Histology & Cytology Lecture Notes PDF

HISTO-CYTOTECH – SCOPE OF STUDY SIGNIFICANCE OF EXFOLIATIVE CYTOLOGY 1. Cytologic 1. for assessing cancerous conditions 2. Histopathologic...

HISTO-CYTOTECH – SCOPE OF STUDY SIGNIFICANCE OF EXFOLIATIVE CYTOLOGY 1. Cytologic 1. for assessing cancerous conditions 2. Histopathologic 2. for assessing female hormonal activity 3. for determination of genetic sex HISTOPATHOLOGIC TECHNIQUES 4. to differentiate cancer from other - Involves the different procedures that have diseases like infection, inflammation and been adopted for the preparation of others materials and tissues for microscopic 5. to determine the different cytologic examination, whether these tissues are indices normal (histologic) or abnormal HORMONAL INDEX / HORMONAL STATUS (histopathologic). - These include the preparation and (HI) examination of smears, the preservation and - ratio between gonadotrophic hormone of a processing of tissues, cutting and staining; person. prior to tissue detail evaluation. - in male – testosterone/ androgen - in female – estrogen / progesterone SIGNIFICANCE OF HORMONAL INDEX CYTOLOGIC TECHNIQUES 1. to determine if the reproductive system - preparation of cells in form of smears for is functioning. microscopic diagnosis. 2. to determine the fertility of sterility. - in relation with cytology (study of normal 3. to diagnose tumor especially cancer. cells) and cytopathology (study of abnormal cells) LABORATORY DIAGNOSIS OF HI: 1. Smear preparation EXFOLIATIVE CYTOLOGY 2. Direct microscopic examination - study of cells that are removed riff horn different parts of the body which includes the RESULT OF LABORATORY DIAGNOSIS OF HI: following: 1. Formation of palm-leaf or ferning crystal 1. lining epithelium is a sure sign of fertility 2. mucosal surfaces  Estrogen,  Progesterone = Fertility 3. fluid aspirates 2. Absence of fern Examples:  Estrogen,  Progesterone = Sterility female scrapings – vagina cervix, uterus male – prostatic tissue CANCER IN HI: respiratory organ – sputum and bronchial - There is persistent high level of hormone washing (estrogen/progesterone) when examined GIT – gastric and duodenal fluid for 28 consecutive days; same case as in Bone marrow male. CSF Body cavity - transudate and exudates Skin scrapings RAMOS, A.B Page 1 of 20  HISTO-CYTOTECH – CYTOLOGIC INDICES IMPORTANT LABORATORY METHODS IN MATURATION INDEX (MI) CYTOLOGY: - comparison between the number of 1. Papanicolaou/Pap’s smear - routine immature and mature cells and cytologic procedure and most common determines the location and extend of method used in cytology. the disease in the body. PAP’S STAINING PROCEDURE: SIGNIFICANCE OF MI: Wash the fixed smear in water 1. Increase in cases of cancer Dip in descending ethyl alcohol – done for 2. Increase in cases of abnormal hormone rehydration Example: 95% EA, 70% EA, 50% EA PYKNOTIC INDEX (PI Wash again with water - Percentage of cells with small, shrunken, Dip in Harris Hematoxylin for 3-5mins – structureless nuclei. serve as the primary or basic stain SIGNIFICANCE OF PI: Wash with water thoroughly (usually 3 1. Increase in cases of cancer changes) 2. Increases in cases of neurosis on Dip in Acid alcohol and wash with water degeneration immediately – serve as the decolorizer BASOPHILIC INDEX/ACIDOPHILIC INDEX (AI) Dip in Ammonia Water 3x and wash with - Percentage of cell staining capacity. water immediately SIGNIFICANCE OF AI: Dip in ascending ethyl alcohol – for 1. Normal staining of cytoplasm is acidic dehydration (pinkish) Dip in OG6 for 2 mins – serve as 2. Normal staining of nucleus is basic intensifier (blueish) Wash by means of ethyl alcohol VAGINAL INDEX (VI) Dip in EA50 for 3-5 mins – serve as the - comparison of different types of cells. acidic counterstain TYPES OF CELLS ARE: Wash with ethyl alcohol thoroughly 1. Mature superficial cells Dip in xylol for few minutes – serve as a 2. Intermediate cells clearing agent 3. Parabasal cells 4. Basal cells 2. Cell Block Method – combination of cyto- technique and histopathologic technique. SIGNIFICANCE OF VI: 3. Millipore Filter Technique – same 1. To determine the location and extend of method or procedure as in Pap’s smear disease but differs in sedimentation, instead it 2. To differentiate cancer from infection uses a special filter apparatus for 3. To detect pregnancy ultrafiltration for collecting sediments 4. Pre-cone or Mini-cone - a special procedure which is done by a direct biopsy with a pre-cone apparatus RAMOS, A.B Page 2 of 20  HISTO-CYTOTECH – to collect the specimen, this is well fitted to the female cervix. 8. Skin - Its purpose is to screen the possibility of a. By simple scraping cancer of the cervix. - If in this method no cancer cells are seen, CYTOLOGIC PROCEDURE: an alternative method is Pap’s smear I. SPECIMEN SMEAR PREPARATION - If it is still negative to Pap’s smear the last - Make use of an adhesive resort is surgical biopsy which is the II. FIXATION punch biopsy. - Preservation of cell integrity - PUNCH BIOPSY – a special biopsy method - To prevent distortion and destruction for cervix. Said to be the best method of of specimen collection. REQUIREMENTS FOR FIXATION OF SPECIMEN: 1. Fixative of choice must be previously COLLECTION OF SPECIMENS: prepared 1. Female specimen scrapings are collected 2. Place in a coupling jar with cover or in two ways: container with cover to prevent a. By simple vaginal aspiration evaporation b. By simple curettage 3. Volume requirements is 1:1 that means, 1 2. Male specimen part of specimen to 1 part of fixative a. Prostatic massage 3. Bronchial washing CHOICE OF FIXATION FOR CYTOLOGY a. By aspiration of fluid known as 1. Ether-alcohol mixture – the primary Bronchoscopy and the apparatus reagent is alcohol. used is Bronchoscope - The secondary reagents are ether, 4. Gastric/Duodenal Fluid acetone, chloroform, glacial acetic a. By nasogastric intubation acid - Purpose is to detect GIT cancer 2. 95% ethyl alcohol - If the test for reaction is 3. Carney’s fluid – contains absolute o Acidic – it’s from gastric alcohol, chloroform and glacial acetic acid o Basic – it’s from duodenal 5. Bone marrow EXAMPLES OF FIXATIVE FOR SUCH SPECIMEN: a. Puncture is done commonly in 1. 50% Alcohol – for pleural and peritoneal ilium by using a needle fluid 6. Cerebrospinal Fluid 2. 70% Alcohol – for sputum a. By lumbar tap 3. 95% Alcohol – for urine, gastric aspirates, 7. Transudates and Exudates – needle CSF puncture aspiration can be from: a. Lungs – pleural fluid III. STAINING b. Heart – pericardial fluid - Reaction between dye and the cell to c. Abdomen – ascetic fluid produce the desired color. RAMOS, A.B Page 3 of 20  HISTO-CYTOTECH – OTHER STAINING METHODS: CLASS III – doubtful cancer; cells are either a 1. Modification of H & E – shorter typical or cancer cells; it ranges from (+) or (-) procedure than Pap’s stain 50%. 2. Polychrome Methylene Blue Stain CLASS IV – True or definite cancer; it ranges from 3. Beef and Pundell – for identification of 51-75%. microorganisms in smear (like in vaginal CLASS V – metastatic cancer; plenty of cancer smear and sputum) cells that ranges from 76-100%. 4. Cresyl Violet Method 5. Periodic Acid Schiff Staining (PAS) CLINICAL REPORT: 6. Feulgen reaction STAGE I – organs 7. Wet Film Method – utilizing supravital STAGE II – surrounding tissues stain STAGE III – blood vessel and lymphatic vessels 8. Giemsa stain – for staining blood STAGE IV – spread to distant organs 9. Immunofluorescent – for cancer, enzymes and hormones. PROCEDURE USED IN CYTOLOGIC - Principles involved is antigen- EXAMINATION: antibody reaction. OLD PROCEDURE: FLUOROCHROME DYES: 1. Simple Microscopy a. Rhodamine dye – Red a. Interference microscopy b. Fluorescein – variety of yellow b. Phase contrast microscopy c. Acridine – pinkish c. Dark field microscopy – identify IV. MOUNTING certain organism - Placing of cover glass on the slide plus the d. Cytochemistry – study of use of a medium for permanent important elements adhesion. e. Electron microscopy – identify Types of Mountant: details of cell Temp or Wet – uses glycerine-jelly f. X-ray – uses gamma radiation Pern or Dry – uses Canada Balsam V. LABELING RECENT PROCEDURE: 1. RAS (Radio Active Scan) – ex. Brain scan, VI. READING AND INTERPRETATION liver scan, bone scan - Scanning of smears and determine the 2. UST (Ultrasound Technique) – for percentage of normal and abnormal cells. reproductive organ PAP’S CYTOLOGIC REPORT: (REPORT FOR 3. RIA (Radio Immune Assay) – for CYTOLOGIC SMEARS) enzymes, hormones, drugs CLASS I — normal cytology; absence of abnormal 4. IF (Immunofluorescence) – uses special cells rind if present it only ranges from 0-25%. stain CLASS II – a typical cytology but no evidence of malignancy; a typical cell ranges from 26-29%. RAMOS, A.B Page 4 of 20  HISTO-CYTOTECH – HISTOPATHOLOGIC TECHNIQUE slide and spread in such a manner; this is - deals with the preparation of solid tissue most commonly used in cytological coming from different specimen from a examination. living or dead body for microscopic a. Streaking — with the aid of an examination. applicator sick or wire loop, the specimen is applied in a zigzag SOURCE OF SPECIMEN manner. 1. Surgical specimen – specimen obtained b. Spreading - a selected portion of after biopsy or operation from live person the specimen is placed to a slide 2. Autopsy specimen – specimen obtained and spread out by teasing mucus from dead bodies after post-mortem strands apart with an applicator examination. stick. c. Pull-Apart — a drop of sediment is HISTOPATHOLOGIC PROCEDURE placed on a slide and covered with I. IDENTIFICATION OF SPECIMENT another slide to let the sediments dispersed, then the two slides are a. Checking the label and content of the pulled apart on opposite direction. specimen d. Touch Preparation (Impression b. Classify to what type of specimen they smear) — a special method belong whereby the surface of a cut piece c. Check the data about the specimen and of tissue is brought into contact record it and pressed unto the surface of a clean slide. METHODS OF FRESH TISSUE EXAMINATION: e. Frozen Section — normally utilized 1. Teasing Or Dissociation — a process when rapid diagnosis of the tissue whereby a selected tissue specimen is in question is required. immersed in a watch glass containing NSS, carefully dissected or separated and - cut at around 10-15u in examined either unstained with the aid of thickness which is kept at a Phase Contrast or Bright Field Microscopy cold chamber on a microtome and stained with differential dyes. with CO2 or Cryostat with an atmospheric temperature of — 2. Squash separation (Crushing) — a l0°C to —20°C. process whereby small pieces of tissues, II. PROCESSING OF TISSUES not more than 1 mm. in diameter are A. FIXATION placed in a microscopic slide and forcibly - procedure adopted to kill, harden and compressed with another slide or with a preserve tissues for microscopic cover glass, or use vital stain placed at examination. the junction of slide and cover glass. - tissues are preserved to prevent: o necrosis — cellular destruction 3. Smear preparation — process where o putrefaction — formation of foul sections or sediments are placed on a odor RAMOS, A.B Page 5 of 20  HISTO-CYTOTECH – o autolysis — action of proteolytic - it preserves plasma protein and enzymes on dead tissues recommended for enzyme o decomposition — action of histochemistry. saprophytic bacteria on dead - Less irritating to nose and does not tissues cause dermatitis. 2. Metallic Fixative CHARACTERISTICS OF A GOOD FIXATIVE: a.Mercuric Chloride - frequently used in 1. It must be cheap saturated aqueous solutions of 5-7%. 2. It must be stable preserves renal tissues, fibrin, 3. It must be safe to handle connective tissues and muscles 4. It must be inhibiting bacterial decomposition and autolysis b. Chromate fixatives: 5. It must produce minimum shrinkage of ▪ Potassium dichromate — used in 3% tissue concentration; it preserves lipids and 6. It must be isotonic mitochondria. ▪ Chronic acid — used in 1-2 % TYPES OF FIXATIVES: concentration; it preserves I. ACCORDING TO COMPOSITIONS: carbohydrates and is a strong oxidizing A. SIMPLE FIXATIVES – made up of only one agent. component substance. c. Lead Fixatives – used in 4% 1. Aldehydes concentration of basic Lead Acetate a.Formaldehyde – gas produced by recommended for acid oxidation of methyl alcohol, pure stock mucopolysaccharides and connective solution is usually 40% and must be tissue mucin. diluted with water. - it preserves fat, mucin, glycogen, 3. Picric Acid Fixatives nervous tissue. ▪ Bouin’s solution – for fixing embryos o 10% Formol Saline – made up of saturated ▪ Brasil’s Alcoholic picro-formol fixative Formaldehyde and 10% NaCl; it preserves central nervous tissues and post-mortem 4. Glacial Acetic Acid – it solidifies at 17’C; tissues (like cadaver). it fixes and precipitates nucleoproteins. o 10% Buffered Neutral Formalin (BNF) — it is 5. Alcohol Fixatives used to preserve surgical, post-mortem, o Methyl Alcohol (100%) – for fixing blood elastic fibers, tissues containing iron and bone marrow smear pigments and research specimens. o Ethyl Alcohol (70-100%) o Formol-Corrosive (Formol-Mercuric Chloride o Carnoy’s fluid – for fixing chromosomes, Solution) – it preserves blood cells, neutral lymph glands and urgent biopsies fats and phospholipids. o Gendres fixative (Alcoholic Formalin) – b. Glutaraldehyde – utilize for preserves glycogen and coagulates routine Light Microscopy and Electron mucus in sputum. Microscopy. o Newcomers’ fluid – nuclear proteins RAMOS, A.B Page 6 of 20  HISTO-CYTOTECH – 6. Osmium Tetroxide (Osmic Acid) – it b. Cytoplasmic Fixatives – those that preserves myelin and peripheral nerves. preserves cytoplasmic structures in o Flemming’s solution – most common particular. - They must never contain glacial HAC chrome-osmium acetic acid fixative which destroys mitochondria and Golgi used. bodies. o Flemming’s solution without HAC – - With pH of more than 4.6 made up of chromic and osmic acid and o Flemming’s fluid without Acetic used in fixing mitochondria. Acid 7. Acetone – used at iced cold temperature o Helly’s fluid from -5°C to 4°C; for fixing brain tissues for o Formalin with “Post-Chroming” – diagnosis of Rabies. secondary fixation where a 8. Heat Fixation – involves thermal primarily fixed tissue is placed in coagulation of tissues proteins for rapid solution of 2.5-3% potassium diagnosis, usually employed for Frozen dichromate for 24 hours and act Section and preparation of bacteriologic as mordant for better staining. smears. o Regaud’s fluid (Moller’s fluid) B. COMPOUND FIXATIVES – made up of two o Orth’s fluid or more fixatives which have been added 3. Histochemical Fixatives – those which together to obtain the optimal combined preserve the chemical constituents of effect of their individual actions on cells. cells and tissues. 10% Formol Saline II. ACCORDING TO ACTION: Absolute Ethyl Alcohol 1. Microanatomical Fixatives — those which Acetone permit the general microscopic study of Newcomer’s Fluid tissue structures. a. 10% Formol Saline DECALCIFICATION b. 10% Buffered Neutral Formalin - procedure where Calcium or Lime salts c. Heidenhain’s Susa - recommended for are removed from tissues following rumor biopsies specially skin and an fixation. excellent cytologic fixative - selected pieces of tissues taken from d. Formol Sublimate (Formol Corrosive) specimens like bones, teeth and calcified e. Zenker’s solution tissues are being subjected to this f. Zenker-Formol (Helly’s Solution) — method. preservative for pituitary gland bone - a good decalcifying agent must be marrow and blood containing organs capable of completely removing Calcium like spleen salt from tissues without destruction of g. Bourn’s Solution cells and tissue components. h. Brasil’s Solution I. ACID DECALCIFYING AGENT 2. Cytological Fixatives – those which - most widely used decalcifying agent for preserve specific parts and particular routine decalcification of large amounts elements of the cell itself. of bony tissues. a. Nuclear Fixatives – those which - they are stable, readily available and preserve the nuclear structure like inexpensive as compared to another chromosomes. decalcifying agent. RAMOS, A.B Page 7 of 20  HISTO-CYTOTECH – - Usual or ideal time required for recommended for detailed microscopic decalcification is 24-48 hrs. except in studies. dense bone tissues which requires more - Decalcification takes 1-3 weeks, the days to complete the process. solution is changed every 3 days, and in the final stage, every day. A. Nitric Acid – most common decalcifying agent and acts rapidly. III. ION EXCHANGE RESIN (AMMONIUM FORM o 10% Aqueous Nitric Acid solution - OF POLYSTRENE RESIN) decalcification time: 12-24 hrs. - hastens decalcification by removing o Formol- Nitric Acid- decalcification Calcium ions from Formic-Acid containing time: 1-3 days decalcifying solution, thereby increasing o Perenyi's Fluid - decalcification time: solubility from the tissue. 2-7 days - not recommended for fluids containing o Phloroglucin-Nitric Acid - mineral acids like Nitric Acid and decalcification time: 12-24 hrs Hydrochloric Acid. B. Hydrochloric Acid – slower action than Nitric Acid and produce greater distortion ***PROCEDURE OF DECALCIFICATION*** of tissue C. Formic Acid – moderate acting IV. ELECTROPHORESIS (ELECTRIC IONIZATION) decalcifying agent produce better - process where positively charged Calcium decalcification, decalcification time: 2-7 ions are attracted to a negative electrode days and removed from the decalcifying Nuclear staining solution. 1 Formic Acid-Sodium Citrate Solution - - same principle as in chelating agent but decalcification time: 3-14 days. differs in the utilization of electricity and D. Trichloroacetic Acid – decalcification time: dependent upon the supply of direct 4-8 days current to remove Calcium salt. E. Sulfurous Acid – very weak decalcifying - solution used for electrolytic agent, suitable only for a minute pieces of decalcification is a mixture of 88% Formic bones Acid, conc. HCL and distilled water F. Chromic Acid (Flemming's Fluid) – recommended for minute bone spicules WAYS IN MEASURING THE EXTENT OF G. Citric Acid-Citrate Buffer Solution (pH 4.5) DECALCIFICATION – permits excellent nuclear and 1. Physical or Mechanical Test cytoplasmic staining and does not produce - done by touching or bending the tissue cell or tissue distortion but its action is too with fingers to determine the consistency slow for routine purposes. of tissue. H. Von Ebner's Fluid - an alternative method of evaluating tissue is by mechanically whereby a II. CHELATING AGENT needle or a probe is used to prick the - substances which combine with Calcium tissue, but this method may produce ions and other salts like Iron and needle tract artifacts and destroy Magnesium deposits to form a weakly important cellular details. dissociated complexes and facilitate the 2. X-ray or Radiological Method removal of Calcium salt. - very expensive but most ideal and ▪ EDTA – most common chelating agent reliable method due to its ability to with a commercial name of Versene and RAMOS, A.B Page 8 of 20  HISTO-CYTOTECH – detect even the smallest focus of Calcium B. DEHYDRATION which appears opaque in X- may plate. - process of removing intercellular and - not recommended for Mercuric Chloride extra cellular water from tissue. fixed tissue due to characteristics radio - solutions utilized to make this possible is opacity that interfere in correct called dehydrating agents. interpretation of plate. COMMONLY USED DEHYDRATING AGENTS 3. Chemical Method 1. Alcohol – most commonly used - simple, reliable and convenient method - tissue is passed thru a series of for routine purposes, which detect the progressively increasing concentration of presence of Calcium in decalcifying Alcohol. solution. a. Ethyl alcohol - is the alcohol - utilizes the used decalcifying fluid and recommended for routine dehydration of done every 24-48 hrs. tissue. - when using this method, the decalcifying - considered to be the best, fast acting, not agent should be prepared in distilled expensive, not poisonous agent which water, because Calcium ions may cause mixes water and penetrates tissue easily false positive result which is present in b. Methyl alcohol - toxic dehydrating agent, tap water. used for blood and tissue films and smear ***PROCEDURE OF CHEMICAL METHOD*** preparation. c. Butyl alcohol - use in plant and animal SOFTENING OF TISSUES micro technique. - Performed when hard tissues are to be processed aside from decalcification. 2. Acetone – cheap, rapid acting dehydrating agent and use for urgent biopsies which TISSUE SOFTENERS dehydrates in 4 to 2 hrs. 1. Perenyi's fluid – act both as decalcifying - limited to small pieces of tissue due to agent and tissue softener. extreme volatility and inflammability done by immersing the hard tissue in fluid for 12-24 hrs or the cut surface of 3. Dioxane (Diethylene Dioxide) – excellent the block may be tampered in the fluid dehydrating and clearing agent which is for 1-2 hrs before sectioning. miscible in water, melted paraffin, alcohol and 2. Phenol solution – wash the immersed xylol. fixed tissue and place in the solution for - tissues can be left in this reagent for long 1-3 days. periods of time without affecting the 3. Molliflex – tissue immersed in this consistency or staining properties of the solution may appear swollen and soapy specimen. but it does not affect the processing and - it is expensive and dangerous for its high staining of tissue section. toxic action to man that it should not be used 4. 2% Hydrochloric Acid routinely 5. 1% Hydrochloric Acid in 70% Alcohol 4. Cellosolve (Ethylene Glycol Monoethyl Ether) – dehydrates rapidly and is not harmful to tissue. - tissues can be transferred directly to cellosolve from water or NSS and stored for RAMOS, A.B Page 9 of 20  HISTO-CYTOTECH – months without causing hardening or smooth muscles and skin which distortion requires 2 changes of clearing solution. Clearing time: 2 – 3 days. 5. Triethyl Phosphate – it removes water 6. Aniline Oil – not normally used as a routine rapidly and produces very little distortion and clearing agent but is recommended for hardening of tissue it is soluble in alcohol, clearing embryos, insects and delicate water, ether, benzene, chloroform, acetone specimen due to its ability to clear 70% and xylene. alcohol. 7. Clove Oli – causes minimum shrinkage of 6. Tetrahydrofuran (THF) – can dissolve many tissues but its quality is not guaranteed due substances including fats and is miscible with to its tendency to become adulterated. lower alcohols, ether, chloroform, acetone, - tissues become brittle, aniline dyes benzene and xylene are removed, celloidin is dissolved - it is non-toxic although prolong exposure (up plus its expensiveness makes it to 6 months) may cause conjunctival unsuitable for routine purposes. irritation. 8. Carbon-Tetrachloride – its properties and disadvantages are the same as in Chloroform. C. CLEARING (DE-ALCOHOLIZATION) 9. Tetrahydrofuran (THF) – superior to ordinary dehydrating and clearing agent due to its - process where alcohol is removed from ability to perform two processes at the same tissue and replaced with a substance that time making the total processing shorter and will dissolve the way with which the allows more time for fixation. tissue is to be impregnated or a medium - Non-toxic but has an offensive odor. on which the tissue is to be mounted. COMMONLY USED CLEARING AGENTS: D. CLEARING (DE-ALCOHOLIZATION) 1. Xylene – most commonly used clearing agent - process where the clearing agent is and also for embedding and mounting completely removed from the tissue procedures. Clearing time: ½ - 1 hr. and replaced by a medium that will 2. Toluene – may be used as a substitute for completely fill all the tissue cavities Xylene or Benzene. Clearing time: 1 – 2 hrs. giving a firm consistency to the 3. Benzene – preferred in embedding process specimen for easier handling and of tissue because it penetrates and clears cutting. tissue rapidly. - - medium used to infiltrate the tissue - Excessive exposure to this reagent is usually the same medium used in may become carcinogenic to health or impregnation. may damage that bone marrow resulting in Aplastic Anemia. Clearing TYPES OF TISSUE IMPREGNATION time: 15 – 60 mins. A. Paraffin Wax Impregnation - the simplest 4. Chloroform – recommended for routine most common and best impregnating and work and for tough tissues, nervous tissues, embedding medium used for routine tissue lymph nodes and embryos. Clearing time: 6 – processes 24 hrs. 5. Cedarwood Oil – use to clear both paraffin SUBSTITUTES FOR PARAFFIN WAX and celloidin sections during embedding 1. Paraplast - a mixture of highly purified process. paraffin and synthetic plastic polymers - Recommended for CNS tissues and with a melting point of 56-57°C. cytological studies particularly the RAMOS, A.B Page 10 of 20  HISTO-CYTOTECH – - more elastic and resilient than paraffin B. Celloidin Impregnation (Collodion) - is a wax which permits large tissue blocks like purified form of nitrocellulose, soluble in bone and brain to be cut easily. many solvents a. Embeddol - a synthetic wax - suitable for specimens with large hollow substitute, similar to paraplast with cavities which tend to collapse like bones, a melting point of 56-58°. teeth and large tissues sections of the - it is less brittle and less whole embryo. compressible than paraplast. - it is supplied in thin (2%), medium (4%), b. Bioloid - a semisynthetic wax or thick (8%) solutions of cellulose recommended for embedding eyes. dissolved in equal parts of ether and c. Tissue Mat - a product of paraffin alcohol. containing rubber and with the same property as paraplast. TWO MEDTHODS OF CELLOIDIN IMPREGNATION 1. Wet Celloidin Method – recommended for 2. Ester Wax - has a lower melting point of bones, teeth, large brain section and whole 46-48°C, but is harder than paraffin. organs. - it is not soluble in water, but is soluble in **PROCEUDRE OF WET CELLOIDIN METHOD** 95% ethyl alcohol and other clearing 2. Dry Celloidin Method – preferred for agents. processing whole eye section. - impregnated tissue to this medium - procedure is similar with wet celloidin should be cut on a heavy-duty microtome method except 70% Alcohol is not used, like sliding or sledge type of microtome instead it uses Gilson's mixture made due to the relative hardness of the wax. from equal parts of chloroform and cedarwood oil and added to celloidin 3. Water Soluble Waxes - are mostly block before hardening. Polyethylene Glycols with melting points of 38-42°C or 45-56°C NITROCELLULOSE METHOD/LOW VISCOSITY a. Carbowax – most commonly used NITROCELLULOSE (L.V.N) Polyethylene Glycol - another form of celloidin which is - it does not remove fats and lipids. soluble in equal concentration of - suitable for enzyme histochemical ether and alcohol which allows to be studies due to less exposure to too used in higher concentration and still much heat. - care must be taken to avoid contact penetrate tissues rapidly. of block to water or ice specially - it forms a harder tissue block and tissue sections in floatation bath makes cutting of thinner tissue due to its solubility to water adding sections possible. soap to water or using 10% - it is more explosive than celloidin and Polyethylene Glycol 900 in water, should be handled carefully that reduces tissue distortion, promotes container should be kept tightly flattening and floating out of covered, protect from sunlight and sections. avoid evaporation of alcohol. - when dry, striking or dropping the container will cause the substance to explode it is usually marketed while wet with alcohol since material is RAMOS, A.B Page 11 of 20  HISTO-CYTOTECH – increasingly dangerous as the alcohol E. EMBEDDING (CASTING OR BLOCKING) continuous to evaporate. - process by which the impregnated tissue is placed into a precisely arranged C. Gelatin Impregnation - rarely used except position in a mold containing a medium when dehydration is to be avoided and when and is allowed to solidify. tissues are subjected to histochemical and - -tissues are arranged at the bottom of the enzyme studies. mold together with the paraffin and - used as an embedding medium for proper label and is cooled rapidly in a delicate specimens and frozen section refrigerator at -5°C or immersed in cold because it prevents fragmentation of water to solidify. tough and friable tissues. - the process by which tissue is arranged in - tissues cut should not be more than 2-3 a precise position in the mold during mm thick since they are harder to freeze. embedding is known as Orientation. THREE WAYS WHICH PARAFFIN WAX TYPES OF BLOCKING-OUT METHODS IMPREGNATION AND EMBEDDING OF TISSUES IS 1. Leuckhart’s Embedding Mold – consist of two PERFORMED L-shaped strips of heavy brass or metal 1. By Manual Processing - requires at least four arranged on a flat metal plate which can be changes of wax at 15 mins. Interval to ensure moved to adjust the size of the mold to the size complete removal of clearing agent from the of specimen. tissue. 2. Compound Embedding Unit – made up of a - specimen is then immersed in fresh series of interlocking plates resting on a flag solution of melted paraffin for 3 hrs. to metal base, forming several compartments. ensue complete embedding or casting of 3. Plastic Embedding Rings and Base Mold – tissue. consist of a special stainless steel base mold fitted with a plastic embedding ring which later 2. By Automatic Processing – it uses an serves as the block holder during cutting. automatic tissue processing machine like the 4. Disposable Embedding Mold Autotechnicon which fixes, dehydrates, a. Peel-Away – disposable thin plastic clears and infiltrates tissues which decreases embedding molds that are simply peeled the time and labor needed for tissue off one at a time, as soon as the wax has processing. solidified. It can be placed directly in the - only 2-3 changes of wax are required to chuck or block holder of the microtome. remove the clearing agent and properly b. Plastic Ice Trays – those are used in impregnate the specimen du to constant ordinary refrigerators and is tissue agitation which accelerates tissue recommended for busy routine penetration. laboratories. Tissue block can be removed by bending the plastic tray 3. By Vacuum Embedding - involves the wax once the wax solidified or by smearing impregnation under negative atmospheric the inner mold with glycerin or liquid pressure inside an embedding oven to hasten paraffin before embedding. removal of air bubbles and clearing agent c. Paper Boats – normally utilized for from tissue block promoting rapid. embedding celloidin blocks but are - tissue is not over-exposed to heat, equally useful for paraffin wax. They are brittleness, shrinkage and hardening. cheap and easy to make. RAMOS, A.B Page 12 of 20  HISTO-CYTOTECH – OTHER EMEDDING METHOD USED TWO STAGES OF SHARPENING OF KNIFE 1. Celloidin or Nitrocellulose Method – 1. Honing (Hard Sharpening) – involves the recommended for embedding hard tissues removal of gross nicks on knife edge (Coarse and large sections of whole organs like eyes. Honing) to remove blemishes and grinding - tissues are embedded in shallow tins or the cutting edge of knife on a stone (Honing enamel pans and covered by sheets of Proper) to acquire an even edge. weighted glass or bell bars to control the - makes use of hone or hard grinding surface rate of evaporation. (Carborundum) to remove nicks and irregularities on knife edge. 2. Double-Embedding Method – process in - the motion of sharpening is edge first, which tissues are first infiltrated with with a heel to toe direction. celloidin and followed by embedding to a paraffin mass. TYPES OF HONES USED a. Belgium Yellow – for manual sharpening F. TRIMMING when cutting edge has been nicked, this - when solidified paraffin block is removed type gives the best result. from mold, the excess wax is cut off from b. Arkansas – gives more polishing effect the block to expose the tissue surface in than Belgium Yellow. preparation for actual cutting. c. Fine Carborundum – much coarser than Belgium Yellow and Arkansas and is used TYPES OF MICROTOME KNIVES: only for badly nicked knives followed by 1. Plane-Concave Knife – usually 25 mm in either any of the first two knife length sharpeners. - one side of the knife is flat while the other is concave. 2. Stropping – involves the removal of "burr" or - usually, concave side type of knife is used irregularities that have been formed during to cut paraffin sections on base-sledge, honing and is the final polishing of knife edge rotary or rocking microtome. - also applied when knife becomes dull and blunt but is free from nicks or teeth 2. Biconcave Knife – usually 120 mm. in makes use of a paddle strop made up of length. the best quality horse leather, firmly - with both sides concave, recommended attached to a solid back to prevent for cutting paraffin embedded sections sagging. on rotary microtome. - the motion of sharpening is edge last, with a toe to heel direction. 3. Plane-Wedge Knife – usually 100 mm. in length. G. SECTION-CUTTING - have both sides straight, recommended - process whereby tissues are cut into for frozen hard and tough specimens in uniformly thin slices or "sections" with paraffin blocks, using a base-sledge or the aid of a machine. sliding microtome. - the machine or instrument used for cutting sections is known as microtome. Bevel Angle – is the angle formed between the cutting edge and the sharpener or the block. normally about 27° to 32°. RAMOS, A.B Page 13 of 20  HISTO-CYTOTECH – ESSENTIAL PARTS OF A MICROTOME D. Freezing Microtome - for cutting 1. Block Holder – where tissue is held in unembedded frozen sections this was position. invented by Queckett in 1848. 2. Knife Carrier and knife – actual cutting of - use to cut undehydrated tissues in frozen tissue sections. state, especially when rapid diagnosis is 3. Pawl, Ratchet Feed Wheel and Adjustment required. Screws – to line up the tissue block in proper position with the knife. E. Cryostat or Cold Microtome – apparatus used in fresh tissue microtomy capable of KINDS OF MICROTOME freezing the tissue into the block holder to A. Rocking Microtome – for cutting serial correct degree of hardness. sections of large blocks of paraffin embedded - kept in cold chamber at a temperature tissues. between 5°C to 30°C - it was invented by Paldwell Trefall in 1881, and is said to be the simplest microtome F. Ultrathin Microtome – for cutting sections among its type. for Electron Microscopy. - knife used for cutting ultrathin section B. Rotary Microtome – for cutting paraffin consists of selected fragments of broken embedded sections. plate glass. - it was invented by Minot in 1885-1886, and is - specimen used is small, fixed in Osmium said to be the most common type use for Tetroxide and embedded in plastic. both routine and research laboratories at present. SECTION CUTTING PROPER: 1. Serial cutting and Ribbon formation. C. Sliding Microtome – for cutting celloidin embedded sections. 2. Floating out - sections are floated out on a - this was developed by Adams in 1789. water bath or floater bath with temperature TWO TYPES OF SLIDING MICROTOME lower than the melting point of wax. 1. Base-Sledge Microtome – suited for sectioning specimens embedded in all 3. Use of adhesives - mixture of solution or forms of media, especially for cutting reagent for attachment of ribbon to the slide sections from tough tissue blocks. COMMON ADHESIVES USED - the block holder is raised towards the a. Mayer's Egg Albumin knife at a thickness predetermined by a b. Dried Albumin micrometer gauge upon turning the c. Gelatin operating handle. d. Starch paste - more stable than the ordinary sliding e. Plasma microtome. 2. Standard Sliding Microtome – different 4. Fishing out - transferring of ribbon from the from the base-sledge microtome because floater bath to slide by rapid using a Camel the block remains stationary while the brush or applicator stick movement knife is moved backward and forward during the process of sectioning. 5. Orientation - correct positioning of ribbon on the slide 6. Deparaffinization-removal of paraffin wax on the slide, so that the tissue will remain only - makes use of an alcohol lamp or oven RAMOS, A.B Page 14 of 20  HISTO-CYTOTECH – H. STAINING 3. Progressive Staining - process whereby - application of dye to the tissue in order tissue elements are stained in a definite to produce a chemical reaction, affinity sequence at a given time. for better contact and differentiation for batter microscopic visualization. 4. Regressive staining - the tissue is first overstained and excess stain is removed or CATEGORIES OF BIOLOGICAL STAINS: decolorized from unwanted parts of the 1. Natural dyes - those obtained from plants tissue. and animals. 5. Metachromatic Staining - use of specific dyes a. Hematoxylin - derived by extraction of which differentiate particular substances by heartwood of a Mexican tree known as staining with a color that is different from "Hematoxylin Campechianum". that of the stain itself. b. Cochineal dyes and its derivatives - - tissue components combine with these dyes derived from extract of a female to form a different color from the Cochineal Bug (Coccus Cacti) and surrounding tissue. treated with alum to produce the dye, - metachromatic dyes are basic dyes belonging carmine. to the Thizine and Triphenylmethane groups c. Orcein - derived from vegetable dye like. extracted from certain Lichens. Methyl Violet or Crystal Violet Cresyl Blue (for reticulocytes) Safranin 2. Synthetic dyes - also known as "Coal Tar Bismarck Brown Basic Fuschin Dyes", they have been taken from Coal Tar Methylene Blue Thionine which is derived from hydrocarbon Benzene Toluidine Blue (C6H6) known as Aniline Dyes. Azure A, B, C GENERAL METHODS OF STAINING 6. Counterstaining - application of a different 1. Direct Staining - process by which sections color or stain to provide contrast and are stained with simple aqueous alcoholic background to the staining of the structural solutions of dye. components to be demonstrated. - examples are Methylene Blue and Eosin. COMMON COUNTERSTAIN USED IN THE LABORATORY: A. Cytoplasmic Stains 2. Indirect Staining - makes use of a mordant as Red well as accentuator. o Eosin Y - the mordant serve as a link to make staining o Eosin B reaction possible, examples of these are o Phloxine B Potassium Alum in Erlich's Hematoxylin and o Rose Bongal iron in Wiegert’s Hematoxylin. Yellow - the accentuator does not participate in o Picric Acid staining reaction but it hastens the speed of o Orange G staining reaction by increasing the staining Green power of the dye. examples are Potassium o Light Green SF Hydroxide in Loeffler's Methylene Blue and o Lissamine Green Phenol in Carbol Thionine and Carbol Fuchsin. RAMOS, A.B Page 15 of 20  HISTO-CYTOTECH – B. Nuclear Stains - thin slices of tissues are placed in staining Red dishes and enough staining solution is added o Neutral Red to cover the tissue. o Safranin O o Carmine COMMON DYES USED Blue a. Neutral Red - best vital dye o Methylene Blue b. Janus Green - recommended for o Toluidine Blue mitochondria o Celestine Blue c. Trypan Blue o Hematoxylin d. Nile Blue e. Thionine 7. Microanatomical Staining - use to f. Toluidine Blue demonstrate the general relationship of tissues and cells with general differentiation Metallic Impregnation - process where specific of nucleus and cytoplasm. tissue elements are demonstrated, not by stains a. Cytoplasmic Staining - demonstrate but by colorless solutions of metallic salts which minute specific structures found in are reduced by the tissue, producing black the cytoplasm and nucleus without deposit on the surface of tissue or bacteria. differentiating tissue structures. b. Negative Staining - use to H&E STAINING PROCEDURE: demonstrate bacterial morphology 1. Deparaffinize the cut tissue section in Xylene the substance and the organism are for 5-10 mins. mixed on a slide and examined 2. Dip in 95% Ethyl Alcohol for 5-10 times. microscopically, the unstained 3. Wash with water (make 3 changes of water) organism in a black background (Dark 4. Immerse in Hematoxylin for 5-10 mins. field Microscopy). 5. Wash with water (make 3 changes of water) 6. Make a quick dip in Acid Alcohol and 8. Vital Staining - selective staining of living cell immediately rinsed with water. constituents, demonstrating cytoplasmic 7. Immersed in Eosin solution for 3 mins. structures by phagocytosis of the dye particle 8. Rinse in 2 coupling jars of 95% Ethyl Alcohol (Cytoplasmic Phagocytosis). by several dipping. - the nucleus of a living cell is resistant to vital 9. Clean the excess stain and tissue marks, then stains, and is not therefore demonstrated let it dry to be ready from the next upon staining. procedure. 9. Intravital Staining - staining of living cells is SPECIAL STAINS: done by injecting the dye into any part of the FOR CONNECTIVE TISSUES: animal body, producing specific coloration of 1. Van Gieson's Stain certain cells. 2. Masson's Trichrome Stain - common dyes used are Lithium, Carmine and 3. Mallory's Aniline Blue Stain India Ink. 4. Azocarmine Stain 5. Krajian's Aniline Blue Stain 10. Supravital Staining - use to stain living cells immediately after removal from the living FOR ELASTIC FIBERS: body. 1. Weigert's Elastic Tissue Stain 2. Taenzer-Unna Orcein Method 3. Verhoeff's Stain RAMOS, A.B Page 16 of 20  HISTO-CYTOTECH – 4. Masson's Trichrome Stain 4. Dilute Giemsa - done by overnight staining, 5. Krajian's Method especially if parasites such as Toxoplasma are suspected FOR RETICULUM FIBERS: 5. Bielschowsky's Technique 1. Bielschowsky's method 2. Periodic Acid-Leuco fuschin Technique FOR ASTROCYTES: 3. Gold Method (Lynch 1964) 1. Mallory's PTAH Stain 4. Periodic Acid Schiff (PAS) 2. Cajal's Gold Sublimate Method FOR GLYCOGEN: FOR MYELIN SHEATH: 1. PAS with Diastase Control 1. Osmic Acid Technique 2. Best Carmine Method 2. Weigert-Pal Technique 3. Langhan's Iodine Stain 3. Sudan Black Method FOR MUCIN (ACID MUCOPOLYSACCHARIDES): 1. Metachromatic Staining FOR PHOSPHOLIPID: a. Toluidine Blue and Azure A 1. Sudan Black B b. Uranyl Nitrate, Azure Method 2. Acid Hematin 3. Colloidal Iron Technique 4. Gomorri's Aldehyde Fuchsin Stain FOR GLYCOLIPID: 5. Fluorescent Acridine Orange Technique 1. Sudan Black B 2. PAS Technique FOR FIBRIN: 1. Hematoxylin and Eosin Stain FOR LIPID: 2. Mallory's Phosphotungstic Acid Hematoxylin 1. Sudan Black Method 3. Gram's Staining (Positive) 2. Sudan IV (Scharlach R) Stain 4. Periodic Acid Schiff Stain FOR FATS: FOR AMYLOID: 1. Osmic Acid Stain 1. Gram's Iodine Stain 2. Nile Blue Sulfate Method (Lorraine-Smith) 2. Krajian's Amyloid Stain 3. Congo Red Method FOR HEMOSIDERIN: 4. Metachromatic Staining 1. Perl's Prissian Blue Method 5. Induced Fluorescence Staining with 2. Turnbull's Blue Reaction Thioflavine FOR BILE PIGMENTS: FOR MUSCLES: 1. Gmelin's Test 1. Hematoxylin and Eosin Method 2. Stein's lodine Test 2. Masson's Trichrome Stain FOR MELANIN: 3. Van Gieson Stain 1. Masson Fontana Technique (Silver 4. Phosphotungstic Acid Hematoxylin (PTAH) Modification) FOR NERVE CELL BODIES OR NEURONS: FOR CALCIUM: 1. H & E Method 1. Von Kossa's Silver Nitrate Method 2. Hematoxylin and Van Gieson Staining Technique 3. Mallory's PTAH-most useful stain RAMOS, A.B Page 17 of 20  HISTO-CYTOTECH – FOR BACTERIA: DIVISIONS OF MOUNTING MEDIA: 1. Gram's Method (Jensen's Modification) A. Aqueous Media - designed to mount water- 2. Ziehl Neelsen method miscible preparations directly from water in cases where the stain is removed or FOR SPIROCHETES (TREPONEMA PALLIDUM): decolorized with Alcohol or Xylene like in 1. Rapid Giemsa Stain Frozen Section and Metachromatic Staining. 2. Levaditi's Method COMMON AQUEOUS MOUNTING MEDIA USED: FOR FUNGI: 1. Water - has low refractive index, moderately 1. Hematoxylin transparent and evaporates easily it does not 2. Gram Staining allow tissues to be examined under OIO. 3. Giemsa Stain - good only for temporary mounting. 4. Ziehl Neelsen's Method 5. Metachromasia of Capsule 2. Glycerin - it has a high refractive index and 6. Special Stains lasts for a few minutes. a. Hotchkiss-McManus PAS Technique - may also be used as a preservative b. Bauer Fuelgen Technique provides greater visibility if slightly c. Alcian Blue method diluted with water (for moist section). d. Gridley's Stain 3. Gum Arabic (Farrant's Medium) - has a refractive index of 1.43. I. MOUNTING - this medium does not solidify upon - placing of cover glass over the slide and storage and may therefore require use of a medium known as mountant to ringing. facilitate permanency of specimen. - it protects the specimen from physical 4. Apathy's Medium - has a high refractive injury such as bleaching and deterioration index of 1.52. the transparent medium should have a - use for Methylene Blue stained nerve good refractive index which is close to preparations. that of the glass and the tissue for better - the medium sets quite hard that it does viewing of microscopic detail. not require ringing. MOUNTING MEDIUM - syrupy fluid applied 5. Brun's Fluid - recommended for mounting between the section and the cover slip, setting Frozen Sections from water or paraffin the section firmly to prevent the movement of sections which require dehydration and the cover slip the refractive index should be as clearing near as that of the glass which is 1.518. 6. Karo Corn Syrup RINGING - process of sealing the margins of the cover slip to prevent escapage of fluid mounts B. Resinous Media - use for preparations that have been dehydrated and cleared in Xylene RINGING MEDIA USED: or Toluene. 1. Kronig cement - recommended for majority of staining 2. Durofix methods. RAMOS, A.B Page 18 of 20  HISTO-CYTOTECH – TYPES OF RESINOUS MEDIA: J. LABELLING 1. Natural Resins - label is placed at one margin of glass a. Canada Balsam - media that can be slide. made neutral or acid by adding excess amounts of Calcium Carbonate or LABELS USED: Salicylic Acid. 1. Gum label - its refractive index is 1.524. 2. India ink with colorless nail polish - use to - a natural resin extracted from Canadian cover the label temporarily Tree (Abus Balsamea), and is miscible in FILING OF SLIDES Xylene. - done for future study - this method is temporarily neutralized that it should be kept in dark glass bottle SPECIAL PROCESSING TECHNIQUES to prevent exposure to sunlight and color - done when histochemical evaluation, changes into brown upon storage and particularly enzyme studies is to be addition of Calcium Carbonate chips may examined. maintain its neutral reaction. - used when chemical fixation of tissue block is - recommended for whole mounts and to be avoided because once the tissue has thick sections because it does not shrink been fixed and processed, important much. chemical constituents can be removed, altered or displaced. b. Gum gummar - all the techniques have a common principle 2. Synthetic Resins- important for embedding of rapidly preserving the tissue block by decalcified bones, electron microscopy and freezing (Quenching, to produce instant light microscopy. cessation of cellular activity thereby a. DPX - has a refractive index of 1.532. preventing chemical alteration. - recommended for small tissue - freezing must be rapid to prevent formation sections but not for whole mounts of ice crystals artifacts and produce optimum because shrinkage is produced upon tissue preservation. drying that it should be used in - commonly employed freezing agent is Liquid excess amount. Nitrogen. - it is available in neutral colorless - using isopentane, Pentane, Propane and solution which dries up rapidly. Dichlorodifluoromethane which can be called to a very low temperature, retains the b. Xam - has a refractive index of 1.52. fluidity of the freezing agents. - a synthetic resin mixture in Xylene, available in a pale yellow or A. FREEZE-DRYING colorless solution. - a special way of preserving tissues by rapid - it dries quickly without retraction freezing (Quenching) and removing water and preserves stain well. (Desiccation) by physical process from the still frozen tissue block without using any c. Clarite - its refractive index is 1.544 chemical fixative. - It is diluted in 60% Xylene. - this technique is time-consuming and expensive and freeze-dried materials are d. Permount generally more difficult to section than ordinary paraffin block. ***PROCEDURE OF FREEZE-DRYING*** RAMOS, A.B Page 19 of 20  HISTO-CYTOTECH – B. FREEZE-SUBSTITUTION tissue in the body like liver, spleen, intestine - similar to freeze-drying in preparing and and others. preserving tissue blocks because both involves rapid freezing of tissues and B. AUTOPSY infiltration and embedding of frozen tissues - examination of tissues from dead bodies for are in paraffin or celloidin. microscopic study in order to determine the - the difference of this technique to freeze- cause of death drying is that it is fixed in Rossman's Fluid or TYPES OF AUTOPSIES Osmium Tetroxide in 1% Acetone for 1 to 6 1. Based on purpose - to determine the cause days at a temperature of - 60°C to -70°C, of death. instead to subjecting it to dehydration in an a. Ordinary hospital - for further study. expensive vacuum drying apparatus. b. Medico-legal - to investigate and involves - infiltration and embedding are done in the prosecution directly or indirectly. same way as in paraffin section. - this method is more economical and suitable for routine purposes. 2. Based on the technique of Pathologist a. Y-shape autopsy - for adult or old C. FRESH FROZEN TISSUE SECTIONING cadaver - this technique requires that tissue should be 3. Straight median autopsy - for small children maintained at its frozen solid state during and incision is made in the middle of the cutting of section to prevent compression body and displacement of all tissue structures as 4. Based on the request of the doctor the knife passes thru it a. Complete - for whole body - the excess of fresh tissue sectioning depends b. Partial - only certain part of the body is upon the large extent of the temperature, opened both of the tissue and the knife ***PROCEDURE OF FRESH FROZEN TISSUE REQUIREMENTS FOR AUTOPSY. SECTIONING*** 1. Written consent from the nearest 2. Clinical record of the patient SPECIAL PROCEDURES IN HISTOPATHOLOGIC 3. Place of autopsy TECHNIQUE 4. Instruments/Apparatuses A. BIOPSY 5. Pathologist and Med Tech - the prosecutors - examination of tissues from a living patient. who perform the autopsy procedure GENERAL TYPES OF BIOPSIES: 1. Aspiration Biopsy - performed for obtaining fluid specimens - applicable for cytotechnology. 2. Surgical Biopsy - performed for obtaining solid specimens. - applicable for histopath technique. TYPES OF SURGICAL BIOPSY: 1. Excision Biopsy - done to tissue coming from the surface of the body. 2. Incision Biopsy - requires operation and opens the body in order to penetrate the RAMOS, A.B Page 20 of 20