Histopathology, Histotechniques, Cytology, and General Pathology PDF

#MTAPNotes HISTOPATHOLOGY, HISTOTECHNIQUES, CYTOLOGY AND GENERAL PATHOLOGY DEFINITION OF TERMS HISTOPATHOLOGY- branch of anatomical pathology that deals on the study of diseases in a give...

#MTAPNotes HISTOPATHOLOGY, HISTOTECHNIQUES, CYTOLOGY AND GENERAL PATHOLOGY DEFINITION OF TERMS HISTOPATHOLOGY- branch of anatomical pathology that deals on the study of diseases in a given tissue or organ HISTOTECHNIQUES- includes the preparation, processing and staining of tissue sections for microscopic study to be interpreted by the pathologist either malignant (can cause death) or benign (cannot cause death) RECORD KEEPING AND SPECIMEN RETENTION o Specimen: 1 month – 1 year o Tissue blocks: 3 years – 10 years o Slides: Indefinite o Records: Permanent o Result forms: Triplicate TYPES OF HISTOLOGICAL PREPARATION/METHODS 1. WHOLE MOUNT Preparation for organisms 0.2-0.5 mm thickness 2. SECTIONING Majority of preparation 3.0-5.0 mm thickness 3. SMEARING Derived from blood, bone marrow and bodily fluids (pleural fluid or ascitic fluid) For cytological examination DIAGNOSTIC & EXFOLIATIVE CYTOLOGY DIAGNOSTIC CYTOLOGY EXFOLIATIVE CYTOLOGY ☞ Simply means microscopic examination of cells ☞ Deals with the microscopic study of cells that from different body sites for diagnostic have been desquamated from epithelial purposes surfaces ☞ Exfoliative Cytology and Fine Needle Aspiration ☞ Epithelial & Mucous membrane (ascitic fluid, (FNAB) specimens CSF, urine, sputum, buccal mucosa) FRESH TISSUE EXAMINATIONS (Observation of physiological processes) 1. TEASING/DISSOCIATION ⚫ Also referred to as “Dissection”/”Separation” ⚫ NSS (Normal Saline Solution)/Physiological Saline: 0.85-0.90% NaCl solution ⚫ Examine as stained preparation (Bright field/Light Microscope) or unstained preparation (Phase-Contrast Microscope) ⚫ Disadvantage: anatomical relationship is destroyed 2. SQUASHING/CRUSHING Tissue (< 1mm) is sandwiched between 2 slides Incorporation of vital stain through capillary action 3. MICROINCINERATION Used to locate the presence & position of mineral elements in the tissue 2 or duplicate sections of alcohol-fixed tissues The ash residue of the incinerated section is examined alongside the stained preparation, and the incidence and location of any mineral deposits is noted A polarizing microscope is often used to examine the ash residue of the incinerated section (SPODOGRAM) 4. AUTORADIOGRAPHY Directly injection of radioactive isotopes into organs It determines the relationship and location of the isotopes and cells to be studied REAGENT PREPARATION Basic Equation: V1C1 = V2C2 PROBLEM: Suppose you have a 3 mL stock solution of 100 mg/mL of phenol and you want to prepare a 200 uL of having 25 mg/mL of the solution. Compute for the unknown and convert your answer from uL to mL. STEPS IN ROUTINE TISSUE PROCESSING 1. FIXATION/PRESERVATION: Is the process in adopting to kill, harden and preserve tissue materials (from destruction and post-mortem changes) Examples: ________________________________________________________________ *DECALCIFICATION/DEMINERALIZATION: Process of calcium, phosphate salt or lime salt removal Examples: ________________________________________________________________ 2. DEHYDRATION: Removes water from the tissue in preparation for impregnation (1:10x volume of tissue; High affinity/miscibility with water) Examples: ________________________________________________________________ 3. CLEARING/DEALCOHOLIZATION: Replacement of the dehydrating fluid with a substance that is miscible with the embedding medium to be used (transparent and translucent tissues) Examples: ________________________________________________________________ 4. INFILTRATION/IMPREGNATION: Infiltration of the tissues with a medium that will fill all natural cavities, spaces & interstices of the tissues Paraffin Embedded Sections: MOST COMMON; Routine uses *Paraplast- _____________________________________________________________ *Embeddol-______________________________________________________ *Bioloid-_________________________________________________________ *Tissue Mat-______________________________________________________ Celloidin/Collodion Embedded Sections: Made up of Purified Pyroxylin Nitrocellulose (hollow organs, large embryos, large cavities, brain, whole eyes) *2%-____________________ *4%-____________________ *8%-____________________ 5. EMBEDDING/CASTING/BLOCKING: Is the process by which the impregnated tissue is placed into a precisely arranged position in a mold containing a medium which is then allowed to solidify Types of Embedding Molds *Leuckhart’s embedding mold ☞ 2 L-shaped heavy metal or brass arranged on a flat metal surface (plate) ☞ It can be moved to adjust the embedding box (molds) depending on the size of the specimen ☞ Recommended for routine purposes *Compound E unit ☞ With several interlocking plates making several compartments ☞ Advantage: embedding more specimens at a time (reduced time for embedding of tissue blocks) *Disposable Molds a. Peel-away (thin plastic embedding molds) ☞ It may be placed directly in the chunckholder or blockholder b. Plastic Ice Trays ☞ Uses ordinary refrigerators ☞ Recommended for busy laboratories c. Paper boats ☞ Advantage: easy and cheap, different sizes, avoids confusion *Plastic Embedding Rings & Base Molds ☞ Base mold is fitted to base ring ☞ Base ring serves as the block holder *Tissue –Tek System ☞ Stainless steel in which the tissue block is embedded ☞ Plastic mold is placed on the top ☞ E.g.: Mark I & II Tissue-Tek Systems with warm and cold plates 6. TRIMMING: Removes excessive amount of impregnating agent from the tissue block 7. SECTION-CUTTING: Process by which a previously processed tissue (tissue block), is trimmed and cut into uniformly thin slices or sections to facilitate microscopic study KINDS OF MICROTOME KNIVES MICROTOME KNIVES LENGTH TYPES OF BLOCK MICROTOME PLANE-WEDGE 100 mm Celloidin embedded sections & for Sliding extremely hard tissues; RECOMMENDED for FROZEN SECTIONS PLANE-CONCAVE 25 mm Paraffin & Celloidin embedded sections Rotary Rocking BICONCAVE 120 mm Paraffin embedded sections CO2 Freezing Rotary Rocking BROKEN PLATE GLASS Electron Microscopy Ultrathin KNIFE 8. STAINING: For contrast; Better optical differentiation; Improve aesthetic value 9. MOUNTING Examples: ________________________________________________________________ 10. LABELLING (diamond leaded pencil) LIST OF COMMON HISTOLOGICAL STAINS STAINS USES/IMPORTANCE/CHARACTERISTICS/OTHER PARTICULARS Iodine Oldest stain; Stains amyloid for microscopic study of starch granules Hematoxylin Natural dye derived from the heartwood of a Mexican tree, Hematoxylin Campechianum RIPENING: Hematoxylin ---[o]--- Hematin; karyosome: dark blue, nucleus: blue; cytoplasm: pink Eosin Most valuable stain; used as a counterstain (Eosin B: blue-deep red; Eosin Y: yellow-green) Methylene Blue Common basic nuclear stain; valuable for plasma cells Methylene Violet Metachromatic dye; for leukocytes Toluidine Blue Nuclear stain substitute for thionine for fresh frozen tissue; Nissl/Tigroid granules and chromophilic bodies Crystal Violet For amyloid, fungi, platelets in blood Aniline Blue Counterstain for epithelial cells Basic Fuschin Is a plasma stain; deep staining for acid fast organisms Van Gieson Mixture of picric acid and acid fuschin; for the demonstration of connective tissue; SIMPLEST method of differential staining of collagen Giemsa Used for staining blood to differentiate leukocytes Celestine Blue Resistant to strong acids; Recommended for routine staining of fixed sections; ALTERNATIVE to Iron Hematoxylin nuclear stain Malachite Green Stains Ascaris eggs, RBCs, Bacterial spore (BOTH A DECOLORIZER AND A COUNTERSTAIN) Methyl Green Stains chromatin green; It gives false positive reactions with certain secretions such as mucin Feulgen’s stain Most reliable and specific histochemical staining technique for DNA Bismarck Brown Contrast stain for Gram’s stain, In Acid Fast, Papanicolau method & Diphtheria organism Von Kossa Silver Calcium: BLACK Nitrate Prussian Blue Colored Salt of Ferric ferrocyanide, normally used for the manufacture of paints Orcein Excellent stain for elastic fiber; Orcein + Ammonia ---exposed to air Blue or Violet Picric acid For the demonstration of connective tissue (fixative and stain) Carmine Used as a chromatin stain for fresh materials in smear preparations Alcian Blue Stains mucopolysaccharide; more specific for connective tissue and epithelial mucins Neutral Red Cell granules & vacuole phagocytic cells Congo Red Best known as an indicator; Utilized as a stain for axis cylinder in embryos; (4% aqueous solution: elastic tissues, amyloid, myelin) Janus Green B For demonstrating mitochondria during intravital staining Victoria Blue Used for demonstration of neuroglia in frozen sections Night Blue Is used as a substitute for Carbol Fuschin Methyl Green Pyronin DNA: green to blue green; RNA: rose red Acridine Red 3B Demonstrates deposits of calcium salts and possible sites of phosphatase activities Acridine Orange Permits discrimination between dead and living cells (DNA: green fluorescence; RNA: red fluorescence) Rhodamine B Stains blood and glandular tissues Benzidine Used for staining haemoglobin Sudan Black Phospholipids Sudan IV/Scharlach R Triglycerides and Neutral lipids (deep red) Sudan III Fats (orange) Silver nitrate Used in Spirochetes reticulum and fiber stains Azocarmine Connective tissues Periodic Acid Schiff Histochemical stain used for the demonstration of carbohydrates (GLYCOGEN) (PAS) Aldehyde Fuschin Stain Used for differential staining of pancreatic islets of Langerhans (Gomori) Mallory’s Staining for muscles and bones; Astrocytes Phosphotungstic Acid Hematoxylin (PTAH) Lissamine Fast Red Muscle demonstration Tartrazine Method Osmic Acid/Osmium Used for fats Tetroxide stain Levaditi’s method Spirochetes Masson Fontana Melanin (Silver Modification): BLACK Technique Gmelin’s Test Diagnostic for Bile pigments Perl’s Prussian Blue Hemosiderin (iron-containing pigment of hemoglobin) Benzidine method For Hemoglobin Cajal’s Gold Sublimate Astrocyte Bielschowsky’s Neurons, Axons, Neurofibrils Technique Weigert-Pal Technique Normal Myelin Sheaths Lindquist’s Modified Copper Rhodamine Gram-Twort Bacteria Brown and Brenn Bacteria, Nocardia, Actinomyces (B&B) Cresyl Violet Acetate Helicobacter Dieterle Legionella pneumophilia Warthin-Starry/ Spirochetes Levaditi’s methods Grocott Methamine Fungi Silver (GMS) Orcein method Hepatitis B Surface antigen Gordon and Sweet’s Reticular Fibers method Errors encountered during routine tissue processing and examination & remedy How to test unknown How to deformalinize tissue section? How to remove mercuric chloride strengths of alcohol? -Refix formalin with other fixatives crystals? -Float an alcoholmeter in the (Zenker’s, Helly’s, Bouin’s fluid) -Leave deposits of crystals on the solution -Produces more brilliant stain tissue -Alcoholmeter: measures the -Deparaffinize for 1 hour in ammonia -Deparaffinize the slides with xylene, strength or % of alcohol water and wash for 1 hour absolute alcohol or 95% alcohol -# of scale just at the surface -Place alcoholic iodine solution; then, of the liquid leave for 5-10 minutes -Wash with running water How to remove formalin How to remove melanin pigments? How to remedy improperly pigment crystals -Deparaffinize (0.25% K2MnO4 solution dehydrated tissue blocks? (dark-brown to black? for 1-4 hours) -Remove paraffin and rehydrate the -Deparaffinize the sections -Wash with tap water tissue with water -Bleach: 5% oxalic acid or pyrogallic -Trim all residual paraffin (melted -Place it in solution (1): 30-60 acid for 5-10 minutes paraffin) minutes; (2) 5-10 minutes; (3) -Wash with tap water for 10 minutes 2-3 minutes -Rinse with distilled water -Wash with running water How to remedy improperly How to reclaim tissue ending series How to retain slides? fixed tissue blocks? on wrong solution? -Place in xylene. Check when the -Remove paraffin -Rinse basket of tissue under cold tap coverslip loosens down (noticeable -Run block through xylene, water for several hours to 1 day) absolute alcohol, or 80% to -Empty capsule onto cloth towel -Place 2 changes of xylene. Then, to 95% alcohol -Put capsule back into dry basket the absolute alcohol, 95% alcohol -Re-fix tissue blocks -Return basket of tissue: 15-30 minutes -Decolorize with acid-alcohol -Reprocessed dehydrants, -Embed/Cast & Re-cut -Wash into a running tap water re-infiltrated blocks -Ready for re-staining How to soften tissues? How to cut blood-containing tissues? How to remove folds of pleated -Change microtome knives -Try to soak the block in tap water paraffin sections? -Soak in a small bowl dish -Try to soak with 10% solution of -Use 50% alcohol containing water and glycerin, 60% alcohol for 1-2 hours -Place in the water bath: drain and detergent -Cut few section (acceptable the 3rd and dry -Leave block into solution for 4th section) ½ to 3 hours -Leave on tap water overnight (difficult) Broken slides Collodionization (Keep section from Sections are compressed, -Remove coverslip by soaking float-off) wrinkled, or jammed with xylene -Float, deparaffinize with xylene and -Re-sharpen the knife -Incubate for 30 minutes absolute alcohol -Mount to a clear xylene moist -Place in 80% alcohol for 3-5 minutes slide -Air dry -Drop mounting media (Clarite/Permount) Tissue is opaque Tissue shrinks away from wax when Tissue is soft when blocked is -Repeat clearing (12 hours); trimmed trimmed then, re-embed -Repeat the whole procedure -Repeat fixation Airholes found on the Wax appears crystalline Paraffin block after cooling is tissue during trimming -Re-embed in freshly filtered wax moist and crumbles -Repeat impregnation -Repeat paraffin impregnation; then, re-embed Sections are torn and Selections of unequal thickness are Horizontal/Parallel lines or furrows crumble when cut produced across the section (CHATTERS) -Remove paraffin with -Reduce the knife tilt -Treat with phenol during processing; clearing agent (decrease or collodionize grade alcohol) -Reduce knife tilt -Repeat dehydration, clearing and embedding TERMS RELATED TO CYTOPATHOLOGIC CHANGES IN DISEASE ☞ Aplasia- defective development of tissue or organ ☞ Agenesia- complete non-appearance of an organ ☞ Hypoplasia- refers to the failure of an organ to reach or achieve its full mature or adult size ☞ Atresia- is the failure of an organ to form an opening ☞ Atrophy- refers to a acquired decrease in the size of a normally developed or mature tissue or organ ☞ Hypertrophy- refers to the increase in size of tissues or organs due to an increase in the size of the individual cells ☞ Hyperplasia- refers to the increase in size of an organ or tissue due to increase in the number of cells resulting from the growth of new cells ☞ Metaplasia- is a reversible change involving the transformation in one type of adult cell to another  ☞ Dysplasia- is the regressive alteration in adult cells manifested by variation in size, shape, and orientation ☞ Anaplasia (DIFFERENTIATION)- is a marked regressive change in adult cells towards more primitive or embryonic cell types, usually utilized as a criterion toward malignancy ☞ Neoplasia (TUMOR)- is the continuous abnormal proliferation of cells without control, serving no useful purpose or function, usually accompanied by increase in size and pigmentation, mitosis, number, metaplastic and anaplastic changes of the cells ☞ Necrosis- means “cell death” which is due to disease or injury TYPES OF NECROSIS ACCORDING TO BASIC MORPHOLOGIC CHANGES 1. Coagulation- consists of more or less rapid coagulation of the cytoplasm brought about by intracellular enzymes (Anemic or Ischemic Infarction) 2. Liquefaction/Colliquative- fairly rapid total enzymatic dissolution of the cells with complete destruction of the entire cell (Bacterial Infection) 3. Fat- involves the peculiar destruction of adipose tissue particularly found in pancreatic degenerations 4. Caseous- made up of mixture of coagulated protein and fat (Syphilis, Tularemia, Lymphogranuloma) 5. Gangrenous – massive death or necrosis of tissue, caused by combination of ischemia and superimposed bacterial infection (necrosis plus putrefaction) ☞ Gangrene- refers to the massive death or necrosis of tissue (bacterial infection and anoxia) TYPES OF GANGRENE 1. Dry- usually caused by arterial occlusion, producing ischemic necrosis and consequently dessication or mummification Example: arterial embolism 2. Wet- this is a result of venous occlusion, wherein bacterial infection supervenes in ischemic injury to the tissue Example: Toxemia, Bacterial infection ---------------------------------------------------------------------------------------------------------------------------------------- MEDICAL TECHNOLOGY LAWS, BIOETHICS AND OTHER RELATED LAWS RFGERONIMO2024 HISTORICAL PERSPECTIVES 1. Hippocrates- Father of Medicine 2. Dr. Jesse Umali- First graduate of BSMT 3. Mr. Crisanto Almario- Father of PAMET NOTE: PAMET (Philippine Association of Medical Technologists) MEDICAL TECHNOLOGISTS’ PRAYER O God, who by calling us to the vocation of a medical technologists, has placed upon us the obligation of being a constant help in the scientific care of the sick, grant us by thy divine light a deep insight into the serious responsibilities of our task. By thy divine wisdom awaken in us a growing zeal and determination to increase our knowledge of how to search for the underlying causes of sickness and disease; how to recognize the evidence of physical changes; how to make important chemical analyses, and other valuable test so helpful in caring for the sick. By thy divine love permit us in this way to share with those who directly care for the sick, that thus we may be of constantly working through the eternal physician, Christ our Lord, Amen. CODE OF ETHICS OF MEDICAL TECHNOLOGISTS As I enter into the practice of Medical Technology, I shall: Accept the responsibilities inherent to being a professional. Uphold the law and shall not participate in illegal work. Act in a spirit of fairness to all and in a spirit of brotherhood toward other members of the profession. Accept employment from more than one employer only when there is no conflict of interest. Perform my task with full confidence, absolute reliability, and accuracy. Share my knowledge and expertise with my colleagues. Contribute to the advancement of the professional organization and other allied health organizations. Restrict my praises, criticisms, views, and opinions within constructive limits. Treat any information I acquired in the course of my work as strictly confidential. Uphold the dignity and respect of my profession and conduct myself a reputation of reliability, honesty, and integrity. Be dedicated to the use of clinical laboratory science to promote life and benefit mankind. Report any violations of the above principles of the professional conduct to the authorized agency and to the Ethics Committee of the organization. To these principles, I hereby subscribe and pledge to conduct myself at all times in a manner befitting the dignity of my profession. PANUNUMPA NG PROPESYONAL Ako, si ________ (pangalan) ng ___________________(Pook na Sinilangan, Bayan/Lungsod, Probinsya) ay taimtim na nanunumpa na itataguyod ko at ipagtatanggol ang Saligang Batas ng Pilipinas, na ako ay tunay na mananalig at tatalima rito; na susundin ko ang mga batas, mga utos na legal, at mga atas na ipinahayag ng mga sadyang itinakdang may kapangyarihan ng Republika ng Pilipinas; at kusa kong babalikatin ang pananagutang ito, na walang ano mang pasubali o hangaring umiwas.Taimtim pa rin akong manunumpa na sa lahat ng panahon at pook na kinaroroonan ay mahigpit akong manghahawakan sa mga etikal at tuntuning propesyonal ng mga_________________________________ sa Pilipinas, at marapat kong gagampanan ng buong husay sa abot ng aking makakaya ang mga tungkulin at pananagutang iniatang sa isang itinakdang ____________________. Kasihan Nawa ako ng Diyos. PAMET HYMN MUSIC: FRANCIS JEROTA PEFANCO LYRICS: HECTOR GENTAPANAN HAYARES, JR. From various lands, races and places With grateful hearts we blend our voices This day to our beloved PAMET From whence unity and love cometh We join together in brotherhood To live up to thine ideals we should In fields of advancement and learning Thy noble goals may be our bearing Loyal and true we'll be to thee Beloved PAMET this we say For service to God and humanity With joy we sing for thee till eternity Loyal and true we'll be to thee Beloved PAMET this we say For service to God and humanity With joy we sing for thee till eternity. BIOETHICAL CONSIDERATION  HISTOPATHOLOGICAL TECHNIQUES, MED-TECH LAWS& OTHER RELATED LAWS, AND LABORATORY MANAGEMENT: a reference and review quick guide By Mr. Ryan Famisan Geronimo Copyright© 2018 by Ryan Famisan Geronimo All rights reserved. No part of this reference and review material may be transmitted or utilized in any form ofelectronic, photocopying, recording, mechanical or otherwise, without the prior permission of the author. For reprints and excerpts, please contact [email protected]. To the ideal future medical technologists whom like me have big dreams and more of dreams to come, to them I dedicate this reference and review material RYAN F.GERONIMO  Preface The compilation is intended for use but not limited to those reviewing for the Philippine licensure examination for medical technologists. This is a thoroughly researched board-sensitive terms and related concepts that uses the actual reference textbooks, following the latest trends in medical technology specifically in histopathology, medical technology laws and other related laws including bioethics, and laboratory management, operation and instrumentation. The author provides a quick and accessible important details spanning across the subject matter to cater the need of the examiners and students. I hope you find this compilation useful in your educational journey and to your future endeavour. The data in this compiled material have been properly selected and verified from reliable and up-to-date references and treatment modalities suggested that have been utilized in medical laboratory science/medical technology profession. Readers are still being advised to consult other references of information written or electronic for updates regarding the aforementioned profession.  Acknowledgement The author wholeheartedly acknowledges the assistance and guidance of the following people who have contributed in producing this compilation: To my parents for their love and support during the making of this review material. To the different reference or source of authors of wisdom and knowledge who are essential to the strength and improvement of this future source of knowledge. Above all, to Almighty God, the greatest author of life, for his uncounted blessings and for giving me the strength and wisdom to finish this reference and review material in contributing and crafting ideal future medical technologists. Ryanfg  Table of Contents Dedication Chapter 6- Impregnation or Infiltration, and Embedding……………………………24 Preface I. Definition of terms Acknowledgement II. Kinds of embedding Table of Contents III. Precautions in impregnation IV. Substitute for paraffin wax Chapter 1- Introduction to Histotechnique…………………………………………………………………8 V. Types of embedding molds I. Methods of tissue preparation and examination VI. Tissue-Tek system II. Steps on manual tissue processing VII. Types of embedding medium Chapter 2- Fixation……………………………………………………………………………………………………………………………15 Chapter 7- Microtomy………………………………………………………………………………………………………………………27 I. Effects of fixatives I. Use of microtome; Principle II. Tissue-Fixative requirement II. Types of microtome III. Type of tissue III. The cryostat IV. Factors that influence fixation A. Different tissues V. Summary of different fixatives and their classification B. Ranging Temperature A. Types of fixative IV. Kinds of Microtome Knives B. Classifications A. Length C. Advantages B. Type of blocks D. Disadvantages C. Kind of Microtome VI. Washing-out method V. Microtome Knives: Inclination or Angle VI. Microtome Knives Sharpening Chapter 3- Decalcification…………………………………………………………………………………………………………18 A. Honing I. Decalcification concepts and other special considerations B. Stropping II. Decalcifying agents VII. Others A. Uses A. Care of the microtome B. Other particulars,advantages, disadvantages B. General steps in fixing sections onto the slides III. Chelating agents IV. Post-Decalcification Treatment Chapter 8- Adhesives and Mounting media……………………………………………………………………28 V. Test for complete decalcification I. Adhesives and their uses VI. Tissue Softeners II. Mounting media A. Refractive Index (R.I.) Chapter 4- Dehydration……………………………………………………………………………………………………………………20 B. Uses and importance I. Definition of dehydrations; Effects III. Ringing II. Dehydrating agents A. Uses Chapter 9- Errors encountered during routine tissue processing and B. Other particulars, advantages, disadvantages examination & remedy………………………………………………………………………………………………………………………29 Chapter 5- Clearing/Dealcoholization………………………………………………………………………………21 Chapter 10- Staining/ Dyeing…………………………………………………………………………………………………31 I. Applications I. Importance II. Clearing agents II. Chemical basis and dyestuff A. Uses III. Methods of staining B. Other particulars, advantages, disadvantages IV. Classification of dye V. List of common histological stains and their uses Chapter 11- Immunohistochemical techniques& Cytology………………………………38 I. Introduction II. Polyclonal vs. Monoclonal Antibodies III. Types of tumor markers, classifications, and examples IV. Broder’s Classification of tumor V. Diagnostic and exfoliative cytology VI. Clinical Apparatuses used VII. Vaginal hormonal cytology VIII. Criteria for cytologic diagnosis of normal pregnancy IX. Classes of cytologic evaluation X. Terms related to cytopathologic changes in diseases Bibliography……………………………………………………………………………………………………………………………………39 MEDICAL TECHNOLOGY, BIOETHICS AND OTHER RELATED LAWS………………………40-57 LABORATORY MANAGEMENT, INSTRUMENTATION AND OPERATION………………………58-74 “Success is most often achieved by those who don’t know that failure is inevitable.” -Coco Chanel  HISTOPATHOLOGICAL ANALYSIS Introduction to Histological Techniques WHAT IS HISTOTECHNIQUE? ☺ It includes the preparation, processing and staining of tissue sections for *AUTOLYSIS- dissolution of cells that causes rupture of lysosomes; microscopic study to be interpreted by the pathologist either malignant (can cause it leads in the formation of WASHED OUT CELLS (homogenous, death) or benign (cannot cause death) granular masses of cells) *PUTREFACTION- breakdown of tissuesthrough bacterial DEFINITION OF TERMS: disintegration (saprophytic organisms) *HISTOPATHOLOGY- branch of anatomical pathology that deals on the study of diseases in a given tissue or organ 1. TEASING/DISSOCIATION  Also referred to as “Dissection”/”Separation” BRANCHES OF ANATOMICAL PATHOLOGY  NSS (Normal Saline Solution)/ Physiological Saline: 0.85- -Forensic pathology 0.90% NaCl solution -Immunohistopathology  Examine as stained preparation (Bright field/Light -Surgical pathology Microscope) or unstained preparation (Phase-Contrast -Cytology Microscope) -Autopsy  Disadvantage: anatomical relationship is destroyed 2. SQUASH/CRUSHING *HISTOCHEMISTRY- it deals on the study of chemical nature of tissues  Tissue (< 1mm) is sandwiched between 2 slides  Incorporation of vital stain through capillary action ☺ Specimens: non-living forms of tissues that can be used for advanced medicine  Preparation is viewed under the microscope o surgical materials 3. SMEARING (for cytological studies) o autopsy materials  For sections (organs) or sediments  Wire loop, applicator stick or another slide ☺ Storage: 4. STREAKING o Specimen: 1 month – 1 year  Uniform distribution in a zigzag manner o Tissue blocks: 3 years – 10 years 5. SPREADING o Slides: Indefinite  For thick or mucoid specimens o Records: Permanent  Example: sputum, bronchoalveolar lavage (macrophages), o Result forms: Triplicate (patient, laboratory, medical doctor) pulmonary lesions/foci  Teasing on a slide Methods of tissue preparation & examination  Fresh tissue preparation o Pull-Apart: with a single uninterrupted motion (blood smears & -Observation of physiologic processes thick secretions& urine sample) -Disadvantages:  Not Permanent o Touch/Impression smear: freshly cut tissue is brought in contact &  Immediate changes after death are likely to occur pressed onto the slide (allows direct transfer of cells; maintains cellular interrelationship/anatomy of the cells) 7|P ag e rfgeronimo2018  2. SECTIONING  Frozen Section: Prevents decomposition and distortion  Majority of preparation *for rapid diagnosis/urgent biopsies; for enzymes, lipids & carbohydrate  3.0-5.0 mm thickness (CHO) derivative tissues (CRYOSTAT/COLD MICROTOME) *freezing Agents: Liquid N2; CO2; aerosol spray; isopentane in liquid 3. WHOLE MOUNT nitrogen (most rapid of the commonly available freezing agent)  Preparation for organisms *delicate specimens: enzyme histochemistry, soluble substance (lipids &  0.2-0.5 mm thickness CHO), immunohistochemical staining, specialized silver stain particularly in neuropathy REAGENT PREPARATION  Basic Formula: V1C1 = V2C2  Preserved tissue PROBLEM: Suppose you have 3 mL stock solution of 100 mg/mL of phenol  Microincineration: Used to locate the presence & position of mineral and you want to prepare a 200 uL of having 25 mg/mL of the solution. elements in the tissue Compute for the unknown and convert your answer from uL to mL. : 2 or duplicate sections of alcohol-fixed tissues : The ash residue of the incinerated section is examined SOLUTION: alongside the stained preparation, and the incidence and  Wherein, location of any mineral deposits is noted *3 mL (the word suppose means approximation/estimation) : A polarizing microscope is often used to examine the *Unknown: V1 (initial volume) ash residue of the incinerated section (SPODOGRAM) *V2 (final volume/ total volume): 200 uL *C2 (new concentration): 25 mg/mL  Autoradiography: Injection of radioactive isotopes into organs; it *C1 (initial concentration): 100 mg/mL determines the relationship and location of the isotopes and cells to be studied V1= V2C2/ C1 : Sections are mounted in contact with a photographic V1= (200uL) (25mg/mL)/ 100 mg/mL emulsion which is blackened by the isotopes when it is V1= 5,000 uL / 100 developed V1= 50 uL : Fix  sectionmount on slides in contact with a photographic emulsionsstain 1mL=1000 uL V1= 50 uL x 1mL/100 uL  MICROWAVE PROCESSING:Based on the principle of using microwave V1= 50 / 100 mL energy to speed up diffusion of liquids into and out of the specimen V1= 0.05 mL TYPES OF HISTOLOGICAL PREPARATION/METHODS STEPS ON MANUAL TISSUE PROCESSING 1. SMEARING  Derived from blood, bone marrow and bodily fluids (pleural fluid or ascitic 1. FIXATION/PRESERVATION 6. TRIMMING fluid) 2. DEHYDRATION 7. SECTION-CUTTING  For cytological examination 3. CLEARING/DEALCOHOLIZATION 8. STAINING 4. INFILTRATION/IMPREGNATION 9. MOUNTING  Fix with ethyl ether 5. EMBEDDING/CASTING/BLOCKING Chapter10. 2 LABELLING 8|P ag e rfgeronimo2018   Cholesterol: Digitonin Fixation  CHO: alcoholic fixatives (e.g. Rossman’s fluid)  CHON: Neutral buffered formol saline or formaldehyde FIXATION:Is the process in adopting to kill, harden and preserve tissue materials (from vapor destruction and post-mortem changes)  Electron Microscopy: 2 fixatives/Conjugated fixatives (Osmic acid and Glutaraldehyde) ☺ EFFECTS: o Denaturation of proteins rendering them insoluble ☺ OTHER FACTORS TO BE CONSIDERED: o Tissues become resistant to the effects of subsequent technical  pH for fixation: 6 – 8 processing  Temperature: Room temperature (20 -22OC); 45OC- o Staining will be strongly influenced RNA; 65 OC - DNA o Inhibits bacterial decomposition  Osmolality: slightly hypertonic o Soft and friable tissues are hardened making handling and cutting easier TYPES OF FIXATIVES ACCORDING TO COMPOSITION AND ACTION o Optical differentiation (varying degrees of refractive indices) of cells and tissue components is increased TYPES OF FIXATIVES ACCORDING TO COMPOSITION o Safer handling & processing 1. Simple Fixatives: made up of only one component or substance ☺ REQUIREMENTS: 2. Compound Fixatives: made up of two or more fixatives which have been added o Tissue size: 2cm2/10 mm X 4 mm thick; 1 - 2 mm2(Electron together to obtain the optimal combined effects of their individual actions upon the Microscopy) cells and tissue constituents o Volume of fixative: 20x the volume of the specimen; Osmium tetroxide/Osmic acid: 5 – 10x of the tissue volume; Museum: Not TYPES OF FIXATIVES ACCORDING TO ACTION < 50 – 100x 1. Microanatomical Fixatives: permits general microscopic study of tissue structures o Tissue container: adequate size, wide mouthed and non-rusting without altering the structural pattern and normal intercellular relationship of the tissues in question o TISSUE ORGAN PREPARATION:  Hollow organs (stomach and intestines) should be packed 2. Cytological Fixatives: preserve specific parts and particular micrsocopic elements of with cotton soaked in fixative before immersion the cell itself  Air-filled lungs must be covered with several layers of gauze a. Nuclear : preserves nuclear structures (chromosomes); less than 4.6 pH; contain  Human brains should be washed with Ringer’s Lactate glacial acetic acid solution (Intravascular perfusion) b. Cytoplasmic: preserves cytoplasmic structures; more than 4.6 pH; do not contain  Whole organs (eyes) should be injected with fixative as glacial acetic acid (destroys mitochondria and golgi bodies) well as immersed in it 3. Histochemical Fixatives: preserve chemical constituents of cells and tissues o TYPE OF TISSUE: (enzymes)  Lipids: Mercuric Chloride or K2Cr2O7  Phospholipids: Baker’s formol-calcium 9|P ag e rfgeronimo2018 SUMMARY OF THE DIFFERENT FIXATIVES AND ITS CLASSIFICATION TYPE OF ACCORDING TO ITS NAME OF FIXATIVE DESCRIPTION/ OTHER PARTICULARS/ ADVANTAGES DISADVANTAGES FIXATIVE CLASSIFICATION CONSIDERATIONS SIMPLE COMPOSITION: FORMALDEHYDE -Gas produced by the oxidation of -cheap, easy to prepare -Fumes are irritating (nose & eyes) FIXATIVE ALDEHYDE methanol; soluble in water to the -compatible with many stains -Solutions: allergic dermatitis extent of 37-40% wt. in vol. -does not overharden the tissue -Produces shrinkage of tissues -PURE STOCK: 40% (unsatisfactory for -penetrates tissues well -“soft fixative”; does not harden some routine fixation) -preserves fat, mucin, glycogen cytoplasmic structures -FIXATION TIME: 24 hours -preserves but does not precipitate proteins -Prolonged fixation may produce bleaching -frozen tissue sections are easily prepared of the specimen, and loss of natural tissue color & dispersal of fat -Unbuffered: reduces both basophilic and eosinophilic staining of cells; it forms abundant brown pigment granules on blood containing tissues (spleen) due to blackening of hemoglobin SIMPLE COMPOSITION: GLUTARALDEHYDE -made up of 2 formaldehyde residues, -more stable in tissues (firmer texture) -more expensive FIXATIVE ALDEHYDE linked by 3-C chains -preserves plasma proteins -less stable -USES: routine L.M. works, E.M. -produces less tissue shrinkage -penetrates tissues more slowly -2.5%: used for small tissue fragments -preserves cellular structures -reduces PAS (+) of reactive mucin and needle biopsies (2-4 hrs) (RECOMMENDED FOR ENZYME -4%: recommended for larger tissues HISTOCHEMISTRY & ELECTRON MICROSCOPY) (6-8 hrs to 1 day) -more pleasant and less irritating to nose -does not cause dermatitis METALLIC FIXATIVE MERCURIC -“MOST COMMON FIXATIVE” -penetrates and hardens tissues rapidly and -it causes marked shrinkage of cells CHLORIDE -5-7% saturated aqueous solution well -rapidly harden the outer layer of the tissue -fine detail nuclear components -slow penetration (2-5mm thickness of -has greater affinity to acid dyes tissues) -Trichrome staining is excellent -unduly and harden tissues (>1-2 days) -FIXATIVE OF CHOICE for Tissue Photography -causes lysis of RBCs and removes iron form -permits brilliant metachromatic staining of hemosiderin cells -it is inert to fats and lipids -RECOMMENDED for renal tissues, -formation of black granular deposits in connective tissues, muscles and fibrin tissues; corrosive to metals -reduces amount of glycogen POTASSIUM -3% aqueous solution -does not precipitate cytoplasmic structures DICHROMATE -preserves lipids -preserves mitochondria (pH of 4.5-5.2) CHROMIC ACID -1-2% aqueous solution -adequately preserves CHO -it precipitates all proteins -“strong oxidizing agent” 10 | P a g e rfgeronimo2018  LEAD FIXATIVE -4% aqueous solution -RECOMMENDED for acid mucopolysaccharide -fixes connective tissue mucin -It takes up CO2 to form insoluble lead carbonate on prolonged standing: this can be removed by filtration or adding acetic acid drop by drop to lower the pH and dissolve the residue PICRIC ACID -normally used in strong saturated -excellent & stable fixative for glycogen -Causes RBC hemolysis aqueous solution (1%) demonstration -NOT SUITABLE for frozen sections (crumble) -penetrates tissues well and fixes small -Prolonged fixation causes hard, brittle tissues rapidly tissues -the yellow stain taken in by tissues prevents -Picrates are formed upon protein (never be small fragments from being overlooked washed in water before dehydration) -allows brilliant staining with the Trichrome -Produces excessive staining of tissues method -Highly explosive when dry -suitable for aniline dye (Mallory’s -Dissolves and alter lipids Heidenhain’s or Masson’s method) -Interferes with Azure Eosin method of -precipitates all proteins staining GLACIAL ACETIC -normally used in conjunction with -fixes and precipitates nucleoproteins - When combined with potassium ACID other fixatives to form a compound -precipitates chromosomes and chromatin dichromate, the lipid fixing property is -It SOLIDIFIES at 17OC (Glacial Acetic -causes tissues to swell (collagen-containing destroyed Acid) substances) -CONTRAINDICATED for cytoplasmic fixation (destroys mitochondria and golgi bodies) ACETONE -Used at ice cold temperature ranging -recommended for study of water diffusible -reduces inevitable shrinkage and distortion from -5 OC to 4 OC enzymes (phosphatases and lipases) -dissolves fat -used in fixing brain tissues and for diagnosis -preserves glycogen poorly of RABIES -evaporates rapidly -used as a solvent for certain metallic salts (Freeze Substitution) ALCOHOL -rapidly denatures and precipitates -IDEAL for small tissue fragments -causes RBC hemolysis proteins (H+ bond is destroyed) -BOTH FIXATIVE and DEHYDRATING AGENT -dissolves fats and lipids -70%-100%: usual concentration range -excellent for glycogen preservation -causes glycogen granules to move towards (lesser concentrated solutions -preserves nuclear stains the poles and ends of the cells produce lysis of cells) (POLARIZATION) METHANOL -excellent for fixing dry and wet smears, -penetration is slow blood and bone marrow tissues -If fixed for >48 hrs: overhardened & difficult to cut tissues ETHANOL (70-100%) -preserves glycogen only but does not fix -LOWER concentrations: RBCs become -fixes blood, tissue films and smears hemolyzed and WBCs are inadequately -preserves nucleoproteins and nucleic acids preserved; Strong reducing agent (Histochemistry and Enzyme Studies) -Hemosiderin preservation is less than in buffered formaldehyde 11 | P a g e rfgeronimo2018  OSMIUM -pale yellow powder which dissolves -precipitates and gels proteins -very expensive TETROXIDE/OSMIC water (up to 6% at 20 OC) strong -preserves fats, lipids, cytoplasmic structures -poor penetrating agent ACID oxidizing solution (golgi bodies & mitochondria), myelin and -irritant to the eyes (conjunctivitis and -Osmic acid fixed tissues must be peripheral nerves, some tissues (adrenal corneal damage blindness) washed in running water for at least glands) -inhibits Hematoxylin stain 24 hours to prevent the formation of -fixes materials for ULTRATHIN sectioning in -extremely volatile artifacts Electron Microscopy (E.M.) -Saturated aqueous HgCl2 solution -produces brilliant nuclear staining with (0.5-1 mL/100mL of stock solution): Safranin O prevents the reduction of formed black deposits HEAT -involves thermal coagulation of tissue - fixation is better -produces considerable tissue shrinkage and proteins for rapid diagnosis -preserves both nuclear and cytoplasmic distortion -it usually employed for frozen tissue details -destroys RBCs sections and preparation of -suitable for frozen tissue preparations -dissolves starch and glycogen BACTERIOLOGIC smears COMPOUND MICRO- 10% FORMOL -simple fixative -penetrates and fixes tissue evenly -slow fixative FIXATIVES ANATOMICAL SALINE -COMPOSITION: saturated -preserves both microanatomical and -tissue tend to shrink: alcohol dehydration FIXATIVES formaldehyde (40 grams by wt. vol.) cytological details with minimum shrinkage -reduced metachromatic reaction of amyloid diluted to 10% NaCl and distortion -acid dye stains less brightly -RECOMMENDED for CENTRAL -LARGE SPECIMENS: may be fixed (solution NERVOUS TISSES and GENERAL POST- should be replaced after 3 months) MORTEM tissues -preserves enzymes and nucleoproteins -demonstrates fats and mucin -ideal for most staining techniques (SILVER IMPREGNATION) 10% NEUTRAL -RECOMMENDED for PRESERVATION -prevents precipitation of acid-formalin -longer to prepare BUFFERED AND STORAGE OF SURGICAL, POST- pigments on post-mortem tissues -reduced positivity of mucin to PAS FORMALIN MORTEM, and RESEARCH SPECIMENS -BEST FIXATIVE for tissues containing IRON -produces gradual loss of basophilic staining PIGMENTS and for ELASTIC FIBERS of cells; inert towards lipids HEIDENHAIN’S -RECOMMENDED for TUMOR BIOPSIES of the SKIN SUSA -Excellent cytologic fixative -It permits most staining procedures to be done: SILVER IMPREGNATION BRILLIANT RESULTS: sharp nuclear and cytoplasmic details -It permits easier sectioning of large blocks of fibrous connective tissues -RBC preservation is poor NOTE: After using Heidenhain’s Susa fixative, the tissue should be transferred directly to a high-grade alcohol (96% alcohol/absolute alcohol) to avoid undue swelling of tissues FORMOL -FORMOL MERCURIC CHLORIDE: -small pieces of tissues penetrate rapidly -slow penetration SUBLIMATE recommended for routine post- -produces minimum shrinkage and hardening -forms mercuric chloride (HgCl2) deposits 12 | P a g e rfgeronimo2018  (FORMOL- mortem tissues -excellent for silver reticulum staining -does not allow frozen tissue sections CORROSIVE) method -inhibits the determination of the extent of -brightens cytoplasmic and metachromatic tissue decalcification stains better than with formalin alone -no need for “washing-out” -fixes lipids: neutral fats and phospholipids ZENKER’S FLUID -made up of HgCl2 stock solution to -RECOMMENDED for TRICHROME staining -poor penetration which glacial acetic acid has been -produces fairly rapid and even fixation of -not stable added just before its use tissues -prolonged fixation (>24 hrs): brittle and -mercuric deposits may be removed -permits brilliant staining of nuclear and hard tissues by immersing in ALCOHOLIC IODINE connective tissue fibers -causes lysis of RBCs SOLUTIONS -may act as a MORDANT -does not permit cutting of frozen tissues -RECOMMENDED: liver, spleen, connective tissue fibers and nuclei ZENKER-FORMOL -EXCELLENT FOR MICROANATOMIC FIXATION: pituitary gland, bone marrow & blood containing organs (spleen and liver) (HELLY’S -Brown pigments are produced from blood containing organs (>24hrs of fixation: RBC lysis immerse the tissue in saturated alcoholic SOLUTION) picric acid or sodium hydroxide -Nuclear fixation & staining is better than Zenker’s fluid -It preserves cytoplasmic granules well BOUIN’S -RECOMMENDED FOR EMBRYO -yellow stains is useful when handling -penetrates poorly SOLUTION fixation fragmentary biopsies -NOT SUITABLE for fixing kidney structures -preserves glycogen -reduces or abolishes Feulgen’s reaction due -does not need washing to hydrolysis of nucleoproteins -produces minimal distortion of -produces RBC hemolysis microanatomical structures BRASIL’S -It is better and less messy than Bouin’s solution ALCOHOLIC -Excellent fixative for glycogen PICROFORMOL -AUTOMATIC TISSUE PROCESSING: Overnight tissue fixation 3-4 changes of Brasil’s fixative (1 ½ to 2 hours) succeeded directly to SOLUTION absolute alcohol CYTOLOGICAL FLEMMING’S -most common Chrome-Osmium -permanently fixes fats -solution deteriorates rapidly FIXATIVES: FLUID acetic acid fixative used -excellent for chromosomal details -has the tendency to form artefact pigments NUCLEAR -RECOMMENDED for NUCLEAR FIXATION TIME: 1-2 days preparation CARNOY’S FLUID -RECOMMENDED for fixing -preserves Nissl granules and cytoplasmic -produces RBC hemolysis CHROMOSOMES, LYMPH GLANDS, granules -hardens tissue URGENT BIOPSIES -permits nuclear staining -dissolves fats, lipids, and myelin -“MOST RAPID FIXATIVE” -FIXATIVE AND DEHYDRATING AGENT -leads to polarization unless and very cold -very suitable for small tissue fragments: temperatures (-70OC) biopsy materials and curetting -dissolve acid-soluble cell granule&pigment 13 | P a g e rfgeronimo2018  BOUIN’S FLUID -RECOMMENDED FOR EMBRYO -yellow stains is useful when handling -penetrates poorly fixation fragmentary biopsies -NOT SUITABLE for fixing kidney structures -preserves glycogen -reduces or abolishes Feulgen reaction due -does not need washing to hydrolysis of nucleoproteins -produces minimal distortion of -produces RBC hemolysis microanatomical structures NEWCOMER’S -RECOMMENDED for fixing mucopolysaccharide and nuclear proteins FLUID -It produces better reaction in Feulgen’s stain than Carnoy’s fluid -Acts as a nuclear and histochemical fixatives HEIDENHAIN’S -RECOMMENDED for TUMOR BIOPSIES of the SKIN SUSA -Excellent cytologic fixative -It permits most staining procedures to be done: SILVER IMPREGNATION BRILLIANT RESULTS: sharp nuclear and cytoplasmic details -It permits easier sectioning of large blocks of fibrous connective tissues -RBC preservation is poor NOTE: After using Heidenhain’s Susa fixative, the tissue should be transferred directly to a high-grade alcohol (96% alcohol/absolute alcohol) to avoid undue swelling of tissues CYTOLOGICAL FLEMMING’S -made up only of CHROMIC ACID AND * SAME AS FLEMMING’S SOLUTION FIXATIVES: FLUID WITHOUT OSMIC ACID *FIXATION TIME: 1-2 days CYTOPLASMIC ACETIC ACID -RECOMMENDED for CYTOPLASMIC structure (mitochondria) HELLY’S FLUID -EXCELLENT FOR MICROANATOMIC FIXATION: pituitary gland, bone marrow & blood containing organs (spleen and liver) -Brown pigments are produced from blood containing organs (>24hrs of fixation: RBC lysis immerse the tissue in saturated alcoholic picric acid or sodium hydroxide -Nuclear fixation & staining is better than Zenker’s fluid -It preserves cytoplasmic granules well REGAUD’S FLUID -It hardens tissues better and more rapidly than Orth’s fluid (MOLLER’S FLUID) -RECOMMENDED for the demonstration of chromatin, mitochondria, mitotic figures, golgi bodies, RBCs and colloid-containing tissues -It preserves hemosiderin less than buffered formalin -Glycogen penetration is poor -Does not preserve fats ORTH’S FLUID -RECOMMENDED for study of EARLY -demonstrates RICKETTSIAE and other *Same as Regaud’s fluid DEGENERATIVE processes & TISSUE bacteria NECROSIS -preserves myelin better than buffered formalin FORMALIN WITH “POST- CHROMING” HISTOCHEMICAL 10% FORMOL *Previously described and identified FIXATIVES SALINE 14 | P a g e rfgeronimo2018  ABSOLUTE ETHANOL ACETONE NEWCOMER’S FLUID Reference: Dr. Jocelyn H. Bruce-Gregorios (2012). Histopathologic Techniques. (2nd Ed.). Goodwill TradingCo. Inc.: Makati City, Philippines.pages 17-52. WASHING-OUT: REMOVAL OF EXCESSIVE FIXATIVE 1. Tap Water: removes excess ☺ MELANIN (BROWN TO BLACK PIGMENT)  Chromates (Helly’s, Zenker’s & Flemming’s)  KMnO4 (decolorizer)  Formalin  Pyrogallic acid/Oxalic acid(reducer)  Osmic Acid  Hydrogen Peroxide (H2O2)(bleach & final removal) 2. 50 – 70 % alcohol SPECIAL PROCESSING TECHNIQUES  Picric acid (Bouin’s solution) 1. Quenching  Rapid freezing of tissue blocks to allow instant cessation of cellular activities 3. Alcoholic iodide ☺ Freezing Agent: Liquid Nitrogen  Mercuric fixatives  Isopentane  Pentane WASHING-OUT: REMOVAL OF PIGMENT AFTER FIXATION  Propane ☺ FORMALIN ( white precipitates)  Dichlorodifluoromethane  Sections to water:  Kardasewitsch’s: 28%NH3 H2O (ammonium water) + 70% 2. Freeze-Drying ethyl alcohol  Rapid freezing the tissues at -150 OC  Lillie’s: 28%NH3 H2O, acetone + H2O2  wash in 70% alcohol  Dessication  Alkaline Picrate: Picric acid + 95% ethanol  Formed tiny ice crystals are removed by sublimation in a vacuum at -40 to -70 OC  Dry—RT—fix/process/embedded ☺ PICRIC ACID  Molten paraffin  Sat. Li2CO3 in 70 % EA  Water soluble waxes  70% EA then 5% Na thiosulfate  Celloidin/Collodion  Lenoir’s solution: concentrated Ammonium acetate, 95% ethanol and Distilled water 3. Free Substitution  Similar with freeze-drying ☺ MERCURIC FIXATIVE (DEZENKERIZATION: the removal of excessive  More suitable for routine purposes mercury)  Can be used for both Paraffin and Celloidin sections  0.5% Iodine solution in 70% ethanol (5-10 minutes)  Instead of dehydration in a vacuum, tissue blocks are:  Langeron’s iodine: Iodine crystals, Potassium Iodide, Distilled  Fixed in Rossman’s fluid or 1% acetone water  Dehydrated with absolute alcohol at room temperature 15 | P a g e rfgeronimo2018  Decalcification/Demineralization DECALCIFICATION  Process of calcium, phosphate salt or lime salt removal Routine Acid Techniques  Bones and teeth  All acid solutions (DECALCIFIERS) are injurious to the organic ground  Teratomas containing bony tissue substance of the bone or other tissues  Calcified pathological tissues like tuberculosis foci  Acids should be changed one or twice/day until decalcification is completed  Arteries calcified by atheroma DECALCIFYING AGENTS  Forms of calcium salts 1. Nitric acid  Calcium urates (gout) ☺ Most common & the fastest  Calcium phosphates (bones) ☺ Very rapid decalcifying agent producing minimal distortion   Calcium fluoride (teeth) RECOMMENDED FOR ROUTINE PURPOSES & URGENT BIOPSIES  Calcium oxalate (renal calculi/kidney stones) ☺ Inhibits nuclear stains and destroying tissues (concentrated solutions) ☺ 5 – 10 %: rapid decalcifier GENERAL CONSIDERATIONS/PARTICULARS ☺ Carefully watch for endpoint  Done after fixation and before infiltration ☺ Remove by 70 - 90 % ethanol  Slides are placed in 1% aqueous Lithium carbonate for 1 hour  Tissue preparation ☺ Usually combined with formaldehyde or alcohol  Use of hacksaw, jig saw, or fret saw ☺ Causes spontaneous yellow discoloration (HNO2)  Complete Fixation (longer in formalin) o Neutralize with 5% NaSO4 wash for 12 hours o Add 0.1% urea  Ideal time : 24 – 48 hrs : 14 days or more (for large and dense tissue specimens) ☺ HNO3 formulations: 1. De Castro’s fluid - silver impregnation of nerve fibers(chloral  Volume of reagent: 20x of the tissue volume hydrates dissolved in water & alcohol, added with HNO3)  Concentration of agent used 2. 10 % Aqueous Nitric Acid (12 – 24 hours) *Higher concentration - more rapid, more harmful to the tissue 3. Formol-Nitric Acid (1 – 3days) *Lower concentration – slower in action, less harmful to the tissue 4. Perenyi’s fluid (w/ chromic acid): 2 - 7 days  Agitation & Partial/Complete vacuum : With little accelerating effect 5. Phloroglucin-Nitric acid (12 hours)  Optimum temperature: RT (18 - 30 OC) 2. Formic Acid Heat ☺ Fixative and a decalcifying agent  Accelerates demineralization; Promotes destructive action of acids on matrix ☺ Moderate-acting decalcifying agent: better nuclear staining  At 55OC -Tissues will undergo complete digestion ☺ RECOMMENDED for ROUTINE DECALCIFICATION OF POST-MORTEM  At 37OC -Impair nuclear staining Van Gieson’s stain: reduced effectiveness of RESEARCH TISSUES Trichrome & Periodic Acid Schiff stain ☺ Not suitable for urgent examinations ☺ For small pieces of bones & teeth (2 – 7 days) ☺ For large pieces of bones 16 | P a g e rfgeronimo2018  ☺ Advantages: 8. Citric acid-Citrate Buffer Solution (pH 4.5) o Safer to handle than HNO3& HCl ☺ Permits excellent nuclear and cytoplasmic staining o Gentler on tissues than HNO3 ☺ TOO SLOW for routine purposes o With wider latitude as regards time ☺ It does not produce cell and tissue distortion ☺ Concentrated reagent: 90% o Aqueous Formic Acid (optional + formalin) 9. Phloroglucin-Nitric Acid o Formic Acid-Sodium Citrate (3 – 14 days) ☺ MOST RAPID DECALCIFYING AGENT; RECOMMENDED FOR URGENT o Neutralize with 5% sodium sulfate  washing-out WORKS ☺ Yellow color must be neutralized with 5% sodium sulphate wash with 3. Hydrochloric acid running tap water (24 hours) ☺ Slower action; with greater distortion ☺ Complete decalcification cannot be determined by chemical means ☺ INFERIOR compared to nitric acid produces good nuclear staining ☺ Poor nuclear staining ☺ RECOMMENDED FOR SURFACE DECALCIFICATION OF THE TISSUE BLOCKS ☺ Nuclear staining: 1% with 70% alcohol 10. Perenyi’s Fluid ☺ Cannot be measured by chemical test ☺ It decalcifies and softens tissues ☺ Maceration is avoided due to the presence of Chromic acid and Alcohol 4. Von Ebner’s fluid ☺ Relatively slow for dense bones ☺ RECOMMENDED for teeth & small pieces of bones ☺ Compositions: 0.5% chromic acid; 10% nitric acid; absolute ethyl alcohol ☺ Moderately rapid decalcifying agent ☺ Permits relatively good cytologic staining CHELATING AGENTS  Substances which combine with calcium ions and other salts (iron & 5. Trichloroacetic acid (TCA) magnesium deposits) to form weakly dissociated complexes and facilitate ☺ Weak and very slow acting decalcifying agent, not used for dense removal of calcium salts tissues ☺ It permits good nuclear staining 1. EDTA (Ethylene DiamineTetraacetic Acid) ☺ It does not require washing-out  Decalcifying agent & water softener  5 – 7% disodium salt @ pH of 7.0 – 7.4 6. Sulphurous acid  2 – 4 oz/g of bone ☺ Very weak decalcifying solution  MOST COMMON CHELATING AGENT ☺ Suitable for minute pieces of bone  Commercial Name: Versene  Slow-acting, but does not interfere with staining, does not distort tissues and 7. Chromic acid enzymes (1 – 3 weeks) ☺ Acts both a fixative and decalcifying agent  Requires continuous change (use of transfusion set: 1 – 3 weeks) for small tissues; 6 ☺ RECOMMENDED FOR MINUTE BONE SPICULES – 8 weeks for large ones; or longer ☺ Nuclear staining with Hematoxylin is inhibited  Very slow: NOT RECOMMENDED for urgent and routine purposes 2. Ion Exchange Resin (ammonium form of polysterene resin)  Formic acid and Resin (Ammonium-sulfonated polysterene)  Sequesters liberated calcium 17 | P a g e rfgeronimo2018   NOT RECOMMENDED for fluids containing mineral acids such as nitric acid or  Very vague and inaccurate way of determining if a tissue has been hydrochloric acid (HCl) completely decalcified or not  Ion Exchange Resin Thickness: ½ inch  Alternative way: pricking of the tissue with a fine needle or a probe  Volume: 20-30x the volume of the tissue  Disadvantage: small calcified foci may not even be detected  The tissue may allow to stay in solution for 1-14 days  UNRELIABLE METHOD  Measured by Physical/Radiological (X-Ray) methods  CANNOT BE MEASURED by chemical means 2. X-Ray or Radiological Method  REACTIVATION OF USED RESIN: immersed with N/10 HCl TWICE wash with  Very expensive; MOST IDEAL and MOST RELIABLE METHOD distilled water THRICE  Smallest Calcium deposit can be detected (opaque)  NOT recommended for Mercuric Chloride fixed tissues 3. Commercial Decalcifying Reagents  Cannot be used when radiopaque metallic salts (HgCl2)  Best reserved for sections not requiring more than 4 - 8 hours in decalcifying solutions 3. Chemical Tests  Avoid over decalcification & use of metal containers a. Calcium oxalate test 4. Electrophoresis/Electrical Ionization  Simple, Reliable and Convenient method RECOMMENDED for ROUTINE ☺ Principle: Electrolysis/ Electrical Ionization PURPOSES ☺ Process whereby positively charged calcium ions are attracted to a negative electrode and subsequently removed from the decalcifying solution 1. 5 mL of the used decalcifying agent  Temperature: 30 – 45OC 2. Alkalinize w/ NH3 water  90% formic acid & concentrated HCl 3. Add 0.5 mL saturated aqueous ammonium oxalate (1% Na oxalate)  stand for 15 -  Insoluble calcium salts in the specimen are changed to ionizable salts by the action 30 minutes of the acids  This method is satisfactory for small bone fragments *RESULTS:  CLEAR fluid = COMPLETE decalcification POST-DECALCIFICATION TREATMENT  CLOUDINESS = (+) Calcium oxalate = incomplete decalcification  Washing-out Note: Grossly undercalcified bone: a cloud of Ca(OH)2 upon addition of NH3  Running water: 3 – 8 hours  Neutralization: immerse in b. Perenyi’s fluid test *2% Lithium carbonate or  Perenyi’s + Ammonia it forms precipitates *5 – 10 % Sodium bicarbonate  Remedy: add glacial acetic