Histopathology Practical Book 2i4z14

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The principles of diseases module; histopathology practical conducted in four practical sessions. First practical session starts with demonstration of tissue processing and staining followed by explanation of cytology procedures with more emphasis on papanicolaou smear. Rest of the three session has two parts 1. Gross specimens & clinical image discussions & 2. Microscopic discussions in relation with basic principles of cellular injury, adaptations, inflammation, tissue repair, hemodynamic disorders and neoplasia. 1. Gross specimen discussions: - Demonstration of plastinated and wet (formalin fixed) specimens - Descriptive discussions on standard macroscopic & clinical static images - List of specimens & static images Plastinated specimens

Fatty liver Acute appendicitis Chronic pyelonephritis Amoebic liver abscess Cirrhosis liver CVC – Liver Sub endocardial infarction Infarct spleen Infarct brain Infarct lung Lipoma Mature teratoma – Testes Pleomorphic adenoma Liposarcoma Squamous cell carcinoma – Skin Carcinoma intestine Carcinoma stomach Metastatic tumour – Brain Formalinated (Wet) specimens Leiomyoma Sarcoma – Soft tissue Cyst – Skin Polyp intestine Carcinoma intestine Standard clinical images Healthy & unhealthy wound Pitting edema Ascites Anasarca Periorbital edema Gangrene – Leg Diabetic foot Petechiae Purpura Hematoma Papilloma – Skin Squamous cell carcinoma & basal cell carcinoma – skin Note: Students are permitted to take photograph or video of specimen demonstrations

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2. Microscopic discussions: - Demonstration of robotic scanned digital microscopic slides with scaled microscopic magnification from 2x to 40x objective lens with 10x eye piece lens. - Descriptive discussions on standard microscopic static images - List of robotic digitally scanned microscopic slides Robotic digital scanned microscopic slides

Glandular metaplasia – Esophagus Monckeberg calcification Fatty liver Coagulative necrosis – Kidney Liquefactive necrosis – Brain Caseous necrosis – Lymphnode Fat necrosis Acute appendicitis Cirrhosis liver Pulmonary edema Coronary artery disease Venous thrombosis Pulmonary congestion Dysplasia – Cervix Lipoma Liposarcoma Adenomatous polyp – colon Adenocarcinoma – colon Malignant melanoma Squamous cell carcinoma – Skin Metastatic carcinoma – Lymph node Note: Students are permitted to take photograph or videos of slides. The manual contains photograph, focused on the characteristic histopathological feature of that particular slide. Mode of assessment: - Station based objective structures practical evaluation (examination) - OSPE. - 5 Stations Station 1 [Observer] Instruments Students has to: Identify Indications Specific features

Station 2 [Observer] Reagents & Stains Students has to: Identify Indications Specific features

Station 3 [Non Observer] Spotter Spot diagnosis of gross specimens

Station 4 [Non Observer] Spotter Spot diagnosis of microscopic slides

Station 5 [Observer] Objective reasoning Students has to: Describe & explain about the changes happened in the given scenario

- OSPE will be conducted in multiple batches. All the batch students should come prepared with all the practical content, taught in the session. Students will be evaluated from the entire practical content.

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Pathology Tissue processing & staining

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Definition of pathology

The word ‘Pathology’ is derived from two Greek words—pathos meaning suffering, and logos meaning study. Pathology is the scientific study of structure and function of the body in disease; or in other words, pathology consists of the abnormalities that occur in normal anatomy (including histology) and physiology owing to disease. Another commonly used term to ‘study of diseases’ is ‘pathophysiology’ comprised by two words: patho=suffering; physiology=study of normal function. Pathophysiology, thus, includes study of disordered function or breakdown of homeostasis (homeostasis – being in a state of balance) in diseases.

Pathologists are the diagnosticians of disease. Therefore, knowledge and understanding of pathology is essential for all the doctors, general medical practitioners and specialists since unless they know the causes (etiology – study of causes), mechanisms, nature and type of disease and understand the language spoken by pathologist in the form of laboratory reports, they would not be able to treatment or advice patient. For the student of any system of medicine, the discipline of pathology forms a vital bridge between initial learning phase of preclinical sciences and the final phase of clinical subjects. It is best to quote an eminent founders of modern medicine Sir William Osler statement, “Your practice of medicine will be as good as your understanding of pathology.”

Terminologies for beginner in pathology It is important for students to be familiar with the language used in pathology: 1. Patient is the person affected by disease. 2. Lesions are the characteristic changes in tissues and cells produced by disease in an individual or experimental animal. 3. Pathologic changes or morphology consist of examination of diseased tissues. 4. Pathologic changes can be recognized with the naked eye (gross or macroscopic changes) or studied by microscopic examination of tissues. 5. Causal factors responsible for the lesions are included in etiology of disease (i.e. ‘why’ of disease). 6. Mechanism by which the lesions are produced is termed pathogenesis of disease (i.e. ‘how’ of disease). 7. Functional implications of the lesion felt by the patient are symptoms and those discovered by the clinician are the physical signs. 8. Clinical significance of the morphologic and functional changes together with results of other investigations help to arrive at an answer to what is wrong (diagnosis), what is going to happen (prognosis), what can be done about it (treatment), and finally what should be done to avoid complications and spread (prevention) (i.e. ‘what’ of disease).

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Subdivisions in Pathology Human pathology is the largest branch of pathology. It is conventionally divided into General Pathology dealing with general principles of disease and Systemic Pathology that includes study of diseases pertaining to the specific organs and body systems. With the advancement of diagnostic tools, the specialty of pathology emerged, which include the following subspecialties: 1. Histopathology: Also called as anatomic pathology, pathologic anatomy, or morbid anatomy, is the classic method of study and still most useful one which has stood the test of time. The study includes structural changes observed by naked eye examination referred to as gross or macroscopic changes and the changes detected by light and electron microscopy ed by numerous special staining methods including histochemical and immunological techniques to arrive at the most accurate diagnosis. Modern time anatomic pathology includes super specialties such as cardiac pathology, pulmonary pathology, neuropathology, renal pathology, gynecologic pathology, breast pathology, dermatopathology, gastrointestinal pathology, oral pathology and so on. Anatomic pathology includes the following 3 main subdivisions: a) Surgical pathology - It deals with the study of tissues removed from the living body. It forms the bulk of tissue material for the pathologist and includes study of tissue by paraffin embedding techniques and frozen section for rapid diagnosis. b) Forensic pathology and autopsy work - This includes the study of organs and tissues removed at postmortem for medico legal work and for determining the underlying sequence and cause of death. c) Cytopathology - It includes study of cells shed off from the lesions (exfoliative cytology) and fine needle aspiration cytology (FNAC) of superficial and deep seated lesions for diagnosis. 2. Hematology - Hematology deals with the diseases of blood. It includes laboratory hematology and clinical hematology; the latter covers the management of patient as well. 3. Chemical pathology - Analysis of biochemical constituents of blood, urine, semen, CSF and other body fluids is included in this branch of pathology. 4. Immunology - Detection of abnormalities in the immune system of the body comprises immunology and immunopathology. 5. Experimental pathology - This is defined as production of disease in the experimental animal and its study. 6. Geographic pathology - The study of differences in distribution of frequency and type of diseases in populations in different parts of the world. 7. Medical genetics - This is the branch of human genetics that deals with the relationship between heredity and disease.

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Molecular pathology - The detection and diagnosis of abnormalities at the level of DNA of the cell is included in molecular pathology.

Even though many specialties exist; it is impossible to define their boundaries. Human body is an integrated structure, so the branches evolved and evolving in pathology of diseases are also integrated. Advancement in the field of pathology, expands the knowledge and keep everyone on their life long journey of learning.

Microscope Microscope is the most commonly used apparatus in the laboratory. It magnifies the minute objects according to the desired calibration. It was first invented by Antony Von Leeuwenhoek (1632 – 1723) and also introduced first histochemical staining. There are different types of microscopes, most commonly used are light microscopy. A light microscope can be a simple or a compound microscope. Simple microscope is a simple hand magnifying lens. The magnification power of hand lens is from 2x to 200x. Compound microscope has a battery of lenses which are fitted in a complex instrument. One type of lens remains near the object (objective lens with magnifying power of 4x, 10x, 20x, 40x and 100x) and another type of lens near the observer’s eye (eye piece lens with magnifying power of 5x, 10x or 15x). The eye piece and objective lenses have different magnification. The compound microscope can be monocular having single eye piece (Fig. 1) or, binocular which has two eye pieces (Fig. 2).

Figure 1: Monocular microscope

Figure 2: Binocular microscope

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Other microscope (specific condition) and its application: No Microscope Application 01

Dark ground illumination

Examination of unstained living microbes. Example – Trepenoma palladium

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Polarizing microscope

Demonstration of birefringence Example - Amyloid

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Fluorescent microscope

Demonstration of naturally occurring fluorescent material or substances stained with fluorescent dyes. UV light is used for illumination. Example – Immunoglobulin, Amyloid, Mycobacterium

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Electron microscope

Study of ultra-structural details of the tissues and cells. Example – Glomerular basement membrane structure Digital Pathology

Over the past few years, the evolution of information technology has vastly changed the practice of pathology. New techniques have been implemented, primarily in the form of digital images and telepathology. Digital pathology can be defined as the electronic capture, management, analysis and distribution of gross or microscopic specimens. It encomes the use of digital images.

The applications of digital pathology is wide and includes primary diagnosis, intraoperative diagnosis, consultation, quality assurance, archiving, education, conferences, and research. Widespread adoption of digital pathology applications could expedite second opinion consultation; digital consultation can be performed within hours versus days to weeks for cases sent through the mail. It can also help improve service quality to under serviced areas, facilitate immediate conferencing about a specimen by individuals in different locations, reduce costs of slide storage, limit slide loss, expedite slide retrieval and overcome the problem of slide fading.

There are, however, several challenges to the widespread implementation of digital pathology, including cost, and a lack of standardization. Added to this is the potential negative attitude of pathologists towards the change.

By The Grace and Mercy of The Almighty Allaah, with the help of our academic istration and management, we overcome all hurdles and established the digital pathology teaching to benefit the students to understand and apply the concepts of pathology in their future clinical life.

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Regular Histopathology Slide (Step 1)

Digital Slide Scanner (Step 2)

 Step 1: The regular histopathology slides were scanned by using motic digital slice scanning microscope.  Step 2: The digital images were collected and stored in the computer database.  Step 3: By using motic digital microscopic image software, the digital histopathology images are visualized.

Viewed Through Software (Step 3) Histopathology Technique

Histopathology is examination of tissues for presence or absence of changes in their structure due to disease processes. Both are done by examining thin sections of tissues which are colored differently by different dyes and stains. Total or selected representative part of tissue not more than 4 mm thick is placed in steel or plastic capsules or cassettes and is subjected to the following sequential processing (tissue processing): Step 1: Fixation Step 2: Processing – Dehydration, Clearing, Impregnation Step 3: Embedding and blocking Step 4: Section cutting Step 5: Routine staining Fixation Any tissue removed from the body starts decomposing immediately because of loss of blood supply and oxygen, accumulation of products of metabolism, action of autolytic enzymes and putrefaction by bacteria. This process of decomposition is prevented by fixation. Fixation is the method of preserving cells and tissues in life like conditions as far as possible. Most fixatives act by denaturation or precipitation of cell proteins or by making soluble components of cell insoluble.

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Use of fixatives: Prevents putrefaction and autolysis, hardens the tissue which helps in section cutting, makes cell insensitive to hypertonic or hypotonic solutions, acts as a mordant & induces optical contrast for good morphologic examination. Fixatives may be simple or compound: Simple fixative consists of one substance (e.g. formalin). Compound fixative has two or more substances (e.g. Bouin’s). Commonly used fixatives and its application: No

Fixative

Composition & action

Application

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Formalin

It is a saturated solution of formaldehyde Commonly used for gas in water, 40% by weight/volume (w/v). all the tissues For all practical purposes, this 40% solution is considered as 100% formalin. For fixation of tissues, a 10% solution is used which is prepared by dissolving 10 ml of commercially available formalin in 90 ml of water. It takes 6-8 hours for fixation of a thin piece of tissue 4 mm thick at room temperature. The amount of fixative required is 15 to 20 times the volume of the specimen. Formalin acts by polymerization of cellular proteins by forming methylene bridges between protein molecules.

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Glutaraldehyde

It is used as 4% solution at 40 degree Celsius Electron microscopy for 4 hours for fixation of tissues.

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Bouin’s fixative

Picric acid , Formaldehyde, Glacial acetic Testes & renal tissues acid

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Carnoy’s fixative

Alcohol fixative (Methyl and ethyl alcohol)

Cytology examination

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Osmium tetroxide

2% solution of osmium tetroxide

Electron microscopy CNS tissues & lipids

Dehydration This is a process in which water from cells and tissues is removed so that this space is subsequently taken up by wax. Dehydration is carried out by ing the tissues through a series of ascending grades of alcohol: 70%, 80%, 95% and absolute alcohol. If ethyl alcohol is not available then methyl alcohol, isopropyl alcohol or acetone can be used.

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Clearing This is the process in which alcohol from tissues and cells is removed and is replaced by a fluid in which wax is soluble and it also makes the tissue transparent. Xylene is the most commonly used clearing agent. Toluene, benzene (it is carcinogenic), chloroform (it is poisonous) and cedar wood oil (it is expensive and very viscous) can also be used as clearing agent. Impregnation This is the process in which empty spaces in the tissue and cells after removal of water are taken up by paraffin wax. This hardens the tissue which helps in section cutting. Impregnation is done in molten paraffin wax which has the melting point of 56oC (54-62oC). Tissue processors Nowadays all the processes of fixation, dehydration, clearing and impregnation are carried out in a special equipment which is known as automated tissue processor. Tissue moves automatically by hydraulic mechanism from one jar to another after fixed time schedule and the whole process takes 16-22 hours Embedding and blocking Embedding of tissue is done in molten wax. Wax blocks are conventionally prepared using metallic L (Leuckhart’s mould); nowadays plastic moulds. Molten wax is poured in the cavity in the moulds. The processed tissue pieces are put into wax with number tag and examining surface facing downward. Wax is allowed to solidify. After solidification, if L-moulds are used they are removed while plastic mould remains with the wax block. In either case, each block contains a tissue piece carrying an identification label. Embedding and blocking can also be performed in a special instrument called embedding centre. Section cutting (microtomy) Microtome is an equipment for cutting sections. There are 5 types of microtomes: 1. Rotary, 2. Sliding, 3. Freezing, 4. Rocking, 5. Base sledge. Most commonly used microtome is rotary microtome. The section is cut with a thickness of 5 - 6 μm (microns).

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Routine staining (Hematoxylin and Eosin – H & E Staining) Hematoxylin - Natural dye obtained from log wood tree, Haematoxylon campechianum. The natural extract from the stem of this tree is haematoxylin which is an inactive product. This product is oxidized to an active ingredient which is haematein. This process of oxidation is known as ripening which can be done naturally in sunlight, or chemically by addition of oxidant like sodium iodate, KMnO4 or mercuric oxide. Hematoxylin will not directly stain tissues, but needs a “mordant” or link to the tissues. So mordant is added to it (e.g. potash alum) which helps in attaching the stain particles to the tissue. Variety of hematoxylin available, most commonly used is Harris hematoxylin. They vary in intensity or hue. Hematoxylin is a basic dye gives blue color, has an affinity for the nucleic acids of the cell nucleus. Eosin – Synthetic dye derived from the action of bromine on fluorescein. There are variety of eosin based on their pH. Eosin is an acidic dye gives pink color, with an affinity for cytoplasmic components of the cell and extra cellular components include collagen, elastin, muscle fibers and red blood cells. Perinuclear areas look bluish to pink because of mixture of protein and RNA. There are two types of staining methods "regressive" or "progressive". With a regressive stain, the slides are over stained and excess stain is removed by acid alcohol. This is good for staining large batches of slides. With a progressive stain the slide is dipped in the hematoxylin until the desired intensity of staining is achieved. This is good for small batches of slide (frozen section) Procedure for Staining Step 1: Removing the wax (hydrophobic) – deparaffinization. Place the slide in a jar of xylene (hydrocarbon solvent) for 10-15 minutes. Step 2: Hydration. As haematoxylin is a water-based dye, the sections before staining are rehydrated which is done by ing the sections in a series of descending grades of alcohol and finally bringing the section to water. Step 3: Hematoxylin staining. Place the slide in haematoxylin stain (contains oxidized hematoxylin + Aluminum salt) for 8-10 minutes. The section become reddish purple Step 4: Blueing. Rinse in water. Dip the section in Scott’s tap water (containing sodium bicarbonate and magnesium sulfate) or saturated solution of lithium carbonate for 2-10 minutes to bring required blue color to the section. Step 5: Differentiation. Dip the section in a in a solution of 1% acid alcohol for 10 seconds to selectively remove excess dye from the section. Rinse in water. [Differentiation is used only in regressive staining method] Step 6: Counterstain with eosin. 1% aqueous solution of eosin for 1- 3 minutes. Step 7: Rinse, dehydrate, clear and mount. Rinse in tap water. Before mounting, the sections have to be dehydrated which is done by ing the sections in a series of ascending grades of alcohol and finally cleared in xylene. 2 - 3 dips in each solution. Mount in DPX (dextrene polystyrene xylene) or Canada balsam. Results: Nuclei stain blue (basophilic, hematoxyphilic). Cytoplasm, muscle, collagen & keratin stain pink (acidophilic, eosinophilic). Red blood cells stain red.

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Changes seen in each steps:

Unstained section in glass slides.

Step 1 Removing the wax

Step 2

Step 3

Hydration

Hematoxylin staining

Step 4

Step 5

Blueing

Differentiation

Step 6

Step 7

Eosin Staining

Dehydrate, clear, mount

In the end of staining, section clearly demonstrate the tissue characteristics

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Hematoxylin and Eosin staining in Liver Section:

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Special Histopathology Technique During the tissue examination, some diseases or certain tissues need other than routine technique and staining. Such procedures are grouped under special histopathology technique which includes frozen section to Immunophenotyping. Here only basic and essential special methods are discussed. Decalcification Calcified tissues or bone are extremely hard to cut using microtome. Chemical substances are used to remove the calcium such process is called as decalcification. Mineral acids, organic acids, EDTA and electrolysis are used to remove calcium from the tissues. Mineral acids (nitric and hydrochloric acids) - Used for dense cortical bone because they will remove large quantities of calcium at a rapid rate but these strong acids also damage cellular morphology, are not recommended for delicate tissues such as bone marrow. Organic acids (acetic and formic acid) – Use for bone marrow, since they are not as harsh. Formic acid in a 10% concentration is the best all-around decalcifier. Some commercial solutions are available that combine formic acid with formalin to fix and decalcify tissues at the same time. EDTA can remove calcium and is not harsh (it is not an acid) but it penetrates tissue poorly and works slowly and is expensive in large amounts. Electrolysis has been tried in experimental situations where calcium had to be removed with the least tissue damage. It is slow and not suited for routine daily use. Frozen section At times during performance of surgical procedures, it is necessary to get a rapid diagnosis of a pathologic process. During such situation frozen sections are performed. The piece(s) of tissue to be studied are snap frozen in a cold liquid or cold environment (-20 to 70 Celsius). Freezing makes the tissue solid enough to section with a microtome. It is done by using cryostat. The cryostat is just a refrigerated box containing a microtome. The temperature inside cryostat is about -20 to -30 Celsius. The tissue sections are cut and picked up on a glass slide. The sections are then ready for staining. Frozen sections are commonly done for; - To confirm the margins of resection of a malignant neoplasm are clear before closing.

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- Unexpected disease process may be found and require diagnosis to decide what to do next, or it may be necessary to determine if the appropriate tissue has been obtained for further workup of a disease process and to demonstrate some special substances in cell or tissue such as fat, enzymes etc. Special stains Stains are applied for demonstration of certain specific substances/constituents in cells/tissues. Special stains Tissue Sections Comments Sudan black /

Used to demonstrate of fat.

Oil red stain

Fat stains black (Sudan black) & red (oil red) Muscle with adipose tissue

Van Gieson stain

Used to demonstrate collagen. Muscle stains yellow and collagen stains red

Muscle, adipose & fibrous tissue Masson’s Trichrome stain

Used to demonstrate muscle Muscle stains red, collagen stains blue and nuclei blue

Muscle and fibrous tissue with vessel

Reticulin

Used to fibers.

(Silver stain) silver methanamine periodic acid Schiff stain Glomerulus & renal tubules

Verhoeff (Elastin) stain

demonstrate

reticular

Reticulin fibers stain black. Especially used in renal, liver and lymphnode biopsies and in vascular tumours Used to demonstrate elastic fibres.

Muscular artery (elastic fibers)

The section is counterstained with van Gieson stain. Elastic fibres stain black, collagen stain red, cytoplasm & muscle fibres stain yellow

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Periodic - acid Schiff stain

Used to demonstrate glycogen and mucopolysaccharides. PAS positive substances are glycogen, amyloid, colloid, neutral mucin and hyaline cast, stains bright pink. Goblet cells of colon

Congo red stain

Glomerulus with amyloid

Used to demonstrate amyloid, an extra cellular fibrillar proteinaceous substance. It gives green birefringence when viewed by polarized light and stain red

Cytology Technique Cytology is the study of body cells that are either exfoliated spontaneously from epithelial surfaces or are obtained from various body tissues and organs by different techniques. Currently, cytology has following branches: A. Exfoliative cytology, B. Aspiration cytology & C. Imprint cytology. Exfoliative cytology: This is the study of cells which are spontaneously shed off from epithelial surfaces into body cavities or fluid. The cells can also be obtained by scraping, brushing or wash of body surfaces. The principle of this technique is that in diseased states, rate of exfoliation of cells is increased. Applications of Exfoliative Cytology Exfoliative cytology is applied in diagnosing diseases of the following: 1. 2. 3. 4. 5. 6.

Female genital tract Respiratory tract Gastrointestinal tract Urinary tract Body fluids (pleural, peritoneal, pericardial, CSF and semen) Buccal smears for sex chromatin

Female Genital Tract Smears from female genital tract are known as ‘Pap smears’. These smears are prepared by different methods depending upon the purpose for which they are intended:

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1. Cervical smear: It is obtained by Ayre’s spatula from portio of the cervix by rotating the spatula through 360o to sample the entire cervix. The scraped material is placed on a clean glass slide and smear prepared. It is ideal for detection of cervical carcinoma. 2. Lateral vaginal smear (LVS): It is obtained by scraping upper third of lateral walls of the vagina and is ideal for cytohormonal assessment. 3. Vaginal pool smear: It is obtained by aspirating material from posterior fornix of vagina and is done for detecting endometrial and ovarian carcinoma.

Method of obtaining cervical material for fast smears Respiratory Tract Material from respiratory tract may be obtained during bronchoscopic procedures as expectorant (sputum) or by brushing (BB), washing (BW) and bronchioalveolar lavage (BAL). Sputum examination is advantageous as samples are easily obtained and cellular content is representative of entire respiratory tract. At least three samples of sputum, preferably early morning samples, should be examined. Gastrointestinal Tract Lesions in the oral cavity can be sampled by scraping the surface with a metallic or wooden spatula. Samples can be obtained from the oesophagus, stomach, small and large intestine either by brushing or lavage during fibreoptic endoscopy. Urinary Tract Samples from lesions in the urinary tract are either urinary sediment examined from voided urine/ catheterised urine or washings of the urinary bladder obtained at cystoscopy. Cytospin Body Fluids Fluid from pleural, peritoneal or pericardial cavity is obtained by paracentesis. At least 50-100 ml of fluid is aspirated. The sample is examined fresh but if delay is anticipated then fluid should be anticoagulated either in EDTA 1 mg/ml or 3.8% sodium citrate 1ml/10ml. Fluid should be centrifuged

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and smears are prepared from the sediment. If amount of fluid is less (less than 1 ml), then it can be subjected to cytospin centrifuged smear preparation. Buccal Smears for Sex Chromatin Smears are prepared from the oral cavity after cleaning the area. Vaginal smears can also be used. In normal females, Barr bodies are present in 4-20% nuclei. In males their count is in less than 2% nuclei. Fixation of Smears in Exfoliative Cytology Methods of fixation depend upon type of staining employed. Pap smears are wet-fixed (i.e. smears are immersed in fixative without allowing them to dry). Smears to be stained by Romanowsky stains are air-dried as fixation is affected during the staining procedure. Fixative used is either equal parts of ether and 95% ethanol, or 95% ethanol alone, 100% methanol, or 85% isopropyl alcohol. Fixation time of 10-15 minutes at room temperature is adequate. Smears may be left in fixative for 24 hours or more. Smears should be transported to the laboratory in fixative solution in coplin jars. Staining of Smears in Exfoliative Cytology Three staining procedures are commonly employed: Papanicolaou and H and E stains are used for wet-fixed smears while Romanowsky stains are used for air-dried smears. Papanicolaou Stain This is the best stain for routine cytodiagnostic studies. In this, haematoxylin gives nuclear stain while OG-6 and EA-50 are two cytoplasmic counterstains. H & E Stain This is the same as that used for histological sections. In this, haematoxylin is nuclear stain and eosin is cytoplasmic counterstain. Romanowsky Stain Leishman’s stain, Giemsa and May-Grunwald-Giemsa (MGG) are usually used; the last one is most commonly used. Aspiration cytology: In this study, samples are obtained from diseased tissue by fine needle aspiration (FNA) or aspiration biopsy cytology (ABC). Applications of FNA FNA or ABC is applied for diagnosis of palpable as well as non-palpable lesions. I. Palpable Mass Lesions in: 1. Lymph nodes, 2. Breast, 3. Thyroid, 4. Salivary glands, 5. Soft tissue masses & 6. Bones

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II. Non-Palpable Mass Lesions in: 1. abdominal cavity, 2. Thoracic cavity & 3. Retroperitoneum Procedure for FNA Materials for performing FNA, a Franzen’s handle, syringe with needles, clean glass slides and suitable fixative are required.

Equipment required for transcutaneous FNAC. Method -

No anaesthesia is required. Ask the patient to lie down in comfortable position exposing the target area. Palpate the target area. Clean the overlying skin with spirit. Fix 10/20 ml disposable syringe in Franzen’s handle. Insert 20-25 gauge disposable needle into syringe. Fix the mass by palpating hand and insert needle into target area. Apply suction while moving needle back and forth within the lesion and change the direction of the needle. Terminate the aspiration when aspirated material or blood is visible at the base/hub of the needle. Release the suction before withdrawing the needle to equalize pressure within the syringe. After withdrawal of needle, apply pressure for 2-3 minutes at the site of puncture to arrest bleeding and prevent hematoma formation. Aspirated material from the needle is expressed on to clean glass slides by first detaching the needle and filling the syringe with air and expressing it with pressure. Smears are prepared as for blood smears. If the material is semi-solid, it is first crushed by gentle pressure with a glass slide and smears prepared. Fixation and staining are the same as for exfoliative cytology.

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Procedure of FNAC of palpable masses Needle is introduced into the mass (A). Plunger is retracted after needle enters the mass (B). Suction is maintained while needle is moved back and forth within the mass (C). Suction is released and plunger returned to original position before needle is withdrawn (D).

Preparation of smears

Semisolid aspirates are crush-smeared by flat pressure with cover slip or glass slide (A). Fluid or blood droplet is collected along edge of spreader (B), and pulled as for peripheral blood films (C). Particles at the end of the smear are crush-smeared (D). Advantages of FNA over Surgical Biopsy Outdoor procedure No anaesthesia required Results obtained within hours Procedure can be repeated Low cost procedure Imprint cytology: This is a touch preparations from cut surfaces of fresh unfixed surgically excised tissue are prepared on clean glass slides. These are fixed, stained and examined immediately. It is considered complementary to frozen section.

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PAP smear A normal Pap smear shows the following cells 1. Superficial cells, 2. Intermediate cells and 3. Parabasal cells

Inflammatory smear: In inflammatory smear, the superficial and intermediate cells show features as under: Cytoplasmic eosinophilia, cytoplasmic vacuolisation, large number of leucocytes in the background, perinuclear halos, clumping of chromatin, nuclear enlargement and pyknosis, increase in the number of parabasal cells.

The specific inflammation could be by the following microbial infections: Trichomonas vaginalis, candida albicans & human papilloma virus

Dysplastic and malignant smear: It is one of the key screening procedures for diagnosing precursor lesions as well as for carcinoma of the cervix. The cytological diagnosis of cervical lesions is based commonly on the Bethesda system which divides them into precursor (1. LSIL - low grade squamous intraepithelial lesions & 2. HSIL - high grade squamous intraepithelial lesions) and malignant lesions.

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Macroscopic Specimens & Clinical Images

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Macroscopic Specimens – Work sheet Plastination Specimen Description Fatty liver

Acute appendicitis

Chronic pyelonephritis

Amoebic liver abscess

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Macroscopic Specimens – Work sheet Plastination Specimen Description Cirrhosis liver

Chronic venous congestion – Liver

Sub endocardial infarction

Infarct spleen

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Macroscopic Specimens – Work sheet Plastination Specimen Description Infarct brain

Infarct lung

Lipoma

Mature teratoma - Testes

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Macroscopic Specimens – Work sheet Plastination Specimen Description Pleomorphic adenoma

Squamous cell carcinoma

Carcinoma intestine

Carcinoma stomach

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Macroscopic Specimens – Work sheet Plastination Specimen Description Liposarcoma

Metastatic tumour - Brain

Formalinated (Wet) Specimen Description Leiomyoma

Sarcoma – Soft tissue

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Macroscopic Specimens – Work sheet Formalinated (Wet) Specimen Description Cyst – Skin

Polyp – Intestine

Carcinoma – Intestine

Others

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Clinical Images– Work sheet Clinical Images Healthy wound healing

Unhealthy wound healing

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Clinical Images– Work sheet Clinical Images Gangrene leg

Diabetic ulcer

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Clinical Images– Work sheet Clinical Images Anasarca

Periorbital edema

Pitting edema

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Clinical Images– Work sheet Clinical Images Petechiae

Purpura

Hematoma with ecchymosis

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Clinical Images– Work sheet Clinical Images Papilloma - Skin

Squamous cell carcinoma - Skin

Basal cell carcinoma - Face

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Microscopic Slides

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Microscopic Slide: 1

Microscopic Features

Histopathological Diagnosis

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Microscopic Slide: 2

Microscopic Features

Histopathological Diagnosis

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Microscopic Slide: 3

Microscopic Features

Histopathological Diagnosis

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Microscopic Slide: 4

Microscopic Features

Histopathological Diagnosis

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Microscopic Slide: 5

Microscopic Features

Histopathological Diagnosis

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Microscopic Slide: 6

Microscopic Features

Histopathological Diagnosis

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Microscopic Slide: 7

Microscopic Features

Histopathological Diagnosis

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Microscopic Slide: 8

Microscopic Features

Histopathological Diagnosis

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Microscopic Slide: 9

Microscopic Features

Histopathological Diagnosis

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Microscopic Slide: 10

Microscopic Features

Histopathological Diagnosis

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Microscopic Slide: 11

Microscopic Features

Histopathological Diagnosis

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Microscopic Slide: 12

Microscopic Features

Histopathological Diagnosis

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Microscopic Slide: 13

Microscopic Features

Histopathological Diagnosis

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Microscopic Slide: 14

Microscopic Features

Histopathological Diagnosis

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Microscopic Slide: 15

Microscopic Features

Histopathological Diagnosis

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Microscopic Slide: 16

Microscopic Features

Histopathological Diagnosis

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Microscopic Slide: 17

Microscopic Features

Histopathological Diagnosis

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Microscopic Slide: 18

Microscopic Features

Histopathological Diagnosis

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Microscopic Slide: 19

Microscopic Features

Histopathological Diagnosis

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Microscopic Slide: 20

Microscopic Features

Histopathological Diagnosis

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Microscopic Slide: 21

Microscopic Features

Histopathological Diagnosis