Histopathologic Techniques - 02 Dehydration, Clearing, and Decalcification

HISTOPATHOLOGIC AND CYTOGENETIC TECHNIQUES DEHYDRATION, CLEARING & DECALCIFICATION METHODS OF DECALCIFICATION • OUTLINE • • • • Introduction • Decalcification o Methods of Decalcification o Methods of Testing for the Completeness of Decalcification o Post Decalcification Dehydration o Dehydrating Agents Dealcoholization/Clearing o Clearing Agents • INTRODUCTION • • • • • • • Dehydration is defined as a process in which water is entirely removed from the tissues. It prepares the tissue to be enveloped in a medium (paraffin wax) which is not miscible in water. Clearing which is also known as dealcoholization follows after dehydration. In clearing, alcohol is replaced by a solution (clearing agent) which is more miscible with paraffin wax. At the same time, alcohol renders the tissues "optically clear". The commonly used reagents in this step make the tissues transparent due to their relatively high refractive index, hence the term"clearing". Not a routine step in tissue processing is decalcification. Bone decalcification is the removal of calcium ions or lime salts from the organic extracellular matrix, calcified collagen, and surrounding tissues of the bone. This process make the bone easy for histopathological investigation by producing thin sections using a microtome. Next to fixation is dehydration. However, there are specimens with calcium and lime salts making the hard and difficult to cut. For tissues with calcium and other lime salts, decalcification must be done. Decalcification is not a mandatory step in tissue processing, must be done following . Ideal thickness for decalcification is 1-3 mm. Usually, decalcification is done by place a specimen in a decalcifying agent. Required ratio of decalcifying agent to tissue is 20:1. DECALCIFICATION • • • • • • • • • Removal of calcium ions or lime salts from the organic extracellular matrix, calcified collagen and surrounding tissues of bones Both decalcification and processing depends on bone thickness. Ideal thickness is 1-3 mm Must be done following fixation. Adequate fixation must be achieved prior to decalcification Ratio of fluid to tissue - 20:1 Heat and constant agitation speeds up decalcification More concentrated acid solutions decalcify more rapidly but may destroy tissue Optimal temperature - room temperature Autotechnicon - cannot perform decalcification Decalcification is done manually CEU Malolos BSMT 2023 • • Electrolytic Method o Most rapid method o By electricity, calcium is removed o Dependent upon supply of electric current o The positively charged calcium ions are attracted to negative electrodes from the decalcifying solution facilitating calcium removal Ion Exchange resin o Calcium is rapidly removed by the decalcifying solution containing formic acid, thereby increasingly solubility from tissues o Duration: 1-14 days ▪ Process is longer than electrolytic o Specimen is placed at the bottom of the container with an ammonium form of polystyrene resin which will increase tissue solubility to facilitate removal of calcium Use of Chelating agent o EDTA - potent anticoagulant, considered as weak decalcificying agent, considered as bet decalcifIer for EM and immunohistochemistry o Inactivates alkaline phosphatase activity; remedy: add magnesium chloride o For small specimens - 1-3 weeks o For dense tissues - 6-8 weeks Use of Acid o Most common method of decalcification o Manually performed ▪ Placing decalcifying acid on a container, and immersing specimen in the acid o Nitric acid ▪ Most commonly used acid ▪ Concentrated acids are not used, can destroy tissues • They must be diluted to be used ▪ 10% Aqueous Nitric Acid - commonly used • Duration: 24 hours (depend on type of specimen; larger specimens will require longer decalcification time and small specimens will require shorter time) ▪ Formol nitric acid - with formaldehyde • Duration: 1-3 days • Require fume hood due to formaldehyde ▪ Perenyi’s fluid - nitric acid, chromic acid and ethyl alcohol • Duration: 2-7 days • Used as decalcifying acid and tissue softener ▪ Phloroglucin Nitric - pholoroglucin + nitric acid • Most rapid acting o Hydrochloric acid ▪ Not commonly used because it is weak and slow ▪ Recommended only for minute pieces of bones ▪ Von Ebner’s (HCl + NaCl) – for teeth and small pieces of bones, for surface decalcification of blocks o Formic acid sodium citrate - recommended for decalcifying cartilage, research specimens, autopsy specimen, bone marrow o TCA, Sulfurous acid - weak and slow decalcifying agents HISTOPATH - M2 • 1 Chromic acid/Fleming’s with HAC - not commonly used because it is considered as environmental toxin ▪ Highly corrosive on the skin o Citric acid citrate buffer - contains chloroform as preservative Summary o Electrolytic method - most rapid method o Ion Exchange Resin and EDTA - not commonly used because they are not suited for urgent biopsies, process is longer o Use of Acid - most common method o • • DEHYDRATING AGENTS • METHODS OF TESTING FOR THE COMPLETENESS OF DECALCIFICATION • • • • Physical method/ Mechanical - not a reliable method o Easy to do o Done by bending the tissue, probing the tissue with needle o Bending/poking do not denote completeness X-ray or Radiologic method - most reliable method o Can detect smallest amount of calcium o Look for opaqueness on Xray film ▪ Opaqueness = decalcification is not complete o Disadvantage: not suited for mercuric chloride fixed tissue Chemical Method/ Calcium oxalate test o Procedure: 1. 5 ml of discarded fluid + strong ammonia 2. If alkaline (check with litmus paper) add ammonium oxalate 3. Let it stand for 30 mins o Discarded fluid - fluid where the tissue was immersed o (+) cloudiness – must be decalcified further o Clear - decalcification is complete Bubble Test - adding calcium carbonate o Presence of bubble - decalcification is not complete • • • • POST DECALCIFICATION • • • • Removing excess decalcifying agents from tissues Decalcified tissues are neutralized by o Immersing in saturated lithium carbonate or 5-10% sodium bicarbonate o Rinsing in tap water - most common method o Storing in formol saline with 15% sucrose or PBS with 15-20% sucrose at 4 degC Tissue softeners for unduly hard tissues - 2% HCl, 1% HCl in 70% alcohol, Perenyi’s fluid, Molliflex Lendrum’s method - immersing specimen in 4% aqeuous phenol for 1-3 days to soften hard tissues DEHYDRATION • • • • • • • Following fixation A mandatory step in tissue processing Can be carried out using autotechnicon Removal of intercellular & extracellular water from tissues after fixation & prior to wax impregnation Uses dehydrating agents of alcohol Amount of dehydrating agent should not be less than 10x the volume of the specimen Most commonly used dehydrating agent is alcohol (used in increasing concentrations) CEU Malolos BSMT 2023 Initial concentration o For Routine - 70-80% ▪ 95% as initial concentration will harden only the surface, deeper parts won’t be completely penetrated ▪ 40% as initial; use of lower concentration tend to macerate tissues o For Delicate Tx i.e. embryo - 30% • • • Acetone o Can fix and dehydrate tissues at the same time o Not commonly used because it evaporates easily and it is highly flammable o Fast-acting, suited for urgent biopsies o Not for routine dehydration Ethylene Glycol monoethyl ether o a.k.a cellusol o Combustible at 110-120F o Toxic by ingestion, inhalation, and skin contact (prolonged exposure) o Toxic to fetal, reproductive, urinary, and blood system o Decomposes upon exposure to sunlight Triethyl phosphate - For sections and smears o Not commonly used o Only causes minimum shrinkage Tetrahydrofuran (THF) o Can dehydrate and clear tissues at the same time o Toxic if ingested or inhaled o Vapors causes nausea, dizziness and headache o With offensive odor, room should be well ventilated o May cause conjunctival irritation Diethylene Dioxide / Dioxane o Can act both as dehydrating agent and clearing agent o Tissues tend to ribbon poorly o Main disadvantage: extremely dangerous & toxic to man o Tissues under dioxane tend to ribbon poorly (causes issues during section cutting) o Methods Of Dioxane Dehydration ▪ Graupner’s • Immersion of 3 changes of pure dioxane • Followed by 3 changes of paraffin wax • Embed to cool in water • Do not require clearing phase because dioxane is dual purpose ▪ Weiseberger’s • Tissue wrap in gauze bag and placed in a bottle with dioxane and anhydrous calcium oxide or quicklime • Dioxane - will remove water from tissue • Water is absorbed by anhydrous calcium oxide or quicklime Ethoxyethanol Polyethylene glycol Alcohol o Most commonly used dehydrating agent o Ethanol - used for routine dehydration ▪ Most commonly used ▪ Fast-acting and nontoxic o Methyl alcohol - not commonly used ▪ Toxic ▪ Suited for blood and tissue films HISTOPATH - M2 • 2 o • Butyl alcohol (butanol) - for animal and plant microtechniques o Isopropanol/ IPA - used as ethanol substitute ▪ Used in microwave technique ▪ Used as xylene substitute for clearing phase o Tertiary Butanol o Pentanol – dissolves paraffin o Important considerations when using alcohol: ▪ Use in increasing concentration ▪ For smaller and delicate tissues (30% as initial concentration) ▪ Prolonged storage in low alcohol can macerate tissues (70-80% initial concentration is recommended) ▪ Longer storage in 70-80% can affect staining ▪ Hastens at high temperatures (heat at 37C) ▪ Anhydrous copper sulfate - accelerates dehydration; used as indicator for water saturation • Blue discoloration of copper sulfate - indicate full saturation of dehydrating fluids with water Dehydrating agents for EM: ethanol, propylene oxide, acetonitrile • • • • • • • • DEALCOHOLIZATION/CLEARING • • • • Removal of dehydrating agent from tissues to replaced it with a medium that will dissolve the wax with which the tissues is to be impregnated. The use of clearing agents will make tissues transparent o And will improve their refractive index o Not all clearing agent make tissue clear Incomplete clearing can lead to uneven H&E staining and poor nuclear chromatin patterns Prolonged clearing time will make tissues become brittle CLEARING AGENTS • • • • Cedarwood oil o Extremely slow clearing agent o Clearing time: 2-3 days o For cytological studies like smooth muscles of skin and CNS tissues o For dense tissue like uterus Aniline Oil o For clearing embryos, insects, and other very delicate specimens Clove Oil o Tissues tend to become adulterated Carbon tetrachloride o Similar to chloroform but cheaper Methyl benzoate, amyl acetate & methyl salicylate o Extremely slow clearing agent o Used in double embedding process N-butyl acetate o Used as xylene substitute Limonene o Obtained from citrus fruit Others: o Oil of bergamot - used to clear smooth muscles of the skin o Oil of origanum - used to clear smooth muscles of the skin o Oil of wintergreen – for delicate tissues o Carbon disulfide – for smooth muscles; with foul odor o Carbon xylene – for friable tissues o Terpineol – for eye specimens ▪ Has faint odor ▪ With low evaporation rate ▪ Good substitute for cedarwood oil o Phenol – for o High Test Aviation Lead Free gasoline- excellent clearing agent • Xylene o Routine clearing agent o With rapid clearing time 30 mins – hours o NOT suited for nervous tissues and lymph nodes o It becomes milky when an Incompletely dehydrated tissue is immersed in it Toluene o Substitute for xylene and benzene o 1-2 hours o Disadvantage: acidifies in a partially filled vessel Benzene o Fast-acting o Suited for urgent biopsies o Highly flammable and carcinogenic, can damage BM leading to aplastic anemia (prolonged exposure) Chloroform o Not a routine clearing agent o Recommended for clearing tough tissues (skin, fibroid and decalcified tissues), for nervous tissues, lymph nodes and embryos o Tissues tend to float; remedy: wrap tissue in a gauze to keep at the bottom o Toxic to the liver o Tissues do not become translucent with chloroform o Can lead to wax deterioration o For dense tissue like uterus CEU Malolos BSMT 2023 HISTOPATH - M2 • 3