Processing of Bone Specimen

8/12/2019 Processing of Bone Specimen

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Bones are rigid organs that constitute part ofthe endoskeleton of vertebrates. Theysupport and protect the various organs of the

body, produce red and white blood cells andstore minerals. Bone tissue is a type ofdense connective tissue. Bones come in avariety of shapes and have a complex

internal and external structure, arelightweight yet strong and hard, and servemultiple functions.


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http://en.wikipedia.org/wiki/File:Illu_compact_spongy_bone.jpg


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http://en.wikipedia.org/wiki/File:Caput_femoris_cortex_medulla.jpghttp://en.wikipedia.org/wiki/Trabecular_bonehttp://en.wikipedia.org/wiki/Trabecular_bonehttp://en.wikipedia.org/wiki/Trabecular_bonehttp://en.wikipedia.org/wiki/Cortical_bonehttp://en.wikipedia.org/wiki/Cortex_(anatomy)http://en.wikipedia.org/wiki/Femur


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The hard outer layer of bones is composedof compact bone tissue, so-called due to itsminimal gaps and spaces. Its porosity is 5

30%.This tissue gives bones their smooth,white, and solid appearance, and accountsfor 80% of the total bone mass of anadult skeleton. Compact bone may also bereferred to as dense bone.


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Filling the interior of the bone is the trabecularbone tissue ,which is composed of a network ofrod and plate like elements that make theoverall organ lighter and allow room for blood

vessels and marrow. Trabecular bone accountsfor the remaining 20% of total bone mass but hasnearly ten times the surface area of compactbone. Its porosity is 30 90%. The microscopicdifference between compact and cancellous

bone is that compact bone consists of haversiansites and osteons, while cancellous bones do not.Also, bone surrounds blood in the compact bone,while blood surrounds bone in the cancellousbone.


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There are several types of cells constituting thebone;Osteoblasts are mononucleate bone-forming cellsthat descend from osteoprogenitor cells. They

make a protein mixture known as osteoid, whichmineralizes to become bone.Osteoid is primarilycomposed of Type I collagen. Osteoblsatsrobustly produce alkaline phosphatase,an enzyme that has a role in the mineralisationof bone, as well as many matrix proteins.Osteoblasts are the immature bone cells, andeventually become entrapped in the bone matrixto become osteocytes- the mature bone cell.


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Osteocytes originate from osteoblasts that havemigrated into and become trapped andsurrounded by bone matrix that they themselvesproduce. The spaces they occupy are known

as lacunae. Osteocytes have many processes thatreach out to meet osteoblasts and otherosteocytes probably for the purposes ofcommunication. Their functions include, tovarying degrees: formation of bone; matrix

maintenance; and calcium homeostasis. Theyhave also been shown to act as mechano-sensoryreceptors regulating the bone's response tostress and mechanical load. They are maturebone cells.


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Osteoclasts are the cells responsible for boneresorption, thus they break down bone. New boneis then formed by the osteoblasts (remodeling ofbone to reduce its volume).Osteoclasts are large, multinucleated cells locatedon bone surfaces in what are called Howship'slacunae or resorption pits. These lacunae, orresorption pits, are left behind after thebreakdown of the bone surface. Because theosteoclasts are derived from a monocyte stem-cell lineage, they are equipped with phagocytic-like mechanisms similar tocirculating macrophages.Osteoclasts mature and/or migrate to discretebone surfaces. Upon arrival, active enzymes, suchas tartrate resistant acid phosphatase,are secreted against the mineral substrate.


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MatrixThe majority of bone is made of the bonematrix. It has inorganic and organic parts.

Bone is formed by the hardening of thismatrix entrapping the cells. When these cellsbecome entrapped from osteoblasts theybecome osteocytes.


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http://en.wikipedia.org/wiki/File:Bertazzo_S_SEM_deproteined_bone_-_cranium_rat_-_x10k.jpg


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There are five types of bones in the humanbody: long, short, flat, irregular, andsesamoid.Long bones are characterized by a shaft,the diaphysis, that is much longer than it iswide. They are made up mostly ofcompactbone, with lesser amounts of marrow,located within the medullary cavity,

and spongy bone. Most bones of the limbs,including those of the fingers and toes, arelong bones. The exceptions are those ofthe wrist, ankle and kneecap


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http://en.wikipedia.org/wiki/File:Illu_long_bone.jpg


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Short bones are roughly cube-shaped, and have only a thinlayer of compact bone surrounding a spongy interior. Thebones of the wrist and ankle are short bones, as arethe sesamoid bones.Flat bones are thin and generally curved, with two parallellayers of compact bones sandwiching a layer of spongybone. Most of the bones of the skull are flat bones, as isthe sternum.Sesamoid bones are bones embedded in tendons. Sincethey act to hold the tendon further away from the joint,the angle of the tendon is increased and thus the leverageof the muscle is increased. Examples of sesamoid bonesare the patella and the pisiform.Irregular bones do not fit into the above categories. Theyconsist of thin layers of compact bone surrounding aspongy interior. As implied by the name, their shapes areirregular and complicated. The bones ofthe spine and hips are irregular bones.


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These can be:Biopsies- for diagnosis of tumor,hematopoietic disorders, infection etc.

Amputation specimens: resulting from tumor,chronic osteomyelitis and gangrene.Resection specimens: for benign or low grademalignant tumors and arthritis.


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Radiographs of bone slabs, blocks orfragments are useful for four main purposes:To examine the nature & extent of a lesion.

To provide a diagram of lesion prior to blockselection for processing.To check progress of decalcification i.e.decalcification endpoint test.

To confirm the presence of large foreignmaterials e.g. prosthetic devices, metal orglass fragments implanted by trauma.


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The faxitron cabinet x -ray system is used.It is a tabletop unit measuring 56 cm wide,51 cm deep & 89 m high.

Energy output is 10-110 kV with 3 mA tubecurrent.It has adjustable shelf levels for film-to-source distances of 31-61 mm.

X-ray film used for specimen radiography isKODAK X-OMAT.


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A good band saw is an essential piece ofequipment.Handymans bench band saws are

inexpensive, small & light weight and thesesaws cut through cortical bone slowly withcuts no deeper than 7.5 cm.Wetter or meat cutters saw is used for

cutting wet, fatty bone.it is heavier, rigid,has floor standing construction and strongcoarse blades for deeper cuts.Electric saw is also available.


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Handyman saw Meat cutters saw


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On small saws:0.5 cm width and 12 to 16 teeth per inch(tpi) making finer cleaner cuts.

On large saws:1.25 cm wide with 6 teeth per inch


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Soft tissues and dense connective tissue e.g.tendons should be removed before sawingthe sample.

The first cut is made through the midplane,then approximately 3-5 mm thick slabs arecut parallel to the first cut.A saw guide or wood plank is held against the

first cut edge to ensure an even slice.After sawing, the blades are cleaned by usinga slow sream of water ans a soft brush.


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It protects bone and surrounding soft tissuefrom damaging effects of aciddecalcification.

10% natural buffered formalin is suitable forboth paraffin and non-tetracycline labeledbone in MMA (methyl methacrylate).Alcoholic formalin or 70% ethanol fixation

can be used to fix tetracycline labeledmineralized bone in MMA.Fixation proceeds faster by reducing the sizeof the bone & removing excess of soft tissue.


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Techniques for demonstration of bones and itscomponents are:For decalcified bone: frozen, paraffin, orcelloidin sections and transmission electronmicroscopyFor mineralised bone: frozen, plastic formicrotomed sections, scanning andtansmission EM.

The technique chosen for examination ofbone is influenced by clinical diagnosis, caseurgency and extent of investigation.


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Fixation99% ethyl alcoholTissue

1-2 mm thick bone piecesSolutions2% aqueous silver nitrate

Reducer :Sodium hypophosphite 5 gm0.1M sodium hydroxide 0.2 mlDistilled water 100 ml


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5% aqueous sodium thiosulphate

Decalcifier :

10% aqueous formic acidVan Giesons picro-fuchsin


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Wash in several changes of distilled water, 4 hrs.Place 2% silver nitrate, 48 hrs in dark.Rinse in 3 changes of distilled water, 15-20 sec each.Wash in running tap water, 4 hrs.

Place in reducer, 48 hrs.Wash in running tap water, 1 hr.Place in sodium thiosulphate sol, 24 hrs.Wash in running tap water, 1 hr.Decalcify in 10% formic acid.Process to paraffin wax, cut & mount.Dewax & stain with van giesons stain, 2 min.Dehydrate, clear & mount.


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Edges of mineralized bone - Black.

Bone - Brown toYellow

Osteoid - Red


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It is influenced by four interdependentfactors:Urgency of case

degree of mineralisationExtent of the investigationStaining techniques required.


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These can be divided into 2 groups:Strong (inorganic) acidsWeak (organic) acids


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Strong acids are used for needle and smallbiopsy specimens to permit rapid diagnosiswithin 24 hrs or less.

They can be used for large or heavilymineralised cortical bone specimens withdecalcification progress carefully monitoredby a decalcification endpoint test.

Examples include nitric acid & hydrochloricacid.


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Nitric acid 5-10 mlDistilled water to 100 ml


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10% nitric acid 40 mlAbsolute ethanol 30 ml0.5% chromic acid 30 ml

mix shortly before use.


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Use inside a fume hoodFormaldehyde (37-40%) - 10 mlDistilled water - 80 ml

Nitric acid - 10 ml


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Examples include formic acid, picric acid,acetic acid.Formic acid is the only weak acid used

extensively as a primary decalcifier.Formis acid solutions can be aqueous (5-10%), buffered or combined with formalin.The formain-10% formic acid mixture

simultaneously fixes and decalcifies, & isrecommended for very small bone pieces orjamshidi needle biopsies.


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Formic acid is gentler and slower than HCl ornitric acids.It is suitable for most routine surgical

specimens particularly whenimmunohistochemical staining is needed.Also it should be endpoint tested.Decalcification is complete in 1-10 days,

depending on size, type of bone & acidconcentration.


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90% stock formic acid - 5-10 mlDistilled water to - 100 ml


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90% stock formic acid - 5-10 mlFormaldehyde (37-40%) - 5 mlDistilled water to - 100 ml


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20% aqueous sodium citrate - 65 ml90% stock formic acid - 35 ml

This solution has a pH of approximately 2.3


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Ethylene-diaminetetracetic acid (EDTA) isused for decalcification.It binds metallic ions, notably calcium and

magnesium.EDTA will not bind to calcium below pH 3 andis faster at pH 7-7.4It binds to ionised calcium on the outside of

the apatite crystal and as this layer becomesdepleted, more calcium ions reform fromwithin and the crystal becomes progressivelysmaller.


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EDTA disodium salt - 250 gmDistilled water - 90 mlFormaldehyde (37-40%) 10 ml


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pH 7.0-7.4EDTA, disodium salt - 250 gmDistilled water - 1750 ml

If the solution is cloudy, adjust to pH 7 withapproximately 25 gm sodium hydroxide.


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Concentration and volume of the activereagent.More concentrated acid solutions decalcifymore rapidly but are harmful to tissues.Alcohol or buffers that protect tissue slowdown the decalcification rate.Temperature

Increased temperature accelerates the ratewhile decreased temperature reduces therate of dcalcification.


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Age of the patientType of boneSize of specimen

Solution agitation


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The two most reliable tests are:

Specimen radiography using an X-ray unitChemical method to test acids and EDTA solutions

Other method first used to test nitric acidis a weight loss, weight gain procedure thatprovides good results with all acids andEDTA. But it is considered inaccurate & isdamaging to tissues.


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Probing, needling, slicing, bending orsqueezing tissue can create artifacts e.g.needle tracks, disrupt small tumor from boneor cause false +ve microfractures of finetrabeculae.


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It is the most sensitive test for detectingcalcium in bone.It uses FAXITRON with a manual exposuresetting of approx. 1 minute, 30 kV andKODAK X-OMAT X-ray film.Acid is rinsed from sample and bone is placedon waterproof polyethylene sheet on the top

of x-ray film.The bone is exposed and is left in place untilfilm is developed.It is then examined for calcifications.


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Areas of mineralization are easily identifiedwith tiny calcifications best viewed using ahand-held magnifier.Metal dust particles from saw blades areradio-opaque, sharply delineated fragmentsthat never change in size.they appear as greyspecs & can be easily removed.

Spicules of metal, metallic paint or glassforced deep into tissue by traumatic injuryare also sharply delineated but cannot beremoved without damaging tissue.


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They detect the presence of calciumreleased from the bone.When no calcium is found or the result isnegative, decalcification is said to becomplete.EDTA can be chemically endpoint tested byacidifying the used solution: this forces EDTA

to release calcium for precipitation byammonium oxalate.


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This method detects the calcium in acidsolutions by precipitation of insolublecalcium hydroxide or calcium oxalate.

Solutions: Ammonium hydroxide, concentrated

Saturated aqueous ammonium oxalate.


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Method:1. Take 5 ml of used decalcifying fluid, add a

piece of litmus paper or use a pH meter.

2. Add ammonium hydroxide drop by drop,shaking after each drop, until pH is 7.3. Add 5ml of saturated ammonium oxalate

and shake well.

4. Allow solution to stand for 30 min.


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Acids react with calcium carbonate in boneto produce carbon dioxide, seen as a layer ofbubbles on bone surface.They disperse with agitation but reformbecoming smaller as less calcium carbonateis reduced.It can be used as a guide to check the

progress of decalcification but as anendpoint test it is subjective & unreliable.


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Acids are removed from tissues afterdecalcification is complete.Chemical neutralization is done by immersingdecalcified bone into 5-10% aqueous sodiumbicarbonate solution for several hours.Specimens can also be rinsed with runningtap water in 30 min for small samples & 1-4

hrs for larger samples.Samples needing immediate processing e.g.needle biopsies can be blotted or quicklyrinsed to remove acid from the surface.


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If an endpoint tested decalcified boneappears chalky & crumbles out out of theblock during sectioning then dehydration orparaffin infiltration maybe incomplete.For this blocks can be melted down & re-infiltrated with paraffin or by meltingparaffin from bone & going back through 2changes of xylene, 2 changes of 100 %alcohol to remove residual water.


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Embedding methods using metal molds withplastic tissue cassettes are also available.A labeled cassette contains the tissuethroughout processing & after embedding,the plastic back of a block fits into amicrotome cassette lamp.A large specimen is the limiting factor for

embedding with cassettes.


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Disposable knives are convenient, extremelysharp, single used blades capable ofsectioning properly decalcified and processedparaffin-embedded bones.Newer microtones come equipped withdisposable blade holders.Heavy steel knives range in size from 16 to

18 cm for small microtomes and for 200-300cms for base sledge microtomes withspecially designed blades for the polycuts.


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An optimal thickness for bone sections issame as that for soft tissues, 4 to 5 m.Small bone samples and biopsies aresectioned with knife angles set for routinesoft tissue microtomy.When sectioning, a sharp knife is necessaryto get flat, uncompressed, wrinkle-free

sections.Hard tissues cut more easily if cooled by amelting ice block to allow water penetrationinto the tissue surface.


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While floating on water, cartilage and bonesections can expand more than paraffin andsmall folds may form as sections dry.To prevent this, water bath temperatureshould be lowered to 10-15C below theparaffin melting point.If cartilage curling is a problem, drying

sections flat at 37 C overnight may solve theproblem.


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Bone sections adhere to slides better whenglass surfaces are coated with adhesive.For this, commercially silanized Plus Chargeor poly-l-lysine coated slides are available.Coating can also be done by dipping theslides in a gelatin and potassium dichromatesubbing solution.

If some sections are persistently nonadherent, a solution containing amylopectin,or HMW bloom gelatin in the chrome subbingmixture may be used.


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Celloidin or nitrocellulose embedding methodis useful for large, decalcified bone.But it has been replaced with MMA-embedded undecalcifed bone section

methods.It doesnt harden cortical bone, is pliableand more elastic, binding tissues withdifferent consistencies together and

preventing separation during sectioning.Double embedding method combinescelloidin and paraffin, uses harder paraffinand has extended processing.


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The disadvantages of celloidin areExpense.Reduced availability of nitro cellulose.Chemical safety issues, i.e. toxic chloroform.

Extended preparation time.Sections too thick for good cellular detail.Need for a sledge microtome with a knife suitable forsectioning celloidin.


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Diagnostic immunohistochemical staining isfrequently done on decalcified bone sectionsembedded in paraffin e.g. bone marrowbiopsies, tumors, cartilage.

Care must be taken to fix bone specimensproperly and decalcify with the leastdamaging agent in shortest time possible toprotect antigens from damaging effects ofacids.2 m thick MMA sections after completeremoval of plastic with warm xylene aretaken .


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Glycol methacrylate cannot be removed &may inhibit adequate antibody preparationto the antigenic sites.


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Mineralized bone must be cut with tungstencarbide-tipped knives and need special, hardsupport to avoid cracked tissue sections.Paraffin and celloidin are too soft and fail tomatch the harness of bone,Acrylic resins and plastics are now widelyused and are preferred embedding media forundecalcified bone.Frozen sections provide some support ofcancellous bone but the bone itself tends tolook damaged.


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They have been used to maintain the intactsections of undecalcified double embeddedbone sections during microtomy.2 methods, 1 for undecalcified bone

embedded in MMA and other for decalcifiedparaffin embedded bone are used.Clear adhesive packaging tape is rolled on tothe trimmed block face and the cut section

sticks to the tape during and aftersectioning.The tape section combination is thenattached to an adhesive coated slide.


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During the staining process, the tape releasesxylene, leaving the section transferred.


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These sections provide rapid diagnosis.They are made by rapid freezing of bonesamples in liquid nitrogen-cooled 2-methylbutane (-120 C).It must be used carefully as some bones mayshatter in extremely cold temperatures.A dry ice/isopentane bath snap freezes bone

coated with 4% aq polyvinyl alcohol gentlywithout shattering (-70 C).


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1. Mount bone on cork.2. Snap freeze in isopentane cooled by liquid

nitrogen or with dry ice.3. Place bone in cryostat at -30 to -35 C.

4. Cut sections at 5-10 m, pickup section onslide & fix with fixative. Fixation in 95% alcoholfor 5 min removes fat.

5. Stain in harris Gill II or Gill III for 1 min.6.

Rinse with water.7. 1% alcoholic eosin for 10-30 sec.8. Dehydrate & mount in permanent mounting

medium.


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Fresh frozen sections can be fixed, rinsedthen decalcified in 10% EDTA beforeimmunostainig.For cryoprotection, formalin fixed biopsiesare immersed in 15-20% sucrose for 1-8 hrs toreplace water before freezing.


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FIXATION 1. The specimen is allowed sufficient timefor it to be fully fixed with 10% formolcalcium (at least 24 hours after removal fromthe body) before processing is commenced.Specimens which are labelled withtetracycline (noted on request form) forsubsequent demonstration with fluorescence,must be fixed only in 70% alcohol and notformol calcium, for at least 24 hours afterremoval from the body.


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2. If the specimen is too large, it should betrimmed/cut so that adequate size block(s)are taken from the part(s) required forexamination. Surplus tissue not required forexamination should be cut off. Shouldcutting/trimming be necessary, then advicemust be sought from an experienced MLSO.3. Large specimens especially those whichrequire cutting/trimming may require longerfixation than 24 hours before processing iscommenced.


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Benzoyl peroxide is supplied damped with wateras it is potentially explosive in its dry form.However, it is essential that only dried benzoylperoxide is used in the solutions A and B. It isdried as follows:1. Spoon out approximately 10g of benzoylperoxide into a filter paper bent in the shape ofa cone on a beaker.2. Dry in a 37C oven overnight. Use only the

oven in the laboratory, and place a sign to warnstaff. 3. Excess dried benzoyl peroxide is storedon the shelf in the container provided away fromdirect heat.


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Allow specimen in resin embedding solution Bto stand for 1 hour.Orientate the label using a wooden cocktailstick, so that it is bent at right angles across

the specimen.Secure lid on vial/container.Partially immerse in pre-heated 60C waterinside a staining dish. Ensure lid is replaced

on dish.Polymerise resin overnight at 60C in theoven. Use only the 60C oven in the resinlaboratory.


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Place glass container (minus lid if possible) infreezing compartment of the refrigerator for15 minutes.Wrapping the container well in paper towels,break the container by hitting gently withthe hammer provided. Wear eye protection.Discard both waste paper and broken glass inthe glass bin, and ensure no glass is lying onthe bench.Carefully wash block under a running tap.


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Clamp block in specially designed holder onReichert-Jung Autocut so that the bone isvertical to the knife edge.Using the trimming tungsten carbide knife, triminto block until a representative area is reached,

lubricating both knife and block with 30% alcoholwith the aid of an artist's brush.Remove the trimming knife and replace it withthe cutting tungsten carbide knife and cutsections at 6, lubricating both block and knife

as previously described. Sections that requirefluorescence to examine tetracycline labellingshould be cut at 12 and kept separate fromother sections. Sections can be picked up using apair of fine forceps.


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Processing of Bone Specimen

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