NAMRATHA R

ARTEFACTS

DEFINITIONAn artefact (Latin ‘ars’- art + ‘factum’-

made) in histology means any non-natural feature or structure accidentally introduced into something being observed or studied

According to Bernstein, Artefact refers to “An artificial structure or tissue alteration on a prepared microscopic slide caused by some extraneous factors

RELEVANCEConfusion with normal structure or

pathological changeCompromise accurate diagnosisAlteration of normal morphological and cytological

features and hiding structuresMay even lead to complete uselessness of the

tissue, thus creating serious errors and misdiagnosis of correct histopathological impression

Minor - involving only small portion of the specimen , do not interfere with an accurate diagnosis

Excessive or may involve the entire specimen- useless for diagnostic purposes

From the time of biopsy to the final stage of mounting.

Surgical biopsy procedureFixationTissue processingEmbeddingMicrotomyMountingStainingCover-slipping.

Pre fixation artefactsThese are features and structures that have

been introduced prior to the collection of the tissues.

Surgical procedure or handling prior to dissection

Physical and chemical

BIOPSY PROCEDURE Pressure marks on the surface of the biopsied

specimen- Forceps with excessive force - crush or compression artifacts

Distorted tissue with scalloped serrations (produced by beaks of the forceps)

Crushed cells, appear as dark chromatin strands and may give a false diagnosis of dysplastic lesions.

Split artefacts occur on the surface and at the side of the lesion due to scalpel or foceps which causes multiple cuts in the tissue.

This artefact may result in a split between epithelium and connective tissue, giving a false impression of vesiculo – bullous lesions.

CRUSH ARTEFACTCrush artifact is artificial elongation and

distortion in  cells or tissue Tissue without supporting fibrous stromaSmall cell carcinoma and lymphoma,

thymoma, olfactory neuroblastoma, Cells with high NC ratio. Elongated streaming

basophilic material, Nuclei pushed into each other

Spillage of cytoplasmic contents into stroma

Edges of sections and Small biopsies

Trouble shooting:Use small atraumatic blunt forceps or Adson's forceps without teethA suture should be placed in one edge of the specimen (substitute for forceps for tissue immobilization)

AGONAL CHANGESThe longer the agonal interval in fixation, the

more mitoses apparently progress to completion. Difficulty in recognizing mitoses Anoxia - release of hydrolytic enzymes from

cytoplasmic lysosomesThe hydrolytic enzymes digest the cells, details are

lacking when they are seen under the microscope Autolyzed tissues - nuclear pyknosis, karyolysis

and karyorrhexis, cytoplasmic vacuolation and disintegration of tissue structure.

Storage of tissues at 40 C, but they can be completely avoided by rapid fixation.

CURLING ARTEFACT- Incisional biopsies Small delicate strip of oral

mucosa, the shrinking process caused by formalin fixation

Curling and bending of the tissue, thus making its correct orientation during difficult .

TS: Specimen placed with its mucosal surface up on a piece of the sterile paper (usually that which held the suture material) and allowed to remain unfixed for some time while the incision is being sutured

Adequate depth of the specimens can prevent this artefact

Pseudomicrocysts- lined with surface epithelium, forced inward by the teeth of the instrument, along with compressed surrounding connective tissue, thus making evaluation impossible

Intralesional injection of anaesthetic solution - haemorrhage with extravasation and separation of connective tissue bands with vacuolization

THERMAL INJURYIatrogenic- Electric cautery Laser BeamInfiltration with over heated waxEmbedding using over heated forcepsDrying section mounted slides on hot

plate or incubatorLEEP- Large thermal artefact zone-

uninterpretable margins

Dehydration and denaturation of protein – coagulation and condensation

Increased acidophilia and amorphous appearance to the epithelium and connective tissue.

The epithelium cells appear detached nuclei assume a spindled, palisading configuration. separation of the epithelium from the basement

membraneLoss of nuclear and cytoplasmic detailAlternating zones of coagulation - "Herring bone" appearance.Connective tissue- coagulatedGlandular tissue- vacuolated

Electrocautery in parotid surgery causes oncocytoid changes in the acinar cells 

TROUBLE SHOOTINGThis can be prevented by using the

cutting and not the coagulation electrodes when obtaining the specimen, so that low milliampere current is produced, that will allow cutting and liberation of the specimen

Use of electrocautery is contra-indicated as biopsy technique

The incision margin should be adequaltely away from the interface of the lesion

SURGICAL SUCTIONNegative pressure of suction pulls air and

mobilises tissue fluidsGross- high density tissue with visible air

bubblesMicro- Extracellular round spaces with fluidOral tissue- Basophilic material with acid

mucopolysaccharides- Myxoid material around dental follicles

Nuclei may line up around spacesDDX- Vascular and glandular spacesSoft tissue emphysema, Pneumotosis intestinalis,

oedema

RADIATIONDosage dependantHigh dosages- Cell deathSmall dosages- AcuteIntermediateChronic

RADIATION ARTEFACT Acute1. Tissue oedema2. Endothelial

proliferation3. Swelling of cells4. Proliferation of

collagen fibres5. Ischemic effects Intermediate-

Regeneration1. Fibrosis and

pleomorphic fibroblasts

Chronic 1. Thin and depigmented

epithelium2. Dense fibrosis with

pleomorphic fibroblasts3. Prominent vascularity4. Multinucleate giant cells5. Pleomorphic epithelial

cellsHyperchromasia, irregular nuclear con tours,

6. Macronucleoli7. Vaculoisation of

cytoplasm8. Normal N/C ration

CHEMICAL ARTEFACTS

ChemotherapyLung- Interstitial fibrosis, type II

pneumocyte hyperplasia, Hyaline membrane formation

Liver- Necrosis, fibrosis, Cholestasis, granuloas, vascular changes, GIT and Skin

shrunken and separated from each other, Loss of cellular and nuclear detail, Hypereosinophila and vacuolation, Nuclear pyknosis

MONSEL’S SOLUTIONHemostatic agent

to control bleeding – skin/ mucosal biopsy

Coagulation and necrosis upto a depth of 0.6mm

Ferric sulphate – macrophages with granular material

General basophilia, partial desquamation, cracking of epithelium and necrosis

Restitution stain(0.5% HCL and 70/5 Ethanol at 60 degrees improves image quality

SteroidsIntra articular,soft

tissue,nasalSteroid granulomasBubbly basophilic

material surrounded by histiocytes

Birefringent crystalline particles

DDx - Rhematoid granuloma

Infectious granulomas

MYOSPHERULOSISSac like structures

containing endobodies- Nasal specimens

Hemostatic packing with petroleum based ointment

30-100micromm Foreign body-

type granulomatous reaction to lipid-containing material and blood

DDx - Protothecosis

Spironolactone bodies

Adrenal gland following therapy

In cytoplasm of cells of Z. glomerulosa or fasciculata

Rounded laminated eosinophilic bodies

2-25µmDerivatives of ER

MEDICAL BIOMATERIALSBiological substances- Cellulose, collagen,

fibrin, silkSynthetic polymers- Oriented molecular

structure- Birefringent,orange to red with Sudan stain

DyesMinerals and metalsPhagocytosis, proteolysis and

hydrolysis- AbsorbtionFragmentation- Foreign body reaction,

Calcification, infective nidus, embolus

SUTURES

Isolated fragments or complete fibre bundles- transverse/ oblique/ longtitudinal

Can damage the microtome knife- tears and knife lines in sections

Silk sutures- strong birefringence under polarised light

Silk sutures around which has formed a granulomatous reaction, the so called 'stitch granuloma'.

Cellulose contamination

Luminal surface of GIT

Shredding of sections

Plant cells with strongly staing walls and square shape

Can be mechanically implanted during dissection

Gelfilm/Gelfoam- absorbable gelatin

Thin film/sponge- used to control bleeding

Sections adhering to specimen surface

Slightly basophilic walls of varying thickness surrounding distorted spaces containing blood or cells

No tissue reactionSurround distorted

spaces with blood

Starch - A powder in surgical gloves , contaminate body

tissues during an operation. - Difficult to see in sections under normal bright

field microscopy. Spherical to slightly angular (hexagonal), very

pale in H&E with small central dark spot.Refractile, glassy, polygonal, PAS +ve bodies,

generally 5-20 mm in diameter. Light blue on H and E staining and deep liliac-red with

PAS. Maltose cross birefringence under polarized Starch artefacts can be prevented by the alternate

use of rubber gloves

STARCH CONTAMINATION

Foam pads to prevent escape of small specimens

A fine pattern of local pattern or molding- pore structure of foam

Troubleshooting- Lens or gauze paper by allowing the tissue to fix before placing it into the sponge pad because after fixation the surface is firmer and less prone to indentation.

 

FIXATION

Fixation is a process which attempts to preserve the tissues in a life like condition by preventing autolysis and putrefaction.

The volume of fixative should be 20 times that of the specimen with thickness not exceeding 6 mm.

Fixation Artefacts: During fixation, tissues commonly change in volume.

Inhibition of respiration, Changes in membrane permeability Changes in ion transport through the

membranes

PROLONGED FIXATIONProlonged fixation in formalin - secondary shrinkage.Inaccurate fixation- Shrinkage or crenation with

hypertonic saline and swelling/bursting of cells with hypotonic saline.

Normal phosphate buffered saline (PBS) based fixative corrects such problems.

Alcohol fixatives - tissue sections brittle resulting in microtome sectioning artifacts with chattering and a “venetian blind” appearance

Intraepithelial cleft formation and acantholysis occurs as a result of formation of calcium carbonate residue due to formalin evaporation from unsealed bottles.

TROUBLE SHOOTINGDewax the section  ↓Transfer to absolute alcohol     ↓Place the slide in Coplin jar containing picric

acid solution for 48 hrs     ↓Wash in 90% alcohol    ↓Wash in 70% alcohol     ↓Wash in water

TISSUE PROCESSING ARTEFACTInadequate or incomplete fixation Vessel shrinkage in CNS.Perivascular shrinkage in nerve tissuesPerfusion fixation and gentle prolonged

processingChloroform as a clearing agentCelloidin paraffin double embedding

DELAYED FIXATIONFixation in formalin and embedded in

paraffin shrink by 33%.Delayed fixation- Cell shrinkage and

cytoplasmic clustering. The nuclear chromatin cannot be

distinguished and the nucleoli are sometimes not visualized.

Vascular structures, nerves and glands show a loss of detail and an impression of scarring or loss of cellularity is seen 

TROUBLE SHOOT Fix specimen immediately in 10% formalin solution as soon as

the tissue is removed. Dewax the section     ↓ Transfer to 70% absolute alcohol    ↓ Immerse in iodine solution for 3 min with continous

agitation     ↓ Wash in water ↓ Immerse in sodium thiosulfate solution until the yellow

color of iodine is completely removed     ↓ Wash in water

ZONAL FIXATIONLarge tissues surrounded by a capsule

and rapidly degenerating tissue(glandular tissue)

Fixative penetrates slowly- different degrees of fixation at different levels within tissue

Insufficient time for fixation, too large a specimen and reagent with poor penetration rate

Diffusion of unfixed material

Rest in places other than their original locations and are most commonly seen in glycogen.

This can be prevented by using smaller blocks (Reale and Luciano 1970) or stronger fixatives for larger bits.

Fixation of tissue for glycogen should be prompt, as there is an initial sharp loss of glycogen postmortem and it should be carried out at 40 C in 80% alcohol or in Rossman’s solution.

Troubleshooting- Glycogen fixatives- formal alcohol and Bouin

DIFFUSION ARTEFACTSSmall molecules like inorganic ions and biogenic

amines can be lost from tissues chromogranin, in case of adrenaline and nor

adrenaline. This can be demonstrated by placing the adrenals

in iodate. The catecholamines can be seen leaving the

tissue as a red cloud of aminochromes. Generated can only be retained by precipitation Can be prevented by proper fixation for accurate

localization and also by preventing the leaching of ions from the tissue 

Formalin pigment artefactAs solutions age, formic acid developes from the

formaldehyde content- lowers the pH. Causes crystals of formalin pigment, or acid

formaldehyde hematin tissues. Acid formalin+ Haemoglobin=Acid

formaldehyde haematinBlack to brown finely granular birefringent

deposit in the vicinty of RBCsTissues richly vascular- Spleen, lymph nodeOccur in nearly all tissues eventually, especially

when they are stored in those solutions for extended periods.

The pigment is birefringent in polarized light and will appear as numerous bright white motes on the slide.

Troubleshooting:Bring sections to water via xylene and ethanol.Place into saturated picric acid in absolute

ethanol for 1 hour.Optionally, treat with saturated aqueous lithium

carbonate to remove picric acid discolouration.Wash well with water.Continue with the staining method.

Trouble shootingPrevented by using 10% neutral buffered

formalin (NBF) or phenol formalin as the fixative.

The NBF should be changed every six months at a minimm

If NBF is not available and either 10% formalin or 10% formal saline are being used, they should be stored over marble chips or crushed oyster shells to neutralise any formic acid as it is produced.

Mercuric chloride artefactSeen with fixatives containing

mercuric chloride.Crystalline or amorphous greenish-

brown artefact pigment of mercury is randomly deposited in tissues.

Treatment of specimens with iodine (Lugol’s iodine) during processing or sections prior to staining, will produce mercuric iodide which can be washed out of the tissues.

A subsequent treatment with sodium thiosulphate then removes residual iodine.

Dewax the section     ↓ Transfer to 70% absolute

alcohol     ↓ Immerse in iodine solution

for 3 min with continous agitation

    ↓ Wash in water     ↓ Immerse in sodium

thiosulfate solution until the yellow color of iodine is completely removed

    ↓ Wash in water

FREEZING DAMAGEFreezing during transport before fixation

also causes cytoplasmic condensation and it occurs secondary to cell dehydration

After frozen section- thaw the block in fixative and process to paraffin

Ice crystal damage and freeze thaw changes

Nuclei surrounded by a free spaceSmaller size with darker chromatinNuclear and cytoplasmic detail not

well defined

Ice crystal artefactClefts and vacuoles

in the central region of the specimen

Frozen section chatter

TROUBLE SHOOTINGDue to slow freezing of tissue

● Solution: Freeze fast (flash/snap); the faster the freeze, the smaller the ice crystals, the less tissue damage (best freezing method is arguably liquid nitrogen)● Smaller tissues yield less artifact - optimally tissue should be 0.5 x 0.5 x 0.3 cm or less● Never freeze fragments larger than the diameter of the chuck● Avoid freezing fat around tissue● Blot the outer surface of the tissue dry with gauze before making your block

UNDER AND OVER HEATINGOptimum temperature for microwave

fixation is 45- 550C.Underheating results in poor sectioning

quality, whereas overheating above 650C produces vacuolization, 

Overstained cytoplasm and pyknotic nuclei .

The mechanism whereby microwaves bring about tissue stabilization involves protein denaturation.

The time taken for IHC and in - situ hybridization can be significantly decreased

Chemical changes can also lead to artefacts.

Gluteraldehyde used to fix tissues will add carbonyl groups to tissues in which they were not present and these groups will react with Schiff’s reagent

This can be overcome by using Bouin’s fixation medium for the storage of specimens.

GROSSINGFloaters or cross-contamination artifacts -

Tissue that appear on a slide which do not belong to that particular area and have floated in during grossing, processing or floatation of cut-sections.

Sloppy procedures on the cutting bench such as dirty towels, instruments or gloves that have remnants of tissue that is carried over to the next case.

One cassette to another during processingDuring section embedding via contaminated

forceps,molds, hot plates,casette coversVia section flotation bathsDuring staining – cells are shed from section and

smear onto another slide

Troubleshoot Floater artefact may be suspected if 

A tissue fragment looks different from others by virtue of section thickness and/or staining intensity

A tissue fragment is on a slightly different plane from others, especially if superimposed

A tissue fragment showing pathologic changes totally different from others and of a type that one would not have expected at all under the clinical circumstances of the particular case.

Thorough rinsing of board and instruments between specimens

Flotation baths skimmed samples

PROCESSING ARTEFACTSPoor processingDehydration is the first step in processing - removal of

aqueous fixative fluids from the tissue by using compounds like alcohol

Clearing is replacing the dehydrating agent with fluid that is miscible with dehydrating fluid and embedding medium

Tissues immersed in too great a concentration of alcohol will usually show a high degree of shrinkage due to rapid removal of water. These are referred to as shrinkage artefacts.

Troubleshoot: After fixation, tissue needs to be dehydrated slowly. Starting with 50% alcohol can prevent this artefact

PROCESSING ARTEFACTSTissue placed in acetone – a dehydrating agent, for a

prolonged period of time tissue – brittle affecting subsequent sectioning.

Prolonged immersion in clearing agent also renders the tissues brittle - crumbling and crystallization of tissues during cutting.

Use the proper amount of clearing agent and no clearing agent should be left behind to contaminate the wax.

Incomplete dehydration - water entrapment within the tissues and cause inadequate staining or opacity within the section.

Frequent changing of processing solutions and covering their containers to avoid moisture contamination 

Inadequate infiltration of tissue with paraffin results in wrinkles that run in all directions.

Artifacts during impregnationThe function of wax impregnation is to remove

clearing agent (wax solvent) from the tissue and for them to be completely permeated by the paraffin wax which is subsequently allowed to harden to produce a block from which sections may be cut.

The artifact produced during this procedure is Crystallization:

Thicker the tissue the more clearing agent it will carry and hence it requires more change of wax to be removed it.

Even if a small amount of clearing agents contaminates the wax it will lead to crumbling and crystallization of tissue during cutting.

LOSS OF SOLUBLE SUBSTANCESCholesterol- tapering needle like crystals in

vessel wall- atheroma and old haemorrhageDissolve – characteristic spaceNeutral lipid from adipose cells- ovoid

spaces

ARTEFACTS OF EMBEDDINGEntrapment of air around the tissue is a

common finding during embedding. This causes the tissue to fall out or vibrate during

the cutting procedure leading to venetian blind artifact which appears as zones of compressed tissue separated by open spaces. . 

 Hydrophilic processing fluids may be retained within the embedded block of tissue and result in wrinkled tissue sections.

If the hardness of the embedding medium is greater than the infiltrated tissue, wrinkles or cracks appear in the tissue sections.

ARTEFACTS OF MICROTOMYMicrotomy, the means by which tissues are

sectioned, so that microscopic examination is possible, involves some artefacts that can get incorporated if proper technique is not followed

Venetian blind effectFine parallel cracks in sectionTiny vibrations in the knife as it passses

hard brittle blocks.Disposable bades are not supported in the

holder.Steep angle of the cutting knife, hard tissue

or wax, and presence of calcification in tissues

TS: Cutting thinner sectionsSharpening the knife before cutting.

Knife lines- Scores in sections in the direction of cutting due to a damaged knife edge.

If the tissue is cut tangentially, the connective tissue cores may become entrapped within the epithelium, giving a false impression of invasive squamous cell carcinoma

Alternate thick and thin sections - the wax is too soft for tissue, block or blade is loose, clearance angle is insufficient or mechanism of microtome is faulty.

TS: Cooling the block, tightening the block or blade and increasing the clearance angle.

Wrinkles and folding of tissue sections - very thin paraffin sections are forced to stretch unevenly around other structures which have different consistencies.

TS: Removed by gentle teasing with forceps or by transferring the sections from the slide to another water bath at high temperature 

Scratch lines - small nicks in the knife edge, large knife clearance angle, hard material embedded in the wax or hard material in the tissue 

Crumbling of sections - knife is blunt or the wax is too soft or due to contamination of wax with clearing agent or water or due to slow cooling of wax

Displacement of tissue components - especially bone - use of dull knife, soft embedding medium, rough sectioning, incorrect blotting and poor adhesion of sections to the glass slide

COARSE CHATTERCutting dense

tissue(uterus/ cervix in large blocks)

Movement of holder or specimen or knife- Vibration in the section.

Poorly designed microtomeChatters or chaffers are

thick or thin zones parallel to knife edge as it cuts the tissue

narrow parallel bands, usually evenly spaced across the tissue specimen 

TS: Suitable microtomeBlocks of a sensible

size, changing the orientation

and soaking the block face with detergent or water.

HOLES FROM ROUGHINGLymph nodes, very cellular

organsExcessively rough

trimmingHoles in thin sections

(a) Tangential section of epithelium caused by improper orientation. (b) Thick and thin section formed due to loosely attached microtome knife. (c) Knife scoring appeared in the section due to a small nick in the knife edge. (d) Folds and wrinkles within the histological section produced by a blunt microtome knife

Artifacts during lifting of tissue sections: Artifacts such as Tissue Folds produce when

tissue adheres to the under surface of the blade, seen most commonly with fatty tissues and mainly seen due to dull blade

TS: transferring the sections to a new water bath or by passing light of Bunsen burner over the section and

adding small amount of detergent may be helpful. Tissue

ARTEFACTS OF FLOTATIONAND MOUNTINGAfter sectioning and

mounting.Section dropped rather than

pulled across the water.Freshly boiled water less likely

to produce artefact.Air bubbles trapped in a

section after flotation and mounting can collapse on drying ,leaving zones which cracks and fails to adhere properly to the slide

Increase temperature of water bath results into expansion of tissue beyond its limit and “Parched Earth (crackes)” effect

Contamination by microorganisms (fungi), air borne fibers, hair, cellulose fibers, floaters or bubbles bene

Contamination by exfoliated squamous cells is another common artifact caused by fingers or sneezes/coughs.

Residual wax artefact- prevent penetration of both aqueous and alcoholic dye solutions - area totally devoid of stain, nuclei - muddy and lack detail

Xylene treatment and re-staining

Stain deposits may appear in sections if the dye solutions are old or unfiltered, undisolved stain, open racks

Replace staining solutions, stain in closed vertical jars

Eosin flakes- Above the focal plane of the tissue section- precipitated dye derived from an unfiltered stock solution.Drying up of sections between the last xylene and cover slipping - entrapment of minute bubbles over the nuclei leading to dark nuclei lacking visible detail (corn flake artifact).

Incomplete staining- Automated staining machines

Leaching of substances from tissues into the dye -- weak staining of calcium by alizarin red S, resulting from loss of calcium ions into aqueous fixative

Leaching of the stain into mounting media Washing eosin-stained sections in tap water with an acidic pH-

Eosin bleed - If there is presence of moisture still in the section after cover slipping due to moisture in alcohols and clearing agents.

Section showing eosin leaching.

Contamination by microorganisms- Regular replacement

TS: Preservative- Thymol or merthiolateMucous contamination- Saliva- Failure

to adequately wash after incubation with saliva- strong PAS staining with residual mucus

TS: Diluting with water or saline, comercial lyophilised diastase preparations

ARTEFACTS OF COVER SLIPPINGStained sections are protected from damage by the

application of cover-glasses with appropriate mounting media.

Bubbles are formed under the cover- slip when the mounting medium is too thick.

TS: A clearing agent is compatible with the mounting medium to be used because some solvent may be carried over to the mounting stage.

Mounting medium of adequate thickness Use adequate amount of mounting mediaIf the mounting media has attained greater

viscosity, xylene can be used as a thinning agent

COVER SLIPPING

 Bleaching of stain is an unwanted outcome of prolonged exposure of the sections to light.

TS: Stained sections should be stored in dark storing cabinets

ARTEFACTS OF BONEReprecipitation

artefacts are caused by lack of agitation and inadequate volume of decalcifying fluid.

They are seen as round granules or crystalline masses which lie mainly in the soft tissues and bone marrow., secondarily calcium phosphate and it stains strongly with alum hematoxylin 

Overdecalcification may hamper cutting qualities, affects staining properties and histological details are destroyed.

Neutralizing the bone section by 1% aqueous solution of lithium carbonate

Surface decalcification of the paraffin block can rectify incomplete decalcification

DIAGNOSTICARTEFACTSCrush artefact in

small cell carcinoma-Nuclear molding and azzopardi effect

Ground glass orphan annie nuclei

An artefact of paraffin embedded tissue

Fried egg artefact- Oligodendroglioma, hairy cell leukemia and chromophobe RCC- Autolytic imbibation of water- Delayed fixation. Not seen in FS.

Lacunar cell of NS HL- Formalin fixation artefact

Peritumoural retraction artefact in BCC

Loss of bullous pemphigoid antigen.

Differentiates from SCC, Trichoepithelioma

Tigroid backround FNA of seminoma- Fraagile cytoplas. Lace like background.

Formalin induced flouresence-Amelanotic melanoma-biogenic amines, including DOPA and dopamine, to show fluorescence after exposure to formaldehyde (formalin-induced fluorescence).

Max Joseph space- basal cell degeneration- processing

summaryPre fixation artefacts- Crush, split, agonal

changes and pseudomicrocystThermalSuctionChemical- Chemo,Monsels solution,

myospherulosis, sutures, gelfoam, starch

FIXATIONDelayed, inadequate and prolonged fixationZonal fixation and diffusion/streamingPigment artefactsFreezing and heating artefactsProcessing artefactsEmedding artefactsArtefacts of microtomy- Venetian blind,chatter, folds,

rough holingStaining artefacts- Wax artefact, Staind deposits, Eosin

flakes, Cornflake artefactMounting and coverslippind artefacts- Bubbles, Bleaching Artefacts of boneDiagnostic artefacts

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