95 7858 Rev A Artifacts Handbook

Artifacts in Histological
and Cytological Preparations

Geoffrey O Rolls, Neville J Farmer and John B Hall
Scientia
Leica Microsystems Education Series
Acknowledgements
This work is derived from Laboratory Histopathology: A Complete Reference edited by Anthony E Woods
and Roy C Ellis. It is republished under licence.
Authors
Geoffrey O Rolls BAppSc, FAIMS
Histology Consultant
Leica Microsystems
Former Senior Lecturer Histopathology
Department of Laboratory Medicine
RMIT University
Melbourne
Neville J Farmer DipMLT, FAIMS
Histology Consultant
Leica Microsystems
Former Manager Anatomical Pathology
Dorevitch Pathology
Camberwell Victoria
John B Hall MSc, FAIMS
Senior Scientist (Retired)
Anatomical Pathology Unit
Alfred Hospital
Prahran Victoria
The authors would like to express their appreciation to Ms Debbie Reich (Dorevitch Pathology) and Mrs
Diana Stockman (Victorian Cytology Service) for providing material and advice on the cytology section,
and to Mr Alan Sutton (Queensland Health), Mr Clyde Riley (Womens Cancer Foundation), and Dr Jenny
Penschow (The Howard Florey Institute) for providing examples and supporting information of particularly
interesting material.
Introduction
In histological and cytological terms an artifact can be dened as a structure that is not normally
present in the living tissue. The problem is recognizing artifacts as such when they do occur and
not confusing them with normal tissue components or pathological changes. In some situations the
presence of an artifact can compromise an accurate diagnosis.
The aim of this publication is to promote an awareness of the various artifacts which may be
encountered in histopathology, to provide a guide for their recognition, to explain their causes and
to suggest, where possible, the means by which their occurrence can be avoided. Examples of many
common artifacts and some rare ones have been included but the intention is to provide an overview
of artifacts and not a complete compendium. Alternative names for the artifacts are included in the
list of common artifacts on page 100.
The scheme used to classify the artifacts is modied from that originally devised by Wallington1. It
follows the sequence normally required to produce parafn sections but also includes categories
for more specialized techniques.
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Contents
Pre xation Artifacts ..................................................................................................................................................... 3
Fixation Artifacts ......................................................................................................................................................... 21
Tissue-Processing Artifacts ..................................................................................................................................... 27
Artifacts of Microtomy and Section Mounting...................................................................................................... 31
Staining Artifacts......................................................................................................................................................... 45
Section Preservation Artifacts ................................................................................................................................. 53
Artifacts in Frozen Sections ...................................................................................................................................... 59
Artifacts in Bone and Calcied Tissues .................................................................................................................. 65
Artifacts in Resin Sections ........................................................................................................................................ 71
Histochemistry, Immunohistochemistry and Hybridization Histochemistry ................................................... 77
Miscellaneous Artifacts ............................................................................................................................................. 83
Artifacts in Cytological Preparations ...................................................................................................................... 89
List of Common Artifacts ......................................................................................................................................... 100
References .................................................................................................................................................................. 105
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2 / Leica Microsystems Scientia
Prefixation Artifacts
Pre xation artifacts are produced in tissues before xation. They may take the form of deposits such
as tattoo pigment, or result from a surgical procedure as with laser knife damage or crush artifact.
Contaminants can also be introduced into tissues during surgery or whilst handling prior to, or during
specimen dissection. This type of artifact can only be avoided by ensuring that those involved are fully
aware of the consequences of allowing a specimen to become contaminated or otherwise damaged.
/3
Heat Damage
Heat damage is often seen along the margin of surgical biopsies. It takes the form of strong acidophilic
staining in a local area with loss of nuclear and cytoplasmic detail (Figure 1). Connective tissue
bers become coagulated due to the effects of heat and, if glandular tissue is present, it may become
vacuolated. This artifact is caused by laser-generated heat xation of the tissues. Resultant dehydration
and denaturation of proteins causes coagulation and condensation and leads to increased acidophilia of
connective tissues compared to normally xed tissues.
General or local heat damage to specimens can also occur at any stage following xation but particularly
during inltration with over-heated wax, when embedding using heated forceps and while drying slidemounted sections on a hot plate or in an incubator.
Figure 1
A breast biopsy in which a laser knife has been used during specimen removal. The typical acidophilia of the heat-damaged tissue
can be seen while the adjacent glandular tissue is largely unaffected; H&E.
Prexation Artifacts / 5
Presence of Sutures
Suture material is an occasional inclusion in histological specimens. It may consist of isolated fragments
or complete ber-bundles cut in transverse, oblique or longitudinal planes. Detail of the ber structure can
sometimes be seen upon careful examination of H&E-stained sections (Figure 2A). Silk sutures exhibit a
strong birefringence under polarized light and this can be useful in their identication (Figure 2B).
Whilst the presence of a suture in a histological specimen may not be of any pathological signicance
it can damage the microtome knife leading to tears and knife lines in sections. Visible sutures should be
removed wherever possible.
A
Figure 2
H&E-stained section from a stitch granuloma. A: Brighteld. B: Polarized light.
Cellulose Contamination
Cellulose is commonly present in association with the lumenal surface of gastrointestinal tract tissues
which have not been washed adequately before processing. Whilst not of pathological signicance
cellulose can cause shredding of sections. Occasionally it may be present in an unexpected situation
such as within the mass of a bowel tumor (Figure 3). It is recognized by the characteristic appearance of
plant cells with their strongly staining cell walls and square shape.
Cellulose can also be encountered as a contaminant arising from paper, cotton gauze or a cork board used
during specimen preparation. In this case, it is usually found on the surface of the specimen but can be
implanted mechanically during dissection.
A
Figure 3
Cellulose in an H&E-stained section of intestinal tumor. A: Brighteld. B: Polarized light.
Gelfoam Artifact
Gellm and Gelfoam are made from absorbable gelatin and, in the form of a thin lm or sponge, are used
to control bleeding in various surgical procedures. They may be encountered in sections usually adhering
to a specimen surface (Figure 4). Gelatin foam has a characteristic appearance with slightly basophilic
gelatin walls of varying thickness surrounding distorted spaces which may contain blood or other cell
types. Typically there is no tissue reaction to the presence of this material.
Figure 4
Gelfoam associated with the splenic capsule; H&E.
Starch Contamination
Starch is used as a powder in surgical gloves and can therefore be deposited within or upon the surface
of surgically-acquired tissues. On occasions it may be present within granulomas removed surgically.
Starch contamination can also occur in the laboratory if new gloves are not washed before handling
specimens.
Starch can be difcult to see in H&E-stained sections using normal brighteld microscopy. Granules tend
to be spherical to slightly angular (hexagonal), with some exhibiting a small, central dark spot (Figure
5A). Starch stains very strongly with the periodic acid-Schiff (PAS) reaction2 and shows a characteristic
Maltese cross birefringence with polarized light (Figure 5B).
A
Figure 5
Starch granules in a typical starch granuloma stained with H&E. A: Brighteld. B: Polarized light.
Catheter Damage
Epithelial surfaces are easily damaged in both living tissue and xed specimens. Catheterization performed
before or during surgery may partly or completely remove epithelium and compress underlying tissues. If
tissue xation is prompt this compression and epithelial damage will remain (Figure 6).
Figure 6
A vas deferens from which most of the lining epithelium has been removed and underlying tissue compression is visible; H&E.
Crush Artifact
In the fresh state some tissues (such as lymph node) are highly susceptible to damage from crushing by
forceps or other surgical instruments. This artifact is typically seen at the periphery of specimens and is
usually in small, localized areas (Figure 7). (See also Effect of Crush Artifact on Immunostaining).
Figure 7
Crush artifact in a section of lymph node. At one margin the section shows darkly staining, distorted cell nuclei, some stretched
out and attened, but maintaining an intense basophilia; H&E.
Necrosis due to Monsels Solution

Monsels solution is a topical hemostatic agent used to control bleeding following skin or mucosal biopsy.
The solution is normally painted onto the site after tissue removal where it causes coagulation and
necrosis to a maximum depth of 0.6 mm. The solution contains ferric subsulphate which is deposited
diffusely in the local area and is taken up by macrophages. 3
On occasion Monsels solution is mistakenly applied before the biopsy is taken. This can result in a general
basophilia, partial desquamation and cracking of the epithelium and signs of early necrosis (Figure 8). It
has been reported that treating sections with a restitution solution consisting of 0.5% hydrochloric acid in
70% ethanol at 60C for one hour prior to staining, signicantly improved the image quality of subsequent
H&E stains. 4
A
Figure 8
The effects of Monsels solution in a cervical biopsy section. A: H&E. B: Perls stain illustrating the extensive deposition of iron
within the epithelium.
Tattoo Pigment
Various colored insoluble pigments used in producing decorative tattoos are occasionally encountered
in sections of skin (Figure 9). These deposits are generally unreactive to histochemical tests and
monorefringent under polarized light.
Figure 9
A section of skin in which deposits of nely granular black material lie free in the reticular dermis. There is no tissue reaction to
the presence of the deposits; H&E.
Postmortem Change
Degenerative changes commence immediately tissue is deprived of an effective blood supply. Autolysis
is produced by hydrolytic enzymes released when lysosomal membranes rupture. Autolysed tissue
usually shows nuclear pyknosis, karyorrhexis and karyolysis to varying degrees along with cytoplasmic
vacuolation and eventually disintegration of tissue structure (Figure 10). Tissues show varying
susceptibility to these changes, with glandular epithelial tissue being rapidly affected while connective
tissue and, in particular, connective tissue bers are much more resistant. Autopsy specimens are more
likely to show autolysis than surgical specimens providing the latter are promptly xed.
Microorganisms present in tissues postmortem can be derived from organisms which form the natural
ora during life (such as those of the gastrointestinal tract) or are contaminants arriving from various
sources after death. These organisms often stain weakly with hematoxylin (Figure 10).
Postmortem changes are retarded by storage at 4 C but can only be completely avoided by rapid xation
an unlikely event in most autopsy cases.
Figure 10
An autopsy specimen of liver illustrating the poorly dened nuclei and imprecise cytoplasmic staining which are characteristic of
autolysis. Shown also are many bacteria within large and small blood vessels and free in tissue spaces. There is no tissue reaction
to the presence of these organisms ; H&E.
Desquamation
Shedding of epithelial cells is also a sign of early autolysis (Figure 11).
Figure 11
Autolytic liver tissue in which a large bile duct shows desquamation of the columnar epithelium; H&E.
Specimen Marking Dyes

The excision margins of fresh or xed surgical specimens are sometimes marked with coloring agents
to allow appropriate orientation of the specimen and assessment of these margins microscopically.
Reagents commonly used include India ink, silver nitrate, alcian blue or alcian green and many proprietary
products of various colors. The agent not only colors the surface of the specimen but may also penetrate
the tissue to varying degrees (Figure 12).
Figure 12
The margin of a skin biopsy marked with silver nitrate. The reagent stains the soft tissues and has penetrated into the dermis.
Intense staining of keratin at the edge of the specimen can also be seen; H&E.
Biopsy-Pad Artifact
Foam pads are commonly inserted into plastic tissue cassettes to prevent the escape of small specimens
during processing. Pads are compressed when the cassette is closed compressing, in turn, the enclosed
specimen. If the specimen is fresh or only partially xed, a ne pattern of local pressure effect, or molding,
can occur and will become a permanent feature of the tissue architecture as xation is completed. The
molding reects the pore structure of the foam (Figure 13).
The artifact is avoided if pads are not used until the tissue is thoroughly xed or by wrapping small, soft
specimens in gauze or lens paper.
Figure 13
Biopsy-pad artifact. Triangular spaces are visible within the tissue; a margin of eosinophilic, compressed tissue with attened
cell nuclei can be seen around the perimeter of each space; H&E.
Freezing Damage
It is common in many laboratories after frozen sections are cut to thaw the tissue block in xative and
process to parafn for conrmation of the diagnosis. These specimens may contain ice crystal damage
as well as more subtle freeze-thaw changes.
For instance in frozen, thawed and xed tissue the nuclei may be surrounded by a clear space and appear
slightly smaller with condensed, darker-staining chromatin (Figure 14). Also the general nuclear and
cytoplasmic detail is not as well dened. These effects are not uniformly distributed across the section
but tend to occur in irregular patches.
Figure 14
Section of lymph node which has been frozen, thawed, xed then processed to parafn; H&E.
Specimen-to-Specimen Contamination
Most specimen-to-specimen contamination (Figure 15) probably occurs during dissection where tissue
from a previous specimen is transferred via the instruments used (such as scalpel blades) or from
fragments which remain on the dissecting board surface. Thorough rinsing of the board and instruments
between specimens or covering the dissection board with separate paper sheets will avoid this problem.
Reusable processing components (such as tissue cassette lids), if not thoroughly cleaned, can also carry
fragments of previous specimens.
It is possible for tissue fragments to pass directly from one cassette to another during processing or
from previously used reagents. This risk can be reduced through the judicious selection of cassettes
and by ensuring that foam pads or other enclosures contain the specimen and that only uncontaminated
reagents are used for processing.
Specimen transfer may occur infrequently during other stages of section preparation including:
during specimen embedding via contaminated forceps, moulds, hot plates and cassette covers
via section otation baths (these should always be skimmed between samples to remove any section
fragments from previous specimens which may remain) this form of contaminant can often be
detected from its position on the slide in relation to the actual section
during staining if cells are shed from a section or cytological smear and deposited on another slide
The possibility for undetected contamination of a specimen with tissue of a similar type (for example
endometrial tissue with endometrial fragments) should not be overlooked. The consequences of such
contamination could, at worst, lead to an inaccurate diagnosis. It is clearly important therefore to remove
the potential for specimen-to-specimen contamination as far as possible.
Figure 15
A section of cardiac muscle with a piece of extraneous thyroid tissue present against one surface; H&E.
Fixation Artifacts
The process of xation can produce artifacts in tissues if the procedure is not carried out under
optimal conditions, if xative does not have proper access to the tissues, or because of the nature and
quality of the particular reagent used.
/ 21
Zonal Fixation
This artifact is most commonly seen in large specimens particularly those surrounded by a capsule as well
as in tissue which degenerates rapidly (such as glandular tissue). It occurs when the xative penetrates
slowly producing differing degrees of xation at different levels within the specimen (Figure 16). Common
causes for this are insufcient time in xative, attempting to x a specimen which is too large, or by using
a reagent with a poor penetration rate.
A
Figure 16
Zonal xation. A: A section of marrow aspirate where red blood cell lysis is evident in the left side of the eld whereas the cells
in the adjacent area are intact; H&E. B: Liver trichrome stain showing uneven staining due to xation effects.
Streaming Artifact
Streaming artifact is caused through precipitation and displacement of glycogen by an advancing xation
front. It is often seen in formalin xed liver (Figure 17) but may not be as obvious with preferred glycogen
xatives (such as formal-alcohol or Bouin). It can be completely avoided by freeze-drying.
Figure 17
Streaming artifact in a formalin- xed section of liver; PAS.
Fixation Artifacts / 23
Formalin Pigment
This pigment appears as a brown to black, nely granular, birefringent deposit associated with, or in the
vicinity of, red blood cells (Figure 18). It is most often seen in tissues rich in blood such as spleen and
in tissues which have had prolonged xation as with many postmortem specimens. It forms when acid
formalin reacts with hemoglobin to form acid formaldehyde hematin.
In time formalin naturally decomposes to form formic acid which causes the problem. In non-buffered
solutions the fall in pH can be quite rapid but even in buffered formalin solutions the buffering capacity
can be eventually overcome and acid conditions can result in the appearance of the pigment.
The artifact can be removed by treating the section with saturated alcoholic picric acid solution prior to
staining or prevented by using buffered formalin solutions and restricting xation time.
A
Figure 18
Formalin pigment. A: Typical appearance associated with red cells in a section of kidney. B: Pigment in a section of fatty liver
where it is also deposited in the vicinity of red blood cells in the sinusoids and within fat vacuoles5 in the form of needle crystals;
H&E.
Mercury Pigment
Fixation in mercuric chloride-containing xatives produces a brown to black granular deposit distributed
randomly throughout the tissues (Figure 19). Mercury pigment can be removed by treating the section
with Lugols iodine or alcoholic iodine solution and then bleaching the section with sodium thiosulphate.
The use of mercuric chloride in xatives should be actively discouraged because of its highly toxic nature.
Zinc salts used in xatives as a substitute for mercuric chloride do not produce artifact pigments.
Figure 19
Mercury pigment (B5 xative) in a section of kidney; H&E.
Fixation Artifacts / 25
Tissue-Processing Artifacts
Artifacts that occur during processing to parafn wax may be the result of inadequate or incomplete
xation or some processing fault. It is inevitable that some shrinkage and distortion will occur during
processing.
/ 27
Vessel Shrinkage in Central Nervous System

Perivascular shrinkage in nerve tissue (Figure 20) can largely be avoided by perfusion xation and gentle,
prolonged processing using chloroform as a clearing agent. Celloidin-parafn double embedding may
also be helpful. A similar effect can frequently be seen around large neurons.
Figure 20
Parafn section of brain stem. Perivascular separation of dense nervous tissue at the junction of the adventitia and glial membrane
is due to poor xation; H&E.
Poor Processing
Extensive loss of architectural detail and clarity within loose connective tissue may reect inadequate
xation, but can also be caused by faults in tissue processing (Figure 21). These include too short a
processing cycle (especially wax inltration times), inappropriate choice of reagents, use of exhausted
reagents, a mechanical fault with the processor, or an error in replacing solvents on the processing
machine.
Figure 21
As a result of poor processing this breast specimen shows extensive tissue disruption within the loose connective tissue; H&E.
Tissue-Processing Artifacts / 29
Loss of Soluble Substances

Cholesterol is seen as tapering needle-like crystals in vessel walls in atheroma and various sites of old
hemorrhage. The crystals dissolve during processing leaving spaces of characteristic shape (Figure 22).
A more common example of the loss of soluble substances is seen when neutral lipid is dissolved from
adipose cells leaving regular ovoid spaces surrounded by a rim of cytoplasm.
Figure 22
Cholesterol clefts in a section of oral cyst; H&E.
Artifacts of Microtomy and

Section Mounting
Various forms of mechanical damage produced during section cutting and otation, together with a
range of contaminants from a variety of sources, are commonly encountered in sections.
/ 31
Knife Lines
Scores in sections are the result of a damaged knife edge (Figure 23). Knife lines may extend across the
whole eld but more commonly appear as a single score running across the section in the direction of
cutting. Severe scoring can usually be seen macroscopically.
Knife edges may be faulty initially (disposable blades may be defective as supplied or conventional steel
knives may be inadequately sharpened), or are damaged through careless handling (for example by
contact with forceps) or when sectioning hard material such as foci of calcication within the block. In
the last instance surface decalcication before cutting is recommended.
Figure 23
Knife lines in a section of spleen; H&E.
Displacement of Components
Displacement of tissue components can occur during section cutting, mounting, or at some later stage
and is commonly seen in sections of bone (Figure 24). It can also affect collagen, elastic, or reticular bers
causing them to become re-aligned along the direction of cutting. Displacement can be caused by a dull
knife, excessively rough sectioning, using an embedding medium which is too soft for the density of the
tissue being cut, careless otation technique, incorrect blotting or poor adhesion of sections (leading to
lifting during staining or coverslipping).
Figure 24
Displacement of cancellous bone in a trephine section stained for reticulin.
Artifacts of Microtomy and Section Mounting / 33
Venetian Blind Effect

Fine parallel cracking in sections is usually caused by tiny vibrations in the knife edge as it passes through
hard, brittle blocks (Figure 25). It most often occurs when disposable blades are not properly supported
in the knife holder. Excessive hardness and brittleness in blocks caused by over-processing, over-heating
during inltration and embedding, or over-chilling followed by excessively rough or rapid cutting may also
produce this effect. Cutting thinner sections or softening the block before cutting will normally overcome
micro-chatter.
Figure 25
Micro-chatter in a section of rodent liver stained for reticulin.
Coarse Chatter
Coarse chatter usually occurs when cutting dense tissue (particularly uterus and cervix) in large blocks or
when movement of the specimen holder, knife or knife holder causes vibration in the section (Figure 26).
The effect can also be seen in a poorly designed microtome albeit in perfect mechanical condition.
Coarse chatter can be avoided by using a properly maintained microtome of suitable design and by cutting
appropriately processed blocks of a sensible size. Care must also be taken to clamp the specimen, knife
and knife holder rmly.
Figure 26
Coarse chatter in a section of cervix; H&E.
Holes from Roughing

This effect may occur when sectioning lymph node and other very cellular organs. Excessively rough
trimming pulls tissue fragments from the block face and these appear as holes in subsequent thin sections
(Figure 27). The effect can usually be seen when oating sections on the waterbath.
Figure 27
Roughing effect in a section of lymph node. The parallel alignment seen in this particular example is not always evident; H&E.
Tidemark due to Adhesive

The pale amorphous, hematoxylin-stained deposits in Figure 28 are caused by the pooling and subsequent
evaporation of otation uid containing dichromate-gelatin adhesive. The pools are more likely to occur
in irregular, poor quality sections where protein-based adhesives are used and sections are not drained
of excess adhesive before drying. In this example the section was dried at on a hot-plate.
Figure 28
Adhesive pool artifact; H&E.
Bubbles Under Section

Air bubbles trapped in a section after otation and mounting can collapse on drying leaving zones which
crack and fail to adhere properly to the slide. These regions often display altered staining (Figure 29).
The bubbles may be caused by poor otation technique, where a section is dropped rather than pulled
gently across the water surface. Alternatively, bubbles already present in the bath can be dislodged by
the slide and rise up under the section. Freshly boiled water used in otation baths is less likely to produce
bubbles.
Figure 29
Section of lymph node showing circular darkly stained areas with pale centers and surrounding radial cracks. These faults
indicate collapsed bubble artifact; H&E.
Sneeze Artifact
Squamous cell contamination on sections is not uncommon and usually originates from ngers or scalp
and, less commonly, sneezes (Figure 30) or coughs.
Figure 30
A sneeze-generated deposit (contaminant) which contains squamous cells, cell debris, some bacteria and an amorphous
background that stains weakly with hematoxylin; H&E.
Contamination of Mounted Sections

Mounted, unstained sections left uncovered, can become contaminated with various materials
including:
microorganisms, particularly airborne fungi (Figure 31)

airborne bers (Figure 32)
hair from the brush used to transfer sections from knife edge to otation bath (Figure 33)
cellulose bers from facial tissues used to skim otation baths or wipe slides and coverslips (Figure 34)
dirt (Figure 35).
Figure 31
A semi-thin resin section of kidney which has become contaminated with fungal hyphae and conidia when left, unprotected, on
a hotplate overnight to dry; PAS.
Figure 32
Fiber of unknown origin contaminating
a section of skin. Despite investigation
of a range of potential local sources
the origin of these bers was never
identied; H&E.
Figure 33
Section of vagina showing a hair beneath
the section; H&E.
Figure 34
Cellulose ber contamination in a section
of ureter; H&E.
Figure 35
Liver section showing dirt which was
deposited on the section prior to staining,
probably during drying. This highlights
the importance of absolute cleanliness
at all stages of section preparation.
Folds
Cartilage is one of several problem tissues likely to show folds after otation (Figure 36). The folds are
difcult to avoid unless specialized techniques, such as double-embedding or resin embedding, are
used. Folding is caused by cartilage shrinking less than other tissue types during processing followed by
variable expansion on otation. These faults are readily identied macroscopically. Careful microtomy
and otation techniques minimize this problem.
Figure 36
Folds in an island of hyaline cartilage. Guinea pig trachea; H&E.
Staining Artifacts
Artifacts which arise during staining fall into two main groups: incomplete or patchy staining in
otherwise satisfactorily stained preparations, and precipitates or contaminants derived from the
staining solution(s). Patchy or incomplete staining is most commonly seen as areas devoid of all or
some of the stain components.
/ 45
Residual Wax
Residual wax in a section will prevent the penetration of both aqueous and alcoholic dye solutions leaving
areas totally devoid of stain (Figure 37). The nal clearing of the section before coverslipping removes all
traces of the wax leaving no evidence as to the cause of the patchy staining. Traces of residual wax can
also have a subtle effect on nuclear staining, producing small patches in sections where nuclei appear
muddy and lack detail. Prolonged xylene treatment and re-staining will overcome this problem. These
faults are readily identied macroscopically.
Figure 37
A section of skin in which the supercial epidermis and keratin have failed to stain with either hematoxylin or eosin because of
residual wax; H&E.
Incomplete Staining
Incomplete staining with one dye in a multi-step procedure may result from an inadequately lled staining
dish (Figure 38). This is most commonly observed with automated staining machines.
Figure 38
A liver section stained on a linear stainer shows satisfactory hematoxylin staining throughout but an absence of eosin at one end
of the section. This was due to an inadequate level of eosin in the container.
Staining Artifacts / 47
Unstained Area in Section

Unstained areas in a section may occur when solvent is allowed to accumulate at the top of the slide
during the dewaxing process, either because the dewaxing bath is lled to a level above that of the
alcohol baths, or solvent is trapped between the slide holder and slide. In both situations beads of waterimmiscible solvent collect above the section and may streak down the slide at any stage to prevent
staining by aqueous dyes.
A variant of this artifact is shown in Figure 39. The section of liver, stained by Shikatas orcein method
for hepatitis B antigen, shows a deeply stained focus of cells. This method requires pretreatment of the
section with potassium permanganate and oxalic acid to reduce background staining. Exposure to these
reagents appears to have been prevented, possibly by traces of solvent which may have remained in the
section. The orcein stain, prepared in alcohol, has had full access to the tissue. This phenomenon will
show the reverse effect when the contaminants prevent the access of aqueous dyes to tissues resulting
in pale or unstained areas in the section.
The remedy to these artifacts is essentially good technique, frequent replenishment of dewaxing solvents,
and maintenance of correct levels in the staining baths.
Figure 39
Section of liver showing a deeply stained focus of cells where suppression of the background staining has failed (see text for
details); Shikatas orcein.
Stain Deposit
This type of artifact may arise from undissolved stain, stain precipitate or any solid component in an
unltered staining solution (Figure 40). Precipitation can occur when volatile solvents are used and in
methods which require heat or long staining times. The problem is increased when slides are stained on
open racks. The use of sealed staining jars which hold slides vertically will eliminate most artifacts due
to precipitates.
Figure 40
A precipitate of alcian blue deposited on the tissue section and to a lesser extent on the slide.
Contaminated Staining Solution

Contamination of staining solutions by microorganisms which are subsequently deposited on the section
(Figure 41) is readily identied and prevented by regular replacement of solutions prone to microbial
growth, or by adding a preservative such as thymol or merthiolate. Filtration of the staining solution may
overcome an immediate problem.
Figure 41
The effect of staining with a solution contaminated by microorganisms.
Mucus Contamination
The standard procedure for the demonstration of glycogen in histological sections utilizes the PAS
reaction preceded by diastase digestion as a negative control. Human saliva is a convenient, reliable, and
cost-free source of this enzyme. However, failure to adequately wash sections after incubation with saliva
may lead to strong PAS staining in residual mucus (Figure 42). The potential for mucus contamination
can be minimized by diluting the saliva with water or saline; the use of commercial lyophilized diastase
preparations eliminates the problem.
Figure 42
A bone marrow section treated with saliva prior to PAS staining. Mucus strands, which have not washed off prior to PAS staining
cover the section.
Section Preservation Artifacts

Degeneration artifacts which develop in sections during storage may not be evident for months
or even years. This can cause major problems when reviews of archival material are required.
/ 53
Drying Artifact
The common problem of large air bubbles in the mountant is easily recognized; a less obvious variant is
shown in Figure 43. The appearance of dark nuclei, lacking visible detail occurs when excessive time is
taken between removing the slide from xylene and applying the coverslip. The section starts to dry and
minute bubbles become trapped over the nuclei when the coverslip is applied. Remounting sections will
usually overcome the problem.
Figure 43
Drying artifact in a section of liver; H&E.
Water in Section
Since water is immiscible with clearing agents, sections which have not been adequately dehydrated will
be opaque due to the presence of water (Figure 44). Sometimes small water droplets may also be seen
microscopically. The presence of water masks microscopic detail and causes leaching of stains. If water
is detected in a section the coverslip should be removed, the section washed in clearant then returned to
alcohol. When the residual water has been removed the section can be cleared and remounted.
Figure 44
Dehydration fault in a section of liver; H&E.
Section Preservation Artifacts / 55
Mountant Breakdown
Incorrectly prepared polystyrene-based mountants are prone to degenerate over time (Figure 45). Should
this occur remove the coverslip and defective mountant and remount with a proven product.
A
Figure 45
Polystyrene mountant breakdown. A: Crazing (cracking). B: Crystallization (presence of spherocrystals)
Bleaching of Stain
Bleaching of stains can result from exposing sections to light for excessive periods. Sections affected in
this way can be re-stained. Stained sections are best stored in the dark.
Figure 46 shows a section of liver showing a circular, very pale area from which the stain has been
bleached. This was caused by exposure of the slide for a short period to the very intense light from an
old-fashioned micro-projector.
Figure 46
Liver section showing a bleached area caused by exposure to a high intensity light source; H&E.
Section Preservation Artifacts / 57
Artifacts in Frozen Sections

Although many of the artifacts encountered in parafn sections may also occur in frozen sections,
there are some that are particular to frozen sections.
/ 59
Ice Crystal Artifact

Ice crystal artifact is one of the more common and potentially serious artifacts in frozen sections as
it often makes diagnosis difcult or impossible (and once formed cannot be overcome). The effect
appears as intercellular clefts in highly cellular tissues and as intracellular clefts and vacuoles in skeletal
muscle (Figure 47). Ice crystal artifact results from the slow freezing of tissue, inappropriate quenching
techniques or the selection of tissue samples too large to permit rapid freezing. Consequently damage
is most prominent in the central region of the specimen. While this artifact is usually associated with
unexpected urgent frozen sections where attention to optimal freezing technique is often secondary to
speed, severe ice crystal artifact also occurs in large specimens (particularly amputated limbs) frozen for
preservation or to facilitate sampling a practice not to be recommended.
Figure 47
Ice crystal artifact in a section of skeletal muscle; H&E.
Frozen Section Chatter

Frozen tissues are prone to sectioning artifacts similar to those which affect parafn blocks. Chatter is
one example this occurs because the specimen is too cold and hard (Figure 48), the specimen or knife
is poorly supported or adjusted, or the section thickness is too great.
Each tissue type has an optimal temperature for the preparation of frozen sections and, in the absence
of other causes, chatter indicates the specimen is below its optimal sectioning temperature. Holding a
gloved nger against the frozen tissue face will usually warm the specimen sufciently to allow one or
two sections to be cut free from chatter.
Disposable microtome blades are less robust than conventional knives and their use has resulted in frozen
section chatter becoming a more common problem. The optimal temperature of the tissue is critical with
disposable blades and their low rigidity makes them more suited to small tissue samples and frozen
sections of 5 m in thickness or less.
Figure 48
Frozen section chatter. The temperature at which the specimen was cut was too low for satisfactory sections to be prepared.
Artifacts in Frozen Sections / 61
Fuzzy Staining
Fuzzy or indistinct staining may be observed when frozen sections of fresh tissue are allowed to dry
before xation. Figure 49A shows a cryostat sections of oral mucosa which was picked up on a slide and
immediately wet- xed using Carnoys uid then stained H&E. It shows good nuclear and cytoplasmic
detail. Another section from the same specimen was prepared in the same way except that it was allowed
to dry before xation (Figure 49B). It shows poor nuclear detail, poorly dened cytoplasm and an overall
loss of clarity. At higher magnication nuclear and cytoplasmic vacuolation is evident.
A
Figure 49
Cryostat sections of oral mucosa; H&E. A: Was immediately wet- xed in Carnoys uid. B: Was allowed to dry prior to xation.
Miscellaneous Artifacts
Other common artifacts associated with frozen sections include folds which result from poor section
quality and/or poor mounting technique, fat displacement and air bubble entrapment during section
mounting (Figure 50). Coverslipping with aqueous mountants is technically difcult and bubbles form
readily. Pressing the coverslip to displace the bubbles may dislodge lipid or other components and should
be avoided.
Figure 50
Frozen section showing folds, fat displacement and air bubble entrapment; Herxheimers Sudan method.
Artifacts in Frozen Sections / 63
Artifacts in Bone and Calcified Tissues

Artifacts in calcied tissues may occur as a result of the sampling or collection techniques, from
tissue processing including decalcication, or because of factors related to microtomy.
/ 65
Impacted Bone Fragments

When samples of bone are taken for histological examination, either by needle or trephine, or when using
a saw to remove a small sample from a large specimen, the bone is subjected to signicant trauma which
often leads to displacement of bony fragments and disruption to the adjacent soft tissues 6 (Figure 51). The
remedy is to trim deeply into the specimen to avoid the most traumatized areas close to the surface.
Cracking of Matrix in Resin Sections

Artifacts of undecalcied bone include cracking in the bone matrix (Figure 51). This is difcult to
eliminate but it may be minimized by extending processing times to improve inltration and by slowing
polymerization of epoxy resins to promote linear polymers and provide exibility.7 The artifact is further
reduced if diamond knives are used in preference to glass knives for sectioning.
Figure 51
Bony fragments impacted into the bone marrow during collection of this trephine. Cracks are also evident in the bone matrix; resin
embedded, undecalcied section, von Kossa method.
Bone Dust Artifact

Undecalcied resin sections may contain a ne, grit-like, bone dust or powder seen within or in close
proximity to the bony trabeculae (Figure 52). This material is most apparent in H&E-stained sections
where it stains strongly with hematoxylin but is largely obscured by von Kossa staining. When deposited
in the bone marrow it stains black with von Kossa, suggesting it originates from the calcied trabecular
matrix. The artifact is probably produced during sectioning and becomes more prominent with increasing
section thickness. The bone dust occurs in sections prepared with both glass and diamond knives, and
there appears to be no reliable way of preventing it.
Figure 52
Bone dust artifact; H&E.
Artifacts in Bone and Calcied Tissues / 67
Effects of Over-Decalcication on Tissues

Most artifacts in decalcied tissues relate to poor initial xation, over-decalcication, or incomplete
decalcication. Artifacts arising from the rst two situations are irreversible whereas incomplete
decalcication can be rectied by surface decalcication of the parafn block.
Specimens subjected to decalcication by mineral or organic acids must be protected from the hydrolytic
action of these agents by proper xation. Digestion of cellular and other tissue components occurs more
rapidly when the tissue is un xed or only partially xed 8 . Prolonged exposure to acid decalcifying agents
will eventually damage even well- xed tissues, so determining the end point of decalcication accurately
is essential in all situations. Over-decalcied sections typically stain strongly with eosin and show a
marked loss of nuclear hematoxyphilia. Nuclear and cytoplasmic detail is poorly preserved (Figure 53).
Figure 53
Over-decalcication (nitric acid) in a section of bone; H&E.
Effects of Incomplete Decalcication

Incomplete decalcication of a specimen is clearly apparent when trimming a parafn block. As discussed
previously, this problem is readily corrected, but there are associated problems such as damage to the
microtome knife and to the soft tissues surrounding the calcied areas. If sections can be obtained, the
bony trabeculae stain strongly with hematoxylin (indicating residual calcium) and the adjacent soft tissue
is severely disrupted (Figure 54).
Figure 54
Incomplete decalcication in a section of cancellous bone; H&E.
Artifacts in Bone and Calcied Tissues / 69
Limitations of Parafn Wax Embedding for Dense Bone

Parafn wax may not adequately support dense bone and resultant sections may show holes, folds and
coarse chatter (Figure 55). Soaking the trimmed face of the parafn block in a softening agent for 5 to 15
minutes prior to chilling may improve the section quality.
Figure 55
Parafn section of decalcied dense bone showing holes, folds and coarse chatter; H&E.
Artifacts in Resin Sections

Although polymerizing resins are rarely used as embedding agents in routine histopathology, they are
valuable for high resolution light microscopy and calcied tissue research. Artifacts in resin sections
can be quite subtle and difcult to overcome.
/ 71
Polymerization Artifacts in Epoxy Resin Sections

When used for small tissue samples, epoxy resins polymerize satisfactorily at 60C in 16 hours
(overnight). Large specimens are often not adequately polymerized in this time and may require up to
3 days. Occasionally a specimen may appear to section satisfactorily but on staining is found to show a
chatter-like artifact which is limited to the central zone of the specimen with the tissue at the periphery
well presented (Figure 56). This artifact can be signicantly reduced or abolished by returning the block
to the 60C oven for 24 hours. Failure to thoroughly mix the components of the epoxy resin and omitting
the accelerator from the inltrating solutions will retard polymerization and can cause this problem.
Figure 56
Polymerization artifact in a 2 m epoxy resin section of lymph node; H&E.
Incomplete Removal of Epoxy Resin Prior to Staining

As many epoxy resin formulations are non-degradable, methods have been developed to stain the tissues
with resin intact. Techniques for the removal of epoxy resin are dependent upon the presence of ester
bonds which are sensitive to halogens and alkalis. Epoxy formulations which produce a polymer with a
low ester to ether bond ratio are not susceptible to standard degradation techniques.
Epoxy resin cannot be removed from a section for several reasons. These include:
Where the resin appears totally unaffected by the solvent it is likely to be a batch problem. If the ratio
of anhydride hardener to epoxy resin is raised to increase the proportion of ester bonds in the polymer,
the resin should then be degradable.
Where the residual epoxy resin has a patchy distribution the problem may be caused by incomplete
mixing of the components leading to a variation in the nature of the polymer throughout the specimen.
Incomplete removal may also occur because of old reagents (particularly saturated alcoholic sodium
hydroxide) or inadequate exposure to the resin solvent.
Residual resin in the section will prevent or retard access of many dyes to the tissue (Figure 57) and
occasionally the resin itself may stain slightly.
Figure 57
Bone marrow. The resin in one half of the specimen has been only partially degraded, preventing full access of the dyes to the tissue; H&E.
Artifacts in Resin Sections / 73
Effects of a Dull Knife on Epoxy Resin Sections

Epoxy sections can be cut with a glass or diamond knife. The quality of the cutting edge is critical for the
production of sections suitable for high resolution microscopy. Macroscopically, sections prepared with
a blunt or dull knife have a ground glass or opaque appearance, whereas those prepared using sharp
knives appear glossy and transparent. Microscopically, the sections from a dull knife lack the sharp and
crisp image associated with resin sections (Figure 58). While this is often a subtle artifact it is seen most
frequently with bone specimens where the glass knife edge deteriorates rapidly from the sectioning of
mineralized trabecular bone. Glass knives should be used as soon as possible after preparation and not
stored for prolonged periods.
A
Figure 58
2 m epoxy resin sections of bone marrow. A: Nuclear and cytoplasmic detail is poor because the knife has been dulled by cutting
several sections prior to this one. B: Section cut from the same specimen with a fresh knife shows much better resolution.
Folds in Epoxy Resin Sections

Epoxy resin sections may fail to atten on drying for a number of reasons (Figure 59). These include:
When an epoxy section is heated on a pool of water on a glass slide the resin-inltrated tissue tends
to expand more than the resin alone; the margin of resin surrounding the tissue will restrain the tissue
during expansion causing folds to form. This can be avoided by trimming close to the tissue along the
two longest sides to permit the tissue to fully expand.
A low drying temperature.
Insufcient water on the slide preventing the section from expanding before the water evaporates.
Using a pool of water too small to accommodate the fully expanded section.
Figure 59
A 1.5 m epoxy resin section of kidney showing a fold running across the eld; Jeons stain.
Artifacts in Resin Sections / 75
Background Staining in Acrylic Sections

Acrylic polymers are effectively insoluble and staining methods are performed with the resin intact.
Contamination of the acrylic resin monomer with methacrylic acid from the initial synthesis of the resin or
the breakdown of the monomer during storage will result in background staining with basic dyes (Figure
60). This artifact is easily monitored and overcome. When a signicant level of methacrylic acid is present
the pH of the monomer (usually 7.0-7.4) falls below pH 7.0. The methacrylic acid is removed by passing the
monomer through an ion exchange resin.
Figure 60
Section of mucosa embedded in LR White showing background staining; Toluidine blue.
Histochemistry, Immunohistochemistry
and Hybridization Histochemistry
Many artifacts encountered in routine histopathological techniques may also be seen in the more
specialized preparations of histochemistry, immunohistochemistry and hybridization histochemistry.
In addition there are some artifacts that are unique to these areas.
/ 77
Effect of Crush Artifact on Immunostaining

Tissues subjected to crush artifact may also show displacement of cellular antigens (Figure 61). (See also
Crush Artifact).
Figure 61
A high grade B cell lymphoma stained for the lymphocyte marker L26. The specimen was crushed with forceps prior to formalin
xation and parafn embedding and shows diffuse staining of both cytoplasm and background.
Non-specic Staining in Immunohistochemistry

Endogenous peroxidase and alkaline phosphatase are well recognized causes of non-specic staining
in immunohistochemistry and the methods for blocking these enzymes are extensively documented.
Endogenous peroxidase is localized in erythrocytes and to a lesser extent to granulocytes in both
formalin xed and fresh tissues. Figure 62 shows residual peroxidase staining in laked red cells in a liver
section stained for hepatitis B core antigen. Formalin pigment can be seen associated with these red
cells. Endogenous alkaline phosphatase activity is not preserved in formalin xed parafn sections and is
a problem only in frozen sections and smears.
Background staining of collagen is another common artifact which may be eliminated or reduced by
predigestion with proteolytic enzymes and hyaluronidase, and by blocking with normal serum from the
animal species used for linking or labeling. This artifact can be attributed to the physical trapping of the
primary antibody in collagen and to electrovalent bonding of the antibody to collagen.
Figure 62
Residual endogenous peroxidase staining in laked red cells in a liver section stained for hepatitis B core antigen. Formalin pigment
can also be seen associated with these red cells.
Histochemistry, Immunohistochemistry and Hybridization Histochemistry / 79
Pigments
Localization of antigens in tissues by light microscopy is dependent upon the production of a colored
nal reaction product at the site of the antigen. The chromogen diaminobenzidine, used to localize the
marker enzyme horseradish peroxidase, gives a brown to black nal reaction product which contrasts
poorly with many artifact and endogenous pigments commonly found in tissues. Some examples include
carbon, formalin, tattoo, lipogenic, melanin, hemosiderin, and pseudomelanosis coli pigments. Careful
examination of negative control sections before examination of the test material is essential to avoid
misinterpreting such pigments as positive staining (Figure 63). In many instances pigments can be
removed before staining, or alternatively chromogenic substrates which yield red or blue nal reaction
products can be used.
A
Figure 63
Sections from the same case of melanoma. A: Stained for S100 with DAB as the chromogen. Here it is difcult to differentiate
between the brown melanin pigment and the reaction product. B: Negative control slide showing the natural color of melanin
only.
Eosinophil Artifact in Hybridization Histochemistry

Non-specic labeling of eosinophils can occur when DNA probes incorporating radiolabels are used on
cell smears of peripheral blood or on tissue sections (Figure 64). It is reported that using a stain for
eosinophils (carbol chromotrope) blocks this non-specic binding but does not inhibit hybridization to
specic nucleotide sequences. 9
Figure 64
A frozen section of sheep adrenal hybridized with a 30mer oligonucleotide probe for side-chain cleavage enzyme, 32P labeled.
Non-specic labeling of eosinophils is evident in the medulla.
Histochemistry, Immunohistochemistry and Hybridization Histochemistry / 81
Crystallization Artifact
This artifact has been observed frequently in student preparations where a tetrazolium salt has been
used in a standard method for the enzyme succinate dehydrogenase. This method is performed on fresh,
un xed cryostat sections which are nally xed in formalin after incubation in a substrate solution which
contains a substituted tetrazolium salt (Nitro BT) and the natural substrate sodium succinate. The reaction
product is an intensely colored formazan which is preserved by mounting in an aqueous mountant.
Figure 65, a section of rat kidney, shows the colored needle-crystals that appear in the section when
it is not thoroughly washed in water following xation and prior to mounting. After several weeks the
stained tissue underlying the crystals also becomes bleached. The crystals are probably produced from
residual tetrazolium salt which has not been washed from the section and is concentrated in the aqueous
mountant as it dries out. Thorough washing prior to mounting prevents the occurrence of this artifact.
Figure 65
Cryostat section of rat kidney showing crystalline deposit at reaction site; SDH method.
This category includes artifacts where the cause is not fully understood or where the cause may be
multi-factorial.
/ 83
Nuclear Meltdown
Nuclear meltdown is one of a number of descriptive names given to a group of artifacts which are
characterized by poorly demonstrated cell nuclei. The causes of these artifacts are poorly understood
and have been the subject of ongoing debate for many years.10,11 Because there are a number of possible
contributing factors the precise cause of any particular instance may be impossible to pin down, however
poor xation does not appear to cause this artifact.
Nuclear meltdown may present the following characteristics.
The presence of poorly dened nuclear membranes.
The presence of chromatin that lacks denition (it may appear amorphous, like cut glass, or blurry, and
can range from very pale to quite dense).
The presence of a blue hue or blue haze (more of a royal blue color than the purple/blue of properly
stained nuclei in the same section).
A patchy distribution which may affect only small parts of the whole section (for example, in a section
of intestine or skin it may be present in only some areas of the epithelium with underlying tissues
unaffected).
It may affect a variable number of specimens in a batch ranging from one or two to many.
It may affect particular types of specimens only. Common specimens affected are: gastro-intestinal tract
(particularly endoscopies), prostate, lymphoid tissue and bone marrow, spleen, skin and endometrium.
Epithelial and lymphoid tissue appear to be the most susceptible.
It may occur periodically, troubling a laboratory for a time and then disappearing only to reappear
weeks or months later.
The following are some of the causes of nuclear meltdown that have been suggested.
Allowing a specimen to dry out before xation12 ( eg. by placing fresh, un xed tissue on a dry absorbent
surface). This can certainly be a problem with tiny endoscopic specimens.
Using xylene that is contaminated with water during the clearing step in processing. In this situation it
has been suggested that the problem can be overcome by reprocessing the specimen.13
Using wax which is contaminated with formalin or both formalin and ethanol during processing.13 This
problem can be caused by a faulty tissue processor (particularly a uid-transfer machine) and appears
to permanently damage the tissue.
Failing to completely replace solvent with wax during processing (retained solvent). This may be caused
by using a protocol that is too short for the dimensions and nature of the specimen, using expired or
contaminated reagents for processing or by a tissue processor fault. In this case reprocessing the
specimen may overcome the problem.
Over-heating the section when drying prior to staining (a faulty slide dryer producing hot-spots in the
section).
Ineffective dewaxing of sections prior to staining leaving traces of wax in the section that may impair
nuclear staining. The nuclei will fail to stain properly with hematoxylin and may take up eosin producing
so-called pink disease.14,15 Extended dewaxing time and fresh solvent may overcome this cause of the
problem.
Figure 66 shows the typical features of nuclear meltdown with the chromatin being very poorly dened
with a hazy blue hue. A portion of the mucosa in the intestine (66A) was affected in this way with some
adjacent areas being normally preserved. Nuclei in a small area of the epidermis and dermis in the skin
section (Figure 66B) were similarly affected. In both cases the specimens were well- xed and processed
on a four-hour cycle. Here the problem was most likely caused by retained solvent in the tissue.
A
Figure 66
Examples of typical nuclear meltdown. A: Intestinal mucosa; H&E. B: Skin, H&E.
Miscellaneous Artifacts / 85
Myocardial Fragmentation
Transverse fragmentation of myocardial bers (Figure 67) has long been recognized as an artifact of
cardiac muscle sections although it is not always evident and is not seen in endomyocardial biopsies. The
cause of the artifact remains obscure.
Figure 67
Myocardial fragmentation; H&E.
Perinuclear Shrinkage
A curious phenomenon which is observed in the erythrocyte precursors in bone marrow sections is
perinuclear shrinkage, or nuclear halo (Figure 68). The artifact is most prominent in thin sections (12 m)
and occurs regardless of the xative (formalin, mercuric chloride, glutaraldehyde) or embedding medium
(parafn and epoxy resin) used. This artifact is most likely caused by shrinkage of the nucleus or cytoplasm
during tissue processing, although it may not represent an artifact at all, being merely a reection of the
three dimensional structure of these cells when they are oriented parallel to the plane of section.
Figure 68
Parafn section of bone marrow in which the nuclei are surrounded by a clear zone in the otherwise intact cytoplasm; H&E.
Miscellaneous Artifacts / 87
Artifacts in Cytological Preparations

Various types of artifacts can be encountered in cytological preparations. They are usually caused by
inappropriate handling before xation, the xation process itself, or are due to contaminants present
in the material taken for examination or introduced at some later stage.
/ 89
Cardboard Imprint
Cardboard imprint (also known as packing artifact) is the result of contact between slide holders and
smears which are wet with xative (Figure 69).
Figure 69
A Papanicolaou-stained smear showing a pattern produced by contact with the cellulose bers in the lid of the cardboard slideholder into which the smear was placed while still wet with xative.
Drying Prior to Fixation

Smears to be stained by the Papanicolaou method should be prepared on the slide then immediately wet xed, without drying. Allowing a smear to dry before xation results in a lack of cellular detail and poor
staining (Figure 70).
Figure 70
An ascites uid. The smear was allowed to completely dry before xation and shows marked cellular enlargement and eosinophilia
in addition to a lack of nuclear and cytoplasmic denition; Papanicolaou.
Artifacts in Cytological Preparations / 91
UV Artifact
The methanol xed, May-Grnwald Giemsa stained smear shown in Figure 71 shows an artifact which was
accidentally induced and required careful investigation in order to establish a cause. Cells in the smear
show loss of crisp nuclei and an uneven vacuolated appearance. The smear was one of a group which
were prepared then left overnight in a biohazard cabinet with the ultraviolet lights on before xation and
staining the next morning. The effects observed were apparently due to the irradiation with ultraviolet
light.
Figure 71
Methanol xed smear showing the effects of radiation with ultraviolet light; May-Grnwald Giemsa.
Propellant Contamination
This artifact occurs when pressure-packs (of hair spray) used for xation are almost empty and the spray
consists mostly of propellant (hydrocarbons) and very little alcohol (which acts as the xative). It appears
to be a defect of xation. Hair spray is no longer recommended for xation because of this risk.16 Cells
appear dark with the normal range of colors seen in a Papanicolaou stain and are masked by an overall
grey-blue coloration and some ne granularity (Figure 72 A). The problem can be largely overcome by
soaking smears overnight in 2% aqueous hydrochloric acid then re-staining (Figure 72 B).
A
Figure 72
A: Smear showing pressure pack artifact. B: Smear after acid treatment and re-staining; Papanicolaou.
Meat Fiber Contamination

Occasionally skeletal muscle bers are seen in sputum smears (Figure 73). These represent a contaminant
which was present in the mouth when the specimen was obtained.
Figure 73
Meat ber contamination in a sputum smear; Papanicolaou.
Insect Contaminant
Figure 74 illustrates two different cervical smears collected on the same occasion and stained by the
Papanicolaou method. The smears contain contaminant material which may have arisen from an insect of
some type or other unidentied source. These contaminants could have been present within the vagina
originally or were introduced from an external source as the smear was taken or spread on the slide.
A
Figure 74
Cervical smears showing insect contaminant; Papanicolaou.
Pollen Grains
Pollen grains may be seen in smears of material from the respiratory tract and from other body sites. They
are recognized by their bizarre but symmetrical shapes and relatively large size (Figure 75). They often
appear above the focal plane of the specimen on the slide.
Figure 75
Pollen in a Papanicolaou-stained smear.
Alternaria Contamination
Alternaria, an airborne fungus, may settle on smears at any stage of preparation. The organism displays
a branching mycelium and snowshoe-shaped macro-conidia although the latter are more commonly
seen alone (Figure 76). Storage of smears in covered containers prior to staining prevents this form of
contamination.
Figure 76
Alternaria in a Papanicolaou-stained cervical smear.
Lubricant Contamination
An occasional contaminant seen in cervical smears is derived from lubricant jelly which has been introduced
before taking the smear (Figure 77). The presence of the lubricant interferes with the visualization of the
cells and stains an intense blue-black with the Papanicolaou stain.
A
Figure 77
A and B show the typical appearance of lubricant deposits in smears. Note the cross-like appearance inside the deposits present in A.
Suture in Smear
Short segments of suture material may be encountered in cervical smears. The material stains strongly
purple with the Papanicolaou stain and is also birefringent (Figure 78).
A
Figure 78
Smear containing suture material; Papanicolaou. A: Brighteld image. B: Polarized light microscopy revealing strong birefringence.
List of Common Artifacts

Common Name
Alternative Description
Page
Prexation Artifacts
Heat damage
Laser knife damage
Cauterization damage
Thermal dehydration
Presence of sutures
Silk sutures
Stitch granuloma
Cellulose contamination
Cellulose ber
Gelfoam artifact
Gellm artifact
Surgical sponge contaminant
Surgical packing
Starch contamination
Starch granuloma
Glove powder contamination
Catheter damage
Surgery-induced artifact
Epithelial compression
10
Crush artifact
Compression artifact
Surgical trauma
11
Necrosis due to Monsels solution
Monsels artifact
12
Tattoo pigment
Tattoo artifact
13
Postmortem change
Autolysis
Putrefaction
Microorganisms introduced postmortem
14
Desquamation
Epithelial shedding
15
Autolytic change
Specimen marking dyes
Silver nitrate contamination

India ink contamination
16
Biopsy-pad artifact
Foam-pad damage
17
Enclosure damage
Pressure effects
Freezing damage
Freeze-thaw- x artifact
Freezing artifact
18
/ 101
Common Name
Specimen-to-specimen contamination
Translocation of tissue
Page
19
Tissue transfer
Contamination with tissue debris
Fixation Artifacts
Zonal xation
Penetration artifact
22
Streaming artifact
Glycogen streaming
Polarization displacement
23
Formalin pigment
Acid formaldehyde hematin

Formalin deposit
24
Mercury pigment
Mercuric chloride deposit

Sublimate deposit
25
Vessel shrinkage in central nervous system
Perivascular shrinkage
28
Poor processing
Under-processing
Inltration fault
29
Tissue-Processing Artifacts
Clearing fault
Loss of soluble substances
Cholesterol clefts
30
Knife lines
Scoring
32
Displacement of components
Translocation
33
Artifacts of Microtomy and Section Mounting
Realignment of bers
Venetian blind effect
Micro-chatter
34
Coarse chatter
Mechanical vibration
35
Holes from roughing
Moth-eaten effect
36
Tidemark due to adhesive
Adhesive pools
37
Bubbles under section
Collapsed-bubble artifact
38
Common Name
Contamination of mounted sections
Sneeze artifact
Page
39
Contamination during mounting

Contamination with squames
Airborne contamination
Fungal contamination
Cellulose contamination
Fiber from facial tissue
Unknown bers, hair, dirt
Folds
Wrinkles
43
Residual wax
Dewaxing fault
46
Incomplete staining
Tide-mark
47
Unstained area in section
Contaminant on section
48
Stain deposit
Stain precipitate
49
Contaminated staining solution
Microorganism contamination
50
Mucus contamination
Saliva contamination
51
Corn-aking
54
Staining Artifacts
Section Preservation Artifacts

Drying artifact
Black nuclei
Air bubbles over nuclei
Water in section
Dehydration fault
Water bubbles
55
Mountant breakdown
Crazing
Crystallization of mountant
56
Bleaching of stain
Fading (photofading) of stain
57
Ice crystal artifact
Ice crystal damage

Freezing artifact
60
Frozen section chatter
Fragmentation artifact
61
Blurred staining
62
Artifacts in Frozen Sections
Fuzzy staining
Indistinct staining
/ 103
Common Name
Miscellaneous artifacts
Fat displacement
Page
63
Bubbles in aqueous mountant
Artifacts in Bone and Calcied Tissues

Impacted bone fragments
Bone-saw damage
66
Cracking of matrix in resin sections
Bone fragmentation
66
Bone dust artifact
Bone powder
67
Effect of over-decalcication on tissues
Acid effects
Acid exposure
68
Effects of incomplete decalcication
Residual calcium
69
Limitations of parafn wax embedding for dense bone
Lack of support
70
Polymerization artifacts in epoxy resin sections
Incomplete polymerization
72
Incomplete removal of epoxy resin prior to staining
Residual resin
73
Effects of a dull knife on epoxy resin sections
Ground-glass effect
Artifacts in Resin Sections
Folds in epoxy resin sections

Background staining in acrylic sections
74
75
Resin staining
76
Histochemistry, Immunohistochemistry and Hybridization Histochemistry

Effect of crush artifact on immunostaining
Compression artifact
78
Surgical trauma
Non-specic staining in immunohistochemistry
Endogenous enzyme
79
Pigments
False positive staining
80
Eosinophil artifact in hybridization histochemistry
Non-specic staining
81
Crystallization artifact
Crystal formation
82
Blue-hue
Ground-glass nuclei
84
Nuclear meltdown
Blue-blob effect
Pink disease
Myocardial fragmentation
Broken-heart artifact
Myocardial splitting
86
Perinuclear shrinkage
Nuclear halo
87
Common Name
Page
Artifacts in Cytological Preparations

Cardboard imprint
Cardboard artifact
90
Drying prior to xation
Air-drying artifact
Drying damage
91
UV damage
92
Packing imprint
UV artifact
Radiation artifact
Propellant contamination
Hair spray xation artifact

Pressure-pack artifact
93
Meat ber contamination
Muscle ber contamination
94
Insect contaminant
Foreign body contamination
95
Pollen grains
Pollen contamination
96
Alternaria contamination
Fungal contamination
97
Lubricant contamination
Lubricant jelly artifact
98
Suture in smear
Silk contaminant
Suture contaminant
99
/ 105
References
1.
Wallington EA: Artifacts in tissue sections. Med Lab Sci 1979, 36:3-61
2.
Thompson SW, Luna LG: An atlas of artifacts. Springeld, Charles C Thomas, 1978
3.
Davis JR, Steinbronn KK, Graham AR, Dawson BV: Effects of Monsel's solution in uterine cervix. Am J Clin Pathol 1984,
82:332-335
4.
Schlosshauer PW, Chen W, Chanderdatt D, Antonio LB: Monsel's artifact in gynecologic biopsies: a simple remedy. The
Journal of Histotechnology 2005, 28:161-162
5.
Raife T, Landas SK: Intracellular crystalline material in visceral adipose material: "rediscovery" of a common artifact. The
Journal of Histotechnology 1993, 16:69-70
6.
Kobayashi H, Mahovlic D, Bauer TW: Interpreting and avoiding histologic artifacts in hard-tissue research. The Journal of
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Artifacts in Histological

and Cytological Preparations

2 / Leica Microsystems Scientia

4 / Leica Microsystems Scientia

6 / Leica Microsystems Scientia

8 / Leica Microsystems Scientia

10 / Leica Microsystems Scientia

Necrosis due to Monsels Solution

12 / Leica Microsystems Scientia

14 / Leica Microsystems Scientia

Specimen Marking Dyes

16 / Leica Microsystems Scientia

18 / Leica Microsystems Scientia

20 / Leica Microsystems Scientia

22 / Leica Microsystems Scientia

24 / Leica Microsystems Scientia

26 / Leica Microsystems Scientia

Vessel Shrinkage in Central Nervous System

28 / Leica Microsystems Scientia

Loss of Soluble Substances

30 / Leica Microsystems Scientia

Artifacts of Microtomy and

32 / Leica Microsystems Scientia

Artifacts of Microtomy and Section Mounting / 33

Venetian Blind Effect

34 / Leica Microsystems Scientia

Artifacts of Microtomy and Section Mounting / 35

Holes from Roughing

36 / Leica Microsystems Scientia

Tidemark due to Adhesive

Artifacts of Microtomy and Section Mounting / 37

Bubbles Under Section

38 / Leica Microsystems Scientia

Artifacts of Microtomy and Section Mounting / 39

Contamination of Mounted Sections

microorganisms, particularly airborne fungi (Figure 31)

40 / Leica Microsystems Scientia

Artifacts of Microtomy and Section Mounting / 41

42 / Leica Microsystems Scientia

Artifacts of Microtomy and Section Mounting / 43

44 / Leica Microsystems Scientia

46 / Leica Microsystems Scientia

Unstained Area in Section

48 / Leica Microsystems Scientia

Contaminated Staining Solution

50 / Leica Microsystems Scientia

52 / Leica Microsystems Scientia

Section Preservation Artifacts

54 / Leica Microsystems Scientia

Section Preservation Artifacts / 55

56 / Leica Microsystems Scientia

Section Preservation Artifacts / 57

58 / Leica Microsystems Scientia

Artifacts in Frozen Sections

Ice Crystal Artifact

60 / Leica Microsystems Scientia

Frozen Section Chatter

Artifacts in Frozen Sections / 61

62 / Leica Microsystems Scientia

Artifacts in Frozen Sections / 63

64 / Leica Microsystems Scientia

Artifacts in Bone and Calcified Tissues

Impacted Bone Fragments