Histopathology (BMS4470A) Tissue Preparation - Week 3 PDF

Week 3 HISTOPATHOLOGY (BMS4470A) PREPARATION OF TISSUES FOR STUDY Dr. Merin Thomas [email protected] Office hours : Tuesday & Thursday – 1.00pm to 3.00pm Learning Objectives What is tissue preparation Steps involved in preparation of tissue What is tissue preparation? Why is it needed Most f...

Week 3 HISTOPATHOLOGY (BMS4470A) PREPARATION OF TISSUES FOR STUDY Dr. Merin Thomas [email protected] Office hours : Tuesday & Thursday – 1.00pm to 3.00pm Learning Objectives What is tissue preparation Steps involved in preparation of tissue What is tissue preparation? Why is it needed Most fresh tissue specimens are colorless and squishy. They provide little useful information. For scientific or diagnostic purposes, tissue specimens must undergo substantial alteration in preparation for viewing under a microscope. Important steps prior to initiation of tissue preparation Tissues come to the lab either in fixative or fresh Sample (tissue) received must be cross checked for patient name, history, clinical details and test required. Attached test requisition form must be verified. Sample then delivered to the ”trimming” or “ cut-up” area and is then sectioned to facilitate further processing. Important steps prior to initiation of tissue preparation Apz_Grossing. (2021, January 28). Histology - Grossing station. Grossing station. https://www.grossingstation.com/histology-specimen-grossing/ Klatt, E. C., MD. (n.d.). Histotechniques. Edward C. Klatt MD. https://webpath.med.utah.edu/HISTHTML/HISTOTCH/TECH001.html Steps taken to prepare tissue for histological study 1. Fixation 2. Processing - dehydration, clearing, and infiltration 3. Embedding 4. Sectioning 5. Mounting 6. Staining Fresh lung specimen Formalin container for specimen Tissue comes to the lab fresh or in formalin The specimen is placed in a “cassette” for tissue fixation The specimen is embedded in paraffin… …and then cut into super thin slices Microtome...and then stained and coverslipped. FIXATION Objective of tissue fixation is to preserve cells and tissue components and keep them as close to normal as possible, allow for the preparation of thin and stained sections Fixation is usually the first step in a process to prepare a sample of biological material for microscopy or other analysis. FIXATIVES – HOW DO THEY ACT Fixative usually acts to disable intrinsic biomolecules—particularly proteolytic enzymes—which otherwise digest or damage the sample. Fixative typically protects a sample from extrinsic damage. Fixatives are toxic to most common microorganisms (bacteria in particular) that might exist in a tissue sample Fixatives often alter the cells or tissues on a molecular level to increase their mechanical strength or stability. This helps in preserving the morphology (shape and structure) of the sample as it is processed for further analysis CHOICE OF FIXATIVE & FIXATION PROTOCOL It depends on the additional processing steps and final analyses that are planned. For example, immunohistochemistry uses antibodies that bind to a specific protein target. Prolonged fixation can chemically mask these targets and prevent antibody binding. In these cases, a 'quick fix' method using cold formalin for around 24 hours is typically used THE MOST COMMONLY USED FIXATIVE IN HISTOLOGY Formalin (10% neutral buffered formaldehyde in water) TYPES OF FIXATION Heat fixation: preserves overall morphology but not internal structures. It is used for the fixation of single cell organisms, most commonly bacteria and archaea. The organisms are typically mixed with water or physiological saline which helps to evenly spread out the sample. Once diluted, the sample is spread onto a microscope slide TYPES OF FIXATION Freezing: Used to get rapid results but does not give fine details. Samples are cooled at a rate sufficient for water to be frozen in a vitreous state, without the formation of ice crystals. Fixation occurs in milliseconds, structures are preserved hydrated, and in as close to their native state. TYPES OF FIXATION Chemical Fixation : In this process, structures are preserved in a state (both chemically and structurally) as close to living tissue as possible. This requires a chemical fixative that can stabilize the proteins, nucleic acids and microsubstances of the tissue by making them insoluble TYPES OF FIXATION Target Fixative of Choice Fixative to Avoid Proteins Neutral Buffered Formalin, Paraformaldehyde Osmium Tetroxide Enzymes Frozen Sections Chemical Fixatives Lipids Frozen Sections, Glutaraldehyde/Osmium Tetroxide Alcoholic fixatives, Neutral Buffered Formalin Nucleic Acids Alcoholic fixatives, HOPE (HepesGlutamic acid buffer mediated Organic solvent Protection Effect) Aldehyde fixatives Mucopolysaccharides Frozen Sections Chemical fixatives Biogenic Amines Glycogen Bouin Solution , Neutral Buffered Formalin Alcoholic based fixatives Osmium Tetroxide FACTORS AFFECTING FIXATION 1. pH of the fixative Should be kept in the physiological range, between pH 4 to 9. The pH for the ultrastructure preservation should be buffered between 7.2 to 7.4 2. Osmolarity of the fixative: Try to avoid Hypertonic solutions :give rise to cell shrinkage. Hypotonic solutions : result in cell swelling and poor fixation 3. Size of the Specimen : Ideal thickness is 1-4mm FACTORS AFFECTING FIXATION 4. Volume of the Fixative : At least 15-20 times greater than tissue volume 5. Temperature: High temperature increases the speed of fixation. However, care is required to avoid cooking the specimen. Fixation is routinely carried out at room temperature. 6. Duration: As a general rule 1hr per 1mm FACTORS AFFECTING FIXATION Fixation is a chemical process, and time must be allowed for the process to complete. Although "over fixation" can be harmful, under-fixation has been appreciated as a significant problem and may be responsible for inappropriate results for some assays PROCESSING Processing TISSUE PROCESSING The aim of Tissue Processing is to remove water from tissues and replace with a medium that solidifies to allow thin sections to be cut THREE steps Dehydration Clearing Infiltration and impregnation TISSUE PROCESSING Dehydration: The tissue is transferred through a series of increasingly concentrated alcohol solutions, ending in 100%, which removes all water. Clearing: The ethanol is then replaced by an organic solvent miscible with both alcohol and the embedding medium. The reagents used here gives the tissue a translucent appearance. Infiltration: The tissue is then placed in melted paraffin until it becomes completely infiltrated with this substance. TISSUE PROCESSING Water Molecule is removed from tissue Dehydration Dehydrating agent is replaced by a clearing agent Clearing Tissue is infiltrated supporting medium Infiltration with a EMBEDDING In the embedding process, the tissue is surrounded by a molten medium using a mold The embedding medium has three crucial functions 1. To give support to the tissue 2. To prevent distortion of the tissue during the cutting 3. To preserve the tissue for archival use EMBEDDING The choice of the embedding medium of the tissue depends on the type of tissue, type of microtomy, and type of microscope to examine the tissue Embedding material For light microscopy, paraffin wax is most frequently used. For electron microscopy, resins are the most commonly used EMBEDDING During embedding the tissue samples are placed into molds along with liquid embedding material which is then hardened. Formalin-fixed, paraffin-embedded (FFPE) tissues may be stored indefinitely at room temperature. EMBEDDING Tissue Embedding Machine Dey, P. (2023). Basic and advanced laboratory techniques in histopathology and cytology. Springer Nature. EMBEDDING Dey, P. (2023). Basic and advanced laboratory techniques in histopathology and cytology. Springer Nature. SECTIONING/MICROTOMY After embedding the tissue and preparing the block, the next step is microtomy (Greek. mikros-small; temnein-to cut) i.e., the next step is to cut/ section the tissue into thin sections. SECTIONING/MICROTOMY Microtome – the main instrument using which the embedded tissue in the paraffin block is cut into thin sections SECTIONING/MICROTOMY The type of knife used ,the thickness of sections etc. vary with the type of microscope which will be used for the analysis For light microscopy, a steel knife mounted in a microtome is used to cut 3-5 µm thick tissue sections which are mounted on a glass microscope slide. (1mm = 1000 µm) For transmission electron microscopy, a diamond knife mounted in an ultra microtome is used to cut 0.1-0.5 µm thick tissue sections which are mounted on a 3-mm-diameter copper grid. Then the mounted sections are treated with the appropriate stain. SECTIONING/MICROTOMY Paraffin sectioning using a Microtome MOUNTING Sections are mounted onto clean glass slides and labelled. Sections are floated in a water bath and then mounted on the slides Mounted sections are dried on a hot plate or a hot air oven and then kept overnight at room temperature. Slides with paraffin-embedded sections can be stored either at room temperature or at 2-8 °C for several years in slide storage boxes. STAINING Biological tissue has little contrast under the microscope The tissue section is colorless because the fixed protein has the same refractive index as that of glass. Staining is employed to give both contrast to the tissue as well as highlighting specific features of interest which helps us to understand the morphology of the tissue. So, dyes that have a specific affinity with the different tissue proteins and color them differently are used. Unstained carotid artery H&E stained carotid tissue sections artery tissue sections (Li et al., 2020) STAINING The dye used may be natural or synthetic. Natural dyes are extracted from plants & animals whereas majority of the synthetic dyes are petroleum derivatives. Natural dyes are rarely used now except for haematoxylin and carmine. STAINING The dyes can be classified based on their electrical charge Anionic dye or Acidic dye Cationic dye or basic dye Neutral dye Chelating dye Different types of dyes are used based on the tissue that is being analysed. STAINING Types of dye Anionic dye/ acidic dy Cationic dye/ basic dye Neutral dye Ligand or Chelating dye Charge of the dye Negatively charged Tissue to bind Cytoplasmic proteins Collagen Positively Nucliec acid charged Epithelial mucin Contains both Both nucleus acidic and basic and dye cytoplasm Weak acid so Nucleus and anionic and cytoplasm negatovely charged Example Eosin Methyl Green Ethyl green Alcian Blue Giemsa Al ligand with haematoxylin: Harris’Hematoxylin, and Mayer’s Hematoxylin Fe ligand with hematein: Iron haematoxylin STAINING - Factors that influence staining 1. Dye affinity to the target tissue specimen: Acidic dye binds with positively charged acidophilic tissue and basic dye binds with negatively charged basophilic tissue. 2. Specimen geometry: E.g., Thick tissue: Less penetration of dye 3. Target concentration: More the amount of the target tissue the more intense will be staining. 4. Rate of reaction: Short reaction time often decreases stain intensity 5. Rate of stain loss: Too much differentiation often removes the stain STAINING In a histopathology lab, the term “routine staining” refers to the Hematoxylin and Eosin stain (H&E) that is routinely used with all tissue specimens to reveal the underlying tissue structures and conditions The term “special stains” is usually used to refer to many alternative staining techniques that are used when H&E does NOT provide all the information the pathologist or researcher needs STAINING Most tissue specimens are stained with hematoxylin and eosin. Most blood smears are stained with the Wright-Giemsa stain. Other stains (like silver, PAS (Periodic acid–Schiff), and acid-fast stains) can be used to highlight special cells or features. STAINING – H&E stain Most commonly used; routine stain Hematoxylin stains DNA in the cell nucleus, RNA- rich portions of the cytoplasm producing a dark blue or purple color. Eosin stains other cytoplasmic structures and collagen pink. Eosin is considered a counterstain, which is usually a single dye applied separately to distinguish additional features of a tissue. STAINING – Blood smear Blood smears are routinely stained with mixtures of acidic (eosin) and basic (methylene blue) dyes. In the lab – Leishman Stain Blood smear stained with Wright-Giemsa stain Section of pancreas stained with hematoxylin and eosin Section of pancreas stained with hematoxylin only Section of pancreas stained with eosin only HISTOPATHOLOGY – Interpretation of structures in tissue sections Microscopic preparations are the result of a series of processes that begin with collecting the tissue and ended with mounting a coverslip on the slide. Certain steps in this procedure may distort the tissues slightly, producing minor structural abnormalities called artifacts not present in the living tissue Artifact – an artificial structure or tissue alteration on a prepared microscopic slide because of an extraneous factor. Can occur either before fixation or during any of the steps involved in the preparation of tissue for study Improper prefixation Solutions such as normal saline do not fix the tissue and tissue will undergo autolysis. Microscopically, such a section will show features of autolysis artifact as well as separation of epithelium from the connective tissue (simulate vesiculobullous lesions such as pemphigus) Artifacts related to microtomy Histopathological image shows scoring and tearing of section due to nick in knife edge In preparation for practicals.. STAINING – Blood smear - Leishman Stain Leishman stain, characterized by shades of blue, pink and purple, gives a clear differentiation and understanding of cellular morphology The nuclei take up a blue stain while the cytoplasm takes on a pink or purple stain depending on the type of cell and its composition. These color differences allows for the differentiation of different blood cell types including RBCs, Lymphocytes, Neutrophils, Monocytes, Basophils, Platelets The Leishman stain also provides information about cellular function STAINING – Blood smear - Leishman Stain - PRINCIPLE Leishman stain – ROMANOWSKY stain Chemical components of Leishman Stain Methylene blue – stains the acidic part of the cell i.e., the nuclei (DNA) & the cytoplasm (RNA) of WBCs and granules of basophils Eosin – stains basic part of the cells (eosinophilic granules) and Hb of red cells Methyl alcohol : Fixes the smear to the slide. STAINING – Blood smear - Leishman Stain – IMPORTANT TIPS!! KEEP THE FOLLOWING IN MIND WHILE MAKING A BLOOD SMEAR The size of the drop : If the drop is too large, the smear will be too long or too thick Speed : The speed at which the spreader slide is moved is very important. Too fast, the smear will be short and to slow the smear will be short Angle of the spreader slide: Angle determines the length of the smear. 45° angle is optimal. REFERENCES Mescher, A. L. (2018). Junqueira’s Basic Histology Text and Atlas, FIFTEENTH. In McGrawHill Education eBooks. http://125.212.201.8:6008/handle/DHKTYTHD_123/5904 Aziz, S. J., & Zeman-Pocrnich, C. E. (2022). Tissue Processing. Methods in molecular biology (Clifton, N.J.), 2422, 47–63. https://doi.org/10.1007/978-1-0716-1948-3_4 Dey, P. (2023). Basic and advanced laboratory techniques in histopathology and cytology. Springer Nature. Taqi, S. A., Sami, S. A., Sami, L. B., & Zaki, S. A. (2018). A review of artifacts in histopathology. Journal of oral and maxillofacial pathology : JOMFP, 22(2), 279. Li, D., Hui, H., Zhang, Y., Tong, W., Tian, F., Yang, X., Liu, J., Chen, Y., & Hui, H. (2020). Deep learning for virtual histological staining of Bright-Field microscopic images of unlabeled carotid artery tissue. Molecular Imaging and Biology, 22(5), 1301–1309. https://doi.org/10.1007/s11307020-01508-6