MODULE 1 HISTOPATH INTRODUCTION.docx

PINES CITY COLLEGES COLLEGE OF MEDICAL LABORATORY SCIENCE 1^st^ Semester A.Y. 2024-2025 HISTOPATHOLOGIC & CYTOLOGIC TECHNIQUES **INTRODUCTION TO HISTOPATHOLOGIC TECHNIQUES** **HISTOPATHOLOGY** - Combination of histology and pathology - also called "anatomic pathology"...

PINES CITY COLLEGES COLLEGE OF MEDICAL LABORATORY SCIENCE 1^st^ Semester A.Y. 2024-2025 HISTOPATHOLOGIC & CYTOLOGIC TECHNIQUES **INTRODUCTION TO HISTOPATHOLOGIC TECHNIQUES** **HISTOPATHOLOGY** - Combination of histology and pathology - also called "anatomic pathology" - the art and science of producing a quality tissue section to enable the pathologist to diagnose the presence or absence of disease - autopsy and biopsy specimens **PATHOLOGIST** - A highly specialized physician who identifies diseases by studying cells and tissues under a microscope **HISTOLOGY** - Is a branch of biology that deals with the microscopic anatomy of cells, tissues and organs in relation to their function. It is the microscopic counterpart to gross anatomy. - Also called **[microanatomy]** **PATHOLOGY** - the scientific study of the nature of the disease and its causes, processes, development, and consequences. **HISTOTECHNOLOGY** - is the art and science performed by the histotechnologist to produce a tissue section of good quality that will enable the pathologist to diagnose the presence or absence of disease Additional Information: \- Performed through macroscopic examination \- Produce tissue section in good quality \- Needed to produce good section of tissue to examine and diagnose **DIVISIONS OF PATHOLOGY** I. **Gross Pathology and Microscopic Pathology** **Gross Pathology:** \- The recognition of disease based on macroscopic examination of surgical specimens generated at the time of surgery or at autopsy. **Microscopic Pathology:** \- The recognition of disease based on microscopic examination of surgical specimens generated at the time of surgery or at autopsy. II. **Anatomic Pathology** - The study of changes in the function, structure, or appearance of organs or tissues, including postmortem examinations and the study of biopsy specimens. **A. Surgical Pathology** \- The pathology of disease processes that are surgically accessible for diagnosis or treatment. \- The study of gross appearance and histology of tissues removed during surgery **B. Autopsy Pathology** \- The pathology of disease processes that are surgically accessible for diagnosis or treatment. **C. Exfoliative Cytology** \- A branch of General Cytology which deals with the microscopic study of cells that have been desquamated from the epithelial surfaces III. **Clinical Pathology** - The branch of general pathology directed to the diagnosis and monitoring of diseases through the examination of blood, body fluids, secretions, and tissue biopsy specimens for chemical, morphological, microbiological, and immunological abnormalities. - Identifies and interprets changes that characterize different diseases or disease states in cells, tissues, and fluids of the body **A. Clinical Chemistry (incl. Toxicology)** **B. Hematology** **C. Blood Banking** **D. Microbiology** **E. Clinical Immunology & Serology** **PIONEERS IN HISTOPATHOLOGY** 1. **Marie Francois Xavier Bichat-** Father of modern histology 2. **Johannes Peter Muller-** Father of histopathology and cellular pathology 3. **Ferdinand Blum-** Proposed the use of formaldehyde as a fixative **EXAMINATION OF FRESH TISSUE** Fresh tissues have the advantage of being examined in the living state, thereby allowing protoplasmic activities such as motion, mitosis, and phagocytosis to be observed. Its use is limited, however, because of the fact that tissues examined in the fresh state are not permanent **FINE NEEDLE ASPIRATION** - the simplest, least invasive test and uses the smallest needle to simply remove cells from the area of abnormality. This is not always adequate to obtain a diagnosis, depending on the area to be biopsied. **CORE NEEDLE BIOPSY** - removes not only cells, but also a small amount of the surrounding tissue. This provides additional information to assist in the examination of the lesion. **INCISIONAL BIOPSY** - takes out even more surrounding tissue. It takes out some of the abnormality, but not all. The doctor will slice into the lesion and remove only a portion of it. If the lesion is found to be cancerous, further surgery may be needed to remove or excise the entire lesion. **EXCISIONAL BIOPSY** - generally removes the entire area in question. - also called a **[wide local incision]** - The amount of normal tissue taken (also called the **clinical margin**) depends on the thickness of the tumor. **PUNCH BIOPSY** - considered the primary technique for obtaining diagnostic full-thickness **skin specimens**. It requires basic general surgical and suture-tying skills and is easy to learn. The technique involves the use of a circular blade that is rotated down through the epidermis and dermis, and into the subcutaneous fat, yielding a 3- to 4- mm cylindrical core of tissue sample. **SHAVE BIOPSY** - where small fragments of tissue are "shaved" from a surface (usually skin). **CURETTINGS** - where tissue is scooped or spooned to remove tissue or growths from body cavity such as endometrium or cervical canal.  **NOTE:** - Specimens are usually received in fixative (preservative) but sometimes they arrive fresh and must be immediately fixed - Tissue specimens received in the surgical pathology laboratory should have a request form that lists the patient information and clinical history along with a description of the site of origin - The specimens are accessioned by giving them a number that will identify each specimen for each patient. It is important that specimens are properly identified to minimize the risk of mislabeling. **Methods of Fresh Tissue Examination** 1. **Teasing or Dissociation** - is a process whereby a selected tissue specimen is immersed in isotonic salt solution such as normal saline or Ringer's solution in a petri dish or watch glass, carefully dissected with a needle and separated by direct or zigzag spread using an applicator stick. 2. **Squash Preparation (Crushing)** - is a process whereby small pieces of tissue (not more than **one mm**. in diameter) are placed in a microscopic slide and forcibly compressed with another slide or with a cover glass. 3. **Smear Preparation** - Is the process of examining sections or sediments, whereby cellular materials are spread lightly over a slide by means of wire loop or applicator. - This technique is especially useful in cytological examinations, particularly for cancer diagnosis a. ***Streaking:*** With an applicator stick or a platinum loop, the material is rapidly and gently applied in a direct or zigzag line throughout the slide, attempting to obtain a relatively uniform distribution of secretion. b. ***Spreading*** - A selected portion of the material is transferred to a clean slide and gently spread into a moderately thick film by teasing the mucous strands apart with an applicator stick c. ***Pull-Apart*** -- This is done by placing a drop of secretion or sediment upon one slide and facing it to another clean slide - two slides are then pulled apart with a single uninterrupted motion, and the specimen is placed under the microscope for immediate examination or applied with vital stains. d. **Touch Preparation (Impression Smear)** - special method of smear preparation whereby the surface of a freshly cut piece of tissue is brought into contact and pressed on to the surface of a clean glass slide 4. **Frozen Section** - Normally utilized when a rapid diagnosis of the tissue in question is required and is especially recommended when lipids and nervous tissue elements are to be demonstrated. - usually done on muscle and nerve biopsies as well as on surgically removed tumors - Very thin slices (10-15 micra) are cut from fresh tissue frozen on a microtome with CO~2~ or on a Cryostat and then transferred to a slide, and processed for light microscopic studies **ADVANTAGE:** rapid processing time with less equipment requirement, and less need for ventilation in the laboratory. **DISADVANTAGE**: relatively poor quality of the final slide **Cryostat**- a cold chamber kept at an atmospheric temperature of **-10° to -20° C** **Application of Frozen Section** 1\. Rapid pathologic diagnosis during surgery 2\. Diagnostic and research enzyme histochemistry 3\. Diagnostic and research demonstration of soluble substances such as lipids and carbohydrates 4\. Immunofluorescent and immunohistochemical staining 5\. Some specialized silver stains, particularly in neuropathology The tissue for freezing should be fresh, and freezing should be done as quickly as possible. The more commonly used methods of freezing include: - **Liquid nitrogen** - **Isopentane cooled by liquid nitrogen** - **Carbon dioxide gas** - **Aerosol sprays** **Note:** Slow freezing can cause distortion of tissue due to ice crystal artifacts. 1. **Liquid Nitrogen** - Used in histochemistry and during intraoperative procedures and is the most rapid of the commonly available freezing agents. **Disadvantages:** - Soft tissue is liable to crack due to the rapid expansion of the ice within the tissue - Overcools urgent biopsy blocks, causing damage to both block and blade (if -70°C or below) - Causes Vapor phase to form around the tissue 2. **Isopentane** - Liquid at room temperature - excellent method for freezing muscle tissue. 3. **Carbon Dioxide Gas** - Tissue blocks can be frozen by adapting a conventional freezing microtome gas supply of carbon dioxide gas from a C0~2~ cylinder, or by using a specially made piece of equipment known as cryostat. 4. **Aerosol Sprays** - Adequate for freezing small pieces of tissue except muscle **TWO METHODS OF PREPARING FROZEN SECTIONS** 1. **Cold Knife Procedure** - Tissue blocks can be frozen by adapting a conventional freezing microtome gas supply of carbon dioxide gas from a C0~2~ cylinder - Using a cold knife in a controlled cold environment, optimum condition for sectioning shall be provided for by the following temperatures: - Knife -40° to - 60°C - Tissue -5° to - 10°C - Environment 0° to - 10°C 2. **Cryostat Procedure** - This method makes use of the Cryostat, an apparatus used in fresh tissue microtomy. The cryostat consists of an insulated microtome housed in an electrically driven refrigerated chamber and maintained at temperatures near -20°C, where microtome, knife, specimen and atmosphere are kept at the same temperature - optimum working temperature of cryostat: **-18 to -20°C** **SPECIAL PROCESSING TECHNIQUES** 1. **Freeze-Drying** - Used in specialized or research laboratories - Preserves tissues by rapid freezing (quenching) of fresh tissue at -160°C and subsequently removing ice water molecules (dessication) by transferring the still frozen tissue block into a vacuum chamber at a higher temperature, e.g. -40°C (sublimation) without the use of any chemical fixative. - This method is particularly important as far as enzyme studies are concerned. In addition to demonstrating hydrolytic enzymes, mucous substances, glycogen and proteins, freeze-drying may be used for special studies, including: 1\. Immunocytochemistry 2\. Fluorescent antibody studies of polypeptide and polypeptide hormones 3\. Autoradiography 4\. Microspectrofluorimetry of autofluorescent substances 5\. Formaldehyde-induced fluorescence of biogenic amines (to demonstrate hydroxytryptamine, adrenaline, and other catecholamines) 6\. Scanning electron microscopy 2. **Freeze-substitution** - A process of dehydration, performed at temperatures low enough to avoid the formation of ice crystals and to circumvent the damaging effects observed after ambient-temperature dehydration - Difference with freeze-drying technique: instead of being subjected to dehydration in an expensive vacuum drying apparatus, is fixed in Rossman\'s formula or in 1% Acetone and dehydrated in absolute alcohol **ADVANTAGE:** more economical and less time-consuming than freeze-drying REFERENCES: Bancroft John D. (2010) THEORY AND PRACTICE OF HISTOLOGICAL TECHNIQUES; 6^TH^ edition.Elsevier(Singapore) PTe Ltd Winsland House 1 Singapore 239S19 Bruce-Gregorios --Jocelyn H. HISTOPATHOLOGIC TECHNIQUES Images from Mayoclinic.org COMPILED AND PREPARED BY: Divina D. Demot, RMT Faculty, CMLS