HCT-Lec6-Special Processing Techniques Part 1 and 2 PDF

HISTOPATHOLOGIC & CYTOLOGIC TECHNIQUES | MYT ‘23 - ‘24 Lesson 6 : Special Processing Techniques Part 1 and 2 | Lecturer: Wendell Jeffrey G. Bayron, RMT, DTA, MSPH (c) LECTURE OUTLINE are more harmful to the tissue....

HISTOPATHOLOGIC & CYTOLOGIC TECHNIQUES | MYT ‘23 - ‘24 Lesson 6 : Special Processing Techniques Part 1 and 2 | Lecturer: Wendell Jeffrey G. Bayron, RMT, DTA, MSPH (c) LECTURE OUTLINE are more harmful to the tissue. High concentrations and greater amounts of fluid will increase the I. Introduction speed of the process. II. Conventional Tissue Processing An occurrence that might happen during the calcification is that III. Principle of Decalcification sometimes, the acid or chelator from your decalcifying agent IV. Factors Affecting the Rate of Decalcification deplete rapidly due to the reaction from the calcium in the bone. V. Procedure of Decalcification ○ This can be avoided by: VI. Decalcification ▪ Using large volumes of fluid compared with the volume of the VII. Other Methods of Decalcification tissue I. INTRODUCTION ▪ Change the solution several times during the entire Decalcification is a critical process in histopathology when dealing with decalcification process bones and other calcified tissues. Because the acid might deplete due to the reaction, the This procedure involves the removal of calcium salts to prepare the tissues recommended ratio of fluid to tissue is “20:1” for microscopic examination. ○ The same with fixation. Removal of calcium of ions from a bone or calcified tissue through a Fluid Access histological process. Tissues that must undergo decalcification: ○ Bones ○ Calcified tumors ○ Teeth ○ Calcified heart valves Decalcification allows the histotechnologist to cut decalcified tissues like any other soft tissues in the body. This requires bone and calcified specimen to be submerged in the As with fixation, a fresh decalcifier must have ready and effective 2 decalcifying agents for days or even weeks depending on the size of the access to all surfaces of the specimen. tissue ○ An effective and complete fluid access will enhance diffusion and Conventional Tissue Processing penetration in the specimen thus facilitating in ionization and 1. Fixation removal of calcium. 2. Decalcification (**) Note: As soon as fixation is complete, the selected pieces of tissues are 3. Dehydration usually placed in gauze bag and suspended in a liberal amount of 4. Clearing decalcifier by means of a thread, making sure that it does not touch the 5. Infiltration bottom of the container to protect the tissue from any precipitate that may 6. Embedding have settled in the bottom. 7. Trimming 8. Sectioning Size and Consistency 9. Staining The bigger the size of the tissue, the longer the period for complete 10. Mounting decalcification. 11. Labeling 3 ○ The denser the tissue, the longer the period for complete decalcification LECTURER’S NOTE Dense bone tissues like compact bones usually require up to 14 days However, if we are going to use a calcified tissue such as bone tissue, or longer to complete the decalcification process. then we are going to insert decalcification between fixation and dehydration. Agitation ○ Thus, decalcification is an optional process. 4 Gentle agitation by low speed rotation, rocking, stirring or bubbling A1. BASIC PRINCIPLES OF DECALCIFICATION air into the solution may increase the rate of decalcification. The basic principle of decalcification is the removal of the substance that makes the bone hard which is why we remove the calcium. Temperature In order to do that, we can use the following: 5 Increased temperature will increase the rate of decalcification, but it 1. Strong Mineral Acid or Weak Organic Acid will also increase the damaging effects of acids on tissue. Move calcium from the bone into the decalcifying solution. Optimum temperature recommended is room temperature: 18-30oC Forms soluble calcium salts in anion exchange that moves calcium out of the bone and into the decalcifying solution Decalcification Procedure (TAKE NOTE) 2. Chelating Agents 1. Selection of Tissue Sequesters metallic ions (including calcium) in aqueous solutions. 2. Fixation 3. Decalcification CRITERIA OF A GOOD DECALCIFYING AGENT 4. Acid Neutralization 5. Washing 1 Complete removal of calcium II. SELECTION OF TISSUE 2 Absence of damage to tissue, cells, or fibers Happens after receiving the specimen in the laboratory and during gross examination with the pathologist 3 Non-impairment of subsequent staining technique Ideal Thickness: ○ Dense Bones: 2-5 mm 4 Reasonable speed of decalcification ○ Soft Bones: 4-6 mm A2. FACTORS AFFECTING THE RATE OF DECALCIFICATION Concentration 1 More concentrated acid solutions decalcify bone more rapidly—but Alforque, Antepasado, Banal, Batuan, Bercede, and Ceracas 1  ○ Acidic dyes like eosin are less affected. LARGE SPECIMEN Prolonged use can macerate the tissues. Important that an appropriate end-point test is used to minimize the exposure of the specimen to these agents. Example: ○ Nitric Acid ○ Hydrochloric Acid A large fine-toothed bone saw, or hacksaw is used in cutting the bone. 1. NITRIC ACID Coarse saw is not advisable. Most common. ○ Can cause considerable mechanical damage and force bone fragments or Fastest decalcifying agent used so far. bone dusts into the soft tissue present in the specimen. Produces minimal distortion, therefore, it is recommended for routine ○ Later on, it will cause problems in processing and staining. purposes. However, at higher concentrations (concentrated solution), nitric acid inhibits SMALL SPECIMEN nuclear stain and destroys tissue. ○ Prevented by combining nitric acid with formaldehyde or alcohol. If used alone or as a simple aqueous solution, its recommended concentration is 5-10%. Examples of nitric acid decalcifying agents: ○ Aqueous nitric acid solution 10% ○ Formol-nitric acid A geological cutting machine fitted with a diamond impregnated cutting disc ○ Perenyi’s fluid is used. ○ Phloroglucin-nitric acid III. FIXATION 10% AQUEOUS NITRIC ACID SOLUTION Protects the cellular and fibrous elements of bone from damage caused Decalcification times : 12-24 hours by acids used in decalcifying agents. ○ Tissue damage is four times greater when the tissue is unfixed. ADVANTAGES DISADVANTAGES In fixation, we need to make sure that the tissue is adequately fixed, and it must be rinsed well prior to decalcification. Rapid in action Imparts a yellow color with Poorly fixed specimens become macerated during decalcification and stain Easily removed by 70% alcohol nitrous acid, thereby impairing poorly afterwards. Recommended for urgent the staining reaction of the ○ Therefore, a common practice in the laboratory is to extend fixation time biopsy, and for needle and tissue. for bone specimens before commencing decalcification. small biopsy specimens to Strong acids tend to be more These are the preferred fixatives for bone, bone marrow, tooth samples, and permit a rapid diagnosis within damaging to tissue antigens samples for electron microscopy. 24 hours or less. for immunohistochemical Minimum distortion of tissues. staining, and enzymes may be FIXATIONS USED Produces good nuclear totally lost. staining. Prolonged decalcification may Satisfactory Fixative for 1. Buffered Formalin Can be used for large or lead to tissue distortion. Bone heavily mineralized cortical Can seriously damage tissue 1. Zinc Formalin Mixtures bone specimens. sustainability. Good Preservation of 2. B-5, Formol-Acetic Alcohol (Davidson’s Bone Marrow Fixative) FORMOL-NITRIC ACID 3. Bouin’s Solution Decalcification times : 1-3 days Tooth Specimen 1. 15% Formic Acid ADVANTAGES DISADVANTAGES Electron Microscopy 1. Glutaraldehyde Produces less tissue destruction Imparts a yellow color with than 10% aqueous nitric acid. nitrous acid which impairs NOTE: Memorize and familiarize everything. Rapid in action; hence, it is staining reaction of the cell. IV. DECALCIFICATION recommended for urgent Prevented by: After fixation, decalcification proper will commence. biopsies. - Neutralizing the tissue with Three Main Types of Decalcifying Agents Nuclear staining is relatively 5% sodium sulfate and 1) Those based on strong mineral acids. good. washing in running tap ○ Principle of acid decalcification. water for at least 12 hours. 2) Those based on weaker organic acids. - Addition of 0.1% of urea to ○ Principle of acid decalcification. pure concentrated nitric 3) Those composed of chelating agents. acid. ○ Principle of chelation method Solution should be used inside A4. TWO METHODS OF DECALCIFYING AGENT a fume hood. Acid Method PERENYI’S FLUID ○ Most widely used ○ Stable Decalcification times : 2-7 days ○ Readily available ADVANTAGES DISADVANTAGES ○ Relatively inexpensive Chelation (Chelating Agents) Decalcifies and softens tissue Complete decalcification Two (2) Types of Acid Method: at the same time. cannot be determined by 1. Strong Mineral Acids Maceration is avoided due to chemical test. 2. Weak Organic Acids the presence of chromic acid Slow decalcifying agent for STRONG MINERAL ACIDS and alcohol. dense bones. Most rapid in action but if used longer than necessary will: Recommended for routine ○ Cause a loss of nuclear staining (failure of nuclear chromatin to take up purposes. basic dyes like hematoxylin). Nuclear and Cytoplasmic Alforque, Antepasado, Banal, Batuan, Bercede, and Ceracas 2  Examples: Staining is Hood. ○ 10% formic acid ○ formic acid sodium citrate solution. PHOROGUCIN-NITRIC ACID Decalcification time : 12-24 hours 1. 10% FORMIC ACID Decalcification time : 2-7 days ADVANTAGES DISADVANTAGES ADVANTAGES DISADVANTAGES Most rapid decalcifying agent Nuclear staining is poor. so far. Prolonged decalcification May be used as both fixative Relatively slow Same with your Aqueous produces extreme tissue and decalcifying agent. Requires neutralization with 5% Permits excellent nuclear and sodium sulfate and washing solution. distortion. cytoplasmic staining. Therefore, it is recommended Complete decalcification out to remove the acid from Recommended for small pieces for urgent biopsies cannot be determined by the tissue. of bones and teeth. chemical test. Suitable for most routine Imparts yellow color. surgical specimens particularly when immunohistochemical staining is needed. EDITOR’S NOTE When decalcification is complete, the acid must be removed to prevent 2. FORMIC ACID - SODIUM CITRATE SOLUTION reactions with the chemicals used in the succeeding histological Decalcification time : 3-14 days processes. ADVANTAGES DISADVANTAGES For the 4 preparations of nitric acid solution: when decalcification is complete à the acid must be removed by three changes of 70% - 90% Permits better nuclear staining Relatively slow ethanol than nitric acid method. Requires neutralization with 5% Recommended for autopsy sodium sulfate. 2. HYDROCHLORIC ACID materials, bone marrow, Inferior compared to nitric acid because of its slower action and greater cartilage and tissue studies for distortion of tissue produced on the decalcified section. research purposes. However, it produces good nuclear staining. ○ If used in 1% solution with 70% alcohol, it may be recommended for surface 3. TRICHLOROACETIC ACID decalcification of tissue blocks. Decalcification times : 4-8 days Example: Von Ebner’s fluid VON EBNER’S FLUID ADVANTAGES DISADVANTAGES ADVANTAGES DISADVANTAGES Permits good nuclear staining. Weak decalcifying agent. Does not require washing out. Not used for dense tissues and Permits relatively good Complete decalcification The excess acid may be is suitable only for small cytologic staining. cannot be determined by removed by several changes of spicules of bone. A moderately decalcifying chemical test. 90% alcohol, thus improving Very slow-acting. agent. tissue dehydration. Does not require washing out before dehydration. 4. SULFUROUS ACID Recommended for teeth and Very weak decalcifying solution small pieces of bone. Only suitable for minute pieces of bone 5. CHROMIC ACID (FLEMMINGS FLUID WEAK ORGANIC ACIDS Acetic and Formic Acid (Organic Acids) ADVANTAGES DISADVANTAGES ○ Better suited to bone marrow since they are not as harsh. Act more slowly on dense cortical bone May be used both as a fixative Inhibits nuclear staining. Picric acid and Acetic acid and decalcifying agent. Undergoes reduction. ○ Not used alone as decalcifying agents but are found as components of For minute bone spicules. Form precipitate at the bottom Carnoy’s and Bouin’s fixatives. of the container; thus requiring May act as incidental weak decalcifiers and they are used mostly in urgent frequent changes of solution: it cases when there is only minimal calcification. is not cost-effective. Examples: Forms insoluble pigments when ○ Formic Acid decalcified tissue is dehydrated ▪ 10% Formic Acid with alcohol. ▪ Formic acid - sodium citrate solution End point cannot be ○ Trichloroacetic Acid determined by a chemical test. ○ Sulfurous Acid Environmental toxin and ○ Chromic Acid (Flemming’s Fluid) carcinogenic. ○ Citric Acid - Citrate Buffer Solution (pH 4.5) FORMIC ACID 6. CITRIC ACID - CITRATE BUFFER SOLUTION Moderate-acting decalcifying agent pH = 4.5 Produces better nuclear staining with less tissue distortion. Decalcification Time:: 6 days Safer to handle than HNO3 (Nitric acid) or HCl (Hydrochloric Acid) So formic acid can be used as a simple 10% aqueous solution or combined ADVANTAGES DISADVANTAGES with a formalin or with a buffer. ○ 10% formic acid is the best all around decalcifier. Permits excellent nuclear and Too slow for routine purposes. It is the only weak acid used extensively as a primary decalcifier. cytoplasmic staining. So addition of citrate can accelerate decalcification by chelating the Does not produce cell or tissue calcium. distortion. Alforque, Antepasado, Banal, Batuan, Bercede, and Ceracas 3  B4. CHELATION METHOD ○ So you have to take note of the calcifiers whose endpoints cannot be determined by chemical tests CHELATING AGENTS Substances which combine with calcium ions and other salts (Fe, Mg) to CHEMICAL TEST PROCEDURE 1 form weakly dissociated complexes and facilitate removal of calcium salt. Chelating agents are preferred over acid if preservation of nuclear DNA is Insert pipette into the discarded decalcifying solution. important or if histochemical methods for nucleic acids and enzyme activities are intended. 1 Why withdraw 5ml of the discarded solution? Example: Because decalcifying fluids are usually changed every 24-48 hours, ○ EDTA and the chemical test is performed on the discarded fluid. ETHYLENE DIAMINE TETRACETIC ACID SALT Withdraw approximately 5 ml of the discarded decalcifying solution and Most common chelating agent in the market 2 place it in a test tube. Commercial name: Versene Combines with calcium, forming an insoluble non-ionized complex. Add approximately 10 ml of the ammonium hydroxide/ ammonium oxalate 3 Works by capturing the calcium ions from the surface of the apatite crystal, working solution ammonium slowly reducing its size. Small specimens: 1-3 weeks 4 Mix well Dense cortical bone: 6-8 weeks (or longer) 5 Let it sit overnight Solution should be changed every 3 days Example: REAGENTS USED ○ Neutral EDTA Add equal parts: NEUTRAL EDTA ○ 5% Ammonium hydroxide stock ADVANTAGES DISADVANTAGES ○ 5% Ammonium oxalate stock RESULTS Permits excellent staining Very slow for urgent and results. routine purposes Turbidity or cloudiness (after letting it sit overnight) indicates the presence Produces minimal cell and Causes slight tissue hardening. of calcium. tissue distortion. InactivateS ALP (alkaline Solution: prolong decalcification and change the solutions. Forms minimal cell and tissue phosphatase) actively If there is a clear solution, there is complete decalcification. distortion restored by addition of MgCl) ○ You may now proceed to the next step of tissue processing. Forms minimal histological artifacts CHEMICAL TEST PROCEDURE 2 Endpoint can be measured by chemical test. 1 Take 5 ml of discarded decalcifying fluid Excellent for enzyme or IHC (Immunohistochemical) 2 Add a piece of litmus paper or use a pH meter with magnetic stirrer staining, and for E/M Solutions can be adjusted to Add ammonium hydroxide drop by drop, shaking after each drop, until litmus 3 specific pH for enzyme paper indicators solution is neutral (pH 7) 4 Add 5 ml of saturated ammonium oxalate C4. DETERMINING THE END POINT OF DECALCIFICATION 5 Shake well OVER-DECALCIFICATION UNDER-DECALCIFICATION 6 Allow the solution to stand for 30 minutes Spoils the staining of basophilic When the tissue is allowed to stay in elements (particularly with strong the decalcifying agent for a very REAGENTS USED acids.º short period of time, decalcification may be incomplete, thus interfering Add equal parts: with the normal cutting of sections ○ 5% Ammonium hydroxide stock and staining of specimens. ○ 5% Ammonium oxalate stock RESULTS Causes maceration of the softer - tissue elements Formation of white precipitate (calcium hydroxide) after the addition of ammonium hydroxide indicates a large quantity of calcium. D4. METHODS TO CHECK IF DECALCIFICATION IS COMPLETE ○ This makes it unnecessary to proceed to step #4 as this will surely turn it PHYSICAL TEST positive. If there is no presence of white precipitate, the procedure may continue Unreliable method. with the addition of saturated ammonium oxalate Testing for the pliability of the specimen If the solution is clear after 30 minutes, then decalcification is complete Require manipulation, bending, probing, or trimming of the specimen to E4. BUBBLE TEST "feel" for remaining calcified areas Acid reacts with calcium carbonate in bone to produce carbon dioxide seen In physical testing, you try to bend the specimen. as layers of bubbles at the surface of the bone during acid decalcification. ○ If successful then it is decalcified. Subjective and unreliable Alternative method: pricking the tissue with a fine needle or probe. TINY BUBBLES: indicate less calcium is present CHEMICAL TEST F4. RADIOGRAPHY (XRAY) We also have chemical tests otherwise known as calcium oxalate test. Best method Simple and reliable. Particularly with large specimens (e.g. femoral heads) NOT done for EDTA decalcifiers. Downside: very expensive Cannot be done for some acid decalcifiers. Although the most ideal, most sensitive, and most reliable method due to its ability to detect even the smallest focus of calcium which appears opaque in Alforque, Antepasado, Banal, Batuan, Bercede, and Ceracas 4  an X-ray plate. VIII. MISCELLANEOUS TOPICS A8. SURFACE DECALCIFICATION LECTURER’S NOTE After determining the endpoint of decalcification, you should now This is a method dealing with small unexpected deposits of calcium that proceed to the next step of the decalcification process which is ACID may be encountered in paraffin blocks. NEUTRALIZATION When the paraffin-embedded block has been trimmed, the tissue surface may reveal small foci of calcification that may cause resistance or a grating V. ACID NEUTRALIZATION sensation when sectioned with a microtome knife. ○ If this is encountered, the block can be removed from the chuck and The chemical used in decalcification should be neutralized in order to prevent placed face down on a pad of cotton or gauze saturated with 10% HCl for a reaction of the chemicals to the succeeding pathologic processes. approximately one hour. The block can then be thoroughly rinsed with water to remove residual acid Chemical neutralization is accomplished by immersing the decalcified then chilled and sectioned again. bone in either: ○ Saturated lithium carbonate solution B8. TISSUE SOFTENERS ○ 5-10% aqueous sodium bicarbonate solution Unduly hard tissues are liable to damage the microtome knives and may Done for several hours. require tissue softeners aside from decalcification. Perenyi’s Fluid VI. THOROUGH WASHING ○ Acts as both decalcifier and tissue softener Generally, a short effective wash in tap water should be sufficient. ○ To soften unduly hard tissues, immerse the tissue in the fluid for 12-24 Adequate tap water rinsing can be accomplished in: hours. ○ Small samples = 30 mins C8. ARTIFACTS ○ Larger specimens = 1-4 hours After this, you can proceed to the next step of tissue processing which is UNDER - DECALCIFICATION OVER - DECALCIFICATION dehydration. Nuclear detail lost or severely Inability to section (tissue is still QUICK RECAP compromised. hard). THE GENERAL TECHNIQUES THAT MUST BE FOLLOWED DURING Disruption of cell membrane Incomplete infiltration of the DECALCIFICATION ARE FF: and cytologic properties. Paraffin. 1) Selection of tissue Loss of glycogen. REMEDY: Surface 2) Fixation Swelling of tissue, especially decalcification, redecalcification. 3) Decalcification proper collagen. 4) Acid neutralization 5) Thorough washing LECTURER’S NOTE Just familiarize the effects of under-decalcification and over-decalcification. VII. OTHER METHODS OF DECALCIFICATION - END OF PART 1 - A7. ION EXCHANGE RESIN Most commonly used ion exchange resin: Ammonium form of polystyrene. Uses a mixture of formic acid and ion exchange resin ION EXCHANGE RESIN PROCEDURE 1 Spread 1⁄2 inch-thick ion exchange resin over the bottom of the container 2 Place the specimen on top of it. 3 Add a decalcifying agent 20-30 times the volume of the tissue. 4 Allow the tissue to stay in the solution for 1-14 days 5 Measure end point using physical or the X-ray method B7. ELECTROPHORESIS (ELECTRICAL IONIZATION) Principle: ○ A process whereby positively charged calcium ions are attached to a negative electrode and subsequently removed from the decalcifying solution. Time required is shortened to 8 hours due to the heat and electrolytic reaction produced in the process. Not done at room temperature (RT).. Uses water bath (30 – 45°C). Decalcifying fluid used: combination of 88% formic acid and HCl. ELECTROPHORESIS DISADVANTAGES 1 Electrically decalcified tissues generally do not stain well. 2 Only a limited number of specimens can be processed at one time. C7. MICROWAVE OVEN DECALCIFICATION In this method, hard tissues are placed in a decalcifying agent in a microwave oven for intermittent periods with regular changes of the solution until end-point is reached. Microwave irradiation has been shown to speed up the process of decalcification significantly from days to hours. Temperature restriction: 42-45°C Same microwave used in the automated tissue processing technique. Alforque, Antepasado, Banal, Batuan, Bercede, and Ceracas 5  Used in immunofluorescent and immunohistochemical staining. PART 2 Some specialized silver stains, particularly in neuropathy. LECTURE OUTLINE A4. FROZEN SECTION: DISADVANTAGES I. Definition of Frozen Section Morphological detail and resolution of tissue are usually inferior compared to II. Purpose and Importance of Frozen Section paraffin-embedded tissues. III. Advantages and Disadvantages of Frozen Section ○ During cutting, tissues produced are somewhat distorted because of the IV. Methods of Frozen Section lack of embedding medium. V. Types of Microtome used ○ That is why even after there has been a biopsy result already released, the VI. Staining Methods frozen tissue blocks are still fixed after the procedure and will still go through the routine tissue processing. I. INTRODUCTION: FROZEN SECTION ○ This is done for further confirmation of the diagnosis. During the performance of surgical procedures, it is necessary to get a rapid Staining is not as satisfactory compared to properly fixed tissues resulting in diagnosis of a pathologic process. poor quality of final slides. The surgeon during operation may want to know if the margins of his Thin sections are not obtained. resection are free from tumor before closing up the patient ○ Only about 5-10 um sections are obtained. ○ Frozen section is important in cases where unexpected disease may be ○ Whereas, paraffin sections are about 4-6 um found that requires immediate diagnosis so that the surgeon can decide II. METHODS OF PREPARING FROZEN SECTION what to do next. A1. DEFINITION OF FROZEN SECTION A2. FREEZING MICROTOME OR COLD KNIFE PROCEDURE Frozen section is a specific type of biopsy procedure that allows the surgeon Freezing of tissue blocks is possible through a simple lever operated valve to establish rapid diagnosis during surgery. that allows the release of rapid intermittent bursts of carbon dioxide. It is especially recommended when lipids and nervous tissue elements are to A second cooling device is also incorporated for lowering the temperature of be demonstrated. the knife. ○ This is because of fixatives, except osmium tetroxide that allows fat to be This microtome is firmly clamped onto the edge of the bench for use, or dissolved during processing. mounted on an especially constructed shelf, with a CO2 cylinder below. ○ This is usually done on muscle biopsy, and surgically removed tumors. Success of this procedure depends upon ambient temperature and humidity. ○ Since it is very difficult to cut sections in a hot or humid environment. PROCEDURE: FROZEN SECTION ○ Ease of cutting and quality of sections are always improved if done in a In the laboratory, frozen sections need to be scheduled cold room Once the patient is admitted and his/her surgeon wants to perform a frozen Sections thinner than 6um generally cannot be obtained using this method. section, they will call the Histopathology department and ask for the Using a cold knife in a controlled cold environment, optimum condition for availability of the pathologist. sectioning shall be provided for by the following temperatures: Pathologists will be the ones who will perform the cutting of the specimen, reading of the tissue slide produced, and the ones who will release the OPTIMUM CONDITION frozen section biopsy result. ○ After all of these are cleared and scheduled, on the day of operation, a KNIFE -40 °C to -60 °C runner, usually a senior clerk, is asked to transport the specimen to the TISSUE -05 °C to -10 °C histopathology department of the laboratory. ○ The specimen of the frozen section is a fresh tissue and unfixed. ENVIRONMENT 0 °C to -10 °C Once the tissue arrives, the pathologist will then get a section from the tissue A3. CRYOSTAT/CRYOCUT (COLD MICROTOME) 1 and freeze it inside a cryostat. This method makes use of the cryostat, an apparatus used in fresh tissue microtome. Once frozen, the pathologist will now section the tissue and mount it on a The cryostat consists of an insulated microtome housed in an electrically 2 glass side, fix it briefly, and then proceed to staining. driven refrigerated chamber. Produces thin and even sections After staining, the slides will be mounted with coverslip and submitted to the 3 ○ Since the type of microtome used is a rotary microtome and we can set pathologist. the desired thickness for those kinds of microtomes If the pathologist has made a diagnosis already, it is then immediately Temperature is maintained at -20°C where the microtome, the knife, the relayed to the senior clerk who transported the specimen, and to the specimen, and the atmosphere are kept at the same temperature since they 4 are all housed in one refrigerated chamber. operating room so that the surgeon will be informed of the diagnosis, and it will help him in choosing his next plan of action. OPTIMUM CONDITION 5 All of these happen in around 30 minutes. -18 °C to -20 °C A2. STEP BY STEP PROCESS OF FROZEN SECTION Generally -20 °C 1 Fresh tissue is frozen on a freezing microtome or cryostat. IV. DIFFERENT KINDS OF FREEZING AGENTS 1. LIQUID NITROGEN 2 Cutting of tissue. ○ Generally used, most rapid of the commonly available freezing agents. 3 Mounting frozen sections on a glass slide. 2. ISOPENTANE COOLED BY LIQUID NITROGEN ○ Liquid at room temperature. 4 Fixed briefly. ○ Excellent for muscle tissue. 3. CARBON DIOXIDE GAS 5 Staining. ○ Used in freezing microtome 4. AEROSOL SPRAYS A3. FROZEN SECTION: ADVANTAGES ○ Quick-freezing spray cans of fluorinated hydrocarbons which have Rapid pathologic diagnosis during surgery. become popular in recent years (e.g. Cryokwik) Diagnostic and research for enzyme histochemistry. ○ Since enzymes are generally affected by the chemical used in routine V. SPECIAL PROCESSING TECHNIQUES tissue processing. These techniques are done if some studies are concerned, like evaluation Diagnostic and research demonstration of soluble substances (lipids and involving enzyme studies and if chemical fixation of tissue blocks is to be carbohydrates). avoided. ○ NOTE: Lipids are dissolved during tissue processing. In most instances, frozen section is deemed to be the most ideal and Alforque, Antepasado, Banal, Batuan, Bercede, and Ceracas 6  preferred means of preserving tissues in order to avoid complete or partial loss of enzymes consequent to chemical fixation. A5. SPECIAL PROCESSING TECHNIQUES: TWO METHODS 1. Freeze Drying 2. Freeze Substitution Like fresh frozen sections, these special techniques have the common principle of rapidly preserving the tissue block by freezing or quenching. FREEZE DRYING STEP BY STEP PROCESS OF FREEZE DRYING Fresh tissue is frozen 1 Using liquid nitrogen. Desiccation (Vacuum Chamber) 2 Subsequently, desiccated or the removal of water molecules in a vacuum chamber. Infiltration 3 After desiccation, the tissue is removed and infiltrated. Embedding 4 Embedded with paraffin wax or celloidin. Paraffin-embedded block (end-product) 5 The end product is still a paraffin-embedded block. Except, it does not go through a chemical fixation. FREEZE SUBSTITUTION STEP BY STEP PROCESS OF FREEZE SUBSTITUTION 1 Fresh tissue is frozen 2 Fixation (Rossman’s Formula, 1% Acetone) 3 Dehydration (absolute alcohol) 4 Embedding 5 Paraffin-embedded block (end-product) It is basically the same procedure as freeze drying. The only variation is that the frozen tissue, instead of being subjected to dehydration in an extensive vacuum drying apparatus, it is fixed in Rossman’s Formula or in 1% Acetone and dehydrated in absolute alcohol. B5. STAINING METHODS FOR FROZEN SECTIONS Rapid H&E Stain Most commonly used 1 Permanent stain Basically, the same as the routine H&E stain ○ EXCEPT the time for staining is shortened since frozen section is a stat test Metachromatic Stain Temporary stain 2 Examples: ○ Thionine ○ Toluidine blue Polychrome Stain 3 Examples: ○ Giemsa (temporary stain) - END OF PART 2- Alforque, Antepasado, Banal, Batuan, Bercede, and Ceracas 7