ARTIFACTS IN CYTOLOGY

A SEMINAR PRESENTED

BY

IKECHUKWU CHUKWUEMEKA K.

INTERN MEDICAL LABORATORY SCIENTIST

DEPT. OF HISTOPATHOLOGY

NNAMDI AZIKIWE UNIVERSITY TEACHING HOSPITAL,

NNEWI.

6 JUNE, 2012.
TH

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INTRODUCTION

Artifacts are undesired objects or phenomena influencing the appearance of a

smear and obstructing or even preventing proper microscopic analysis of
important diagnostic factors of the cytological sample (Valente and Schantz,
2003). They are products or formations found in microscopic preparation of a
fixed tissue or cell that is caused by manipulation or reagents and is not
indicative of actual structural relationships. Artifacts are also referred to as
modifications of the appearance or structure of protoplasm produced
artificially. Strictly speaking, all cytologic preparations are standardized
artifacts (Valente and Schantz, 2003).

The microscopic examinations of stained cytologic smears remain the hallmark

of cytodiagnosis (George et al, 1997). However, diagnostic accuracy and
reliability are major issues in cytology practice. Artifacts in cytologic smears
have been considered as a potential diagnostic pitfall in cytodiagnosis
(Geoffrey and Neville, 2008). Several artifacts have been found to be inherent
to the cytological smears. While some of these artifacts are trivial and would
not ultimately affect the final diagnosis, others could result in either
misclassification of the lesion or a false diagnosis. The accuracy of the
cytologic examination from any body site depends greatly on the quality of
collection, preparation, staining and interpretation of the cytological material.
Inadequacy in any of these steps will adversely affect the quality of diagnostic
cytology as a result of the presence of misleading artifacts (Agarwal, 2005).

Consequently, familiarity with these artifacts is essential to avoid

misinterpretations that can lead to false positive or false negative diagnosis.

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DIAGNOSTIC CYTOLOGY

The three pre-requisites for a meaningful cytodiagnosis are;

Proper technique: collection procedure, preparation of smears, fixation

and staining.
Microscopic examination of stained smears.
Correlation of morphology with the clinical picture (history, clinical
features, physical examination, radiological and laboratory findings).

Diagnostic cytology can be carried out by different methods which includes

the following;

Exfoliative cytology: In this method, cells are collected after they have
been spontaneously shed by the body (spontaneous exfoliation) or manually
scrapped, brushed, swabbed, aspirated or washed from the epithelial surface
of various body organs (Agarwal, 2005). Normal cells are cohesive in nature
but exfoliate when they attain maturation. In malignant condition or during
infection, the exfoliation becomes exaggerated and the epithelial cells show
variation in morphology. Such exfoliated cells when collected and
appropriately stained, give information on the epithelium from which they are
derived. These characteristic cellular and nuclear appearances in cells thrown
off from healthy epithelium differ distinctly from those derived from inflamed
or malignant lesions. Thus, by studying the alterations in morphology of the
exfoliated cells and their pattern, the diagnosis of various pathologic
conditions can be made (Biscotti et al, 1995).

Fine needle aspiration cytology: This is now a widely accepted diagnostic

procedure which has largely replaced open biopsy. It is a technique used to
obtain material from organs that do not shed cells spontaneously (Agarwal,
2005). Thus, it is valuable in the diagnosis of palpable lesions of the breast,
thyroid gland, lymph nodes and skin. Guided fine needle aspiration cytology
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can also be performed on deep-seated lesions within the body (e.g liver, lungs
etc) mainly with the aid of ultrasonography and computed tomography.

Sediment cytology: In this method, the sample is collected from the fixative
that was used for processing the biopsy or autopsy specimen. The fixative is
mixed properly in a centrifuge tube and centrifuged. The sediment is used for
smearing. These sediments are cells that are shed by the autopsy and biopsy
specimen during the processing (Diane and Ritu, 2003).

Imprint cytology: This is indicated in the case of tumours especially of

lymph nodes. Soon after an excision biopsy of lymph node, the specimen is
cut using a sharp scalpel blade. If there is blood oozing from the outer
surface, touch the surface with a cotton ball soaked in normal saline. Then
take imprint smears by touching the cut surface with a clean microscope slide
and fix immediately (Agarwal, 2005).

The cytologic smears prepared using any of the above method is fixed
immediately according to the requirement of the staining method to be used.

THE ROLE OF DIAGNOSTIC CYTOLOGY

Diagnostic cytology has been widely applied as a diagnostic tool in the

following areas;

As a screening tool to detect precancerous and cancerous lesions.

It is also commonly used to investigate thyroid lesions and diseases
involving sterile body cavities and a wide range of other body sites.
It is used for hormonal assessment in cases of infertility in females.
It is used for genetic sex determination.
It aids in the diagnosis of certain infectious diseases and other
imflammatory conditions (e.g florid tuberculosis, rheumatoid pleurisy).
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ARTIFACTS IN CYTOLOGICAL SMEARS

The presence of artifacts in cytological smears is important, as this finding

although sometimes accidental may have implications for diagnosis, prognosis
and therapy. However, on numerous occasions the presence of these artifacts
which are varied in nature may give rise to confusion and/or incorrect
interpretation (Martinez-Giron et al, 2005).

Artifacts found in routine cytological smears can result from technical error in
any of the following stages of cytodiagnosis;

Methods of specimen collection.

Fixation and fixatives.
Preservation of fluid specimens prior to processing.
Preparation of material for microscopic examination.
Staining and mounting of the cell sample

The following artifacts can be found in routine cytological smears;

Fixation artifacts

These artifacts occur in too thick cytological smears resulting in inadequate

fixation and stabilization of the cellular structures (Agarwal, 2005).

Drying artifacts

These groups of artifacts are observed in cytological smears stained with

haematological stains like May-Grunwald-Giemsa (MGG). In this method, the
smears are intentionally air-dried, but if the smears are not correctly made
and dried quickly artifacts will result (Agarwal, 2005). These artifacts can be
found in pap smears requiring alcohol fixation when the time between
smearing and wet fixation is prolonged resulting in rapid evaporation and air-
drying. Thus, from the instant the smear is made, air-drying proceed

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extremely rapidly, hence the urgency for fixation. The cytoplasm takes up
more eosin and nuclear details are less clear resulting in pale-stained nuclei,
lack of differential cytoplasmic staining, cytoplasmic and nuclear eosinophilia
(Agarwal, 2005).

Crush/smearing artifacts

These artifacts are found in cytological smears prepared from specimens

diluted with blood. The specimen is spread like a peripheral smear and the
particles tend to come to the edge of the smear. Crushing these larger
particles with undue pressure can result in crush/smearing artifacts (Cibas and
Ducatman, 2009).

Iatrogenic artifacts

These artifacts are a group of physician-related changes in morphologic

pattern involving specimen acquisition (instrumentation) or therapeutic
interventions such as radiation, chemotherapy, tissue ablation and surgery.
Such induced changes may mimic those of neoplasia and cause considerable
diagnostic confusion (Valente and Schantz, 2003). Starch crystals, suture
materials and lubricants are examples of iatrogenic artifacts.

Pollen Grains

These are aerial contaminants. They generally present with a thick refractile
cell wall.

Cornflake artifacts

This brown artifact is thought to occur when there is a delay between

removing the slides from the last xylene and applying the mounting media.
This may be due to air trapped within the surface grooves of mature
squamous cells.

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Stain precipitate artifacts

These are highly amorphous and granular precipitates seen in stained smears.
They result from improper staining procedures. They are common if saturated
solution of stain is allowed to evaporate or dry up on the smear (Ochei and
Kolhatkar, 2000).

Worm-like artifacts

The presence of worms in cytological smears is occasionally reported,

although various other structures exist that may be confused with such
parasites. Recognition of these structures is important to avoid overvaluation
or confusion with true worms. Morphological criteria are required to
differentiate these worm-like artifacts regularly abserved in smears from
clinically significant parasites (Martinez-Giron et al, 2006). These artifacts are
considered sample contaminants (intrinsic contamination) or contaminants of
cytological smears during processing (extrinsic contamination).

According to Martinez-Giron et al (2006), the following worm-like artifacts are

observed in cytological smears;

Curschm anns spiral in sputum sm ear: This structure appears as a cast

of small bronchi and bronchioles from inspissated mucus. A dense central core
can be observed, formed principally by proteins and nuclear debris,
surrounded by mucus. These spirals are frequently observed in bronchial
asthma.

Fragm ent of skeletal m uscle ber in sputum sm ear: In this structure,

various nuclei distributed longitudinally can be observed, together with the
typical transversal striations. Its presence in sputum smears is usually due to
contamination by food in the oral cavity.

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Ferruginous body in sputum sm ear: It is a long, narrow structure with
club-shaped ends, with yellowish deposits of inorganic material (principally an
iron-protein pigment) along the whole of its length (segmented morphology or
bamboo-shaped). These elements are frequently encountered in the sputa of
patients with pneumoconiosis, especially those exposed to asbestos.

P lant hair (trichom e) in sputum sm ear: One end of the hair is rounded in
appearance, while the other is usually tapered. An internal refractile elongated
core is also visible.

Synthetic ber in sputum sm ear: Although the two extremes are rounded
and do not terminate abruptly, there is no recognizable internal structure. The
whole surface is covered by ne dots, characteristic of synthetic bers such as
rayon.

Natural ber in cervicovaginal sm ear: The surface does not show the
dotted pattern characteristic of synthetic bers, but there is a series of lines
forming a fringe along the whole of its length, typical of bers such as cotton.

Algae of Cyanophyta class (Oscillatoria sp.) in cervicovaginal sm ear:

It is lamentous in shape and is quite wide (6070 lm in thickness).

OVERCOMING THE POTENTIAL DIAGNOSTIC PITFALLS OF

ARTIFACTS

In order to ensure accurate and reliable cytodiagnosis from the microscopic

examination of an artifact-free cytological smear, the following procedures are
necessary.

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1. Proper patient preparation prior to sample collection:

Proper patient preparation is the beginning of good cervical cytology. For

instance, prior to cervical smear collection, the patient should be instructed
before coming for smear collection that:

She should not douche the vagina for at least a day before the
examination.

No intravaginal drugs or preparations should be used for at least one

week before the examination.

The patient should abstain from coitus for one day before the
examination.

Smear should not be taken during menstrual bleeding, because of

contamination with blood, endometrial component, debris and
histiocytes.

2. Strict adherence to standard operating procedures in sample

collection, preservation, processing and microscopic
examination:

Immediate fixation of smears is essential.

Smears should never be allowed to dry before placing the coverslip.

Haematoxylin should be filtered every day before use.

All solutions and other stains are filtered daily after use, to keep them
free of sediment.

Avoid contamination from one smear to another.

Keep stains and solutions covered when not in use.

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 All dishes are washed daily.

Stains are discarded and replaced as the quality of the stain

deteriorates.

Avoid contamination during placing of the coverslip, with the dropper

used to dispense the mounting medium.

Place the coverslip on the microslide slowly without trapping air

bubbles.

Agitation of the slides by occasional dipping is necessary to remove

excess dye.

Dipping should be done gently to avoid cell loss and the slide carrier
should not hit the bottom of the staining dish.

3. Preparation and maintenance of stains and fixatives:

Solutions may be used for longer period of time, if the slide carrier is
rested on several layers of tissue paper (paper toweling) for a few
seconds before transferring to the solutions.

Stains keep longer if they are stored in dark coloured, stoppered

bottles.

Haematoxylin keeps relatively constant staining characteristics and

do not require frequent discarding if small amounts of fresh stain are
added to replace stain loss due to evaporation.

Use of coating or spray fixatives may cause contamination making

frequent changes necessary.

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 OG and EA stains lose strength more rapidly than haematoxylin and
should be replaced each week or as soon as the cells appear without
crisp staining colours.

Bluing solution and HCl should be replaced at least once daily.

Water rinses should be changed after each use.

Alcohol used for the process of dehydration prior to the cytoplasmic

stains may be replaced weekly. The alcohol rinses following the
cytoplasmic stains are usually changed on a rotating basis after each
use. The absolute alcohols should be changed weekly and can be
kept water free by adding silica gel pellets.

Xylene should be changed as soon as it becomes tinted with any of

the cytoplasmic stains. Xylene becomes slightly milky if water is
present in it and if so the clearing process may be disturbed. Tiny
drops of water may be seen microscopically on a plane above the
cell on a slide. Addition of silica gel pellets to the absolute alcohol
will minimize water contamination of xylene.

The quality of the stained slide is dependent on timing, solubility and

percentage of dye concentration.

4. Clinical correlation
Correlation of cytomorphology with the clinical picture (history, clinical
features, physical examination, radiological and laboratory findings) is
important in order to avoid misinterpretation of artifacts.

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CONCLUSION

Diagnostic cytology has become a widely accepted diagnostic tool in the

diagnosis of malignant, infectious, inflammatory, genetic and endocrinological
disorders. Artifacts resulting from both intrinsic and extrinsic contamination of
cytological smears remain a major diagnostic pitfall in cytodiagnosis. This has
resulted in increased difficulty in accurate microscopic interpretation of
cytomorphological structures in cytological smears. Medical Laboratory
Scientists have the responsibility of eliciting the maximum possible information
of diagnostic, therapeutic and prognostic use from submitted specimens.
Thus, familiarity with these artifacts and strict adherence to standard
operating procedures is essential to avoid misinterpretations that can lead to
false positive or false negative diagnosis.

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REFERENCES

Agarwal S. P. (2005). Manual for cytology. Ministry of Health and Family

Welfare: India. Pp. 5-30.

Biscotti C.V, Hollow J.A, Toddy S.M, Easley K.A. (1995). ThinPrep versus
conventional smear cytologic preparations in the analysis of thyroid
ne-needle aspiration specimens. Am J Clin Pathol. 104:150153.

Cibas E.S. and Ducatman B.S. (2009). Cytology; Diagnostic Principles and
Clinical Correlates. 3rd edition. Saunder Elsevier: Philadelphia. Pp. 6-18.

Diane S. and Ritu N.(2003). The Bethesda System for Reporting Cervical
Cytology; Definitions, Criteria, and Explanatory notes. 2nd edition.
Springer: New York. Pp. 23-28.

Geoffrey R. and Neville F. (2008). Artifacts in histological and cytological

preparations. Leica Biosystems: Germany. Pp. 34-37.

George L. W, Catherine M. K, Koss L.G, Regan J. W. (1997). Compendium on

Diagnostic Cytology. 8th Edition. Tutorials of Cytology: Chicago. Pp. 23-
27.

Martinez-Giron R, Jodra F.O, Tormo M. R, Guillermo E. J, Ribas B. A. (2005).

Uncommon structures simulating helminth eggs in sputum. Acta Cytol.
49:578580.

Martinez-Giron R, Jose R. G, Maria T. G, Carlos A, Andres R. (2006). Worm-

like artifacts in exfoliative cytology. Diagn. Cytopath. 34(9): 636-639.

Ochei J. O. and Kolhatkar A. A. (2000). Medical Laboratory Science; Theory

and Practice. Tata McGraw-Hill Publishing Company Limited: New Delhi.
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Valente P. T. And Schantz H. D.(2003). Iatrogenic artifacts in cytology. Path
Cas Rev. 8(3): 126-133.

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