MODULE-3-DECALCIFICATION-1.pdf

Evolve. Adapt. Overcome. CEFI is now ready. MODULE 3 / TISSUE PROCESSING MLS 113: GENERAL PATHOLOGY AND HISTOPATHOLOGY HARLEY ROSE B. BAUTISTA, RMT, MPH College of Me...

Evolve. Adapt. Overcome. CEFI is now ready. MODULE 3 / TISSUE PROCESSING MLS 113: GENERAL PATHOLOGY AND HISTOPATHOLOGY HARLEY ROSE B. BAUTISTA, RMT, MPH College of Medical Technology DECALCIFICA TION Calcium may be removed by the following acid Chelating agents Ion exchange resins electrical ionization ACID DECALCIFYING AGENT I. NITRIC ACID - Most common and the fastest decalcifying agent used so far. However it inhibits nuclear stain and destroying tissues especially in concentrated solutions. A. Aqueous nitric add solution 10% Recommended for urgent biopsies and for needle and small biopsy specimens to permit rapid diagnosis within 24 hrs. or less. B. FORMOL-NITRIC ACID Rapid acting; recommended for urgent biopsies Produce yellow color; remedy: neutralizing tissue in 5% sodium sulfate and washing in running tap water for atleast 12 hrs. addition of 0.1% urea to pure concentrated nitric acid will also make discoloration disappear without affecting the efficiency of the decalcifying agent. C. PERENYI’S FLUID Recommended for routine purposes Nuclear and cytoplasmic staining is good. Maceration is avoided due to the presence of chromic acid and alcohol Not recommended for urgent work. Composed of chromic acid, ethyl alcohol and nitric acid D. PHLOROGLUCIN NITRIC ACID Most rapid decalcifying agent II. HYDROCHLORIC ACID A. VON EBNER’S FLUID - Moderately rapid decalcifying agent. Does not require washing out before dehydration Recommended for teeth and small pieces of bone. III. FORMIC ACID May be used as both fixative and decalcifying agent. Recommended for routine decalcification of postmortem research tissues. A. FORMIC ACID-SODIUM CITRATE SOLUTION - Recommended for autopsy materials, bone marrow, cartilage. IV. TRICHLORO ACETIC ACID Very slow acting; weak decalcifying agent Suitable for small spicule of bone. V. SULFOROUS ACID -is a very weak decalcifying solution suitable only for minute pieces of bone. VI. CHROMIC ACID (FLEMMING’S FLUID) May be used as both fixative and decalcifying agent Suggest for minute bone specimens. Consider as carcinogenic VII. CITRIC ACID – CITRATE BUFFER SOL. pH 4.5 excellent nuclear and cytoplasmic staining. Too slow Contains chloroform as preservatives CHELATING AGENTS I. EDTA (VERSENE) Most common chelating agent Recommended for detailed microscopic studies Very slow decalcifying agent For small specimens: 1-3 weeks 6-8 weeks or longer totally decalcify dense cortical bone. Excellent for immunohistochem or enzyme staining and electron microscopy. Inactivates ALP; to restored add magnesium chloride. ION EXCHANGE RESIN Not recommended for fluids containing nitric acid or hydrochloric acid. The decalcifying agent is then added, usually 20-30 time of the volume of the tissue. The tissue will stay in solution 1-14 days. FACTORS INFLUENCE OF DECALCIFICATION 1. Concentration and volume -More concentrated, more rapidly; more harmful 2. Structure and temperature ratio: 20:1 higher concentration and greater amount of fluid will increase the speed of the process. 3. Heat 4. Mechanical Agitation 5. Accelerates the rate of diffusion and speeds up the decalcification process. MEASURING EXTENT OF DECALCIFICATION 1. Physical or mechanical test -Touching or bending the tissue with the fingers. Inaccurate method. 2. X – ray or radiological method - Very expensive; most ideal; most sensitive and most reliable method. 3. Chemical method (calcium oxalate test) - Simple, reliable and convenient method. - Cloudiness will signify incomplete decalcification. POST DECALCIFICATION Involves lithium carbonate to wash the tissue after the decalcification is complete. Also decalcified tissue can rinse in running tap water to re moved acids. TISSUE SOFTENER’S 1. Perenyi’s fluid May act as both decalcifying and tissue softners 2. 4% aqueous sol 3. Molliflex Tissue immersed in molliflex appear swollen and soapy. DEHYDRATION Starts by placing the fixed specimen in 70% to 95% to 100% ethyl alcohol. For delicate tissues, particularly embryonic tissues starting 30% ethyl alcohol The amount of dehydrating agent is should not be less than 10 times the volume of the tissue to ensure complete penetration of the tissue by the dehydrating agent. I. ALCOHOL Anhydrous copper sulfate will accelerate dehydration by removing water from dehydrating fluid. A blue discoloration will indicate a complete dehydration. A. ETHYL ALCOHOL Recommend for routine dehydration of tissues Best dehydrating agent B. METHYL ALCOHOL Toxic dehydrating agent Used for blood and tissue film for smear preparations C. BUTYL ALCOHOL - For plant and animal microtechniques - Slow dehydrating agent 2. ACETONE Cheap, rapid acting dehydrating agent For urgent biopsies which dehydrates 30 mins to 2 hrs Limited for only small specimen Volatility and inflammable Most lipids are removed from tissues with this dehydrating agent. DIOXANE (DIETHYL DIOXIDE) Excellent dehydrating agent and clearing agent Readily miscible with water and paraffin Tends tissue ribbon poorly Highly toxic in man and expensive CELLOSOLVE (EHTYLENE GLYCOL MONOETHYL ETHER) Cellosolve dehydrates rapidly. The tissue may be transferred from water or normal saline directly to cellosolve and stored in it for months without producing hardening or distortion. TRIETHYL PHOSPHATE Used to dehydrate sections and smears following certain stains and produces minimum shrinkage. TETRAHYDROFURAN (THF) Both dehydrates and clears tissue since it is miscible in both water and paraffin. CLEARING De-alcoholization; process whereby alcohol or a dehydrating agent is removed from the tissue and replaced with a substance that will dissolve the wax with which the tissue to be impregnated. Must be miscible with water, paraffin and mounting medium. COMMON CLEARING AGENTS I. XYLENE (XYLOL) Colorless clearing agent, commonly used in histology laboratories. Miscible with absolute alcohol and paraffin It is cheap; used for celloidin sections Highly inflammable Not suitable for nervous tissues and lymph nodes Xylene turns milky when an incompletely dehydrated tissue is immersed in it. II. TOLUENE May be used as a substitute for xylene and benzene. More expensive Miscible with absolute alcohol and paraffin III. BENZENE Preferred by some as clearing agent in the embedding process of tissues because it penetrates and clears tissue rapidly. Carcinogenic, may damage the bone marrow resulting aplastic anemia Miscible with absolute alcohol IV. CHLOROFORM does not make tissue translucent recommended for tough tissues (skin, fibroid, decalcified tissues) toxic to liver tissue tend to float in chloroform to avoid this wrapped the tissues with absorbent cotton gauze to facilitate sinking of the section in solution. Miscible with absolute alcohol V. CEDARWOOD OIL Recommended for central nervous system tissues and cytological studies. (particularly smooth muscles) Requires two changes in clearing solution It makes tissue transparent Extremely slow clearing agent VI. ANILINE OIL Recommended for clearing embryos, insects and very delicate specimens. VII. CLOVE OIL Unsuitable for routine clearing process VIII. CARBON TETRACHLORIDE Properties are very similar to chloroform IX. METHYL BENZOATE AND METHYL SALICYLATE -double embedding technique is needed. IMPREGNATION Process whereby the clearing agent is completely removed from the tissue and replaced by a medium that will completely fill the cavities, thereby giving a firm consistency to the specimen and allowing easier handling and cutting suitably thin sections without any damage to the tissue and its cellular components. I. PARAFFIN WAX IMPREGNATION Simplest, most common and best embedding medium, used for routine processing. Very rapid Prolong impregnation will cause excessive shrinkage and hardening making cutting of sections difficult. Not recommended for fatty tissues  For routine work melting point is : 56-58 degcel For the lab temp between 15-18 deg cel melting point is: 50-54 degcel For the lab temp between 20-24 deg cel melting point is 54-58 degcel METHODS OF PARAFFIN WAX IMPREGNATION A. MANUAL PROCESSING - Four changes of wax are required at 15 minutes interval. B. AUTOMATIC PROCESSING - Use auto technicon which fixes, dehydrates, clears and infiltrates tissues. - 2 -3 changes of wax are required. C. VACCUM EMBEDDING Involves the wax impregnation under negative atmospheric pressure inside an embedding oven to hasten removal of air bubbles and clearing agent. Most rapid Recommended in urgent biopsies SUBSTITUTES FOR PARAFFIN WAX 1. PARAPLAST Mixture of highly purified paraffin and synthetic plastic polymers Melting point: 56-57 degcel More elastic and resilient For bones and brains 2. EMBEDDOL Synthetic wax substitute similar to paraplast Melting point is : 56-58 degcel 3. TISSUE MAT -contains rubber 4. ESTER WAX - Has a lowering melting point 46-48 degcel Harder than paraffin WATER SOLUBLE WAX (CARBO WAX) Melting point: 38-42 degcel or 45-46 degcel II. CELLOIDIN IMPREGNATION Purified form of nitrocellulose soluble in many solvents Suitable for specimens with large hollow cavities which tend to collapse for hard and dense tissues such as bones teeth and for large tissue sections such as whole embryo. Recommended for processing neurological tissue Very slow TWO METHODS FOR CELLOIDIN IMPREGNATION 1. WET CELLOIDIN METHOD -Recommended for bones teeth large brain and whole organs 2. DRY CELLOIDIN METHOD -Recommended for whole eye sections -Does not use alcohol due to the presence of cedarwood oil in the block. III. GELATIN IMPREGNATION Rarely used except for histochem and enzyme studies Used for delicate specimens and frozen tissue Water soluble Tissue should not be more than 2-3mm thick; add 1% phenol to prevent molds Excess gelatin may remove by floating the sections to paper and trimming them with scissors. EMBEDDING After impregnation, the tissue placed into a mold containing the embedding medium and these medium allow solidifying. In this process orientation is very important. Temperature of melted paraffin is 5-10 deg cel above the melting point Immersed in cold or ref temp to solidify 1. Leukhart’s embedding mold -Contains two L- shaped strips of heavy brass. 2. Compound Embedding unit -Made up of a series of interlocking plates resting on a flat metal base. 3. Plastic embedding rings and base molds -Consist of special stainless steel base mold fitted with a plastic embedding ring, which later serves as the block holder during cutting 4. Tissue-tek -Equipped with a warm plate to manage the impregnated specimen. 5. Disposable embedding molds - Peel-away -Plastic ice trays - Paper boats REMEMBER Celloidin or nitrocellulose method – recommended for embedding hard tissues. *double-embedding method-process; in which tissue first infiltrate by celloidin and embedded in paraffin. THANKYOU J