Week 2 - Histology and its methods of study.pdf

Week 2 HISTOPATHOLOGY (BMS4470A) HISTOLOGY AND ITS METHODS OF STUDY Dr. Merin Thomas [email protected] Office hours : Tuesday & Thursday – 1.00pm to 3.00pm Learning Objectives What is histology? History Methods of study Histology The term histology, is derived from the Greek histos, meaning...

Week 2 HISTOPATHOLOGY (BMS4470A) HISTOLOGY AND ITS METHODS OF STUDY Dr. Merin Thomas [email protected] Office hours : Tuesday & Thursday – 1.00pm to 3.00pm Learning Objectives What is histology? History Methods of study Histology The term histology, is derived from the Greek histos, meaning tissue (web) and logia, meaning study of Synonyms: microanatomy/ microscopic anatomy/ Cell & Tissue Biology It is the study of the microscopic anatomy of tissues which involves the examination and architectural description of the microscopic anatomy of normal cells of the body and all their contents and products It is performed by examining a thin slice (section) of tissue under a microscope Why are we studying histology? Understanding the normal structure and function of different tissues is essential for interpreting the changes that occur during disease To develop a mental image of the microscopic appearance of cells, structures, tissues, and organs To correlate microscopic appearance with function in health and disease To provide a basis for later study of anatomy and pathology Because it’s awesome ;) Where does histology fit in? Basic medical Sciences / Pre-clincal Sciences : These are prerequisites for understanding clinical and public health sciences Anatomy Gross Anatomy Biochemistry Embryology Physiology Microscopic Anatomy/Histology Microbiology Radiological Anatomy Pathology Surface Anatomy Pharmacology … History Robert Hooke was the first person to observe cells. He looked at thin slices of cork under a very simple microscope. The cork appeared as little boxes which he called cells In 1883 Mathias Schleiden and Theodor Schwann proposed that all plants and animals were composed of cells which were the basic building blocks of life In 1855 Rudolf Virchow stated that new cells arise from the division of pre-existing cells and that chemical reactions needed for life occurred inside the cell All this work led to the formation of the cell theory Cell theory: The scientific theory that all living organisms are made of cells as the smallest functional unit. History Innovations in science arise when ideas and theories that are popular at the time are combined with new technological methods. The development of the cell theory, which is essential to biology and medicine, is undoubtedly an example of this. Cell biology, cell pathology, and cell injury research are all based on the principles of cell theory. Histological Hierarchy – Structural organization of the body Cells Tissues Organs Organ systems Organism What is a Cell? Defined as the smallest basic structure and function of higher organisms capable of independent existence The study of cells is called Cytology (Cyto – Cell; logos – the study of) A cell contains three structural components: Organelles – metabolically active Cytoskeleton – structural framework (fibrillar elements) Inclusions – cell products, metabolites (metabolically inactive), CYTOSOL – fluid component (cytoplasmic matrix, intracellular fluid) What is a Tissue? It is a group of cells along with extracellular ground substance, of similar function and origin that form functional units Tissues are composed of Cells and Extracellular matrix There are FOUR basic types of human tissue Epithelial tissue Connective tissue Muscle tissue Nervous tissue All tissue types are subtypes of these four basic tissue types What is an Organ? An organ represent an even greater measure of complexity and is composed of various tissues, have special shape, structure and function Organs are composed of: Parenchyma (the cells that perform the main function of organ) Stroma (supporting tissue) What is an Organ System? At an even higher level of organization: An organ system is composed of several organs which have related function (such as the gastrointestinal system, respiratory system, cardiovascular system, endocrine system) What is an Organism? An organism can be seen to be formed of different levels of organization, with increasing levels of complexity and each of which plays important roles homeostasis of the body in the physiological Histology Microscopic study of tissue General Histology – Microscopic study of the 4 basic tissues Systemic Histology – Microscopic study of tissues of the different organs/organ systems STRATIFIED EPITHELIUM BONE General Histology CUBOIDAL EPITHELIUM COLUMNAR EPITHELIUM Systemic Histology (EXAMPLE) Source of Tissue Histological examination of tissues starts with biopsy or autopsy (or necropsy, in the case of animal tissues). Biopsy is an examination of tissue taken from a living body Autopsy is an examination of post-mortem tissue Necropsy is an examination of tissue taken from dead animal Examples of types of Biopsies Preparation of Tissue prior to histopathological study Basic sample preparation techniques prior to histological analysis 1. Fixing 2. Processing - dehydration, clearing, and infiltration 3. Embedding 4. Sectioning 5. Mounting 6. Staining Will be discussed in detail in later classes… Special Techniques Used for Histological Diagnosis Light microscopy Electron microscopy Histochemistry Immunohistochemistry Tissue / Cell Culture Cytogenetics and Molecular Cytogenetics Bone Marrow smear / Blood Smear Fine needle aspiration / biopsy Flow cytometry Autoradiography Histochemistry Refers to the science of using chemical reactions between laboratory chemicals and components within tissue A method of tissue staining that gives information on the presence and location of intracellular and extracellular macromolecules Example: A commonly performed histochemical technique is the Perls Prussian blue (PPB) reaction, used to demonstrate iron deposits in diseases like Hemochromatosis Histochemistry Accumulation of iron in liver Hereditary hemochromatosis is a disorder that causes the body to absorb too much iron from the diet. Iron stain (Prussian blue) Immunohistochemistry A highly specific interaction between macromolecules is that between an antigen and its antibody The body’s immune cells interact with and produce antibodies against other macromolecules—called antigens—that are recognized as “foreign,” not a normal part of the organism, and potentially dangerous. Antibodies belong to the immunoglobulin family of glycoproteins and are secreted by lymphocytes. Antibodies normally bind specifically to their provoking antigens and help eliminate them Immunohistochemistry Every immunohistochemical technique requires an antibody against the protein or other antigen that is to be detected. In immunohistochemistry, a tissue section that contains the protein of interest is incubated in a solution containing antibody against this protein. The antibody binds specifically to the protein, if present, and the protein’s location in the tissue or cells can be seen with either the light or electron microscope by visualizing the antibody bound to its specific antigen. Immunohistochemistry Antibodies are commonly tagged with fluorescent compounds, with peroxidase or alkaline phosphatase for histochemical detection, or with electron-dense gold particles for TEM. Medical Application - cells in some diseases, including many cancer cells, often produce proteins unique to their pathologic condition, immunohistochemistry can be used by pathologists to diagnose many diseases, including certain types of tumors and some virus-infected cells. Immunohistochemistry – Direct and Indirect methods A section of small intestine treated with an antibody against the enzyme lysozyme. The secondary antibody labeled with peroxidase was then applied and the localized brown color produced histochemically with the peroxidase substrate 3,3′diaminoazobenzidine (DAB). The method demonstrates lysozyme-containing structures in scattered macrophages and in the large clusters of cells. Nuclei were counterstained with hematoxylin. (X100) Tissue / Cell Culture Live cells and tissues can be maintained and studied outside the body in culture (in vitro). Cell culture allows the direct observation of cellular behavior under a phase-contrast microscope, and many experiments technically impossible to perform in the intact animal can be accomplished in vitro. Medical Application - Cell culture is very widely used to study molecular changes that occur in cancer; to analyze infectious viruses, mycoplasma, and some protozoa; and for many routine genetic or chromosomal analyses. Hybridization Techniques Hybridization usually implies the specific binding between two single strands of nucleic acid, which occurs under appropriate conditions if the strands are complementary. The greater the similarities of their nucleotide sequences, the more readily the complementary strands form “hybrid” doublestrand molecules. Hybridization at stringent conditions allows the specific identification of sequences in genes or RNA. This can occur with cellular DNA or RNA when nucleic acid sequences in solution are applied directly to prepared cells and tissue sections, a procedure called in situ hybridization Hybridization Techniques Fluorescent labelled probes used – Fluorescence in situ hybridization (FISH) – molecular cytogenetics Common HER2 (red) and CEP17 (green) status demonstrated using fluorescence in situ hybridization. Magnification, 100×10. (A) HER2/CEP17-negative group; no amplification of HER2 or CEP17. (B) HER2/CEP17-positive group; amplification of HER2 and normal CEP17. HER2, human epidermal growth factor receptor 2; CEP17, chromosome enumeration probe 17. Bone Marrow Smear Aspiration of the marrow has been primarily utilized for cytologic assessment, with analysis directed toward assessing the morphology and obtaining a differential cell count. Further sampling allows material to be directed toward other ancillary tests, such as cytogenetics, molecular studies, microbiologic cultures, immunohistochemistry, and flow cytometry. Biopsies, on the other hand, allow evaluation of the marrow’s overall cellularity, detection of focal lesions, and determination of the extent of infiltration by various pathologic entities Blood Smear/ Peripheral blood film A laboratory work-up that involves cytology of peripheral blood cells smeared on a slide It exposes the morphology of peripheral blood cells, which ensures its place in the morphologic diagnosis of various primary and secondary blood and blood related diseases. Let’s Discuss with an example Let’s Discuss with an example Breast Cancer – HER2 gene testing Let’s Discuss with an example Lung Cancer – ALK gene testing Several platforms developed to detect ALK positive cells Fluorescence in situ Hybridization (FISH) Immunohistochemistry (IHC) Polymerase Chain Reaction (RT PCR) Next Generation Sequencing (NGS) FISH Image source: Chia et al., Clinical Epidemiology 2014:6 423–432 IHC A: Score 3: intense granular cytoplasmic staining B: Score 2: moderate, smooth cytoplasmic staining C: Score 1: faint, barely discernable cytoplasmic staining D: Score 0: no staining Let’s Discuss with an example CHRONIC MYELOID LUKEMIA (CML) Bone marrow smear Karyotype (A-B): Chronic myeloid leukemia (CML) showing hypercellular bone marrow on H & E staining at x100 & x400 magnification. (C): Classical karyotype of CML translocation-t(9; 22) & (D): CML Complex translocation 9,6,19,22. Ujjan, I. D., Akhund, A. A., Saboor, M., Qureshi, M. A., & Khan, S. (2015). Cytogenetic and Molecular Analyses of Philadelphia Chromosome Variants in CML (chronic myeloid leukemia) Patients from Sindh using Karyotyping and RT-PCR. Pakistan Journal of Medical Sciences, 31(4). FISH A nucleus lacking the t(9;22) translocation will exhibit the two orange, two green (2O2G) signal pattern. In a nucleus containing a simple balanced t(9;22), one orange and one green signal from the normal 9 and 22 chromosomes and two orange/green (yellow) fusion signals, one each from the derivative 9 and 22 chromosomes, will be observed (1O1G2F) In situ hybridization is a histologic technique used to visualize what type of macromolecule? 1. 2. 3. 4. Proteins Carbohydrates Nucleic acids Lipids To identify and localize a specific protein within cells, which approach would best used? 1. Enzyme histochemistry 2. Immunohistochemistry 3. Transmission Electron Microscope (TEM) 4. Autoradiography 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 Musumeci, G. (2014). Past, present and future: overview on histology and histopathology. Journal of Histology & Histopathology, 1(1), 5. https://doi.org/10.7243/2055-091x-1-5 Mazzarini, M., Falchi, M., Bani, D., & Migliaccio, A. R. (2020). Evolution and new frontiers of histology in bio‐medical research. Microscopy Research and Technique, 84(2), 217– 237. https://doi.org/10.1002/jemt.23579. Buja, L. M. (2021). The cell theory and cellular pathology: Discovery, refinements and applications fundamental to advances in biology and medicine. Experimental and Molecular Pathology, 121, 104660. https://doi.org/10.1016/j.yexmp.2021.104660