Pathology Tutorial 1 PDF

PREPARATION OF BIOLOGICAL MATERIAL FOR FURTHER HISTOPATHOLOGICAL DIAGNOSTICS mgr Amanda Gosk ◦ 1. Histopathological techniques in pathomorphological diagnostics Rules of conducting classes, credit receival (scoring), health and safety regulations and other...

PREPARATION OF BIOLOGICAL MATERIAL FOR FURTHER HISTOPATHOLOGICAL DIAGNOSTICS mgr Amanda Gosk ◦ 1. Histopathological techniques in pathomorphological diagnostics Rules of conducting classes, credit receival (scoring), health and safety regulations and other regulations. Introduction into pathological techniques, types of histopathological procedures (intraoperative, postoperative, autopsy). Pre-laboratory and intra-laboratory preparation of a tissue material for histopathological examination. Fixing of tissue material (types, pros and cons of fixatives). Selection of suitable fixative for a specific tissue and type of examination. Classical and alternative methods for tissue processing. Preparation of special tissue material (bones/biopsy) for histopathological examination). Basic staining of histopathological slides. Exit test. Introduction Pathology is the branch of medical sciences that treats the essential nature of disease, especially the changes of structure and function in tissues and organs of the body that cause or are caused by disease. You need to have a basic knowledge of normal Anatomy (structure) and Physiology (function) to understand Pathology. In the clinical setting, pathologists, histotechnologists, and cytotechnologist study tissues and cells to establish the cause of a disease. Physicians use that information to form a treatment plan. The basic tool of a pathomorphologist is a light microscope which, in correlation with clinical data, results of histochemical, immunohistochemical and molecular tests, determines the pathomorphological diagnosis.  What is the histopathological examination? Histopathology is the study of the signs of the disease using the microscopic examination of a biopsy or surgical specimen that is processed and fixed onto glass slides. To visualize different components of the tissue under a microscope, the sections are dyed with one or more stains. Sample collection Cytological: aspirate, smear, swab, body fluids Tissue material: oligobipsy, surgical specimen Microscopic examination ◦ Pathology, histopathology or histology aims to study the manifestation of disease by microscopic examination of tissue morphology. Through the identification of characteristic features in tissue structures, it is possible to diagnose cancers and many other diseases by comparing changes in the cellular structure between healthy and diseased tissues. ◦ In pathology, the sample to be examined under the microscope usually is the result of a surgery, biopsy or autopsy after fixation, clearing/embedding and sectioning of the tissue specimen. ◦ Alternatively, frozen section processing with a cryostat is done when rapid results are required (e.g. during surgery). Microscopic examination ◦ Cytology (also known as cytopathology) involves examining cells from bodily tissues or fluids to determine a diagnosis. ◦ There are two main kinds, or branches, of cytology: exfoliative cytology and intervention cytology. Examples of exfoliative cytology : ◦ Gynecological samples: A Pap smear, which involves brushing off cells from your cervix using a swab, is the most well-known type of exfoliative cytology. ◦ Respiratory samples: Your provider can collect fluids such as spit and mucus (also called phlegm or sputum) that you cough up for a respiratory cytology test. ◦ Urinary samples: Your provider can collect a urine (pee) sample from you to use for a cytology test. ◦ Discharge or secretion samples: If you experience abnormal bodily discharge, such as from your eye, vagina or nipple, your healthcare provider may collect a sample of the discharge for a cytology test. Microcopic examination ◦ The most common type of intervention cytology is fine-needle aspiration (FNA). Some areas of your body that a healthcare provider may perform a fine-needle aspiration include: Fluid-filled lumps (cysts) under your skin, Solid lumps (nodules or masses) under your skin, Your lymph nodes, Your pericardial fluid, which is the fluid in the sac around your heart, Your pleural fluid, which is in the space between your lung and the inside of your chest wall. Frozen section procedure (Intra-Operative Frozen Section Consultation) ◦ perform rapid microscopic analysis of a specimen ◦ the examination is made while the patient is under anaesthesia on the operating table ◦ the examination report will then be conveyed as soon as possible to the operating surgeon via telephone or intercoms ◦ the result will greatly influence the surgeon’s intra-operational decision ◦ takes max. 20 minutes  ◦ Establish the nature of a lesion To establish whether a lesion that needs to be resected is benign or malignant is very important to the operating surgeon, as this will decide the type of operative procedure or further sampling that he has to make. ◦ Establish the presence of a lesion FS is sometimes utilized to confirm the presence of a lesion or skip lesion in surgically suspicious tissue area. ◦ Confirm the presence of a benign lesion This is quite important in the case of a bony lesion. A benign lesion need to be confirmed for curettage and packing. Malignant bone lesion is usually diagnosed using preoperative biopsy. ◦ Establish the grade of the lesion Grading of a malignant tumour is best done after the tumour is removed. However, sometimes it may be necessary to do so intra-operatively to guide the surgical procedure e.g. during evaluation for the presence or absence of The application endometrial carcinoma. ◦ Determine the presence of synchronous lesions FS may also be utilized to ascertain of frozen section the presence of another lesion spotted unexpectedly during an operation. ◦ Determining the organ of origin using FS in operation should not replaced surgeon’s skill in gross anatomy. However, this procedure is important when dealing with tissue such as parathyroid glands that are too small and difficult to recognize. ◦ Determine the adequacy of margins Adequacy of surgical margins is very important on large resections in a case of malignancy. In a complicated operating site such as in the head and neck, margin clearance of a malignant lesion is very crucial as tumour recurrence can be very aggressive and difficult to treat. ◦ Establish evidence of invasion FS is used to establish the presence of tumour invasion to the lymph nodes and nerve. It is also sometimes used to ascertain metastasis at distant organs. ◦ Determine the presence of infection This is basically looking at the presence of tissue inflammation, granuloma and fungal infection. Procedure ◦ Sending to the laboratory fresh specimen without any fixative, Steps: 1. Examine and palpate all surfaces noting the following: color, texture, consistency, nodules, defects, adherent tissues, marking sutures, anastamotic lines, and any deviations from normal anatomy. 2. Taking representative sections from tissue. 3. Embedding of tissue for frozen sectioning using a gel-like embedding medium. The surgical specimen is placed on a metal tissue disc which is then secured in a chuck and frozen rapidly to about –20 to –30 °C. 4. Cutting the frozen section. It is cut frozen with the microtome portion of the cryostat, the section is picked up on a glass slide. 5. Fixation and staining the frozem section using rapid H&E (hematoxalin and eosin) stain Fine-needle aspiration (FNA) ◦ procedure used to investigate lumps or masses ◦ a lump may be felt during a doctor's examination or it may be discovered on an imaging test such as: ◦ CT scan ◦ mammogram ◦ ultrasound ◦ a thin, hollow needle is inserted into the mass for sampling of cells that, after being stained, are examined under a microscope (biopsy) ◦ used in the diagnosis of cancer and inflammatory conditions ◦ relatively non- invasive, less painful and quicker method when compared to other methods of tissue sampling such as surgical biopsy ◦ most fine needle aspirations are done on these area: thyroid, breast, lymp nodes (in the neck, groin, or armpit) , salivary glands Procedure ◦ holding the mass with one hand or under ultrasound guidance, the doctor will precisely sample the mass with a thin needle held in a needle holder, which provides greater control ◦ usually 2 to 3 samples will be required from the mass to provide an accurate diagnosis ◦ after the needle has been removed from the skin, the material is quickly and gently expressed onto the slides ◦ smears are made by gently spreading the material with another glass slide ◦ alcohol fixation must occur immediately after smears are made ◦ staining according to PAP or with H&E Thick needle biopsy (oligobiopsy) ◦ used for collection of cylindrical tissue specimens with a special cutting needle ◦ a tissue or organ specimen obtained in this way is subjected to histological assessment ◦ formalin fixation must occur immediately ◦ most oligobipsy are done on these area: breast, liver, kidney, prostate Bone marrow trephine biopsy ◦ Collection is performed at local anesthesia, usually from the scapula of the hip bone or breast bone ◦ hollow needle is moved deep into the bone and collects a tiny sample bone with the marrow inside ◦ remove a very thin 1 or 2cm long core of bone marrow in one piece ◦ Similar to bone marrow aspiration, however, in trephine biopsy, a larger sample can be obtained, so it is possible to perform not only cytological (which cells are) but also histological (mutual relationship of cells in the tissue) Bone marrow trephine biopsy ◦ transported and fixed in acetic acid–zinc–formalin fixative, decalcified in 10% formic acid–5% formaldehyde and processed with other specimens to paraffin‐wax embeddingThin sections are cut and are stained, as a minimum, with haematoxylin and eosin and with a reticulin stain; Giemsa stain is also desirable ◦ need for other histochemical or immunohistochemical stains is determined by the clinical circumstances and the preliminary findings  Organ Small piece of changed tissue SURGICAL SPECIMEN Autopsy (post-mortem examination) ◦ to determine the cause of death, mode of death, manner of death, the state of health of the person before he or she died, and whether any medical diagnosis and treatment before death were appropriate ◦ examination of each of the main body systems, including the brain, and all the contents of the chest and abdomen ◦ it can include the removal and retention of small tissue samples for examination under a microscope A B C A - TRANSPORT AND ADMISSION B – DESCRIPTION C - COLLECTING REPRESENTATIVE PIECES FROM SPECIMEN D E F D – TISSUE PROCESSING E – EMBEDDING F – PARAFFIN-EMBEDDED TISSUE BLOCKS G – SECTIONING USING MICROTOME G H I H –STAINING I – MICROSCOPIC EVALUATION  WHAT SHOULD BE INCLUDED IN THE MEDICAL REFERRAL?  SAMPLE COLLECTION AND TRANSPORT Histopathology ◦ Brushing ◦ All tissues should be immersed immediately in fixative Thin layer of smear is attached onto a clean slide, which (10% neutral buffered formalin) in a properly closed has been labeled with patient’s identification. container, labeled with patient identification. Then, the slide is preserved immediately with 95% alcohol ◦ Recommended ratio of specimen size to fixative volume or spray fixative. is 1:10. ◦ Cervical smear ◦ All tissues should be sent to the laboratory as soon as possible. Smear is attached onto a slide which has been labeled with patient’s identification. The slide is preserved immediately ◦ For samples that are in formalin should not be stored in in a container containing 95% alcohol or fixed with spray a refrigerated area as this slow down the fixation rate fixative. ◦ Tissue could also be sent fresh directly from the ◦ FNAC (Fine Needle Aspiration Cytology) operation theatre without delay during office hours if necessary. Sample is taken by the Pathologist/Clinical Specialist/ Medical Officer directly from patients. Cytology: Smearing is done to several slides which has been labeled ◦ Body fluid with patient’s identification. Specimen is collected into sterile containers (preferably 20ml of sample volume), labeled with patient identification. Specimen should be sent immediately to the laboratory. If the specimen cannot be delivered immediately, it should be kept refrigerated at 2-8°C (DO NOT FREEZE). Specimen fixation Specimens for routine histology are required to be placed into 10% neutral buffered formalin, which is available on request from the laboratory. Formalin is used to fix the specimen and preserve the tissue in as life-like state as possible. If there is a delay between the removal of the tissue and fixation in formalin, this can adversely impact the specimen integrity and therefore report. To ensure proper specimen fixation, the following guidelines should be adhered to: Specimen container – should be appropriately sized and large enough to easily accommodate the specimen Formalin – ensure adequate volumes of formalin are used 1:5 tissue to formalin ratio for very large specimens 1:10 tissue to formalin ratio for small specimens where possible Ensure the details on the specimen pot and request card match, are legible and a formalin hazard label is attached Ensure the lid of the specimen container is securely fastened All specimens in formalin should be stored at room temperature and not in the fridge prior to transport to the laboratory.  1 2 3 Preserve the tissue and Prevent putrefaction by Stabilize and protect cells as life-like as bacteria and prevent tissues and cells against possible, without any autolysis by cathepsin- the detrimental effects of shrinking or swelling and containing cells. subsequent processing without distorting or and staining procedures. dissolving cellular constituents. An ideal fixative should: What fixatives do with specimen? The three most commonly employed fixatives for general use being ◦ neutral buffered formalin, ◦ glutaraldehyde ◦ paraformaldehyde Formalin, like other aldehyde fixatives, forms cross linking methylene bridges and Schiff bases between basic amino acid (lysine) residues of proteins. This cross linking stabilizes the proteins in situ, which is the basis of fixation. Formaldehyde produces mild cross linkages when compared to other aldehyde fixatives such as glutaraldehyde. Time of fixation ◦ Fixation time will depend upon the size of the specimen. ◦ In order to achieve adequate and consistent fixation it is essential that the specimens be sliced to a maximum thickness of 3 mm. ◦ Tissue such as lymph node (3 mm slices), skin and bone marrow trephines are routinely fixed for approximately 24 - 48 hours at room temperature. ◦ Dense tissue such as spleen may require extended fixation. ◦ Raising the ambient temperature can increase the rate of fixation. ◦ The pH of the formalin solution is generally between 5 and 7 Pros and cons Its advantages are: ◦ low cost, ◦ simplicity of use ◦ good fixation traits, which are fast tissue penetration, ◦ good preservation of morphological structures ◦ compatibility with downstream histological applications. Formaldehyde disadvantages are: ◦ negative effects on nucleic acids ◦ formaldehyde is present in the air, some individuals may experience adverse effects such as watery eyes; burning sensations in the eyes, nose, and throat; coughing; wheezing; nausea; and skin irritation. Macroscopic examination ◦ Pathology involves both macroscopic and microscopic study of the tissue and its correlation with the clinical and radiographic history, helping in arriving at correct diagnosis. ◦ should be placed on a cutting board in an anatomic position and following information’s are to be recorded: ◦ type of specimen, ◦ structures included, ◦ dimensions, ◦ weight, ◦ shape, ◦ colour, ◦ identification of surgical margins The areas of interest within the tissues are cut into small pieces, numbered and labelled. Cutting sugestions ◦ Do not make blocks too thick. ◦ Blocks should be no larger than 2 x 2 x 0.3 cm. ◦ Do not crowd all lymph nodes into one cassette. ◦ Remove sutures, clips and bone fragments. ◦ Try to cut blocks with flat surface and squared corners. The length of the tissue block should be no more than 20 mm and the thickness ideally about 2-3 mm. It is absolutely mandatory that sections be thin or these will not properly be fixed. ◦ Mark area to be cut with India ink. (Skin, epithelium, vessels, etc.)  DIFFEREN T TYPES A OF B C D CASSETTE A – standard cassette S B – for small biopsy such as liver biopsy C – for small specimen such as small skin biopsy or biopsies from gastrointestinal endoscopy D – for specimen which need higher circulation during tissue processing (adipose tissue) Tissue processing machines The modern processors have a chamber in which the specimens are held, and the different solutions are pumped in and out of the chamber. 1. Dehydratation Removing the water is carried out by immersing tissue in a series of ethanol solutions of increasing concentrations until 100%, water-free alcohol is reached. 2. Cleaning Although the tissue reaches the final stage of dehydration in 100% ethanol, it’s not possible to proceed straight to wax embedding, as ethanol and wax don’t mix! The term clearing refers to the property of the solvents used—they have a relatively high refractive index, and when tissue is immersed in it, it becomes transparent and clear. The solvent used for this intermediate stage is usually xylene. 3. Wax infiltration Infiltration is when the final xylene is replaced with molten wax, which infiltrates the tissue. Embedding After the final infiltration, the tissue cassettes are transferred to an embedding station. This machine has reservoirs of molten wax, hotplates, and a cold plate for setting the tissue blocks. The infiltrated tissue is removed from the cassette and orientated within a suitably sized metal mold. The mold is filled with molten wax, the main part of the labeled cassette is placed on top, and this is also filled with wax. The whole mold is transferred to the cold plate to finally set. Section cutting ◦ Microtomy or section cutting is the technique of making the very thin slices of tissue specimens for the microscopic examination ◦ Nowadays, the most commonly used microtome is rotary microtome which allows the perfect sectioning of the paraffin-embedded tissue specimens and serial sections can easily be obtained. ◦ Most commonly the section cutting is done at the thickness of 4-6μ (microns). ◦ These sections are mounted on slides for hematoxylin-eosin (H&E) staining and further immunohistochemistry if needed. Staining The choice of the type of staining depends on: ◦ The purpose of the study ◦ Type of biological material ◦ Type of fixative Routin hematoxylin and eosin stain Preparation for staining a paraffin sections: Deparaffization: ◦ xylen I ◦ xylen II ◦ xylen III ◦ xylen IV Hydration: ◦ Alcohol 99,8% ◦ Alcohol 96% 80% ◦ Distilled water Staining steps: Action Reagent Time Staining I Hematoxylin 5 min Rinsing Running water 2 min Staining II Eosin 2 min Rinsing Running water 2 min Dehydration Alcohol from 80% to 99,9% 2 min Clearing Xylen I 2 min per each Xylen Xylen II Xylen III Xylen IV Mount in medium ARCHIVING BLOCKS AND GLASS SLIDES THANK YOU FOR YOUR ATTENTION