e-report The Flow of Process in Histopathology Laboratory Humaira Syahirah Binti Hamizi (2022612534) Nur Nasuha Syuhada Binti Herman Bakhtiar (2022478028) Muhd Farqhan Bin Syamsul Bahrain (2022608202) Iarina Syamimi Binti Ishak (2022611344) Nur Afiqah Binti Mohd Zahari (2022604766) Class : HS241 3B Lecturer : Dr Hartini Binti Yusof
INTRODUCTION Histology pathology is a crucial aspect of medical diagnostics, focusing on the microscopic examination of tissues to identify diseases and understand their underlying mechanisms. The workflow typically involves several key steps and essential in diagnosing diseases ranging from cancers to infectious and inflammatory conditions, playing a vital role in patient care and treatment planning. It combines precise laboratory techniques with the expertise of pathologists to provide critical insights into disease pathology ✨ HISTOLOGICAL TECHNIQUES Purpose: Detailed, long-term preservation and analysis of tissue samples. ✨ Preserves tissue structure for longterm storage and analysis. ✨ Allows for a detailed examination of tissue morphology. Purpose: Rapid assessment of tissues during surgical procedures to aid in immediate diagnosis. ✨ Rapid results: Offers quick evaluation during surgery to guide further procedures. ✨ Does not require fixation and embedding, enabling immediate analysis. Tissue grossing Figure 1.0 Grossing the sample Figure 1.1 Processing the sample Tissue processing Figure 1.2 Paraffin embedding centre Tissue embedding Tissue sectioning Figure 1.3 Serial section of tissue ribbon Tissue staining Figure 1.4 Staining tissue on slides Mounting & labeling Figure 1.5 Lowering the slides onto the mounting medium Microscopic examination Figure 1.6 Uterus tissue observed under light microscope
TISSUE GROSSING PROCEDURES: The principle of tissue grossing involves the careful identification, orientation, dissection, and documentation of tissue specimens to ensure accurate pathological examination at a microscopic level. PRINCIPLE MATERIALS: Human tissue specimens fixed with commercially prepared 10% formalin APPARATUS: Scalpel holder & blades Forceps Tissue cassettes Containers Rulers Dissecting boards Medisheet Gloves Aprons Mask EQUIPMENT Ducted Fume Hood: Iryas 3 Set up the grossing workstation and verify that the instruments are prepared and ready for use RESULT Tissue blocks Assign identification numbers and dates to appropriately label the cassette(s) based on the type of specimen 1 Wear personal protective equipment (PPE) 2 C gr o o n s d s u in c g t o th f e 4 specimens sequentially, one at a time 5 Provide an account of the macroscopic characteristics of the specimen 6 Immerse the tissue cassette in 10% Neutral Buffered Formalin 7 Dispose of formalin waste in the designated formalin waste container Figure 2.0 : Fume Hood Figure 2.1 : Complete PPE Attire Figure 2.2 : Cassette Figure 2.3 : Grossing instruments Figure 2.4 : Grossing Station Figure 2.5 : Sample and cassette Figure 2.6 :Cassette Figure 2.7 : Formalin Container Figure 2.8 : Formalin Container, Cassette
TISSUE PROCESSING procedure 2. Dehydration 4. Infiltration PROBLEM TROUBLESHOOT Process removal of clearing agent completely and replaced by medium (wax). 5. Embedding 3. Clearing Tissue processing helps to preserve cells and tissue by remove all extractable water from tissue, replacing it with certain chemical reagent called as fixatives. Fixatives will preserve the shape, size, structure, relationship and chemical composition of the tissue after death. PRINCIPLE MATERIALS Figure 3.0 : Tissue Processor Figure 3.1 : Tissue Processor Plastic Container Tissue hardening and loss of antigenicity Inadequate fixation Adhere to recommended fixation times Follow recommended fixation times and use fresh fixative 1. Fixation and gross section Process that allow water, aqueous fixative and lipid to be removed. Process removal of dehydrating agent from tissue and replace it with clearing agent. Inactivates lysosomal enzyme. Re su l t Re su l t Re su l t The tissue specimen fixed into paraffin before embedding.
MATERIALS Processed tissue blocks 1 2 3 4 8 Paraffin Wax Tissue Embedding Centre EQUIPMENT Paraffin Wax REAGENT APPARATUS: Gauze Tissue paper Gloves Forceps Scraper Steel block mould PROCEDURES -The mold was promptly cooled by placing it on the cryo console. After the paraffin wax solidified, the resulting tissue block was detached from its mold. -A cassette holding treated tissue was unsealed to observe the tissue sample. -A small quantity of liquid paraffin was poured into the mold, and then the mold was placed onto a chilled plate. -The treated tissue was promptly moved to the mold using forceps as soon as the paraffin wax reached a partially solid state. -The tissue was softly compressed to secure it in a stable position. 5 -A cassette with appropriate labeling, along with labeled paper, was positioned above the mold. 6 7 -Liquid paraffin was subsequently poured into the mold until it completely filled the mold. -Once more, the paraffin wax was poured into the mold to completely cover the surface of the cassette. PRINCIPLE Embedding is the process in which the infiltrated tissues or the specimens are enclosed in a mass of the embedding medium using a mould. Known as casting or blocking. Since the tissue blocks are very thin in thickness, they need a supporting medium in which the tissue blocks are embedded. This supporting medium is called the embedding medium. TISSUE EMBEDDING Figure 4.0 : Cassettes Figure 4.1 : Tissue Embedding Machine Figure 4.2 : Paraffin Wax Figure 4.3 : Instruments Figure 4.4 : Cassettes Figure 4.5 : Wax Dispenser Figure 4.10 : Cold Station Figure 4.7 : Tissue and Mold Figure 4.8 : Embedding Station Figure 4.9 : Wax Dispenser Figure 4.9 : Wax Dispenser Figure 4.6 : Chilled Plate Figure 4.11 : Tissue Blocks
To expose the suitable area of tissue forsectioning- thickness ar 10um On exposing- section thicknessis setto 0.5-4um Trimuntilthe complete tissue section appears on the tissue ribbon 5) Floating out section 4) Cutting sections Slides racks Scrapers Clean brush Clean gauze Paper Tissue paper Pasteur pippets Applicator sticks Pencils Biohazard bags Glass slides Microtome blade Angle of slope of the knife at 90 Tighten knife clamp screws securely and block clamp screwmustfirmly Clearance angle : 5-10 3) Trimming of the tissue block 6) Picking up section 7) Place the slide containing the tissue section onto the slide rack Tissue sectioning is a process done to produce thin slice section by using extremely sharp knife or metals with a rotary microtome. Once ribbons are produced, its is then placed into floatation bath and pick up on slides. The section thickness range is from 2 to 3 um Principle Paraffin blocks Rotary microtome: Slee Mainz CUT5062 Tissue floatation bath: XH-1001, Thermo Scientific 3120058 Freezer Oven 1) Setting up the microtome Procedure 2) Place the tissue block vertically onto the standard object clamp To produce ribbon section Optimalspeed is onbtained through experience Softertissue- at a slowerrate Transferribbon towater bath (45 °C) Action in floating outmust be smooth When the ribbon has come to rest on water bath,the remainingwrinkles are removes by teasing a part using forceps or applicatorsticks Using a glassslide Vertically in the water bath Position the section into contactwith the slide 8) Drying sections Place into the oven Dried in oven for overnight at 37°C Result Problems and Solutions Example Materials Apparatus Equipments Figure 5.1 Tissue block Figure 5.3 Biohazard bags Figure 5.4 Glass slides Figure 5.2 Microtome blade Figure 5.5 Rotary microtome Figure 5.8 Freezer Figure 5.7 Tissue flotation bath Figure 5.8 Oven Figure 5.9 Rotary microtome Figure 5.10 Tissue block on microtome Figure 5.11 Ribbon section on microtome Figure 5.13 Transferring to Figure 5.12 Ribbon section water bath Figure 5.14 Fishing technique Figure 5.15 Slides on slide rack Figure 5.16 Drying slides in the oven Figure 5.18 Tissue sectioning Figure 5.17 Knife lines on tissue section TISSUE SECTIONING
H&E staining is the chemical interaction betweem the sample tissue and dye. Hemotoxylin, a basic dye present blue-purple contrast on basophilic components, mainly those with nucleic acid such as chromatin, ribosomes and RNA-rich cytoplasmic regions. An acidic eosin counterstains the basic elements like RBC, cytoplasm, muscle and collagen in multiple shades of pink, orange and red After the cycle has completed, the stained slide is ready to be covered by cover-slipped Result Unstained tissue slides Hematoxylin Xylene Eosin Absolute alcohol 95% alcohol Distilled water Tap water Staining sections Slide staining rack Oven Stopwatch Take the slide from the oven and allow it to cool for 3-5 mins Transfer the slides to the slide rack to be delivered to staining section By following the table given, Stain the slide by dipping it completely in the reagents according to the time given to each reagent Table 1: Staining sequence Principle Materials Reagents Figure 6.1 Unstained tissue slides Apparatus Figure 6.2 Oven Figure 6.3 Slide staining rack Figure 6.4 Staining sections Figure 6.5 Stopwatch Figure 6.6 Slides in the oven Figure 6.7 Transferring slides Procedure Figure 6.8 Staining process Figure 6.9 Stained tissue on a slide Problems and Solutions H&E STAINING
place a drop of mountant on the center of cleaned coverslip mounting medium spread quickly over the section Excess mounting medium PPrroobblleemmss Air bubbles trapped beneath the coverslips Remove the traped air by gently squeezing Wet the slide in xylene and remove it with tissue SSoolluuttiioonnss invent the slide and slowly lower the slide on the coverslip until the mounting medium touches the section Remove surplus clearant from the back of the slide and around the section using alcohol pad select an appropriate sized coverslip, and use alcohol pad to clean the coverslip The final step of histological preparation of a slide is mounting. This protects the cell film from damage, air drying and stain fading. The refractive index (RI) of the glass, cellular material, coverslip and mounting medium should closely match each other for proper visualization of cellular characteristics. Also, labeling is a common practice to affix to the slide after mounting. Labels needs to be fixed to the the frosted-end and orientated so the details can be read with the slide in this upright position (Fig. 7.7) MOUNTING & LABELING Principle Principle Principle MMaatteerriiaallss RReeaaggeennttss AAppppaarraattuuss CCoovveerrsslliippss ((2222xx2222mmmm//2222xx4400mmmm)) aallccoohhooll ppaadd ppeenncciill SSlliiddee rraacckkss HH&&EE ssttaaiinneedd sslliiddee ((FFiigguurree 77..11)) MMoouunnttiinngg mmeeddiiuumm ((CCoovveerrSSeeaall--XX)) XXyylleennee LLaabbeelliinngg SSeeccttiioonn Slide RReessuulltt CCoovveerrsslliipp Figure 7.7 Labeling of slide FFiigguurree 77..11 HH&&EE ssttaaiinneedd slide Histopathology is a branch of pathology that involves the examination of tissues for the diagnosis of disease. In a histopathology lab, pathologists analyze tissue samples to identify abnormalities and make diagnoses. CONCLUSION FFiigguurree 77..22 2222xx2222mmmm ccoovveerrsslliippss FFiigguurree 77..33 cclleeaann tthhee sslliiddee FFiigguurree 77..44 oonnee ddrroopp ooff mmoouunnttiinngg mmeeddiiuumm FFiigguurree 77..55 sseeccttiioonn ttoouucchheess tthhee ssuurrffaaccee ooff mmoouunnttiinngg mmeeddiiuumm FFiigguurree 77..66 ccoommpplleetteedd sstteeppss ooff hhiissttoollooggiiccaall pprreeppaarraattiioonn
Jujenum Duodenum Mucosa Submucosa Brunner’s glands Villi Location: small intestine Function: Brunner's glands in the submucosa secrete alkaline mucus, protecting the intestinal lining from acidic damage and aiding in digestion by neutralizing stomach acids. Regulating the release of hormones that aid in digestion. Ileum Interstitium & small cappilaries Location: seminiferous tubules Characteristic: Spermatogenic series cells Contains sertoli cells Function: Production of male gametes, spermatzoa Support cells for spermatogenesis Seminiferous tubules Ducts of the epididiymis Tunica albuginea Smooth muscle Spermatozoa Testis Thyroid Colloid Blood vessels Slides Interpretation Submucosa Mucosa Muscularis mucosae Location: small intestine Function: Peyer's patches help to spot trouble, train defenses, and maintain a balanced immune response. A major site for nutrient absorption. Finger-like projections called villi, which increase the surface area available for nutrient absorption. Serosa Villi Peyer’s patches Submucosa Muscularis interna Villi Location: small intestine Function: Completes the absorption of nutrients that were not fully absorbed in the preceding parts of the small intestine. Peyer’s patches play a critical role in immune surveillance, detecting and responding to pathogens in the gut, thus supporting the body's immune defenses. Peyer’s patches Ovary Stroma Blood vessels Location: Medulla Characteristics: Contains hilus cells Function: Production of female gametes, the ova, by the process of oogenesis endocrine organs that produce the hormones oestrogen and progesterone. Smooth muscles Location: Thyroid gland characteristics: thyroid follicles composed of a single layer of cuboidal epithelial cells Variable in size and contains homogenous colloid Function: the synthesis, storage and secretion of hormone thyroxine Parafollicular cells loose connective tissue Follicle
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