were obtained. The tissue pieces were then processed in automated tissue processor, well labelled paraffin blocks were made. Approximately 2-3μ thickness sections were cut with the help of microtome and were stained routinely by Hematoxylin &amp;amp; Eosin stain and special stains like PAS or other were used wherever necessary. The histopathological findings of uterus and cervix were noted and these findings were then correlated with clinical diagnosis. &lt;strong&gt;Results:&lt;/strong&gt; Overall clinicopathological correlation was noted in all 150 cases. In cases of uterine fibroid it was 69.7% and 100% in case of endometrial carcinoma and cervical dysplasia. Most common pathology found was uterine leiomyomas in 48 cases and next to it was adenomyosis in 30 cases. &lt;strong&gt;Conclusion:&lt;/strong&gt; It can be concluded that clinico-pathological correlation in case of endometrial cancer and uterine fibroid is excellent, but in case of DUB and prolapse uterus it varies. This signifies the importance of clinico-pathological correlation in all cases of hysterectomy to improve the clinical outcome and post-operative management.

Histopathological Findings in Uterus and Cervix of Hysterectomy Specimens

Background: For centuries, the female reproductive system has been affected by various abnormalities and diseases and hence has been the subject of interest and the basis for the gynaecological practice. The uterus being a vital reproductive and hormone-responsive organ, is subjected to a variety of physiological changes and benign and malignant disorders. Aims and Objectives: To study the indications of hysterectomy, to study gross and histopathological features of uterus and cervix in hysterectomy specimens and correlate with clinical findings. Material and Methods: This was a descriptive study of the gross and histopathological findings of uterus and cervix in 150 hysterectomy specimens received in the department. The hysterectomy specimens received were fixed in 10% formalin for 24 hours, were examined grossly and necessary sections were obtained. The tissue pieces were then processed in automated tissue processor, well labelled paraffin blocks were made. Approximately 2-3μ thickness sections were cut with the help of microtome and were stained routinely by Hematoxylin & Eosin stain and special stains like PAS or other were used wherever necessary. The histopathological findings of uterus and cervix were noted and these findings were then correlated with clinical diagnosis. Results: Overall clinicopathological correlation was noted in all 150 cases. In cases of uterine fibroid it was 69.7% and 100% in case of endometrial carcinoma and cervical dysplasia. Most common pathology found was uterine leiomyomas in 48 cases and next to it was adenomyosis in 30 cases. Conclusion: It can be concluded that clinico-pathological correlation in case of endometrial cancer and uterine fibroid is excellent, but in case of DUB and prolapse uterus it varies. This signifies the importance of clinico-pathological correlation in all cases of hysterectomy to improve the clinical outcome and post-operative management.

Histomorphology Spectrum of Gall Bladder Pathology in Cholecystectomy Specimens with Clinical Diagnosis of Chronic Cholecystitis

Chronic cholecystitis is a common disease in surgery where majority of cholecystectomy are performed. This study was carried out to see histomorphological pattern of gallbladder diseases, staining pattern with various stains and prevalence of carcinoma. This prospective study was conducted in the department of Pathology, B.P. Koirala Institute of Health Sciences, Dharan, Nepal from January to December 2008. A minimum of fifty random samples were included in study. Clinical details and pathological data were retrieved from case sheets and patient's history. All specimens were fixed in 10% formalin. Three sections each from neck, body and fundus were taken. Tissues were processed in an automated tissue processor and paraffin blocks made. The tissue sections were examined under light microscope after hematoxylin-eosin, neutral mucin, sulfated mucin and sialomucin stain. Chronic cholecystitis without cholelithiasis constituted majority of 25(50.0%) cases with M:F of 1:7.1. The gastric and intestinal metaplasias were present in 33.0% and 8.0% of the cases respectively. The neutral, sulfated and sailomucin were positive in 35.0%, 21.0%, 1.0% and 34.0%, 21.0%, 1.0% cases respectively in gastric and intestinal metaplasia. Both gastric and intestinal metaplasia showed significant p-value on sulfated mucin. The increase risk factors for developing of chronic cholecystitis were seen in female gender (86%). The gastric and intestinal metaplasia showed positivity more with neutral followed by sulfated mucin.

Changes in the Chondrocyte and Extracellular Matrix Proteome during Post-natal Mouse Cartilage Development

Skeletal growth by endochondral ossification involves tightly coordinated chondrocyte differentiation that creates reserve, proliferating, prehypertrophic, and hypertrophic cartilage zones in the growth plate. Many human skeletal disorders result from mutations in cartilage extracellular matrix (ECM) components that compromise both ECM architecture and chondrocyte function. Understanding normal cartilage development, composition, and structure is therefore vital to unravel these disease mechanisms. To study this intricate process in vivo by proteomics, we analyzed mouse femoral head cartilage at developmental stages enriched in either immature chondrocytes or maturing/hypertrophic chondrocytes (post-natal days 3 and 21, respectively). Using LTQ-Orbitrap tandem mass spectrometry, we identified 703 cartilage proteins. Differentially abundant proteins (q < 0.01) included prototypic markers for both early and late chondrocyte differentiation (epiphycan and collagen X, respectively) and novel ECM and cell adhesion proteins with no previously described roles in cartilage development (tenascin X, vitrin, Urb, emilin-1, and the sushi repeat-containing proteins SRPX and SRPX2). Meta-analysis of cartilage development in vivo and an in vitro chondrocyte culture model (Wilson, R., Diseberg, A. F., Gordon, L., Zivkovic, S., Tatarczuch, L., Mackie, E. J., Gorman, J. J., and Bateman, J. F. (2010) Comprehensive profiling of cartilage extracellular matrix formation and maturation using sequential extraction and label-free quantitative proteomics. Mol. Cell. Proteomics 9, 1296-1313) identified components involved in both systems, such as Urb, and components with specific roles in vivo, including vitrin and CILP-2 (cartilage intermediate layer protein-2). Immunolocalization of Urb, vitrin, and CILP-2 indicated specific roles at different maturation stages. In addition to ECM-related changes, we provide the first biochemical evidence of changing endoplasmic reticulum function during cartilage development. Although the multifunctional chaperone BiP was not differentially expressed, enzymes and chaperones required specifically for collagen biosynthesis, such as the prolyl 3-hydroxylase 1, cartilage-associated protein, and peptidyl prolyl cis-trans isomerase B complex, were down-regulated during maturation. Conversely, the lumenal proteins calumenin, reticulocalbin-1, and reticulocalbin-2 were significantly increased, signifying a shift toward calcium binding functions. This first proteomic analysis of cartilage development in vivo reveals the breadth of protein expression changes during chondrocyte maturation and ECM remodeling in the mouse femoral head.

Protein Profiling of Human Breast Tumor Cells Identifies Novel Biomarkers Associated with Molecular Subtypes

Molecular subtypes of breast cancer with relevant biological and clinical features have been defined recently, notably ERBB2-overexpressing, basal-like, and luminal-like subtypes. To investigate the ability of mass spectrometry-based proteomics technologies to analyze the molecular complexity of human breast cancer, we performed a SELDI-TOF MS-based protein profiling of human breast cell lines (BCLs). Triton-soluble proteins from 27 BCLs were incubated with ProteinChip arrays and subjected to SELDI analysis. Unsupervised global hierarchical clustering spontaneously discriminated two groups of BCLs corresponding to "luminal-like" cell lines and to "basal-like" cell lines, respectively. These groups of BCLs were also different in terms of estrogen receptor status as well as expression of epidermal growth factor receptor and other basal markers. Supervised analysis revealed various protein biomarkers with differential expression in basal-like versus luminal-like cell lines. We identified two of them as a carboxyl terminus-truncated form of ubiquitin and S100A9. In a small series of frozen human breast tumors, we confirmed that carboxyl terminus-truncated ubiquitin is observed in primary breast samples, and our results suggest its higher expression in luminal-like tumors. S100A9 up-regulation was found as part of the transcriptionally defined basal-like cluster in DNA microarrays analysis of human tumors. S100A9 association with basal subtypes as well as its poor prognosis value was demonstrated on a series of 547 tumor samples from early breast cancer deposited in a tissue microarray. Our study shows the potential of integrated genomics and proteomics profiling to improve molecular knowledge of complex tumor phenotypes and identify biomarkers with valuable diagnostic or prognostic values.

Microwave-assisted tissue processing for same-day EM-diagnosis of potential bioterrorism and clinical samples

The purpose of this study was to explore the turnaround times, section and image quality of a number of more “difficult” specimens destined for rapid diagnostic electron microscopy (EM) after microwave-assisted processing. The results were assessed and compared with those of conventionally processed samples. A variety of infectious agents, some with a potential for bioterrorism, and liver biopsies serving as an example for routine histopathology samples were studied. The samples represented virus-producing cell cultures (such as SARS-coronavirus, West Nile virus, Orthopox virus), bacteria suspensions (cultures of Escherichia coli and genetically knockout apathogenic Bacillus anthracis), suspensions of parasites (malaria Plasmodium falciparum, Leishmania major, Microsporidia cuniculi, Caenorhabditis elegans), and whole Drosophila melanogaster flies infected with microsporidia. Fresh liver samples and infected flies were fixed in Karnovsky-fixative by microwaving (20 min), all other samples were fixed in buffered glutaraldehyde or Karnovsky-fixative overnight or longer. Subsequently, all samples were divided to evaluate alternative processing protocols: one part of the sample was OsO 4-postfixed, ethanol-dehydrated, Epon-infiltrated (overnight) in an automated tissue processor (LYNX, Leica), and polymerized at 60 °C for 48 h; in parallel the other part was microwave-assisted processed in the bench microwave device (REM, Milestone), including post-osmication and the resin block polymerization. The microwave-assisted processing protocol required at minimum 3 h 20 min: the respective epon resin blocks were uniformly polymerized allowing an easy sectioning of semi- and ultrathin sections. Sections collected on non-coated 200 mesh grids were stable in the electron beam and showed an excellent preservation of the ultrastructure and high contrast, thus allowing an easy, unequivocal and rapid assessment of specimens. Compared with conventional routine methods, microwave technology facilitates a significant reduction in sample processing time from days to hours without any loss in ultrastructural details. Microwave-assisted processing could, therefore, be a substantial benefit for the routine electron microscopic diagnostic workload. Due to its speed and robust performance it could be applied wherever a rapid electron microscopy diagnosis is required, e.g., if bioterrorism or emerging agents are suspected. Combining microwave technology with digital image acquisition, the 1-day diagnosis based on ultrathin section electron microscopy will become possible, with crucial or interesting findings being consulted or shared worldwide with experts using modern telemicroscopy tools via Internet.