Histo-Lec-Finals PDF

UNIVERSITY OF SAN AGUSTIN BS MEDICAL LABORATORY SCIENCE MLS15 1ST SEMESTER | 2024 - 2025 | PROF. MARK ALEXAN...

UNIVERSITY OF SAN AGUSTIN BS MEDICAL LABORATORY SCIENCE MLS15 1ST SEMESTER | 2024 - 2025 | PROF. MARK ALEXANDER TOBIAS, RMT, (ASCPi)CM, MSMT (CAR) POSTMORTEM EXAMINATION 1870s Rudolf Virchow AUTOPSY ○ Father of Modern Pathology Necropsy, post-mortem examination ○ Realized the importance of microscopes when “Autopsiaˮ - to see with oneʼs own eyes conducting pathological research to uncover minute Process of taking pieces of the tissue from a dead person for details the purpose of examination or investigation ○ Characterized as a case of leukemia - one of the ○ Determine the cause of death earliest formal reports in cancer ○ Extent if injury leading to the death of the person ○ Requirement: Consent from next of kin PREPARATIONS BEFORE POSTMORTEM EXAMINATION 1. Administrative preparations NOTE:. ○ obtain and confirm consent for autopsy Can an autopsy be done without the consent of the next of kin? Yes, if the death is deemed suspicious or falls under circumstances ○ obtain and review clinical records requiring legal or forensic investigation, such as in cases of ○ contact clinical team and staff pathologist homicide, unexplained death, or public safety concerns. 2. Preparation of autopsy rooms ○ set up dissection instruments and tools PURPOSE 3. Confirmation of the decedent identity decedent 1. Establish the cause of death ○ most important step in the autopsy procedure 2. Interpret and correlate facts surrounding death ○ identifiers on the body must be confirmed and matched 3. Collect evidence in cases of questionable death with the autopsy consent form 4. Identify the decedent (deceased) 5. Establish the time of death PRELIMINARIES FOR POSTMORTEM EXAMINATION 6. Reconstruct the events surrounding the infliction of injury Written consent form from the next of kin ○ Spouse ROLE OF MEDICAL TECHNOLOGISTS ○ Adult children 1. Assist in the collection of specimen ○ Adult grandchildren 2. Process the collected specimen ○ Parent 3. Maintain the quality control in the records and specimen ○ Siblings 4. Dispose the analytes ○ Nephew/Niece HISTORY OF AUTOPSY ○ Grandparents ○ Uncles/Aunts 44 BC - First Recorded Autopsy ○ Cousins Antistius ○ Step children ○ Examines Julius Caesarʼs body after assassination Death certificates determining which of 23 stab wounds proved fatal Medical abstract clinical data ○ It was one wound to the chest that ruptured Caesar's Medicolegal clearance aorta. PME is permitted WITHOUT consent in the following circumstances: NOTE:. 1. Ordered by the police or coroner La Mort de Cèsar, the death of Caesar Vincenzo Camuccini, 1806 2. Necessary to complete the death certificate 3. Advanced directive 4. Deceased military personnel who dies in the active duty/training in the military service THREE LEVELS OF AUTOPSY Complete: all body cavities are examined Limited (partial): all body cavities EXCLUDING head or brain Selective: specific organs only are examined ○ Minimal invasive autopsy No dead Julius Caesar, No History of Autopsy. NOTE:. Death Certificate Color: 1247 - Hsi Yuan Lu The Washing Away of the Wrongs) ○ Current: White The washing away of the wrongs ○ Previously: Blue ○ Important Section: Cause of Death ○ an instruction manual on how to conduct medico-legal investigations, examine corpses, and determine the time Cause of Death and cause of death. Immediate Hypovolemic Shock) ○ other forward-thinking forensic issues were illustrated, Antecedent: Multiple fractures) such as poisoning, decomposition, wounds from various Underlying Cause of Death: Pedestrian hit by a truck) weapons, strangulation, and fake wounds Song Ci TYPES OF AUTOPSIES DONE IN PH ○ author of “the washing away of the wrongsˮ According to Purpose 1302 1. Routine Hospital Autopsy Bartolomeo de Varignana ○ Done in private hospitals for the purpose of ascertaining ○ influenced by the washing away of the wrongs the cause of death of the person ○ conducted the first known legal autopsy ○ Cause is cannot be determined clinically or the cause of Medicolegal autopsy - death was investigated explicitly to death is problematic to the clinician determine if there was a fault 2. Medico-legal Autopsy The investigation was requested by a magistrate in Bologna. ○ Done in the NBI or other government institutions RENAISSANCE PERIOD ○ Purpose: prosecution Leonardo da Vinci and Michelangelo ○ Congregate in an operating theater and cadavers were opened by a “lay dissectorsˮ MLS 15: Histopathology (Lec) GALLEGO, A.A | MLS 3A 1  UNIVERSITY OF SAN AGUSTIN BS MEDICAL LABORATORY SCIENCE MLS15 1ST SEMESTER | 2024 - 2025 | PROF. MARK ALEXANDER TOBIAS, RMT, (ASCPi)CM, MSMT (CAR) JURISDICTION OF MEDICOLEGAL AUTOPSY SOMATIC DEATH I. All natural deaths occurring in the hospital within 24 hours of admission, bodily death unless the case was attended by a private physician within 36 hours of death II. Newborn in the first 24 hours of life Death or complete cessation of metabolic and functional III. All injury cases, old or recent activities of the organism or body as a whole IV. All deaths due to unknown cases V. All death due to suspicious cases Refers to a continuum of changes that occur in a dead body VI. All abortion cases, whether self-induced or otherwise following death VII. All violent deaths VIII. All accidental deaths ○ Livor mortis, rigor mortis, decomposition, and IX. All sudden deaths taphonomy X. All cases without medical attendance within 36 hours prior to the hour of death PRIMARY CHANGES/SIGNS IN DEATH XI. All deaths due to drowning, hanging, or strangulation XII. All deaths due to shooting stab wounds, burns, electricity, lighting, tetanus, A. CIRCULATORY etc. XIII. All homicides ○ Implies immediate death XIV. All suicides ○ For medicolegal purposes, death is said to occur when XV. All cases in which there is suspicion poisoning XVI. Stillborn cardiac function ceases XVII. Prematures Absence of pulse rate and heartbeat B. RESPIRATORY According to Completeness or Procedure of Technique ○ Leads to death due to the absence of oxygen and 1. Partial accumulation of carbon dioxide ○ Autopsy request involved only examines region or ○ Loss of all oxidative processes regions of body C. NERVOUS ○ Head only, thorax only, abdomen only ○ Loss of coordination of various body functions 2. Complete ○ Characterized chiefly by loss of reflexes ○ Autopsy request involved in the examination of the SECONDARY CHANGES whole body from head to foot A. ALGOR MORTIS “coolness of deathˮ ○ Complete diagnosis and investigation ○ First demonstrable change observed According to Manner of Incision or Opening of Cadaver ○ Characterized by cooling of the body caused by the 1. Y-shaped incision absence of circulation - equalize that of the temperature The cadaver is open from both shoulder regions down to the surrounding environment 7⁰F/hr the xiphoid area and the incised down to the pubis RT: 22.5 °F/hr during the first hour Done in adult female cadaver 12 hours: 1.52 °F 2. Straight-cut incision 1218 hours: 1 °F The cadaver is open from the midline of the body from ○ Important in establishing the approximate time of death suprasternal notch down to the pubis Commonly done in children and infants B. RIGOR MORTIS “stiffness of deathˮ ○ Rigidity or stiffening the muscles TECHNIQUES IN AUTOPSY due to lack of ATP and accumulation of lactic 1. A Y-shaped incision is made on the chest acid 2. The ribs are cut along the costochondral junction 3. The brain is removed by the following sequence NOTE:. In a dead body, glycogen stores are depleted preventing the energy dependent breakage of sarcomere contraction. Initially notable in small muscles like jaw followed by larger muscles like the legs Technique of R. Virchow Most widely used method ○ Occurring about 6 to 12 hours after death, persisting for The organs are removed one by one (individually) 3 to 4 days Dissected as they are removed ○ The position of the cadaver is usually affected by the The order of organ removal: muscular activity at the time of death ○ Brain, spinal cord, abdominal cavity organs & thoracic Initial: 2 hours cavity organs Complete rigor mortis: 612 hours Dissipates: 36 hours Technique of C. Rokitansky C. LIVOR MORTIS/LOSTMORTEM LIVIDITY “color of deathˮ Student of R. Virchow ○ Purplish discoloration of the skin in the dependent Organs are removed based on convenience portion of the body Removal is done partly en bloc ○ Due to stasis and eventual settling down of blood into Dissection is done in situ vessels Pathologies are correlated ○ Helps determine if the body has been moved from a Technique of A. Ghon different position Visualize: 20 mins after death Zenker technique Evident: 4 hours The physically related organs are removed at the same time Completely: 812 hours Preserves the anatomical relationship among organs without the ○ Blanching: occurs in gravity dependent areas of the unwieldy mass of organs body that come into contact with firm surfaces like floor or tight clothing Technique of M. Le Tulle ○ Appearance of “Tardieu spotsˮ En masse technique subpleural and subpericardial petechiae or All at one time removal of organs of the thoracic cavity, ecchymosis (or both) ecchymosis observed in abdominal cavity, and pelvic area and subsequently dissected the tissues of persons who have been strangled into organ blocks or otherwise asphyxiated Preserves the blood supply as well as the relationships of the organs to each other Quickest MLS 15: Histopathology (Lec) GALLEGO, A.A | MLS 3A 2  UNIVERSITY OF SAN AGUSTIN BS MEDICAL LABORATORY SCIENCE MLS15 1ST SEMESTER | 2024 - 2025 | PROF. MARK ALEXANDER TOBIAS, RMT, (ASCPi)CM, MSMT (CAR) Livor Mortis Found Ecchymosis Found in blood vessels) in tissues) Discoloration due to Stasis and eventual Blood that escaped settling into blood into the tissues from vessels ruptured blood vessels Application of Discoloration No change Pressure disappears upon pressure; reappears upon release Oozing of blood yes no upon incision D. POSTMORTEM CLOT ○ Occurs slowly immediately after death ○ Definite settling and separation of RBCs from fluid phase ○ Characterized as: Portions of clot assume a yellow chicken fat appearance Sediments of blood cells are called currant jelly that assumes the shape of the blood vessels and a rubbery consistency E. ANTEMORTEM CLOT ○ Occurred before death ○ Characterized as : Friable Irregular precipitation in tangled, irregular fashion Usually granular, not readily detachable , and do not have rubbery consistency F. DESICCATION/DRYING ○ Drying and wrinkling of the cornea and anterior chamber of the eyes ○ “Tache noir de sclérotiqueˮ G. PUTREFACTION ○ Production foul-smelling gases of due to the invasion of the tissue by multiplying saprophytic organisms Greenish-blue discoloration in the belly - iron sulphide Softening of the muscles - autodigestion Retraction of the cornea - absorption of aqueous humor Loss of rigor mortis - liquefaction of coagulated myosin Peeling off the skin with crepitation in the subQ tissues and swelling of the face ESTABLISHING THE TIME OF DEATH Rigor mortis - 6 to 12 hours after death Algor mortis - 7ºF per hour after death Skin color ○ Marble color with visible veins: 4 to 7 hours after death ○ Greenish tinge over the abdomen: 48 hours after death Livor mortis - 6 hours after death Gastric emptying - presence or absence of food MLS 15: Histopathology (Lec) GALLEGO, A.A | MLS 3A 3  UNIVERSITY OF SAN AGUSTIN BS MEDICAL LABORATORY SCIENCE MLS15 1ST SEMESTER | 2024 - 2025 | PROF. MARK ALEXANDER TOBIAS, RMT, (ASCPi)CM, MSMT (CAR) SPECIAL STAINING TECHNIQUES Lead method for 5'N Wachstein and Meisel) ○ 5-nucleotidase blackish-brown deposits NUCLEIC ACIDS Feulgen technique for nuclear DNA ANAE or NSE ○ demonstrates sugar ○ α- Napththyl acetate method for nonspecific esterase ○ DNA: red purple ○ Esterase: reddish brown ○ Cytoplasm: green ○ Nuclei: green Methyl green - pyronin for RNA and DNA Indoxyl acetate method for NSE Holt & Withers) ○ demonstrates phosphate ○ Esterase activity: blue ○ DNA Chromatin): Green or blue-green ○ Nuclei: red ○ RNA Nucleoli): Rose-red ○ Granules: Dark rose-red Tetrazolium method for monoamine oxidase Glenner, et al) ○ Plasma cell cytoplasm: Purple ○ Monoamine oxidase activity: bluish black Acridine orange fluorescent staining for RNA and DNA CARBOHYDRATES ○ DNA: yellow- green fluorescence Periodic acid Schiff ○ RNA: brick to orange-red ○ PAS-positive substances: red or magenta red ○ Nuclei: blue FATS AND LIPIDS PAS with diastase for glycogen Sudan IV Scharlach R for lipids ○ Nuclei: blue ○ most commonly used stain, producing a rapid and ○ Glycogen: red permanent coloration of lipid ○ Lipids (mainly triglycerides): red Best carmine ○ Nuclei: blue/black ○ Celloidin sections are the best. ○ Nuclei: blue or grayish blue Oil red O method in dextrin ○ Glycogen: pink to red ○ the intensity of its red coloration makes it the preferred ○ Mucin: weak red choice Langhan's iodine method for glycogen Carleton's ○ Lipid: red modification) ○ Nuclei: blue Fresh frozen azure A metachromatic staining Metachromatic toluidine blue staining Osmic acid stain Combined aldehyde fuchsin-alcian blue ○ Nuclei: yellow-orange Mucicarmine stain ○ Fats: black Hale's dialyzed (colloidal) iron technique Fluorescent acridine orange technique Nile blue sulfate ○ 2 components: CONNECTIVE TISSUE red oxazone: dissolves neutral lipids Gomori silver impregnation stain for reticulin blue oxazine: basic and reacts with ○ Reticulin fibers: black phospholipids and free fatty acids Van Gieson's stain for collagen Toluidine blue - acetone method for sulfatide ○ simplest method of differential staining of collagen and Borohydride - periodic - Schiff BHPS methods for glycosides other connective tissue ○ Nuclei: brownish black to black BONE MARROW AND BLOOD ELEMENTS ○ Collagen (fibrous connective tissue): pink or deep red Rapid toluidine - eosin stain for glycol-methacrylate sections ○ Muscle, Cytoplasm, RBC and Fibrin: Yellow Wright-Giemsa or Jenner-Giemsa stain Masson's trichrome stain Peroxidase reaction for myeloid stains ○ Muscle, RBC and keratin: red MUSCLE AND BONE ○ Nuclei: blue-black Modified Gomori's trichrome stain ○ Collagen and mucus: blue Mallory's PTAH Weigert's elastic tissue stain Heidenhan's iron hematoxylin ○ Elastic fibers: brown to purple or with blue-black with Lissamine fast red - tartrazine method for muscles methyl violet on a clear background. Schmorl's picro-thionin method Other structures are colored depending on the counterstain used. PROTEINS AND ENZYMES ○ Nuclei: red with carmine before or after staining of Alkaline fast - green method for basic proteins fibers. ○ especially protamines and histones Carmine stain is quite hard to obtain after Zenker fixation. Peracetic acid - alcian blue for cystine and cysteine A light stain with alum hematoxylin is preferable. ○ strong cysteic acid: stained blue-green by a basic dye Orcein Tanzer-Unna-Orcein) Sakaguchi's test for arginine ○ orcein - naturally occurring vegetable dye ○ orange-red color on objects containing arginine ○ Elastic fibers: dark-brown ○ Nuclei: blue Gomori calcium method for ALP ○ Alkaline phosphatase activity: brownish-black Kraijan's technique Congo red) ○ Nuclei: green ○ rapid method for staining elastic fibers, fibrin and amyloid, employing Congo red. Gomori lead method for ACP ○ Elastic fibers: bright red ○ Acid phosphatase activity: black ○ fibrin and connective tissues: dark blue ○ Nuclei: green ○ RBC: orange-yellow MLS 15: Histopathology (Lec) GALLEGO, A.A | MLS 3A 4  UNIVERSITY OF SAN AGUSTIN BS MEDICAL LABORATORY SCIENCE MLS15 1ST SEMESTER | 2024 - 2025 | PROF. MARK ALEXANDER TOBIAS, RMT, (ASCPi)CM, MSMT (CAR) PTAH Mallory's) FROZEN SECTIONS ○ fibers are demonstrated using a tungsten mordant provided by 1% aqueous phosphotungstic acid Applications mordant binds hematein and stains selective 1. Rapid diagnosis during surgery tissue components blue 2. Diagnostic and research enzyme histochemistry phosphotungstic acid is believed to stain other 3. Diagnostic and research demonstration of soluble substances tissue components a red-brown color (lipids and carbohydrates) 4. Immunofluorescent and immunohistochemical stain High pH Congo red technique 5. Silver stains in neuropathology ○ Amyloid, elastic fibers, eosinophil granules: red ○ Nuclei: blue Methods of Preparing Frozen Sections A. COLD KNIFE PROCEDURE Alkaline Congo red ○ Different temperature is employed between tissue and ○ Amyloid, elastic fibers, eosinophil granules: red knife ○ Nuclei: blue ○ Carbon dioxide gas - commonly used freezing agent Kraijan's amyloid stain Modified Benhold method) ○ Optimum condition for cold knife section: Knife: 40°C to - 60°C Methyl violet - crystal violet method Tissue: 5°C to - 10°C ○ Amyloid: Purplish red Environment: 0°C to 10°C ○ Nuclei, cytoplasm, connective tissue: shades of violet B. CRYOSTAT ○ Refrigerated apparatus in fresh tissue microtomy CENTRAL NERVOUS TISSUES ○ Air controls for the microtome - operated outside the Bielchowsky's techniques for neurons, axons and neurofibrils cabinet Sevier-Munger technique for neural tissues ○ Sections are cut - isothermic conditions Cresyl fast violet Nissl stain for paraffin sections ○ Cold chamber maintained at: 5 to 30°C Weigert-Pal technique for staining normal myelin sheath ○ Optimum working temperature: 18 to 20°C (near Kluver and Barrer's Luxol fast blue stain for myelin with Nissl 20°C counterstain Luxol fast blue - H&E stain for myelin METHODS OF FREEZING Luxol fast blue - PAS stain for myelin LIQUID NITROGEN Weil's method for myelin sheath Most rapid among the available freezing agents Cajal's gold sublimate method for astrocytes Used in histochemistry during intraoperative procedures Modified Holzer's method for astrocytic process Disadvantages: TISSUE PIGMENTS AND DEPOSITS ○ Ice crystals/artifacts - soft tissues is liable to crack due to rapid expansion of the ice within tissue Perl's Prussian blue for hemosiderin (ferric iron) ○ Damage to block and knife - overcooling of biopsy Gomori's Prussian blue stain for iron blocks Turnbull's blue reaction for ferrous iron (hemosiderin) ○ Size & amount of ice formed - DP to speed of Benzidine - nitroprusside stain for hemoglobin & oxidase processing granules ○ Uneven cooling of tissues - vapor phase to form around Modified Fouchet's technique for liver bile pigments the tissues Schmorl's ferric-ferricyanide method for reducing substances Gomori's aldehyde fuchsin technique for lipofuscin ISOPENTANE Mallory fuchsin stain for hemofuchsin pigment Liquid n room temperature Masson-Fontana technique of staining melanin and Isopentane in a Pyrex glass beaker suspended in the liquid argentaffin cell granules nitrogen to cool (approximately 170°C Von Kossa's silver nitrate method for calcium Linquist's modified rhodamine technique for copper CARBON DIOXIDE commonly used in cold knife procedure MICROORGANISMS AEROSOL SPRAYS Gram-Twort stain for bacteria Brown and Benn B&B method for bacteria, Nocardia and quick freezing spray cans with fluorinated hydrocarbons Actinomycetes Cryokwik) Ziehl-Nelsen method for AFB commonly used nowadays Wade-Fite for leprosy bacilli and Nocardia SPECIAL PROCESSING TECHNIQUES Auramine-Rhodamine stain for mycobacteria (fluorescent method) FREEZEDRYING Dieterle's method for Legionella pneumophila Levaditi's method for spirochetes Quenching special way of preserving tissues by rapid freezing Warthin-Starry method for spirochetes 160 to 180 C Modified Steiner & Steiner technique for spirochetes Desiccation removing ice water molecules Grocott methenamine silver GMS, modified) stain for fungi Lendrum's phloxine - tartrazine method for viral inclusions Sublimation physical process of transferring the still frozen tissue block Orcein method for HBSAg in vacuum at higher temperature Rapid Giemsa stain 30 to 40 C Demonstration of hydrolytic enzymes, mucous substance, ENDOGENOUS TISSUE PIGMENTS glycogen and proteins Hemosiderin - iron-containing pigment of hemoglobin Special studies: Hematoidin - iron-free pigment of hemoglobin ○ Immunocytochemistry, autoradiography, scanning EM, Hematin - hemoglobin without the globin molecule microspectrofluormetry of autofluorescent substances Hemozoin - black granule formed by malarial parasites Advantages: Hemofuscin - iron-free brownish yellow pigment ○ Minimum tissue shrinkage ○ Allows processing of tissue in fresh state MLS 15: Histopathology (Lec) GALLEGO, A.A | MLS 3A 5  UNIVERSITY OF SAN AGUSTIN BS MEDICAL LABORATORY SCIENCE MLS15 1ST SEMESTER | 2024 - 2025 | PROF. MARK ALEXANDER TOBIAS, RMT, (ASCPi)CM, MSMT (CAR) Disadvantages: CHARACTERISTICS OF TUMOR CELLS ○ Time consuming and expensive ○ More difficult to section than ordinary paraffin blocks Changes in intercellular structural pattern ○ Not advisable in routine manner Increase in size Irregular shape FREEZESUBSTITUTION Irregular pattern Fixation in: Anisocytosis and anisokaryosis observed in clusters ○ Rossman's formula ○ Anisocytosis: size ○ 1% acetone and dehydrated in absolute alcohol ○ Anisokaryosis: nucleus Indistinct cell membrane BIOPSY Excessive grouping and crowding of cells to form clusters Examination of cells or tissues form a living organism and Cytoplasmic changes studied in order to diagnose disease or to confirm findings or normality Acidophilia marked orangeophilia Excessive cytoplasmic inclusion bodies EXCISION BIOPSY Abnormal vacuolation Surgical removal of a tumor and some normal tissue around it Nuclear changes The amount of normal tissues taken (surgical margin) depends o the size, histologic type and thickness of the tumor Larger nucleus Irregular nucleus INCISION BIOPSY More deeply pigmented Partial removal of small protein of tissues in form of wedges, Multinucleated cylindrical pieces cores, punch, or scrapings o the suspected Increase in number and size of nuclei tissue or organs Increased distribution and irregular size of chromatin materials ○ Representation of the portion of interest Markedly thickened nuclear membrane Necrotic or degenerative changes EXAMINATION OF FRESH TISSUE TUMOR BASIC COMPONENTS FINE NEEDLE ASPIRATION Parenchyma Simplest, least invasive test ○ Active elements of the tumor Uses the smallest needle to simply remove cells from the area ○ Composed of transformed neoplastic cells of abnormality ○ Basic cellular component of a tumor ○ Not always adequate to obtain a diagnosis, depending Stroma on the area to be biopsied ○ Connective tissue framework with lymphatic and vascular channels CORE NEEDLE BIOPSY ○ Interstitium Removes not only cells, but also a small amount of the DIFFERENTIATION OF TUMORS surrounding tissue ○ Provides additional information to assist in the Capacity to produce death examination of the lesion Benign Do not produce death Gross and microscopic appearances are considered INCISIONAL BIOPSY relatively innocent The doctor will slice into the lesion and remove only a portion Remains localized ○ Will not spread to other sites of it ○ Amenable to local surgical removal ○ If the lesion is found to be cancerous, further surgery Made up of well-differentiated cells may be needed to remove or excise the entire lesion ○ Benign tumor mimics appearance or structure of tissues and cells in a specific EXCISIONAL BIOPSY region of the body Generally removes the entire area in question Malignant Will produce death Poorly differentiated cells PUNCH BIOPSY Cancer (crab) Primary technique - diagnostic full thickness skin specimen ○ Adhere to any part that they seize on in n 3 to 4 mm cylindrical core of the sample obstinate matter Malignant tumors can invade and destroy adjacent ○ Involves the use of a circular blade that is rotated down structures through the epidermis and dermis & subcutaneous fat ○ Spread to distant sites Requires general surgical & suture-tying skills ○ Causes metastasis ○ Cause death SHAVE BIOPSY Small fragments of tissue are shaved from a surface particularly Benign Malignant the skin Behavior Autonomous CURETTINGS Tumor Encapsulated Not capsulated Tissue is scooped or spooned to remove tissue or growth from Well-defined Amorphous Palpable body cavity ○ Endometrium or cervical canal Manner of Growth Expansile growth only Expansile and invasive growth Metastasis NEOPLASM Histology Resembles cell of origin May show failure of Abnormal mass of cells/tissues (well-differentiated) cellular Few mitosis differentiation State of poorly regulated cell division Normal or slight increase Many mitosis Failure of the mechanisms which control cellular proliferation in NC ratio Cellular or nuclear Cells are uniform through pleomorphism and maturation the tumor Abnormal proliferation of autonomous cells Metastatic Growth Via lymphatics draining the site ○ No useful purpose Vascular (venules and veins draining the site, portal vein, neoplasm trapped in the lungs) ○ Persisting at the expense of the surrounding cells Local invasion ○ Excessive mitosis because there is no stimuli Trans-coelomic spread ○ across peritoneal and pleural cavities MLS 15: Histopathology (Lec) GALLEGO, A.A | MLS 3A 6  UNIVERSITY OF SAN AGUSTIN BS MEDICAL LABORATORY SCIENCE MLS15 1ST SEMESTER | 2024 - 2025 | PROF. MARK ALEXANDER TOBIAS, RMT, (ASCPi)CM, MSMT (CAR) Depending on histological characteristics CIN Cervical Intraepithelial Neoplasia) System Medullary ○ More cells than supporting tissues CIN0 Presence of well-differentiated cells ○ Soft and very malignant CIN1 Evidence of mild dysplasia Scirrhous ○ More CT than cells CIN2 Evidence of moderate dysplasia ○ The cells are trapped within the fibrous tissue CIN3 Evidence of severe dysplasia NOMENCLATURE OF NEOPLASMS CIN4a Evidence of carcinoma in situ Based on what tissue type is present “Omaˮ- benign CIN4b Evidence of invasive carcinoma “Carcinomaˮ - malignant Papanicolaou Papʼs Grading Benign Malignant Screen for the presence of possible malignancy Surface epithelium Papilloma (finger-like, warty Carcinoma (squamous cell Exfoliated epithelial cells present in the samples collected from projections) carcinoma) sites are examined for atypical cells Solid glandular Adenoma (glands and ducts) ADENOcarcinoma ○ Dysplasia - reversible epithelium ○ Anaplasia Connective/Mesenchymal Tissues Grade I Absence of atypical cells ˮOmaˮ- benign Grade Il Presence of atypical cells but no evidence of malignancy “Sarcomaˮ - malignant Grade III Presence of atypical cells strongly suggestive but not Benign Malignant conclusive of malignancy Fibrous FIBRoma FIBROsarcoma Grade IVa Carcinoma in situ Bone OSTEoma OSTEOsarcoma Grade IVb Invasive carcinoma Cartilage CHONDRoma CHONDROsarcoma The Bethesda System TBS Adipose LIPoma LIPOsarcoma tell whether infection or reactive reparative changes are present Smooth muscle LEIOMYoma LEIOMYOsarcoma general categorization of the specimen used for hormonal evaluation Skeletal muscle RHABDOMYoma RHABDOMYOsarcoma Group I Exclusively normal cells (negative results) GRADING AND STAGING OF TUMORS ↑ differentiation = ↑ stage Group II Normal variant but not suspicious for malignancy Broderʼs Classification Group III Moderate to severe dysplasia based on histologic picture as interpreted from the degree of Group IV Carcinoma in situ cellular differentiation Group V Invasive carcinoma Grade Differentiated cells Undifferentiated cells Grade I 75 - 100% 0 - 25% Grade Il 50 - 75% 25 - 50% Grade III 2550% 50 - 75% Grade IV 0 - 25% 75 - 100% Union Internationale Contre Center Applicable to the grading of tumor found close to the inguinal or regional lymph nodes ○ TNM SYSTEM: most commonly used T0 "in situ" tumor T1 1 cm diameter T T2 2 cm diameter Primary tumor T3 3 cm diameter T4 4 cm diameter N0 LN are not involved N N1 LN are involved Mobile LN Nodal Involvement N2 Fixed nodes are involved M0 "in situ" tumor No distant metastases M Mx 1 cm diameter Suspected metastases Presence of distant M1 2 cm diameter metastasis Evidence of distant metastases M2 3 cm diameter Sometimes, blood-borne/vascular metastases MLS 15: Histopathology (Lec) GALLEGO, A.A | MLS 3A 7  UNIVERSITY OF SAN AGUSTIN BS MEDICAL LABORATORY SCIENCE MLS15 1ST SEMESTER | 2024 - 2025 | PROF. MARK ALEXANDER TOBIAS, RMT, (ASCPi)CM, MSMT (CAR) IMMUNOHISTOCHEMISTRY TISSUE PREPARATION Identification of specific or highly selective cellular CRYOSTAT SECTION epitopes (antigens) Fixed in ethanol or acetone Combines anatomical, immunological and biochemical ○ Preserve immunological activity techniques ○ Prevent destruction of labile antigenic sites ○ Interaction of target antigens with specific IMMUNOFLUORESCENCE AND IMMUNOPEROXIDASE TECHNIQUE antibodies tagged with a visible label Formaldehyde-fixed Applications: Paraffin-embedded 1. Detect organisms in cytologic preparations MASKED ANTIGENS 2. Disease diagnosis, drug development and biological research PROTEOLYTIC ENZYME DIGESTION ANTIGENANTIBODY REACTION & DEFINITION OF TERMS ○ Heavy chain immunoglobulins, complement and specific antigens ANTIGENANTIBODY INTERACTION Trypsin 0.1% trypsin + 0.1 CaC in distilled H2O Epitopes pH adjusted to 7.8 using NaOH; preheated in 37°C ○ Structural part of the antigen that reacts with an antibody Protease 0.05% to 0.1% protease in distilled H2O pH adjusted to 7.8 using NaOH (faster rate of digestion) Antibody labels ○ Site of antigen binding MICROWAVE ANTIGEN RETRIEVAL ○ Optimal length of exposure - 10 to 60 mins. ○ Most satisfactory period - 20 minutes (most fixation 8 and fixation protocol) Boiling of 0.01 M citrate buffer (pH = 6 formalin-fixed EDTA (pH = 8 deparaffinized Tris-EDTA (pH 9.9 or 10 sections METHODOLOGY MICROWAVE AND TRYPSIN ANTIGEN RETRIEVAL SAMPLE PREPARATION PRESSURE COOKER ANTIGEN RETRIEVAL ○ Alternative that appears to be less time consuming ↓ ○ Allows for more consistent recovery of many antigens. LABELLING DIRECT TECHNIQUE POLYCLONAL ANTIBODIES Traditional technique Conjugate the primary antibody to the label Immunizing an animal with a purified specific molecule ○ Fluorochrome that contains the antigen of interest ○ Horseradish peroxidase Produced by different cells of animals ○ Immunochemically not identical to each other ○ React with various epitopes on the antigen Some of the polyclonal antibodies may cross-react with other molecules—non-specific staining ○ Purification by absorption with the appropriate antigen ○ Antibody dilution to eliminate the unwanted reaction Rabbit INDIRECT TECHNIQUE ○ most frequently used animal for the production of polyclonal antibodies More sensitive goat, pig, sheep, horse, guinea pig Most commonly used enzyme - horseradish peroxidase 2 or 3-step procedure: MONOCLONAL ANTIBODIES ○ Application of unconjugated primary antibody ○ Followed a labelled antibody directed against the Animals immunized with the specific immunogen will first antibody produce numerous clones of plasma cells that in turn will produce the antibody Products of an individual clone of plasma cells Only one cell (plasma cell) produced antibody Hybridoma - do not cross react with other molecules 1. Immunochemically identical 2. React with a specific epitope on the antigen Mice - production of monoclonal antibodies MLS 15: Histopathology (Lec) GALLEGO, A.A | MLS 3A 8  UNIVERSITY OF SAN AGUSTIN BS MEDICAL LABORATORY SCIENCE MLS15 1ST SEMESTER | 2024 - 2025 | PROF. MARK ALEXANDER TOBIAS, RMT, (ASCPi)CM, MSMT (CAR) AVIDINBIOTIN COMPLEX EPITHELIAL MEMBRANE ANTIGEN Positive: adenocarcinoma of the breast, lungs and kidneys Uses avidin (derived from egg white) - marked affinity for Negative: sarcomas, lymphomas and melanomas, biotin (low molecular weight vitamin) meningiomas, mesotheliomas, ALCL, plasma cell tumor Consists of: ○ Primary antibody CARCINOEMBRYONIC ANTIGEN ○ Biotinylated secondary antibody Oncofetal antigen present in cardinomas of Gl tract, ○ Preformed (strept) avidin-biotin-complex of the pancreas, lung, breast, ovary, uterus and cervix ABC technique or by labelled streptavidin CEA Labelled Streptavidin Avidin-Biotin Technique Adenocarcinoma + 4 to 8 more times sensitive than the old ABC method Mesothelioma - Staining sequence: ○ Primary rabbit or mouse antibody THYROID TRANSCRIPTION FACTOR1 ○ Biotinylated secondary antibody (ant-rabbit or Used to distinguish lung adenocarcinomas from mouse Ab) mesotheliomas ○ Streptavidin-enzyme conjugate ○ Positive in: thyroid, lung and neuroendocrine tumors (medullary thyroid carcinomas, carcinoid tumors and small cell tumors of the lung) PROSTATE SPECIFIC ANTIGEN Useful in the diagnosis of prostatic adenocarcinoma ○ Positive in: pancreatic and salivary gland tumors INTERMEDIATE FILAMENT MARKERS Actin PEROXIDASEANTIPEROXIDASE TECHNIQUE ○ Contractile protein present in muscle ○ Sensitive marker for muscle differentiation Indirect antibody-enzyme complex ○ Smooth, skeletal and cardiac muscle tumors The soluble PAP complex is bound to unconjugated primary antibody (rabbit antihuman lgG by a second Vimentin 57 kD layer of "bridging antibody" (swine anti-rabbit antibody) ○ Present in normal mesenchymal cells ○ Binds to both the primary antibody and the rabbl ○ Sarcoma, melanoma, lymphoma, leukemia, PAR complex seminoma and some neural tumors Most commonly used enzyme for labelling - horseradish Melanomas and Schwannomas - always peroxidase stain positive for vimentin Most commonly used chromogen - diaminobenzidine ○ Stable, insoluble dark brown reaction product Desmin 53 kD when the antigen is present ○ Expressed by skeletal and cardiac muscles ○ Highly specific for myogenic tumors (leiomyoma and rhabdomyosarcoma) ○ Mixed tumors with myogenic components (carcinosarcomas or malignant mixed tumors) Glial fibrillary acidic protein 51 kD ○ Expressed by nervous system glial cells - astrocytes ○ Confirms diagnosis of astrocytoma ANTIGEN/MARKERS Neurofilament ○ Expressed in cells of neural origin - neurons, EPITHELIAL CELL TUMOR MARKERS neuronal processes, peripheral nerves, sympathetic ganglia, adrenal medulla and KERATIN neuroendocrine cells Sensitive marker for epithelial cells Tumors that show neuronal or ○ Epithelial tumors neuroendocrine differentiation will stain ○ Mesotheliomas positive ○ Non-seminomatous germ cell tumors S100 protein CK7 CK20 ○ Low molecular weight calcium-binding protein Serous tumors + - ○ CNS glial cells, Schwann cells, melanotes, histotes, (carcinomas of breast, uterus and ovaries) chondrocytes, skeletal and cardiac muscle, Carcinomas of colon and stomach - + myoepithelial cells and some epithelial cells Transitional cell carcinomas + + (bladder and mucinous ovarian tumors) Renal cell, hepatocellular, prostatic, thyroid, squamous - - cells (skin, lungs and esophagus) carcinomas MLS 15: Histopathology (Lec) GALLEGO, A.A | MLS 3A 9  UNIVERSITY OF SAN AGUSTIN BS MEDICAL LABORATORY SCIENCE MLS15 1ST SEMESTER | 2024 - 2025 | PROF. MARK ALEXANDER TOBIAS, RMT, (ASCPi)CM, MSMT (CAR) NEUROENDOCRINE MARKERS INFECTIOUS AGENTS MARKERS Neuron specific enolase Hepatitis A virus Toxoplasma ○ Provides strong evidence of neural or Hepatitis B surface and core Pneumocystis carinii antigens Helicobacter pylori neuroendocrine differentiation Hepatitis C virus Cryptosporidium Chromogranin HPV Cryptococcus neoformans ○ Found in endocrine tissues CMV Histoplasma ○ Marker for neuroendocrine differentiation EBV Entamoeba histolytica Mycobacteria Chromogranin Keratin Neuroendocrine carcinoma + + CONTROLS IN IHC Paraganglioma - - Test for specificity of the antibodies involved Avoid misinterpretations due to false positive or false Synaptophysin negative results ○ Presynaptic vesicles of neurons POSITIVE CONTROL GERM CELL TUMOR MARKERS ○ A section that is known and proven to contain the antigen in question Human chorionic gonadotropin ○ Synthesized by placental syncytiotrophoblasts NEGATIVE CONTROL ○ Marker for choriocarcinoma ○ Done using a parallel section from the tissue Alpha-fetoprotein Omitting the primary antibody from the ○ Synthesized by hepatocytes staining schedule ○ Marker for endodermal sinus tumors Replacing the specific primary antibody by ○ Positive for AFP: Embryonal carcinomas and an immunoglobulin that is directed against teratomas & hepatocellular carcinomas an unrelated antigen Placenta-like alkaline phosphatase ○ Produced placental syncytiotrophoblasts INTERNAL TISSUE CONTROL ○ Marker for germ cell tumors (germinomas) ○ Eliminates the variable of tissue fixation between ○ Positive: carcinomas, choriocarcinomas, specimens and controls but it contains the target endo-dermal sinus tumors and seminomas antigen ○ The tissue elements under investigation MESENCHYMAL TUMOR MARKERS ○ Adjacent normal tissue element Myogenic tumors Actin, desmin, myo-D1, myoglobin and myogenin Malignant CD68, FAM 56, alpha-1-antitrypsin and fibrohistiocytic tumors alpha-1-antichymotrypsin Sarcoma Vimentin Vascular tumors Factor VIl-related antigen, CD31 and Ulex (angiosarcoma) Europaeus 1 Melanomas S100 protein, melanosome HMB45, Melan-A MART1 Lymphomas Leukocyte common antigen CD 45 T cells - CD3, CD4, CDS B cells - CD 19, CD 20, CD 23 Reed-Sternberg cells - CD15, CD30 Immunoglobulin heavy and light chains OTHER MARKERS CELL PROLIFERATION MARKERS Ki-67 & proliferating cell nuclear antigen ○ Most common immunohistochemical markers - assess proliferation of tumor cells ○ Increased expression - greater aggressiveness and higher likelihood of recurrence of metastasis CANCER ASSOCIATED GENES Abnormalities of structure or activity of proto-oncogenes Mutation of tumor suppressor genes - p53 ○ Breast cancer oncogenes: c-erbB2, c-myc & ras MLS 15: Histopathology (Lec) GALLEGO, A.A | MLS 3A 10  UNIVERSITY OF SAN AGUSTIN BS MEDICAL LABORATORY SCIENCE MLS15 1ST SEMESTER | 2024 - 2025 | PROF. MARK ALEXANDER TOBIAS, RMT, (ASCPi)CM, MSMT (CAR) AUTOMATED TISSUE PROCESSING SPECIMEN TECHNIQUES Steps involved: ○ Fixation, dehydration, clearing and infiltration 1. Cervicovaginal 1. Streaking ○ Most critical stage: dehydration smear/Pap's smear 2. Spreading ○ Advantage: constant agitation 2. Urine sediments 3. Pull-apart 3. CSF 4. Touch impression Stations 12 10% formalin 4. Sputum (glass beaker - 1L 5. Serous fluids Stations 36 Ascending grades of ethyl alcohol 6. Gastric/bronchial (glass beaker - 1L 7095% secretions 7. Nipple discharge Stations 78 2 changes of acetone (glass beaker - 1L PRECAUTIONS Stations 910 2 changes of chloroform or xylol (glass beaker - 1L FOR AN OPTIMAL SPECIMEN Stations 1112 2 changes of liquid paraffin Smears - fixed immediately (wax bath - 3° higher than the melting point of paraffin) ○ Quick immersion in a fixative ○ Spray fixative DIAGNOSTIC CYTOLOGY IF SMEARS CANNOT BE MADE IMMEDIATELY Microscopic examination of cells from different sites of Placed in 50% alcohol the body for diagnostic purposes Replaced by Sacommano's fixative ○ Branches: ○ 50% alcohol and 2% carbowax Exfoliative cytology SPECIMENS THAT REQUIRE ADHESIVES Fine needle aspiration Urinary sediments Bronchial lavage FINE NEEDLE ASPIRATION Concentrated sputum Deep-seated tumors or tumors in abdominal cavity Lavage sample from GIT Simple, safe and rapid cytologic technique - diagnosis of NOTE:. cancer Adhesives that can be used: Pooled human serum/plasma Palpable Mass Non-palpable Celloidin ether Leuconostoc culture Superficial Mass Deeply-seated lesions (lung, mediastinum, abdominal & peritoneal organs) FIXATIVES Clinicians or pathologists Laparoscopy, CT and/or ultrasound 1. Ether alcohol (best fixative) ○ abandoned because flammable and extremely EXFOLIATIVE CYTOLOGY volatile Applicable on surface tumors 2. 95% alcohol (most common fixative) Microscopic study of desquamated cells from epithelial 3. Carnoy's fixative (bloody smears) surfaces 4. 100% methanol Exfoliated epithelial cells (brushing, swabbing, scraping, ○ substitute for 95% ethanol aspiration of body fluids) 5. Commercial aerosol spray (hairspray) APPLICATIONS PRECAUTIONS DURING FIXATION ○ Assessing malignant or cancerous conditions 1. Identify slides before preparing smears ○ Vaginal cytology 2. Use paper clips to identify the end of the slide ○ Assessment of female hormonal activity (sterility & 3. Smears should be placed into the fixative container endocrine disorders) immediately after preparation ○ Determination of the presence of possible infection 4. Place each smear in fixative by a single uninterrupted ○ Determination of genetic sex motion to avoid rippling of smeared material Barr body - conglomeration of chromatin in 5. Avoid striking the bottom of the fixative container the nuclei of female demonstr