Histotechniques Dr Mulazim Hussain Bukhari MBBS DCP MPhil

Histotechniques Dr Mulazim Hussain Bukhari MBBS, DCP, MPhil, FCPS, Ph. D Associate Prof Pathology King Edward Medical University, Lahore 25 th October 2008, Saturday CME, DEpartment of Pathology, King Edward Medical University, Lahore 1

Tissue Processing n n n Specimen Accessioning Gross Examination Fixation Tissue Processing Sectioning Frozen Sections Staining H and E staining Cover slipping Decalcification Artifacts in Histologic Sections Problems in Tissue Processing

Safety in the Lab n n The lab should be well illuminated and well-ventilated. Rules and Regulations governing n n formalin and hydrocarbonds n such as xylene n and toluene. Limits set by the Occupational Safety and Health Administration (OSHA) that should not be exceeded. These limits should be revised and revived to reduced any mishap

Cont. n Every chemical compound used in the laboratory should have a materials safety data sheet on file n n that specifies the nature, toxicity, and safety precautions to be taken when handling the compound. The laboratory must have a method for disposal of hazardous wastes. n n Health care facilities processing tissues often contract this to a waste management company. Tissues that are collected should be stored in formalin and may be disposed by incineration or by putting them through a "tissue grinder" attached to a large sink (similar to a large garbage disposal unit).

Cont. n n n Check the sharpness of scalpel, scissors and quality of other ones like ruler, probes weighing machines Every instrument used in the laboratory should meet electrical safety specifications and have written instructions regarding its use. Flammable materials may only be stored in approved rooms and only in storage cabinets that are designed for this purpose.

Cont. n Fire safety procedures are to be posted. n n Safety equipment including fire extinguishers, fire blankets, and fire alarms should be within easy access. A shower and eyewash should be readily available. No smoking, eating or movements in the labs n Use disposable gloves n

Cont. Laboratory accidents must be documented and investigated with incident reports and industrial accident reports. n Specific hazards that you should know about include: n n Bouin's solution is made with picric acid. This acid is only sold in the aqueous state. When it dries out, it becomes explosive.

Sodium azide n Many reagent kits have sodium azide as a preservative. n n You are supposed to flush solutions containing sodium azide down the drain n with lots of water, or n there is a tendency for the azide to form metal azides in the plumbing. These are also explosive.

Drainage n n Benzidine, benzene, anthracene, and napthol containing compounds are carcinogens and should not be used. Mercury-containing solutions (Zenker's or B-5) should always be discarded into proper containers. Mercury, if poured down a drain, will form amalgams with the metal that build up and cannot be removed. Hazards of usually used formalin

Objective n n n Tissues from the body taken for diagnosis of disease processes must be processed in the histology laboratory to produce microscopic slides that are viewed under the microscope by pathologists. The techniques for processing the tissues, whether biopsies, larger specimens removed at surgery, or tissues from autopsy The persons who do the tissue processing and make the glass microscopic slides are histotechnologists

Specimen Accessioning n n Tissue specimens received in the surgical pathology laboratory have a request form that lists the patient information and history along with a description of the site of origin. The specimens are accessioned by giving them a number that will identify each specimen for each patient

Grossing Describing the specimen n Placing all or parts of it into a small plastic cassette n When a malignancy is suspected n n Inking a gross specimen for margins

Fixation Types of fixatives (AMAPO) n Aldehydes n Mercurials n Alcohols n Picrates n Oxidizing agents

Fixation - factors affecting fixation There a number of factors that will affect the fixation process: n Buffering n Penetration n Volume n Temperature n Concentration n Time interval n Position of tissue

Buffering n n n Fixation is best carried out close to neutral p. H, in the range of 6 -8. Hypoxia of tissues lowers the p. H, so there must be buffering capacity in the fixative to prevent excessive acidity. Acidity favors formation of formalin-heme pigment that appears as black, polarizable deposits in tissue. Common buffers include phosphate, bicarbonate, cacodylate, and veronal. Commercial formalin is buffered with phosphate at a p. H of 7.

Penetration n Penetration of tissues depends upon the n n diffusability of each individual fixative, which is a constant. Formalin and alcohol penetrate the best, and glutaraldehyde the worst. Mercurials and others are somewhere in between. One way to get around this problem is sectioning the tissues thinly (2 to 3 mm). Penetration into a thin section will occur more rapidly than for a thick section

Volume n The volume of fixative is important. n n There should be a 10: 1 ratio of fixative to tissue. Obviously, we often get away with less than this, but may not get ideal fixation. One way to partially solve the problem is to change the fixative at intervals to avoid exhaustion of the fixative. Agitation of the specimen in the fixative will also enhance fixation.

Temperature Increasing the temperature, as with all chemical reactions, will increase the speed of fixation, as long as you don't cook the tissue. n Hot formalin will fix tissues faster, and this is often the first step on an automated tissue processor. n

Concentration of fixative n Concentration of fixative should be n n n adjusted down to the lowest level possible, because you will expend less money for the fixative. Formalin is best at 10%; glutaraldehyde is generally made up at 0. 25% to 4%. Too high a concentration may adversely affect the tissues and produce artefact similar to excessive heat.

Time interval n n Also very important is time interval from of removal of tissues to fixation. The faster you can get the tissue and fix it, the better. Artefact will be introduced by drying, so if tissue is left out, please keep it moist with saline. The longer you wait, the more cellular organelles will be lost and the more nuclear shrinkage and artefactual clumping will occur

Kaiserling formula for preservation for surgical specimens for museum n n n Formalin pure Distilled water Potassium acetate(CH 3 COOK ) Chloral hydrate 5 liter 22. 5 liter 250 gm 50 gm § 27 liter Potassium acetate is used in mixtures applied for tissue preservation, fixation, and mummification. Most museums today use the formaldehyde-based method recommended by Kaiserling in 1897 and containing potassium acetate. For example, Lenin's mummy was soaked in a bath containing potassium acetate

Gough Sections: Whole organs may be sectioned on paper by the methods of Gough and Wentworth. n These sections provide valuable information on whole organ structure and serve as links between mounted museum specimens and histologic sections. n

Colour restoration n Small amount of sodium hydrosulphite to preserve the colour. n If the container is properly sealed, the colour restoration is then permanent. n For photography, the procedure is to first wash and clean the specimen. n It is then soaked in an excess of 60% ethanol until the colour has been restored satisfactorily

Characteristics of Fixatives n n n Chemical Fixatives Freeze Substitution Microwave Fixation

Ideal Fixative n n n Penetrate cells or tissue rapidly Preserve cellular structure before cell can react to produce structural artifacts Not cause autofluorescence, and act as an antifade reagent

Chemical Fixation Coagulating Fixatives n Crosslinking Fixatives n

Coagulating Fixatives Ethanol n Methanol n Acetone n

Coagulating Fixatives Advantages n n n Fix specimens by rapidly changing hydration state of cellular components Proteins are either coagulated or extracted Preserve antigen recognition often Disadvantages • Cause significant shrinkage of specimens • Difficult to do accurate 3 D confocal images • Can shrink cells to 50% size (height) • Commercial preparations of formaldehyde contain methanol as a stabilizing agent

Crosslinking Fixatives Glutaraldehyde n Formaldehyde n Ethelene glycol-bis-succinimidyl succinate (EGS) n

Cross-linking Fixatives n Form covalent crosslinks that are determined by the active groups of each compound

Principles of Fixation n Once tissues are removed from the body, they undergo a process of self-destruction or autolysis n which is initiated soon after cell death by the action of intracellular enzymes causing the breakdown of protein and eventual liquefaction of the cell.

Principles of Fixation n Autolysis is independent of any bacterial action, n n n retarded by cold, greatly accelerated at temperatures of about 30°C and almost inhibited by heating to 50°C

Cont. n Autolysis is more severe in tissues which are rich in enzymes, n n n such as the liver, brain and kidney, and is less rapid in tissues such as elastic fibre and collagen.

Cont. n By light microscopy, autolysed tissue presents a `washed-out' appearance with swelling of cytoplasm, n eventually converting to a granular, homogeneous mass which fails to take up stains.

How Autolysed tissue looks like n The nuclei of autolytic cells may show some of the changes of necrosis n n including condensation (pyknosis), fragmentation (karyorrhexis) and lysis (karyolysis) D/D these are not accompanied by an inflammatory or cellular response.

How Autolysed tissue looks like There may be diffusion of intracellular substances of diagnostic significance, such as glycogen which is lost from the cells in the absence of prompt and suitable fixation. n Autolysis also causes desquamation of epithelium which separates from its basement membranes. n

Bacterial Action on dead tissue n Bacterial decomposition can also produce changes in tissues that mimic those of autolysis and is brought about by bacterial proliferation in the dead tissue.

Bacterial Action on dead tissue n Such bacteria may normally be present in the body during life such as the non-pathogenetic organisms present in the bowel, or may be present in diseased tissues at the time of death such as in septicaemia.

The objective of fixation is to preserve cells and tissue constituents in as close n n n a life-like state as possible and to allow them to undergo further preparative procedures without change. Fixation arrests autolysis and bacterial decomposition and stabilizes the cellular and tissue constituents so that they withstand the subsequent stages of tissue processing. Aside from these requirements for the production of tissue sections, increasing interest in cell constituents and the extensive use of immunohistochemistry to augment histological diagnosis has imposed additional requirements.

Cont. Fixation should also provide for the preservation of tissue substances and proteins. n Fixation is, therefore, the first step and the foundation in a sequence of events that culminates in the final examination of a tissue section. n

Common pitfalls of fixation n n It is relevant to point out that fixation in itself constitutes a major artifact. The living cell is fluid or in a semi-fluid state, Whereas fixation produces coagulation of tissue proteins and constituents, a necessary event to prevent their loss or diffusion during tissue processing; the passage through hypertonic and hypotonic solutions during tissue processing would otherwise disrupt the cells. n n For example, if fresh unfixed tissues were washed for prolonged periods in running water, severe and irreparable damage and cell lysis would result. In contrast, if the tissues were first fixed in formalin, subsequent immersion in water is generally harmless.

Summary of objective n Fixation: Confers chemical stability on the tissue n Hardens the tissue (helps further handling) n Halts enzyme autolysis n Halts bacterial putrefaction n May enhance later staining techniques n Introduces a 'consistent artifact' n

Aldehydes include formaldehyde (formalin) and glutaraldehyde. n Tissue is fixed by cross-linkages formed in the proteins, particularly between lysine residues. n This cross-linkage does not harm the structure of proteins greatly, so that antigenicity is not lost. n

Cont. Therefore, formaldehyde is good for immunoperoxidase techniques. Formalin penetrates tissue well, but is relatively slow. n The standard solution is 10% neutral buffered formalin. n A buffer prevents acidity that would promote autolysis and cause precipitation of formolheme pigment in the tissues. n

Formaldehyde n n Formaldehyde, as 4% buffered formaldehyde (10% buffered formalin), is the most widely employed universal fixative particularly for routine paraffin embedded sections. It is a gas with a very pungent odor, soluble in water to a maximum extent of 40% by weight and is sold as such under the name of formaldehyde (40%) or formalin (a colorless liquid).

Formaldehyde n Formaldehyde is also obtainable in a stable solid form composed of high molecular weight polymers known as paraformaldehyde.

Cont. n n n Heated paraformaldehyde generates pure gaseous formaldehyde which, when dissolved in water, reverts mostly to the monomeric form. Aqueous formaldehyde exists principally in the form of its monohydrate, methylene glycol, CH 2(OH)2, and as low molecular weight polymeric hydrates or polyoxymethylene glycols. It has been suggested that the hydrated form, methylene glycol, is the reactive component of formaldehyde but this has been disputed.

Preparation of 10% n Four per cent formaldehyde or 10% buffered formalin is commonly prepared by adding 100 ml of 40% formaldehyde to 900 ml distilled water with 4 g sodium phosphatase, monobasic and 6. 5 g sodium phosphate, dibasic (anhydrous).

Formaldehyde solutions n n 10% neutral buffer formalin (4% formaldehyde) REAGENTS REQUIRED 1 40% formaldehyde 100 ml 2 Distilled water 900 ml 3 Sodium dihydrogen orthophosphate 4 g 4 Disodium hydrogen orthophosphate (anhydrous) 6. 5 g (sodium hydrosulphite) METHOD Prepare, using quantities indicated. Fixation time: 24 -72 hours

Buffered formaldehyde-glutaraldehyde 200 m. Osm 38 n REAGENTS REQUIRED 1 -Sodium dihydrogen orthophosphate 1. 6 g 2 -Sodium hydroxide 0. 27 g 3 -Distilled water 88 ml 4 -40% formaldehyde 10 ml 5 -50% glutaraldehyde 2 ml METHOD Prepare, using quantities indicated. Fixation time: 16 -24 hours.

Formol saline n REAGENTS REQUIRED 1 - 40% formaldehyde 100 ml 2 -Sodium chloride 9 g 3 -Tap water 900 ml n METHOD Prepare, using quantities indicated

Some other forms of Fixatives n Baker's formol-calcium (modified) REAGENTS REQUIRED 1 40% formaldehyde 100 ml 2 Distilled water 900 ml 3 10% calcium chloride 100 ml 4 7 g of cadmium chloride is sometimes added to the mixture n METHOD Prepare, using quantities indicated. Fixation time: 16 -24 hours. Formol saline REAGENTS REQUIRED 1 40% formaldehyde 100 ml 2 Sodium chloride 9 g 3 Tap water 900 ml n n METHOD Prepare, using quantities indicated. Alcoholic formaldehyde REAGENTS REQUIRED 1 40% formaldehyde 100 ml 2 95% alcohol 900 ml 3 0. 5 g calcium acetate may be added to this mixture to ensure neutrality METHOD Prepare, using quantities indicated. Fixation time: 16 -24 hours.

Fixatives - general usage Formalin is used for all routine surgical pathology and autopsy tissues when an H and E slide is to be produced. n Formalin is the most forgiving of all fixatives when conditions are not ideal, and there is no tissue that it will harm significantly. n

Zenker's fixatives n Zenker's fixatives are recommended for reticuloendothelial tissues including n n n lymph nodes, spleen, thymus, and bone marrow. Zenker's fixes nuclei very well and gives good detail. However, the mercury deposits must be removed (dezenkerized) before staining or black deposits will result in the sections

Bouin's solution n Bouin's solution is sometimes recommended for fixation of n n testis, GI tract, and endocrine tissue. It does not do a bad job on hematopoietic tissues either, and doesn't require dezenkerizing before staining

Glutaraldehyde n n Glutaraldehyde is recommended for fixation of tissues for electron microscopy. The glutaraldehyde must be cold and buffered and not more than 3 months old. The tissue must be as fresh as possible Preferably sectioned within the glutaraldehyde at a thickness no more than 1 mm to enhance fixation

Alcohols n n Alcohols, specifically ethanol, are used primarily for cytologic smears. Ethanol (95%) is fast and cheap. Since smears are only a cell or so thick, there is no great problem from shrinkage, and since smears are not sectioned, there is no problem from induced brittleness. Note: For fixing frozen sections, you can use just about anything--though methanol and ethanol are the best

Glutaraldehyde n n Glutaraldehyde causes deformation of alpha-helix structure in proteins so is not good for immunoperoxidase staining. However, it fixes very quickly so is good for electron microscopy. It penetrates very poorly, but gives best overall cytoplasmic and nuclear detail. The standard solution is a 2% buffered glutaraldehyde

Glutaraldehyde n n First used in 1962 by Sabatini et al* Shown to preserve properties of subcellular structures by EM Renders tissue autofluorescent so less useful for fluorescence microscopy, but fluorescence can be attenuated by Na. BH 4. Forms a Schiff’s base with amino groups on proteins and polymerizes via Schiff’s base catalyzed reactions

n n n Forms extensive crosslinks - reacts with the �-amino group of lysine, �-amino group of amino acids - reacts with tyrosine, tryptophan, histidine, phenylalanine and cysteine Fixes proteins rapidly, but has slow penetration rate Can cause cells to form membrane blebs

Glutaraldehyde n n Supplied commercially as either 25% or 8% solution Must be careful of the impurities - can change fixation properties - best product from Polysciences (Worthington, PA) As solution ages, it polymerizes and turns yellow. Store at -20 °C and thaw for daily use. Discard.

Summary of Formaldehyde n n Crosslinks proteins by forming methelene bridges between reactive groups The ratelimiting step is the deprotonation of amino groups, thus the p. H dependence of the crosslinking Functional groups that are reactive are amido, guanidino, thiol, phenol, imidazole and indolyl groups Can crosslink nucleic acids

Cont. n n n Therefore the preferred fixative for in situ hybridization Does not crosslink lipids but can produce extensive vesiculation of the plasma membrane which can be averted by addition of Ca. Cl 2 Not good preservative for microtubules at physiologic p. H

Protein crosslinking is slower than for glutaraldehyde, but formaldehyde penetrates 10 times faster. n It is possible to mix the two and there may be some advantage for preservation of the 3 D nature of some structures. n

Mercurials n n n fix tissue by an unknown mechanism. They contain mercuric chloride and include such well-known fixatives as B-5 and Zenker's. These fixatives penetrate relatively poorly and cause some tissue hardness, but are fast and give excellent nuclear detail.

Their best application is for fixation of hematopoietic and reticuloendothelial tissues. n Since they contain mercury, they must be disposed of carefully n

Alcoholsn including methyl alcohol (methanol) n n and ethyl alcohol (ethanol), are protein denaturants and are not used routinely for tissues because they cause too much brittleness and hardness. However, they are very good for cytologic smears because they act quickly and give good nuclear detail. Spray cans of alcohol fixatives are marketed to physicians doing PAP smears, but cheap hairsprays do just as well

Oxidizing agents n include permanganate fixatives n n Potassium permanganate, dichromate fixatives n n Potassium dichromate, Osmium tetroxide. They cross-link proteins, but cause extensive denaturation. n Some of them have specialized applications, but are used very infrequently. n

Bouin’s Solution (Picrates) n n n include fixatives with picric acid. Foremost among these is Bouin's solution. It has an unknown mechanism of action. It does almost as well as mercurials with nuclear detail but does not cause as much hardness. Picric acid is an explosion hazard in dry form. As a solution, it stains everything it touches yellow, including skin.

On the left with H&E staining black mercuric chloride precipitate is seen in this lymphoma fixed in B-5 and not properly dezenkerized. This precipitate is seen on the right under polarized light microscopy.

Removal of Pig n n n n Formalin pigment 1. Dewax the sections, rinse in 100% alcohol, rinse in 70% alcohol, rinse in distilled water. 2. Treat in saturated alcoholic picric acid for 30 minutes to 2 hours. 3. Wash well in running tap water. 4. If yellow staining of the section persists rinse in dilute lithium carbonate. 5. Rinse in tap water. 6. Continue with method.

Mercury pigment 1. Dewax the sections, rinse in 100% alcohol, rinse in 70% alcohol, rinse in distilled water. n 2. Treat in Lugol's iodine for 2 minutes. n 3. Decolourise in 5% sodium thiosulphate for 5 minutes. n 4. Wash well in running tap water. n 5. Continue with method. n

Dichromate pigment 1. Dewax the sections, rinse in 100% alcohol, rinse in 70% alcohol, rinse in distilled water. n 2. Treat in 2% HCl in 70% alcohol 16 -24 hours. n 3. Rinse in tap water. n 4. Continue with method. n

Tissue Processing n Once the tissue has been fixed, it must n n n be processed into a form in which it can be made into thin microscopic sections. The usual way this is done is with paraffin. Tissues embedded in paraffin, which is similar in density to tissue, can be sectioned at anywhere from 3 to 10 microns, usually 6 -8 routinely. The technique of getting fixed tissue into paraffin is called tissue processing n n Dehydration Clearing

Dehydration Wet fixed tissues (in aqueous solutions) cannot n n n be directly infiltrated with paraffin. First, the water from the tissues must be removed by dehydration. This is usually done with a series of alcohols, say 70% to 95% to 100%. Sometimes the first step is a mixture of formalin and alcohol. Other dehydrants can be used, but have major disadvantages. n n Acetone is very fast, but a fire hazard, so is safe only for small, hand-processed sets of tissues. Dioxane can be used without clearing, but has toxic fumes

Clearing n n n Removal of the dehydrant with a substance that will be miscible with the embedding medium (paraffin). The commonest clearing agent is xylene. Toluene works well, and is more tolerant of small amounts of water left in the tissues, but is 3 times more expensive than xylene. Chloroform used to be used, but is a health hazard, and is slow. Methyl salicylate is rarely used because it is expensive, but it smells nice (it is oil of wintergreen).

Embedding n n n Finally, the tissue is infiltrated with the embedding agent, almost always paraffin. Paraffins can be purchased that differ in melting point, for various hardnesses, depending upon the way the histotechnologist likes them and upon the climate (warm vs. cold). Wax hardness (viscosity) depends upon the molecular weight of the components and the ambient temperature. High molecular weight mixtures melt at higher temperatures than waxes comprised of lower molecular weight fractions. Paraffin wax is traditionally marketed by its melting points which range from 39°C to 68°C.

Other agents n n n A product called paraplast contains added plasticizers that make the paraffin blocks easier for some technicians to cut. A vacuum can be applied inside the tissue processor to assist penetration of the embedding agent. methyl methacrylate, glycol methacrylate, araldite, and epon. Methyl methacrylate

General Embedding Procedure n METHOD 1 Open the tissue cassette, check against worksheet entry to ensure the correct number of tissue pieces are present. n 2 Select the mould, there should be sufficient room for the tissue with allowance for at least a 2 mm surrounding margin of wax. n 3 Fill the mould with paraffin wax. n 4 Using warm forceps select the tissue, taking care that it does not cool in the air; at the same time.

n 5 Chill the mould on the cold plate, orienting the tissue and firming it into the wax with warmed forceps. This ensures that the correct orientation is maintained and the tissue surface to be sectioned is kept flat. n 6 Insert the identifying label or place the labelled embedding ring or cassette base onto the mould. n 7 Cool the block on the cold plate, or carefully submerge it under water when a thin skin has formed over the wax surface. n 8 Remove the block from the mould. n 9 Cross check block, label and worksheet

Sectioning tissues n n n Turn on the water bath and check that the temp is 35 -37ºC. Use fresh deionized water (DEPC treated water must be used if in situ hybridization will be performed on the sections). Blocks to be sectioned are placed face down on an ice block or heat sink for 10 minutes. Place a fresh blade on the microtome. Insert the block into the microtome chuck so the wax block faces the blade and is aligned in the vertical plane. Set the dial to cut 4 -10 µM sections. The blade should angled 4 -6º.

n n n n Face the block by cutting it down to the desired tissue plane and discard the paraffin ribbon. If the block is ribboning well then cut another four sections and pick them up with forceps or a fine paint brush and float them on the surface of the 37ºC water bath. Float the sections onto the surface of clean glass slides. If the block is not ribboning well then place it back on the ice block to cool off firm up the wax. If the specimens fragment when placed on the water bath then it may be too hot. Place the slides with paraffin sections in a 65°C oven for 20 minutes (so the wax just starts to melt) to bond the tissue to the glass. Slides can be stored overnight at room temperature

H and E staining n n Hematoxylin is the oxidized product of the logwood tree known as hematein. Since this tree is very rare nowadays, most hematein is of the synthetic variety. In order to use it as a stain it must be "ripened" or oxidized. This can be done naturally by putting the hematein solution on the shelf and waiting several months, or by buying commercially ripened hematoxylin or by putting ripening agents in the hematein solution.

Cont. n n n Hematoxylin will not directly stain tissues, but needs a "mordant" or link to the tissues. This is provided by a metal cat ion such as iron, aluminum, or tungsten. The variety of hematoxylins available for use is based partially on choice of metal ion used. They vary in intensity or hue. Hematoxylin, being a basic dye, has an affinity for the nucleic acids of the cell nucleus.

Cont. n n n Hematoxylin stains are either "regressive" or "progressive". With a regressive stain, the slides are left in the solution for a set period of time and then taken back through a solution such as acid-alcohol that removes part of the stain. This method works best for large batches of slides to be stained and is more predictable on a day to day basis. With a progressive stain the slide is dipped in the hematoxylin until the desired intensity of staining is achieved, such as with a frozen section. This is simple for a single slide, but lends itself poorly to batch processing.

Eosin n n Eosin is an acidic dye with an affinity for cytoplasmic components of the cell. There a variety of eosins that can be synthesized for use, varying in their hue, but they all work about the same. Eosin is much more forgiving than hematoxylin and is less of a problem in the lab. About the only problem you will see is overstaining, especially with decalcified tissues

Decalcification n n Some tissues contain calcium deposits which are extremely firm and which will not section properly with paraffin embedding owing to the difference in densities between calcium and parffin. Bone specimens are the most likely type here, but other tissues may contain calcified areas as well. This calcium must be removed prior to embedding to allow sectioning. A variety of agents or techniques have been used to decalcify tissue and none of them work perfectly. n n Mineral acids, organic acids, EDTA, and electrolysis have all been used.

Strong mineral acids nitric and n hydrochloric acids n n rapid damage cellular morphology, so are not recommended for delicate tissues such as bone marrow.

Organic acids n n n acetic and formic acid are better suited to bone marrow, since they are not as harsh. However, they act more slowly on dense cortical bone. Formic acid in a 10% concentration is the best all-around decalcifier. Some commercial solutions are available that combine formic acid with formalin to fix and decalcify tissues at the same time.

EDTA and Electrolysis n EDTA can remove calcium and is not harsh (it is not an acid) n n but it penetrates tissue poorly and works slowly and is expensive in large amounts. Electrolysis has been tried in experimental situations where calcium had to be removed with the least tissue damage. n It is slow and not suited for routine daily use.

Procedure: n n Specimens should be decalcified in hydrochloric acid/formic acid working solution 20 times their volume. Change to fresh solution each day until decalcification is complete. n n n It may take 24 hours up to days or months depending on size of the specimens. See below for the testing procedures Once the decalcification is complete, rinse specimens in water briefly and transfer to ammonia solution to neutralize acids left in specimens for 30 minutes. Wash specimens in running tap water thoroughly up to 24 hours. Routine paraffin embedding.

End-Point of Decalcification X-ray (the most accurate way) n Chemical testing (accurate) n Physical testing (less accurate and potentially damage of specimen) n

Chemical Test: n 5% Ammonium Hydroxide Stock: n n n n n Ammonium hydroxide, 28% ---------- 5 ml Distilled water ----------------- 95 ml Mix well 5% Ammonium Oxalate Stock: Ammonium oxalate -------------- 5 ml Distilled water ----------------- 95 ml Mix well Ammonium Hydroxide/Ammonium Oxalate Working Solution: Use equal parts of the 5% ammonium hydroxide solution and the 5% ammonium oxalate solution.

Procedure: n n Insert a pipette into the decalcifying solution containing the specimen. Withdraw approximately 5 ml of the hydrochloric acid/formic acid decalcification solution from under the specimen and place it in a test tube. Add approximately 10 ml of the ammonium hydroxice/ammonium oxalate working solution, mix well and let stand overnight. Decalcification is complete when no precipitate is observed on two consecutive days of testing. Repeat this test every two or three days.

Physical Tests: n n n The physical tests include bending the specimen or inserting a pin, razor, or scalpel directly into the tissue. The disadvantage of inserting a pin, razor, or scalpel is the introduction of tears and pinhole artifacts. Slightly bending the specimen is safer and less disruptive but will not conclusively determine if all calcium salts have been removed. After checking for rigidity, wash thoroughly prior to processing.