HCT Lecture Notes - Midterm - Fixation PDF

HCT LECTURE NOTES / MIDTERM u WEEK 7.1: FIXATION OSMOLALITY Prof. Jillian Audrey Angeles CONCENTRATION OVERVIEW 1. FIXATION 2. DECALCIFICATION (if spx has Calcium) 3. DEHYDRATION 4. CLEARING 5. INFILTRATION / IMPREGNATION 6. EMBEDDING ● 7. TRIMMING 8. CUTTING 9. STAINING 10. MOUNTING 11. LA...

HCT LECTURE NOTES / MIDTERM u WEEK 7.1: FIXATION OSMOLALITY Prof. Jillian Audrey Angeles CONCENTRATION OVERVIEW 1. FIXATION 2. DECALCIFICATION (if spx has Calcium) 3. DEHYDRATION 4. CLEARING 5. INFILTRATION / IMPREGNATION 6. EMBEDDING ● 7. TRIMMING 8. CUTTING 9. STAINING 10. MOUNTING 11. LABELLING DURATION OF FIXATION FIXATION - process by which the constituents of the cells and the tissues are fixed in a physical and chemical state. ○ GOAL: to preserve the morphologic appearance of the tissue. FIXATION FIXATIVE The process The chemical introduced in the tissue TIME INTERVAL PRACTICAL CONSIDERATION OF FIXATION SPEED DEFINITION AND PURPOSE ● ● ● First and most critical step ○ This is the starting point of preservation of tissue for examination. ○ If fixation is not adequate, the other processes that follow, such as dehydration, clearing, infiltration, embedding, microtomy and staining, will also be inadequate; Tissue will get brittle at the start of dehydration and the sample will change in color, meaning fixation process was not done properly. PRIMARY PURPOSE: to preserve the morphological and chemical integrity of the cell ○ Like-manner as possible kung paano ang hitsura niya inside the body ○ Pag linalabas sya, nagkakaroon ng putrefaction ○ Fixation prevents degeneration, decomposition, putrefaction, and distortion of tissues after removal from the body. ○ All vital cellular processes stop when the tissue is placed in a fixative. SECONDARY PURPOSE: to harden and protect the tissue, thus easier to cut ○ insufficient fixation = malambot, madudurog, walang makukuhang tissue sample for cutting. Pag hindi na-fix, nagiging brittle or nagiging soft ang tissue (reject sample) Correct fixative to tissue ratio Usual fixation time Usual fixation temperature (surgical specimen) Isotonic solution ● Ex: PBS (phosphate buffer saline solution) Low concentration ● Ex: 10% formaldehyde; and ● 3% glutaraldehyde 24 hours without agitation ● Depends on the fixative used, normally 24 hours for formaldehyde and formalin. ● It is important not to prolong fixation. There will be shrinkage and hardening of the specimen or tissue, so do not overfix your specimen. Fixed immediately ● After the removal of the tissue or organ from the body ● To prevent cell death, autolysis, and putrefaction PENETRATION VOLUME DURATION Specimens should be transferred to fixative quickly (<1 hour) after surgery as deterioration will commence with the loss of blood supply Normally 1 mm/hr 20:1 (optimum ratio of fixative:tissue) ● Sa ibang book, 15:1, 25:1 (tama pa rin naman) pero para hindi na malito, 20:1 na 24 hours without agitation EFFECTS OF FIXATION ● ● ● ● ● Hardens soft and friable tissues Makes cells resistant to damage and distortion Inhibit bacterial decomposition Increase optical differentiation of cells Acts as mordant or accentuator thereby facilitating staining process Reduce the risk of infection ● CHARACTERISTICS OF GOOD FIXATIVES ● ● ● ● ● Cheap Stable Safe to handle Kill the cell quickly Inhibit bacterial decomposition and autolysis ● ● ● ● Produce minimum shrinkage of tissue Rapid and even penetration of tissues Isotonic Harden tissues TYPES OF FIXATIVES 20:1 (fixative:tissue) At least 24 hours (optimum time) Stored at room temperature IMPORTANT FACTORS TO BE CONSIDERED VOLUME HYDROGEN ION CONCENTRATION TEMPERATURE THICKNESS OF SECTION Amount of fixative: 10-20 times ● The most common error in histotechnology is insufficient ratio of tissue volume to fixative volume ● Mas ok kung sobra yung fixative kaysa sa kulang, kasi may part ng organ yung hindi mappreserve kung kulang General pH of fixative: 6-8 ● For nuclear: < 4.6 ● For cytoplasmic: > 4.6 Surgical spx / routine tests: Room temperature ● Electron microscopy / histochemistry: 0-4°C ≤ 3 mm thick ● ● SIMPLE - made up of only one component substance. ○ Formaldehyde COMPOUND - two or more fixatives ○ Zenker’s solution - Has mercuric chloride and glacial acetic acid ○ Mercuric chloride causes cell/tissue shrinking, while glacial acetic acid causes cell/tissue swelling ○ These two have effects that counter each other which results to a better morphologic results BALEROS. BELOCURA. CHEUNG. S, DEL ROSARIO. FORTUNO. FREKING. GAGUI. J, GARCIA. GUINTAPAN. LEAÑO. LOVERIA. MABUNGA. PACLEB | MT’25 | A.Y. 2023 - 2024 1 ○ ○ ○ MICROANATOMICAL permit the general microscopic study of tissue structures without altering the structural pattern and normal intercellular relationship of the tissues in question. ■ 10% Formol saline ■ 10% NBF ■ Heidenhain’s Susa ■ Bouin’s ■ Formol Sublimate ■ Zenker’s solution ■ Zenker-Formol ■ Brasi’s CYTOLOGICAL ■ NUCLEAR (< 4.6 pH) - there is presence of glacial acetic acid ❖ Flemming’s ❖ Carnoy’s ❖ Bouin’s ❖ Newcomer’s ❖ Heidenhain’s SuSa ■ CYTOPLASMIC (> 4.6 pH) - no presence of glacial acetic acid because it destroys the mitochondria and golgi bodies ❖ Flemming’s Fluid w/o acetic acid ❖ Helly’s Fluid ❖ Formalin with post chroming ❖ Regaud’s (Muller’s) ❖ Orth’s HISTOCHEMICAL preserve the chemical constituents of cells and tissues; mas broad ■ ■ ■ ■ 10% Formol Saline Abs. ETOH 1. 2. 3. 4. 5. 6. 7. 8. 9. ● ● ● ● Irritating fumes prolonged fixation may bleach tissues Pure stock solution Unsatisfactory for routine fixation because it over hardens the tissue (high concentration), making the tissue difficult to cut during sectioning 10% FORMALDEHYDE ● ● Working solution Paraformaldehyde and formic acid is formed in prolonged fixation in 10% Formaldehyde (also applicable in Formalin) ○ Paraformaldehyde – white crystalline crystals / white precipitate ○ Formic Acid – reacts with hemoglobin which leads to acid formaldehyde hematin PRECAUTIONS ● ● ● ● ● Newcomer’s Fluid Paraformaldehyde formation – avoid prolonged fixation Prolonged storage may induce precipitation – filter or add 10% methanol to remove crystals/precipitate Well ventilated room – causes irritation to skin, eyes, etc. Not neutralized if concentrated – explosion Buffered or neutralized by adding magnesium carbonate/CaCO3 – wide mouth bottle Bleaching prevented by changing formalin (every 3 months) 10% NEUTRAL BUFFERED FORMALIN PROBLEM CAUSE Failure to arrest early autolysis of cells Removal if substances soluble in fixing agent Presence of artifacts pigments on tissue sections Tissues are soft and feather-like in consistency Loss or inactivation of enzymes needed for study Shrinkage and swelling of cells and tissue structure Tissue blocks are brittle and hard Failure to fix immediately; Insufficient fixative Wrong choice of fixative ● ● ● ● Incomplete washing of fixative Incomplete fixation Wrong choice of fixative Overfixation or prolonged fixation ● ● NOTE: Lipid – Frozen section Carbohydrates – Alcoholic fixative Protein – Formaldehyde or Neutral buffered formol-saline ● ● ● ● ● ALDEHYDE FIXATIVES ● According to other/old books, formaldehyde and formalin are synonyms According to new books/latest edition, formaldehyde and formalin are different especially in terms of their chemical weight/component ○ Formalin is a product from formaldehyde Gas produced by oxidation of methanol 10% recommended, buffered to 7.0 ph FORMALDEHYDE & FORMALIN Most widely used fixative for routine histology Formalin is the lower concentration of formaldehyde Recommended for the preservation and storage of surgical, post-mortem, and research specimen Fixative of choice for: ○ Paraffin embedding, ○ Immunohistochemistry ○ Fluorescent In-Situ Hybridization (FISH). Fixation time: 4-24 hours Neutral because it has a pH 7 ADVANTAGES Overfixation or prolonged fixation Satisfactory for routine paraffin sections For electron microscopy For Histochemical and enzyme studies ● ● Cheap Readily available Easy to prepare Stable Compatible w/ stains Penetrates tissues well Preserves fat Mucin Glycogen for tissue photography DISADVANTAGES FORMALDEHYDE 40% FORMALDEHYDE ● ● ● ● ● Fixation time: 24 hrs ADVANTAGES Acetone DIFFICULTIES CAUSED BY IMPROPER FIXATION ● ● ● ● Almost same with formaldehyde ● Penetrates and fixes tissues well ● Minimum shrinkage & distortion ● Does not over harden tissues Best fixative for Iron pigments and elastic fibers DISADVANTAGES Longer to prepare Time consuming Inert towards lipids 10% FORMOL SALINE ● ● ● ● ● Made up of formaldehyde diluted to 10% NaCl Recommended for CNS tissue and post-mortem tissues for histochemical exam Preservation of lipids ○ Specifically phospholipids Fixation time (from latest edition): 12-24 hours Formal Saline is a simple microanatomical fixative NOTE: From the old/2nd edition, fixation time is: ● 24 hrs: 35C BALEROS. BELOCURA. CHEUNG. S, DEL ROSARIO. FORTUNO. FREKING. GAGUI. J, GARCIA. GUINTAPAN. LEAÑO. LOVERIA. MABUNGA. PACLEB | MT’25 | A.Y. 2023 - 2024 2 ● ADVANTAGES ● ● ● ○ 24 hrs: 20-25C (room temperature) DISADVANTAGES Penetrates and fixes tissues well ● Minimum shrinkage & distortion Does not over harden tissues Slow (>24 h) FORMOL CORROSIVE (FORMAL SUBLIMATE) ● Recommended for routine post-mortem tissues ○ 10% buffered formalin, formol saline, formol corrosive – all of these can be used in post- mortem tissues ■ But for ROUTINE post-mortem tissue, Formol Corrosive is used Fixation time: 3-24 hours ● ADVANTAGES ● ● Minimum shrinkage and hardening No need for washing out from fixative to alcohol DISADVANTAGES ● ● Slow Forms mercuric chloride deposits - results to a colored fixative (normally black) UNBUFFERED ZINC FORMALIN ● ● ● Alternatives to mercuric chloride formulations Improved results with Immunohistochemistry (IHC) Fixation time: 4-8 hours ● If the washing process of a tissues with formalin is improper or insufficient, it will result to a change in color during dehydration REMOVAL OF FORMALIN PIGMENT KARDASEWITCHLS METHOD ● ● Bring down to water Place in mixture of 70% ethyl alcohol (ETOH) and 28% Ammonia water for 5 mins-3 hrs Wash with water ● ● ● ● Bring down to water Place in mixture of Acetone, H2O2, 28% Ammonia water for 1-5 mins Wash with 70% ETOH Wash with water ● ● PICRIC ACID METHOD ● ● ● Bring down to water Place in saturated alcoholic picric acid for 5 mins-2 hrs Wash in running water for 10-15 mins GLUTARALDEHYDE ● Made up of 2 formaldehyde residues linked by 3 carbon chains ● For Light Microscopy & Electron Microscopy ● 2 Concentration: ○ 2.5 % Glutaraldehyde ■ Fixation Time: 2 – 4 hours RT ■ Small tissues ■ FNAB ○ 4 % Glutaraldehyde ■ Fixation Time: 6 – 24 hours RT ■ Larger tissues HCHO - Formaldehyde ADVANTAGES vs. HCHO DISADVANTAGES vs. HCHO ● ● More stable effect Less tissue shrinkage, less irritating ● ● More expensive Slow penetration ● Mixture of paraformaldehyde and glutaraldehyde KARNOVKY’S FIXATIVE NOTE: Must be fresh when preparing this fixative, unlike Formaldehyde that you only change every 3 months Uses 4% paraformaldehyde and 1% glutaraldehyde in a 0.1M phosphate buffer ALCOHOLIC FIXATIVES ● ● ● ● Acts as both fixative and dehydrating agent Rapidly denatures and precipitates proteins NOTE: It is NOT usually used in Tissue Processing because it may cause too much brittleness and hardness of tissues Another disadvantage: polarization = movement of glycogen granules towards the end of the cells METHYL ALCOHOL ● ● ● ● Advantage - Excellent for fixing dry and wet smear Disadvantage - Slow penetration Fixation Time: 6 – 24 hours NOTE: 100% Methyl Alcohol can also be used in Bone Marrow Tissue ○ it is also used as fixation in the Hematology Section ISOPROPYL ALCOHOL ● ● ● LILIE’S METHOD ● ● Paraformaldehyde – there are white precipitate crystals / white crystalline crystals due to long storage Suitable for use when preparing samples for light microscopy in resin embedding and sectioning, and for electron microscopy Used for fixing touch preparations. Used in staining procedures using Wright Giemsa Stain ○ Combination of Romanowsky Stain NOTE: 95% Isopropyl Alcohol is normally used to prepare air dried specimen ETHYL ALCOHOL ● ● ● ● ● ● ● ● Advantage - fixes blood, tissue films and smears, It preserves but does not fix glycogen Disadvantage - strong reducing agent Fixation Time: 18 – 24 hours NOTE: Ethyl alcohol as a strong reducing agent is its disadvantage ○ Because it cannot be mixed with chromic acid, potassium dichromate— you most likely won’t be able to preserve the smears (and tissue films) well It is a commonly used cytosmear fixative It may be used as a simple fixative however it is more frequently incorporated into compound fixatives for better results Uses 70-100% ethyl alcohol, but 95% is the most commonly used alcoholic fixative 70-80% not usually used especially in blood smears, because they cause hemolysis and does not preserve leukocytes CARNOY’S FIXATIVE ● ● ● ● ● ● Advantage - most rapid fixative, fixation of chromosomes and fixation of brain tissues for the diagnosis of rabies. Following fixation for one hour, tissues may be transferred directly to absolute alcohol-chloroform mixture, thereby shortening processing time Disadvantage - RBC hemolysis, causes considerable tissue shrinkage Fixation Time: 1 – 3 hours NOTE: Brain tissues are examined for the diagnosis of rabies Components: absolute alcohol, chloroform, and glacial acetic acid GENDRE’S FIXATIVE ● Advantage: ○ Coagulation of mucus BALEROS. BELOCURA. CHEUNG. S, DEL ROSARIO. FORTUNO. FREKING. GAGUI. J, GARCIA. GUINTAPAN. LEAÑO. LOVERIA. MABUNGA. PACLEB | MT’25 | A.Y. 2023 - 2024 3 ○ ○ Fixation is faster (fixation time reduced to ½) Can be used for rapid diagnosis because it fixes and dehydrates at the same time ■ in the frozen section room Can be used to fix sputum ○ Fixation Time: 4 – 18 hours ● ● preservation and include such well-known fixatives as B-5 and Zenker's solution Produces black precipitates of mercury – removed by 0.5 % iodine solution in 70% ethanol then decolorize iodine using 5% sodium thiosulfate ADVANTAGES ● ● NOTE: Components: ethyl alcohol, formaldehyde, and glacial acetic acid (it is the routine fixative of choice) for tissue photography Permits brilliant metachromatic staining of cells DISADVANTAGES ● ● ● NEWCOMER’S FLUID ● ● Both nuclear and histochemical fixative Recommended for fixing mucopolysaccharides nuclear proteins Fixation Time: 12 – 18 hours at 3°C ● ● and ● ● Used on Frozen section and smear It can produce fair results after conventional processing if fixation time is kept very short. It preserves nucleic acids but extracts lipids. Tissues can be transferred directly into 95% ethanol. Components: ○ Ethyl alcohol ○ Glacial acetic acid Fixation Time: 3 – 4 hours ● ● ● ● ALCOHOLIC FORMALIN ● Used for fixation or post-fixation of large fatty specimens (e.g. breast) Alcoholic-formalin combines a denaturing fixative with the additive and cross-linking effects of formalin Fixation Time: 12 -24 hours ● ● FORMOL-ACETIC ACID ● ● Faster acting agent than alcoholic formalin Sometimes used to fix diagnostic cryostat sections (rotary microtome inside the chamber) ○ Frozen section is used for breast cancer (if malignant or benign) as it can produce results within 15 minutes Fixation Time: 1 – 6 hours ● ● ● NOTE: Formol-Acetic Acid is an alternative of Clarke’s solution because the frozen section is almost the same as cryostat ○ You will put it in a cold chamber with a rotary microtome and then dun pa sa loob After the Primary Fixation, the Clarke Alcoholic and Formol-acetic Alcohol can be directly put in a 95% ethyl alcohol for processing METALLIC FIXATIVES ● ● ● Best application for fixation of hematopoietic samples and reticuloendothelial tissues Excellent nuclear detail Increases the brightness of the staining MERCURIC CHLORIDE ● ● EXAMPLES OF MERCURIC CHLORIDE ZENKER’S FLUID (HgCl2 + Glacial Hac) ● NOTE: Components: isopropyl alcohol, propionic acid, petroleum, ether, acetone, and dioxin CLARKE’S SOLUTION Most common metallic fixative; 5 – 7 % Recommended for renal tissue, fibrin, connective tissues and muscles ● ● ● Mercuric chloride is widely used as a secondary fixative reacting with a number of amino acid residues and accompanied by spectroscopic changes, probably due to reaction with histidine residues. Mercuric chloride-based fixatives are used as an alternative to formaldehyde-based fixatives to overcome poor cytological Advantage: Recommended for liver, spleen, CT fibers, nuclei. Recommended for trichrome staining o Trichrome staining - usually used for connective tissue samples Disadvantage: Poor penetration Fixation Time: 12 – 24 hours ZENKER-FORMOL (HELLY’S) SOLUTION ● ● ● Advantage: Excellent micro anatomic fixative for pituitary, BM, spleen, liver. Disadvantage: brown pigment produced if fixed for more than 24 hours due to lysis of RBC – removed by saturated picric acid or NaOH Fixation Time: 4 – 24 hours HEIDENHAIN’S SUSA (HgCl2, NaCl, TCA, HCHO) ● ● ● ● ● ● Recommended for tumor biopsies (skin) Excellent cytological fixative Susa came from the two words: sublimate (su) and saure (sa), meaning. acid. Composed of mercuric chloride, sodium trichloroacetic acid, and formaldehyde. Disadvantage: black precipitate produced – immerse in alcoholic iodine Fixation Time: 3 – 12 hours LILLIE’S B-5 FIXATIVE ● ● ● ● ● ● Component: sodium acetate anhydrous and formaldehyde 4 % aqueous formaldehyde with 0.22M mercuric chloride and 0.22M acetic acid A dirty looking brown crystalline precipitate, probably mercurous chloride (Hg2Cl2) forms in all parts of tissues fixed in mixtures containing HgCl2. It is called mercury pigment and before staining must be removed by sequential treatments with iodine and sodium thiosulfate solutions. Recommended for the use of BM biopsy ○ Normal and abnormal cell types of bone marrow Fixation Time: 4 – 8 hours CHROMATE FIXATIVES 1 - 2% CHROMIC ACID ● ● Preserves carbohydrates Fixation Time: 24 – 48 hours ● Preserves ○ Lipids ○ mitochondria Fixation Time: 24 – 48 hours POTASSIUM DICHROMATE PRECAUTIONS ● Hardens outer layers only Black granular deposits formed (removed by adding iodine) Corrosive to metals (all types of metal except monel) ○ Monel – a nickel alloy that causes the tissue to shrink and lyse the RBC. It also produces black granules. ● REGARD’S / MULLER’S FLUID ● Recommended for the fixation of: ○ Chromatin BALEROS. BELOCURA. CHEUNG. S, DEL ROSARIO. FORTUNO. FREKING. GAGUI. J, GARCIA. GUINTAPAN. LEAÑO. LOVERIA. MABUNGA. PACLEB | MT’25 | A.Y. 2023 - 2024 4 ○ Golgi ○ RBC ○ Mitochondria Fixation Time: 12 – 48 hours ● GLACIAL ACETIC ACID ADVANTAGES ● ORTH’S FLUID ● Recommended for the ○ Rickettsia ○ Bacteria ○ Myelin ○ Early degenerative processes and tissue necrosis Fixation Time: 36 – 72 hours ● LEAD FIXATIVES ADVANTAGES ● ● ● For acid mucopolysaccharide (MPS) and fixes connective tissue mucin when you stain it it turns into alcian blue ● ● ● ● ● PRECAUTIONS NOTE: Insoluble lead carbonate accumulates carbon oxide because of prolonged standing, that’s why we need to either filter it or add HAc— drop by drop, until the pH lowers and eventually dissolves the residue o ○ Residue that was due to prolonged standing ADVANTAGES ● ● ● ● ● ● EXAMPLES OF PICRIC ACID Commonly used picric acid Advantage: recommended for fixation of embryos and pituitary biopsies. ○ Excellent for glycogen preservation Disadvantage: not good for kidneys, mitochondria, hemolyzes RBC Components: ○ Picric acid ○ Formaldehyde ○ Glacial acetic acid Fixation Time: 4 – 18 hours at RT ● ● ● ● NOTE: Fixation time in old/2nd edition is 6 – 24 hours ● ● ● ● Advantage: recommended for GIT specimens, endocrine tissues ○ Produces less lysis than bouin ○ Has decalcifying properties Fixation Time: 4 – 18 hours ● ● Expensive Poor penetration Reduced with sunlight – black precipitate Acid vapor - conjunctivitis, osmic oxide in cornea – blindness Inhibits hematoxylin and extremely volatile o ○ Which makes the staining difficult Most common chrome-osmium acetic acid fixative used Recommended for nuclear preparation (chromosomes) Component: ○ Aq. chromic acid ○ Aq. osmium tetraoxide ○ Glacial acetic acid Fixation Time: 24 – 48 hours FLEMMING’S SOLUTION WITHOUT ACETIC ACID ● ● ● Made up only of chromic and osmic acid Recommended for cytoplasmic structures (mitochondria) Fixation Time: 24 – 48 hours ● TRICHLOROACETIC ACID (TCA) ● ● ● ● Used for precipitation of proteins and nucleic acids Incorporated into compound fixatives Both decalcifier and fixative in microscopy addition of TCA to a final concentration of 10% (w/v) will precipitate most proteins from solution ADVANTAGES ● ● ● Advantage: better & less messy than bouin. ○ Excellent for glycogen preservation Fixation Time: 24 hours HOLLANDE’S FIXATIVE ● ● ● ● BRASIL’S ALCOHOL PICROFORMOL (W/ TCA) ● DISADVANTAGES Fixes conjugated fats and lipids Preserves cytoplasmic structures It precipitates and gels proteins. It shows uniformly granular nuclei with clear cytoplasmic background. Some tissues (e.g. adrenal glands) are better fixed in vapor form of osmium tetroxide. This eliminates "washing out" of the fixed tissues. Osmium tetroxide completely permeabilizes cell membranes. The osmolarity of the fixative vehicle or solute is relatively unimportant. FLEMMING’S SOLUTION ● ● ● BOUIN’S ● ● Used in Electron Microscopy Can be used as a fixative and heavy metal stain This is a pale yellow powder which dissolves in water up to 6% at 20C to form a strong oxidizing solution Once vapored, may irritate the eyes With direct contact, may cause blindness Should be kept in a dark colored, chemically clean bottle to prevent evaporation and reduction by sunlight or organic matter ○ Nagkakaroon ng discoloration since Osmium Tetroxide is pale yellow na dinidissolve sa water ○ Prolonged exposure may cause black precipitate due to mercuric chloride ● Prevention: add saturated aqueous mercuric chloride ● Remedy: black osmic oxide crystals may be dissolved on cold water NOTE: ‘pag may prolonged storage, may precipitation or excess discoloration Forms insoluble lead carbonate – removed by filtering or adding HAc NOTE: Excessive use of picric acid fixatives can cause yellow stains on tissues Picric acid should never be directly washed with water before dehydration. o ○ So in order to remove its yellow/excess color (which can interfere with tissue processing) follow this order: ■ Adding 70% ETOH → 5% sodium thiosulfate → running water Destroys mitochondria and Golgi bodies ● Highly explosive when dry Normally used in strong aqueous solution (1%) Yellow in color (removed by dipping 70% ETOH followed by 5% sodium thiosulfate & running water) Excellent for glycogen demonstration Do not wash before dipping into ethyl alcohol to avoid destroying the morphology of the tissue ● ● ● OSMIUM TETROXIDE (OSMIC ACID) FIXATIVES ● ● ● PICRIC ACID FIXATIVES ● ● ● Fixes nucleoproteins (chromosomes & chromatin material) Causes tissues to swell DISADVANTAGES ● DISADVANTAGES DISADVANTAGES Precipitates proteins ● Weak decalcifying agent ● marked swelling effect on tissues serves to counteract shrinkage produced by other fixatives Poor penetration Suitable only for small pieces of tissues or bones ● ACETONE ● ● Not recommended as morphological fixative for tissue blocks because it can cause shrinkage and poor preservation effects Fixation of cryostat sections BALEROS. BELOCURA. CHEUNG. S, DEL ROSARIO. FORTUNO. FREKING. GAGUI. J, GARCIA. GUINTAPAN. LEAÑO. LOVERIA. MABUNGA. PACLEB | MT’25 | A.Y. 2023 - 2024 5 ● ● Use at ice cold temp (0°C to 4°C) almost always used alone and without dilution, it fixes by dehydration and precipitation ● ● NOTE: Temperature sa old/2nd edition: -5°C to 4°C FIXATION TIME ○ Several minutes: cell smears, cryostat sections ○ Several hours: 1 – 24 hours for small tissue blocks ADVANTAGES ● ● For enzyme studies (lipase & phosphatase) Fixes brain tissues (diagnosis of rabies) DISADVANTAGES ● ● ● ● ● ● ● Cold Temperature ● some enzymes may become inactivated WEEK 7.2: DECALCIFICATION Prof. Jiego DECALCIFICATION PROCESS ● Done before the dehydration and on deparaffinize section before staining 10 % formalin or 10 % formol saline as the primary fixative Process of placing an already fixed tissue in a second fixative in order ○ To facilitate and improve the demonstration of particular substances ○ To make special staining techniques possible (with secondary fixative acting as a mordant) ○ To ensure further and complete hardening and preservation of tissues Ex: 10% formalin > connective tissue sample > use masons stain > secondary fixation with zenker’s solution acts as a mordant Process of removing Calcium or Lime Salts from tissues ○ Calcium and lime salts make tissues hard. If tissues are hard, it will be harder to cut it into smaller pieces which will make it harder to process, making it harder to make good slides to be stained properly and to be presented to the pathologist ○ Basically, decalcification = pagpapalambot ng mga tissues that contain calcium or lime salts ● ● ● Primarily fixed tissue is placed in aqueous solution of 2.5 – 3 % potassium dichromate for 24 hours To act as mordant for better staining effect WASHING OUT ● Removing excess fixative o ○ Tap water – for chromates, formalin, osmic acid ○ 50 – 70 % alcohol – for picric acid ○ Alcoholic iodine – for mercuric fixatives ● ● ● ● DECALCIFYING AGENTS ACIDS CHELATING AGENTS ION EXCHANGE RESINS HEAT FIXATION ● ● ● ● Microwave fixation (optimum temp: 45 – 55°C) To speed up the process Disadvantage: for small tissues only PROCEDURES 1. Fix tissue (no more than 5 mm) in formalin solution for 4 hours 2. Soak blocks in water at room temperature for 1 minute in 100 ml of formalin 3. Place in microwave, 450 watts, at 55°C for 1.5 to 4 minutes 4. Removes blocks and slice tissue to 2 mm thick 5. Place directly in alcohol ELECTRICAL IONIZATION (ELECTROPHORESIS) ● ● ● FACTORS THAT AFFECT FIXATION RETARDED BY: (PROLONGED PROCESSING) BOOK DEFINITION DECALCIFICATION: removal of calcium ions from a bone or calcified tissue through a histological process that makes them flexible and easier to cut. Performed on: ○ Bones ○ Teeth ○ Calcified tissues ■ Tuberculous lungs ■ Arteriosclerotic vessels Prevents poor cutting of hard tissues / knife damage If tissue size is very large – use saw (fine-fret) Decalcifying agent should be changed regularly “GRATING” sensation during cutting = place block in 10% HCl for 1 hour POST-CHROMATIZATION ● processor Moderate heat (37-56℃, accelerates fixation) Fatty Tissues ● manner of cutting should be thin for faster penetration of the fixative into the tissue it produces inevitable shrinkage and distortion Dissolves fat Preserves glycogen poorly Evaporates rapidly SECONDARY FIXATION ● NSS) Blood ● difficult for the fixative to penetrate, wash it with NSS HNO3, HCl, Formic, TCA, Sulfurous, Chromic Citric EDTA (Versene) Ammonium form of polystyrene resin 1-14 days - spread on bottom of container Attraction of Ca to negative electrode All four decalcifying agents have one goal: to decalcify tissues. The only difference between them is HOW they do it Used for different tissues depending on the need of the tissue ○ Ex: if one tissue sample has not been fully decalcified by acids, use ion exchange resins, etc. NOTE: “Tandaan lang first yung uniqueness nila, saang tissue sila ginagamit, malaki or maliit na tissues ba yung ginagamit, are they specifically used for certain tissues, are they specifically used for certain special tests… hindi need i-memorize lahat pero remember distinct characteristics.” ENHANCED BY: Size & Thickness Size & Thickness ● Pag malaki ang tissue, malaki rin ang ● faster fixation time fixative require lesser fixative Mucus Agitation ● Prevents the complete penetration of ● only happens fixative (can be prevented by when using washing mucus off the tissue with automated ACIDS NITRIC ACID ● ● Fastest and most common Inhibits nuclear stain ○ Prevented by combining formaldehyde or alcohol NITRIC ACID BASED DECALCIFYING AGENTS BALEROS. BELOCURA. CHEUNG. S, DEL ROSARIO. FORTUNO. FREKING. GAGUI. J, GARCIA. GUINTAPAN. LEAÑO. LOVERIA. MABUNGA. PACLEB | MT’25 | A.Y. 2023 - 2024 6 10% AQUEOUS NITRIC ACID (12-24 hours) FORMOL-NITRIC ACID (1-3 days) PERENYI’S FLUID (2-7 days) PHLOROGLUCINNITRIC ACID (12-24 hours) SULFUROUS ACID 10 mL conc. nitric acid + 100 mL dH2O ● Rapid, with minimal tissue distortion (if prolonged) ○ Decalcification = digesting calcium and lime salts, which can also digest part of the tissue (disadvantage) ● Recommended for urgent biopsy, needle biopsy* ● YELLOW color imparted conc. nitric acid + 40% formalin + dH2O ● Rapid acting, not as rapid as 10% ANA ● Good nuclear staining ● Less tissue destruction than 10% ANA ○ Allows for prolonged exposure to formol-nitric acid ● Uses fume hood ○ Toxic when inhaled ● LESSENS YELLOW tissue discoloration by 5% sodium sulfate or 0.1% urea* 10% nitric acid + 0.5% chromic acid + absolute ETOH ● Decalcifies and softens* ○ Other decalcifying agents only decalcify and do not soften ● Good nuclear and cytoplasmic staining ● Maceration avoided by chromic/ethyl ● Slow, Difficult to assess* complete decalcification by chemical means conc. nitric + phloroglucin + nitric acid 10% ● Most rapid ○ Puro acid, walang dH2O unlike 10% ANA ● Poor nuclear staining ● When decalcification is complete, acid must be removed by 3 changes of 70% to 90% ethanol (70% → 80% → 90%) SULFUROUS ACID CHROMIC ACID chromic acid + osmium tetroxide + glacial HAc (acetic acid) ● Fixative and decalcifying agent ● Nuclear staining with hematoxylin is inhibited ● Forms precipitate at the bottom ● Carcinogenic, corrosive to skin CITRIC ACID ACID BUFFER (ph 4.5) 7% citric acid + 7.4% ammonium citrate + 1% zinc sulfate + chloroform (preservative) ● slow ● Permits good nuclear and cytoplasmic staining CITRIC ACID (6 days) CHELATING AGENTS CHELATING AGENTS (1-3 weeks) Inferior compared to Nitric Acid as decalcifying agent Slower action, greater tissue distortion Good nuclear staining Recommended for surface decalcification* ○ Merong tissues na kapag cinucut using microtome, may grating sensation (matigas / makunat sensation). yung tissue block na yon, pwedeng ipatong sa HCl para ma-decalcify yung surface, para lumambot. (1-14 days) NaCl + HCl + dH2O ● Good cytologic staining ● Recommended for teeth and small pieces of bone ● extent of decalcification cannot be measured by a chemical test. Better nuclear staining with less tissue distortion Safer to handle than Nitric acid and HCl Recommended for postmortem research tissues* upon the addition of sodium citrate ○ To preserve the tissues of patients who died na possible pang gamitin for research purposes (ELECTRICAL IONIZATION) (2-7 days) FORMIC ACID-SODIUM CITRATE SOLUTION (3-14 days) formic acid (SG 1.20) + 10% formol saline ● Slow ● Fixative and decalcifying agent ● Permits excellent nuclear and cytoplasmic staining 45% formic acid + 20% Na Citrate ● Slow, not recommended for routine purposes ● Permits better nuclear staining than Nitric Acid ● Requires neutralization with 5% Na sulfate 1. 2. 3. TRICHLOROACETIC ACID (TCA) TRICHLOROACE TIC ACID (TCA) (4-8 days) TCA + 10% formol saline ● Slow, not recommended for routine purposes ● Permits good nuclear staining ● Weak decalcifying agent ● Used pag kailangan matagal naka-expose sa decalcifying agent; Pag hindi pa clear kung anong gagawin further sa tissue, isstore muna sya sa TCA ● ● Ammonia form of Polystrene Resin Hastens decalcification by removing Ca ions from Formic acid-containing decalcifying solutions Disadvantages: ● Artifacts produced, usually caused by CO2 bubbles ● Slow ● Degree of calcification cannot be measured by chemical means formic acid 88%, conc. HCl, dH2O ● Satisfactory for small bone fragments ● Uses electricity FACTORS INFLUENCING RATE OF DECALCIFICATION FORMIC ACID BASED DECALCIFYING AGENTS 10% FORMIC ACID Substances which combine with calcium ions and other salts (Fe, Mg) ● Acids digests calcium while chelating agents bind calcium. ● EDTA (Versene) ○ Most common chelating agent, will not bind Ca at pH below 3.0 ● Permits excellent staining Disadvantages: ● Very slow ● Slight tissue hardening produced ● EDTA inactivates alkaline phosphatase ○ Add magnesium chloride ELECTROPHORESIS ELECTROPHORESIS FORMIC ACID ● ● ● ● ION EXCHANGE RESINS ION EXCHANGE RESINS HCl BASED DECALCIFYING AGENTS VON EBNER’S FLUID Very weak decalcifying agent Suitable only for minute pieces of bone Ginagamit pag may sample na need na nakababad sa decalcifying agent for an extended period of time (like TCA) CHROMIC ACID (FLEMMING’S FLUID) HYDROCHLORIC ACID ● ● ● ● ● ● ● 4. Concentration and volume of decalcifying agent ○ ↑ concentrated = faster rate of decalcification ○ ↑ volume = faster rate of decalcification Temperature ○ ↑ temperature = faster rate of decalcification ■ at 37°C - impaired nuclear staining of Van Gieson's stain for collagen fibers ■ at 55°C - tissue will undergo complete digestion within 24-48 hours ○ Optimum: room temperature (18°C -30°C). Mechanical agitation ○ ↑ gentle agitation = faster rate of decalcification ○ Specifically, shaking motion ○ Tissue samples that are submerged in decalcifying agent can be placed on top of certain machines that operate in a continuous rocking motion which speeds up yung pag-seep or pagpasok ng decalcifying agent dun sa buong tissue ○ Increases rate of decalcification Size of the tissue ○ ↓ size = faster rate of decalcification BALEROS. BELOCURA. CHEUNG. S, DEL ROSARIO. FORTUNO. FREKING. GAGUI. J, GARCIA. GUINTAPAN. LEAÑO. LOVERIA. MABUNGA. PACLEB | MT’25 | A.Y. 2023 - 2024 7 ○ ● ↑ size = slower rate of decalcification EXTRA INFO FROM BOOK Factors influencing decalcification rate: ○ CONCENTRATION, FLUID ACCESS, SIZE AND CONSISTENCY, AGITATION, TEMPERATURE ○ FLUID ACCESS - Decalcification may be hastened by suspending the tissue in decalcifying solution for greater fluid access TEST FOR COMPLETENESS OF DECALCIFICATION ● To see/check the completeness of the removal of calcium and limestones of the tissue ● ● POST DECALCIFICATION ● ● NOTE: Descriptions of complete decalcification tests are extra info from book. PHYSICAL / MECHANICAL ● ● ● Manipulation, bending, probing or trimming of the specimen to “feel” for remaining calcified areas. Mechanical damage can occur during bending or probing and small deposits of calcium can easily be missed. PROCEDURE ○ A method of determining the endpoint by carefully weighing the ● ● specimen after rinsing and blotting has also been described, and may be an effective method for large specimens. ○ ● An alternate method of evaluating tissues mechanically is by pricking the tissue with a fine needle or a probe. This method is apt to produce needle tract artifacts and destroy important cellular details. DISADVANTAGE ○ May be successful in experienced hands; generally considered to be unreliable. ○ Can disrupt soft tumor from the bone ○ Cause false positive microfractures of fine trabeculae, leading to a potential misdiagnosis. ○ Small calcified foci may not even be detected. CHEMICAL (CALCIUM OXALATE TEST) ● ● ● ● Applied when some acid decalcifiers are used (particularly formic acid). Decalcifying fluid: usually changed every 24-48 hours More objective than the bubble method (observation of CO2 bubbles at the surface of the bone during acid decalcification). PROCEDURE: ○ The chemical test is performed on the discarded fluid. A piece of blue ○ Can allow further treatment if required. DISADVANTAGE ○ Not recommended for mercuric chloride-fixed tissues. This tissue’s radio-opacity will interfere with the correct interpretation of the plate. ○ Very expensive Remove acid by saturated lithium carbonate solution or 5-10% aqueous NaHCO3 (sodium bicarbonate) for several hours Use running tap water for rinsing ○ To wash out the solution ○ 30 minutes: small samples ○ 1-4 hours: large samples EXTRA INFO FROM BOOK SURFACE DECALCIFICATION: method of dealing with small unexpected deposits of calcium that may be encountered in paraffin blocks. TISSUE SOFTENERS: Unduly hard tissues which are liable to damage the microtome knives may require tissue softeners, aside from decalcification. ○ PERENYI’S FLUID - both decalcifying agent and tissue softener ■ Tissues are left in the fluid for 12-24 hours and dehydrated in the usual manner; or ■ the cut surface of the block may be submerged in the fluid for 1-2 hours before sectioning, to facilitate easier cutting of tissues ○ 4% AQUEOUS PHENOL SOLUTION ■ Washing out and immersion of fixed tissues in 4% aqueous phenol solution for 1-3 days ■ no marked deleterious effects; no tissue distortion. ○ OTHERS (Molliflex, 2% HCl, 1% HCl in 70% alcohol) ■ Molliflex - may appear swollen and soapy but does not affect the normalizing and subsequent staining of tissue sections. WEEK 8.1: DEHYDRATION Dr. King Estrella formaldehyde 1:20 parts - 37% in a bucket of water, place spx immediately and directly label (most important, date person and specimen). OVERVIEW TISSUE PROCESSING ● Dehydration ● Clearing ● Infiltration/Impregnation ● Embedding DEHYDRATION it is a term which pertains to removal of water or anything that will allow water to disappear rewatch litmus paper is added to a test tube containing 5 ml of the discarded ● Removal Of intercellular and extracellular water from the decalcifying agent (the litmus paper will turn red due to the acidity of the fluid). tissue ○ Strong ammonia is then added drop by drop until the fluid is ● Increasing concentration of alcohol starting from 50% up neutralized (this can be detected by the change in color of the litmus to 100% paper from red to blue, indicating alkalinity). The presence of ○ Routine - starts with 70% usually ethyl alcohol cloudiness indicates that there is still calcium found in the solution. ■ ethyl alcohol (comes from plants) and isopropyl ○ The tissue is then immersed in a new solution of decalcifying agent. If alcohol (comes from petroleum) can be used on the solution remains clear after neutralization with concentrated skin ammonia, 0.5 ml. of saturated aqueous solution of ammonium butanol ■ butanol cannot be used and denatured alcohol oxalate is added and the solution is allowed to stand for 30 minutes. alcohol and methanol (tendency to explode) Cloudiness will signify incomplete calcium removal; hence, the need denatured ■ NOT IDEAL METHANOL ALWAYS ETHANOL for further decalcification. If the solution remains clear after 30 methanol ○ Embryonic - tissues - starts with 30% ethyl alcohol minutes, decalcification is considered to be complete. ■ emergency abortion 30% isopropanol or ethanol ● DISADVANTAGE ■ hindi sasabihin na fetus or embryo, term that is ○ Cumbersome and useless in the everyday practice of used to denote sac is gestational sac surgical pathology ● 10:1 ratio of dehydrating agent and tissue X-RAY / RADIOLOGICAL ○ most is made up of alcohol water ? alcohol ○ water is added to lessen alcohol lessening tendency ● Best method for large specimens (ex: femoral heads) of rapid evaporation ● Most ideal, most sensitive and most reliable method ○ ○ Able to detect even the smallest focus of calcium which appears opaque in an X-ray plate Clearly reveal tiny residual calcium deposits ● QUALITIES OF AN IDEAL DEHYDRATING SOLUTION ● Rapid acting w/ no tissue shrinkage or distortion ○ ideal BALEROS. BELOCURA. CHEUNG. S, DEL ROSARIO. FORTUNO. FREKING. GAGUI. J, GARCIA. GUINTAPAN. LEAÑO. LOVERIA. MABUNGA. PACLEB | MT’25 | A.Y. 2023 - 2024 8 ● ● ● Should not evaporate fast ○ Most used are more alcohol to water ratio ■ water is added to reduce dehydration Can dehydrate even tatty tissues ○ Bakit mo lalagyan chicharon ng alcohol it would turn turbid ○ Bula bula na milky miscibility ○ like dissolves like; non polar si alcohol ■ mix with water ■ miscible with water ■ non poisonous ■ penetrates easily ○ Si chicharon needs gasoline ether ○ Slightly miscible Non toxic ○ most important (BAKIT MO GAGAMITIN? -prof) Not fire hazard Should not harden tissues excessively ● ● ● ● ● ● Alcohol - most common, least expensive Acetone - bigger tendency to evaporate rapidly Dioxane Cellosolve Triethyl phosphate Tetrahydrofuran (THF) - least common, most expensive ● ● ALCOHOL - PROGRESSIVELY INCREASING ● ● ● ● ● ● ● ● ● ● ● minimum ● ● ● Minimum shrinkage Minimum distortion and hardening of tissue Used to dehydrate sections and smears following certain stains papanicolau smear or vaginal swab increase solution we utilize triethy phosphate as dehydrant TETRAHYDROFURAN - THF ● ● ● ● Prolonged storage in 70 % alcohol - macerates tissues ○ slowly penetrates tissues Directly placed in high grade alcohol - shrinkage & hardening of tissues ○ Because it does not only remove the water content, but a lot of the cellular components/ part of the cytoplasm or the cells ○ Hardening of tissue will result to difficulty in tissue sectioning Dehydrates and clears Less shrinkage Easier cutting w/ fewer artifacts Non toxic; 6 months exposure – conjunctival irritation (like sore eyes) Offensive odor (use well ventilated room) anesthsia Called someone on lab histopathology Formaldehyde used to from the laboratory 1 is to 20 parts a liter vs @37 percent in a bucket of water Do not forget to label Date is indicated the person and specimen as all of them appear the same grayish in color even if it is a liver….. WEEK 8.2: CLEARING CLEARING / DEALCOHOLIZATION ● Readily available because it is used to remove stains or paints Cheap Rapid acting Volatile Tissue shrinkage ○ not recommended because of the last two bullets ● ● ● ● ● DIOXIDE (DIETHYLENE DIOXIDE) ● ● ● ● Ethylene Glycol Monoethyl Ether - other name Dehydrates rapidly Storage without producing hardening or distortion ○ Rarely used in dehydration TRIETHYL PHOSPHATE ACETONE ● w/ CELLOSOLVE ETHYL ○ best, fast, mixes with water and penetrates easily, not poisonous, cheap ○ Considered the fastest ● ○ Reacts automatically ADDITIVES TO DEHYDRATING AGENTS ○ Mixes with water and penetrates easily ● 4 % phenol added to 95 % ethanol – softens hard tissues 490 phenol ○ Readily available in pharmaceuticals ● Hard tissues – immerse in glycerol/alcohol mixture or in 95W ethanol Molliflex ○ Widely utilized as a disinfectant ○ A must have in everyone’s pocket Most chemicals used in decalcification is an acidic in a certain ○ magsisimula sa mababa then pataas ng pataas concentration mol (CASINO tubo sugar cane) Miss x bile sludge vs neoplasm on process METHYL Attending physician is not not normal neoplasm in progress surgeon ct scan 95% accurate 5% inaccurate ○ Toxic, for blood films 2.5 cm mass ○ Recommended for blood films Allowable mass is 1 cm only BUTYL Consultation with relatives to diagnose Operation close or open surgery ○ Slow action, does not dehydrate rapidly Close holed out then specialzed instruments laparoscomy ○ Utilized for Plant and Animal microtechnique Forcep 3 strings only ○ Utilized for the organic substance retained Open open cut then whole organ is observed similar to autopsy but under ALCOHOL ● ● Expensive Dangerous - vapor - cumulative ○ Dangerous because once inhaled, the vapor is carcinogenic –it will cause cumulative effects in the human body Graupner' s method Dioxane w/ paraffin wax ○ Dioxane in combination with paraffin wax Weiseberger' s method ○ Dioxane in combination with Ca oxide placed in a gauze Dioxane ( a oxide tylertk-DAAC.TT DEHYDRATING AGENTS ● ● ● Excellent dehydrating & clearing agent Less tissue shrinkage Tissues can be left here for a long time Ribbons poorly ○ Ribbons - tissue sections that are cut ○ Ribbons are poorly stained Process where we try to remove excess alcohol and excess water in order for the histopathologist to see the tissues clearly without any interference or any debris that will interfere with microscopy Removal of dehydrating solutions, making the tissue components receptive to the infiltrating medium After staining - transparent tissues Improve refractive index of the tissue ○ Refractive index is the ability of the tissues to be closely monitored or closely seen if it is focused under HPO or LPO fililinaw CHARACTERISTICS OF GOOD CLEARING AGENTS ● ● Miscible with alcohol and paraffin &/or mounting medium Should not produce excessive tissue shrinkage & hardening BALEROS. BELOCURA. CHEUNG. S, DEL ROSARIO. FORTUNO. FREKING. GAGUI. J, GARCIA. GUINTAPAN. LEAÑO. LOVERIA. MABUNGA. PACLEB | MT’25 | A.Y. 2023 - 2024 9 ● ● Should not evaporate quickly in a waterbath Should make tissues transparent ○ Important because clearing agents remove unwanted debris so the tissues will appear naturally without any artifacts or debris which will interfere with microscopy CLEARING AGENTS ● ● ● ● ● Xylene Toluene Benzene Chloroform Cedarwood oil ● ● ● ● ● ● Aniline oil Clove oil Carbon Tetrachloride Methyl benzoate Methyl salicylate THF XYLENE (XYLOL) ● ● ● ● ● ● ● Most common, colorless Most rapid Cheap Milky if incomplete dehydration Hardens/shrinks tissues ○ Not recommended for ■ Nervous system & lymph nodes ■ Because it is highly volatile and carcinogenic Hard/brittle tissues if > 3 hours Banned - difficult for the laboratory to secure xylene in the market CLOVE OIL ● ● ● ● CARBON TETRACHLORIDE (CCL4) ● ● ● ● ● ● ● ● Not carcinogenic: but will emit fumes that are toxic upon prolonged exposure Rapid acting Tissues do not become excessively hard and brittle even if left here for 24 hours Slower than xylene & benzene expensive ● ● ● ● ● Totally banned - organic chemical that is no longer available commercially Rapid acting Volatilizes rapidly in paraffin oven Tissue shrinkage if left for a long time Carcinogenic; may damage bone marrow resulting to aplastic anemia ● ● Dehydrating and clearing agent Non toxic but with offensive odor and should be used in a well-ventilated room ● ● Permeating the tissue with a support medium Clearing medium is completely removed from the tissue and replaced by a medium that will completely fill all the tissue cavities ○ Lahat ng maliliit na spaces within the tissue ○ This helps in the tissue processing in the context of the tissue being hard enough for it to be cut ■ Easier to cut ■ hindi madaling masira during the process of cutting or tissue ribbon WEEK 8: INFILTRATION/ IMPREGNATION ● ● ● CHLOROFORM ● ● ● ● ● For tough tissues – decalcified, nervous, lymph nodes, embryos – minimum shrinkage and hardening For large specimens Toxic to liver (prolonged inhalation) Does not make tissues transparent Tissues tend to float CEDARWOOD OIL ● ● ● ● ● ● ● Very penetrating Extremely slow For CNS, smooth muscles, skin, cytology Milky on prolonged storage (filter) May produce crystals – heat to 200 C Very expensive Maybe used for paraffin and celloidin sections ANILINE OIL ● ● For clearing embryos, insects and delicate specimens due to its ability to clear 70% alcohol without excessive tissue shrinkage and hardening Preserves egg of insects Slow acting – used for double embedding TETRAHYDROFURAN (THF) BENZENE ● Totally banned Carcinogenic Properties and disadvantages are like chloroform but cheaper Hepatotoxic toxic to the liver METHYL BENZOATE/METHYL SALICYLATE TOLUENE ● Causes minimum shrinkage of tissues Wax impregnation here – slow / difficult ○ If it is used for a longer period of time, it will be difficult to remove Tissues become brittle - longer period of time Expensive NOTE: Usually during the process of clearing, dehydration, and infiltration, there are some leftover clearing materials that fill up the gaps of the tissues o We need to remove them for us to fill the gaps with other materials that will solidify at a lower temperature During the tissue processing procedure, when clearing is done, the vacant areas were usually left behind by the clearing agent o The purpose of clearing is to allow tissues to become transparent o The purpose of dehydration is to remove the water content ■ combined with alcohol, (alcohol and water) will then evaporate thus leaving vacant tissue areas Waxes or any semi solid materials that eventually solidify, will be allowed to permeate/penetrate the inner cavity of the tissues to establish solid materials which will then facilitate easy sectioning/cutting TYPES OF TISSUE IMPREGNATION/EMBEDDING ● ● ● ● Paraffin Wax ○ Manual – 4 changes of wax 15 mins interval ○ Automatic – Autotechnicon, Elliott Bench type ○ Vacuum – fastest, negative atmospheric pressure, gives the fastest result Celloidin (Collodion) ○ Wet – bones, teeth, large brain sections ○ Dry – whole eye sections Gelatin Plastic PARAFFIN WAX IMPREGNATION ● ● ● ● ● ● Melting Point: 54°C – 58°C 1- 9 Remains solid or slowly solidifies at 20°C – 24°C 4 Simplest, most common, best Melting point: 54c-58c at 20c-24c Ease in cutting or Eary cuttig ○ More malleable Permanent paraffin blocks ○ Can be stored for several years BALEROS. BELOCURA. CHEUNG. S, DEL ROSARIO. FORTUNO. FREKING. GAGUI. J, GARCIA. GUINTAPAN. LEAÑO. LOVERIA. MABUNGA. PACLEB | MT’25 | A.Y. 2023 - 2024 10 ● ● ● ● ○ becomes permanent once impregnated with paraffin Good staining results ○ can be viewed under microscope under Hematoxylin & Eosin staining procedure Not recommended for fatty tissues ○ Madaling nadudurog when using paraffin wax ○ Madaling nadudurog when using paraffin wax ○ Eventually, fatty tissues when mixed with paraffin, will combine easily because they are highly miscible— it doesn’t serve its purpose of filling in the parts which are then cleared by the clearing agents Overheated paraffin -> brittle specimen Inadequate impregnation -> clearing agent retained ○ Usually visible if the clearing agent is not completely removed during clearing procedure ■ Saline (clearing agent) was still left during the procedure hence, once combined with paraffin, paraffin will not impregnate/penetrate the gaps of the tissue since the clearing agent is still present SUBSTITUTE PARAFFIN WAX ● PE Biti ● ● Paraplast – more elastic / resilient (56-57C) ○ Embedded – less brittle (56-58C) ○ Bioloid – for eyes ○ Tissue Mat – with rubber Ester Wax – low melting point (46-48C) ○ Harder than paraffin; water insoluble, can be used for impregnation without prior clearing ○ Known organic substances that form an esterified ○ wax, usual byproducts of aldehydes and ketones ○ Harder than paraffin; water insoluble, can be used for impregnation without prior clearing ○ Assuming that there is leftover saline during The clearing procedure, we recommend the use of ester wax for impregnation Water Soluble Wax ○ Recommended for enzyme histochemistry (special staining/procedures in Histopath) ■ Because it is highly hygroscopic ■ Water combined with wax, is said to be miscible ○ Carbowax – water soluble (no dehydration/clearing); for enzyme histochemistry; hygroscopic ● ● ● ● ● ● WEEK 8.2: EMBEDDING ● ● ● ● ● ● ● ● ● ● DOUBLE EMBEDDING 2% Celloidin for 3 days and subsequent paraffin for large blocks of dense firm tissues – brain Obsolete – due to paraffin waxes with different types of resins ● ● ● ● Epoxy, polyester, acrylic For hard tissues, renal and bone marrow biopsies For ultrathin sections requiring minimal distortion With VCD – vinylcyclohexene dioxide-carcinogenic PLASTIC (RESIN) EMBEDDING WEEK 9.1: IMPREGNATION AND EMBEDDING Therese R. Suratos, RMT DEFINITION IMPREGNATION process whereby the clearing agent is completely removed from the tissue and replaced by a medium that will completely fill all the tissue cavities and give a firm consistency to the specimen. Permits cutting of thicker tissue sections Crumbling of tissues avoided Very slow Very volatile; it easily evaporates ○ esp. if not kept properly Thin sections difficult to cut Serial sections difficult to prepare - difficult to prepare Photomicrographs - difficult to obtain Very volatile Suitable for specimens with large hollow cavities which tend to collapse Recommended for osseus tissues or calcified tissues like bones, teeth, large brain sections and whole organs placed in celloidin materials + DRY CELLOIDIN METHODS Dry eyes " ● Preferred for whole eye section ● ● ● ● ● LVN – low viscosity nitrocellulose soft to harder A form of celloidin but more expensive y Used for softer tissues since it forms harder tissue blocks Forms harder tissue block; makes thinner sections When dry, striking or dropping the container will cause explosion. ● The use of polysaccharide agar material NITROCELLULOSE METHOD GELATIN IMPREGNATION Leuckhart’s Embedding Mold Compound Embedding Unit Plastic Embedding Rings & Base Mold ○ Tissue Tek – machine w/ warm plate Disposable Embedding Molds ○ Peel Away ○ Plastic Ice Trays ○ Paper boats ○ TIMS tissue processing and embedding system ● ● ● - WET CELLOIDIN METHODS Casting /Blocking Place tissue in a precisely arranged position in a mold with a medium which is then allowed to solidify (orientation) Orientation – process by which tissue is arranged in precise positions in the mold during embedding, on the microtome before cutting, and on the slide before staining BLOCKING OUT MOLDS ● ● ● CELLOIDIN ● ● ● ● Rarely used – when dehydration is avoided; for histochemical and enzyme studies Recommended for special occasions/procedures only: ○ histochemical and enzyme studies Does not require dehydration & clearing Embedding medium for delicate specimen and frozen sections Low melting point Can be used as substitute for paraffin wax EMBEDDING 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. QUALITIES OF A GOOD INFILTRATING AND EMBEDDING MEDIUM ● ● ● ● ● ● ● Soluble in processing fluids Suitable for sectioning and ribboning molten between 30°C and 60°C translucent or transparent; colorless stable Homogeneous Capable of flattening after ribboning non-toxic Odorless Easy to handle Inexpensive EMBEDDING MEDIUM ● ● The medium used to infiltrate the tissue is usually the same medium utilized for impregnation, and for general purposes There are generally four types of impregnation and embedding medium, namely: 1. Paraffin wax 2. Celloidin (colloidion) 3. Gelatin 4. Plastic BALEROS. BELOCURA. CHEUNG. S, DEL ROSARIO. FORTUNO. FREKING. GAGUI. J, GARCIA. GUINTAPAN. LEAÑO. LOVERIA. MABUNGA. PACLEB | MT’25 | A.Y. 2023 - 2024 11 PARAFFIN WAX IMPREGNATION PARAFFIN - simplest, most common, and best embedding medium used for routine tissue processing. PARAFFIN WAX - a polycrystalline mixture of solid hydrocarbons produced during the refining of coal and mineral oils. ● It is solid at room temperature but melts at temperatures up to about 65°C or 70°C. ● Paraffin wax can be purchased with melting points at different temperatures,the most common for histological use being about 56°C to 58°C ● At its melting point, it tends to be slightly viscous, but this decreases as the temperature is increased. ● The traditional advice with paraffin wax is to use this about 2°C above its melting point. ● Wax hardness (viscosity) depends upon the molecular weight of the components and the ambient temperature. ● To decrease viscosity and improve infiltration of the tissue, technologists often increase the temperature to above 60°C or 65°C. ○ ↑ temperature = ↓ viscosity ● High molecular weight mixtures melt at higher temperatures than waxes composed of lower molecular weight fractions. ○ ↑ MW mixtures will met at ↑ temperature. ● Paraffin wax is traditionally marketed by its melting points which range from 39°C to 68°C ● Tissue-wax adhesion depends upon the crystal morphology of the embedding medium. ● Small, uniform sized crystals provide better physical support for specimens through close packing. ● Crystalline morphology of paraffin wax can be altered by incorporating additives which result in a less brittle, more homogeneous wax with good cutting characteristics. ● There is consequently less deformation during thin sectioning. Make a list for temperatures ADVANTAGES AND DISADVANTAGES OF PARAFFIN WAX IMPREGNATION ADVANTAGES 1. 2. 3. 4. 5. ● ● ● Thin individual serial sections may be cut with ease from the majority of tissues without distortion The process is very rapid, allowing sections to be prepared within 24 hours Tissue blocks and unstained mounted sections may be stored in paraffin for an indefinite period of time after impregnation without considerable tissue destruction Because formalin-fixed, paraffin-embedded tissues may be stored indefinitely at room temperature, and nucleic acids (both DNA and RNA) may be recovered from them decades after fixation, they are an important resource for historical studies in medicine Many staining procedures are permitted with good results. DISADVANTAGES 1. 2. 3. 4. 5. Overheated paraffin makes the specimen brittle. Prolonged impregnation will cause excessive tissue shrinkage and hardening, making the cutting of sections difficult. Inadequate impregnation will promote retention of the clearing agent. Tissues become soft and shrunken, and tissue blocks crumble when sectioned and break up when floated out in a water bath. Tissues that are difficult to infiltrate, e.g. bones, teeth, brains and eyes, need long immersion for proper support; otherwise, they will crumble on sectioning. Prolonged immersion in paraffin, on the other hand, is not advisable Paraffin processing is not recommended for fatty tissues. The dehydrating and clearing agents used in the process dissolve and remove fat from the tissues NOTE: 10 years na nakatago sa cabinet, merong drawers for each block in case need ulit i-test etc. pwede sya i-cut ulit. Once gumamit na ng formalin, indefinite time na yung pag-use nya In every hosp, there are certain protocols for histopath technologies, 1 h