Exfoliative cytology with pap smear

2. DEFINITION :A branch of General Cytology which deals with the
microscopic study of cells that have been desquamated
from the epithelial surfaces.
SPONTANEOUS EXFOLIATION: In this method, cells are collected after
they have been spontaneously shed
by the body .
MECHANICAL EXFOLIATION: Cells are manually scraped/brushed off of
a surface in the body .

3. INDICATIONS
1. Detection of malignant cells or precancerous lesions in the
body
2. Detection of asymptomatic or precancerous cervical lesions in
women
3. Assessment of female hormonal status in case of sterility and
endocrine disorders
4. Determination of genetic (phenotypic) sex
5. Detection of the presence of infectious microorganisms

4. PRINCIPAL FEATURES OF EXFOLIATIVE
CYTOLOGY
 The technique is applicable to organs with easy clinical access whence the
samples can be obtained.
 The samples often contain a great variety of cells of various types from many
different sources.
 The cellular constituents are sometimes poorly preserved.
 The samples may contain inflammatory cells, macrophages, microorganisms,
and material of extraneous origin.
 The signal advantage of exfoliative cytology is the facility with which
multiple samples can be obtained.

5. PRINCIPAL TARGETS OF EXFOLIATIVE CYTOLOGY
Target Organ techniques Principle lesions to
be identified
Incidental benefits
Female genital
tract
Smear of material
from the vaginal
pool obtained by
pipette or a dull
instrument.
Fixation in alcohol
or by spray fixative.
Precancerous
lesions and cancer
of the vagina,
uterine cervix,
endometrium,
rarely fallopian
tubes, ovaries
Identification of
infectious agents,
such as bacteria,
viruses, fungi, or
parasites
Respiratory tract Sputum: either
fresh or collected in
fixative (smears and
cell blocks)
Precancerous states
mainly carcinoma in
situ and lung
cancers
Identification of
infectious agents,
such as bacteria,
viruses, fungi, or
parasites

6. Continued..
Urinary tract Voided urine; fresh
or collected in
fixative (smears
and cytocentrifuge
preparations)
Precancerous
states, mainly flat
carcinoma in situ
and high grade
cancers
Identification of
viral infections and
effect of drugs
Effusions (pleural,
peritoneal, or
pericardial)
Collection of fluid:
fresh or in fixative
(smears and cell
blocks)
Metastatic cancer
and primary
mesotheliomas
Other fluids
(cerebrospinal
fluid, synovial
fluid, etc.)
Collection in
fixative
Cytocentrifuge
preparations
Differential
diagnosis between
inflammatory
processes and
metastatic cancer
Identification of
infectious agents
(viruses, fungi)

7. ADVANTAGES
 Rapid diagnosis - Inexpensive - Simple
 It is better in evaluating the infectious diseases.
 Supplement or replace frozen section or biopsy
 No injury to tissue allowing repeated sampling
 It is better for hormonal assay
 Cytopathological smear cover a wider surface than that involved in surgical
biopsy.

8. DISADVANTAGES
 Interpretation of the morphological cellular changes is based only on
individual cell observation.
 Not always fully diagnostic, so it is confirmed by histopathology in some
cases.
 Does not determine the size and type of lesion of some cases.

9. FACTORS THAT DETERMINE THE
APPEARANCE OF CELLS
 Type of the technique used.
 Level of cell maturation at the time of cell collection.
 Nature of the parent tissue: soft tissue, cyst, or solid organ.
 Medium of the exfoliated cells.
 Interval between the stain of the exfoliated cells and collection of samples.
 Type of fixative, stain, and processing technique used.

10. PAP SMEAR

11. HISTORY
The current resurgence of diagnostic cytology is the result of the achievements
of DR. GEORGE N. PAPANICOLAOU (1883-1962), an American of Greek descent .
He developed a small glass pipette that allowed him to obtain cell samples from the
vagina of rodents.
In smears, he could determine that, during the menstrual cycle, squamous cells
derived from the vaginal epithelium of these animals followed a pattern of
maturation and atrophy corresponding to maturation of ova.

12.  He made major contributions to the understanding of the hormonal
mechanisms of ovulation and menstruation and is considered to be
one of the pioneering contributors to reproductive endocrinology.
 However, his fame is based on an incidental observation of cancer
cells in vaginal smears of women whose menstrual cycle he was
studying.

13. Dr George N. Papanicolaou

14. ADVANTAGES AS A SCREENING TOOL
 Relative accessibility of cervix to take the smear
 Long natural history of cervical carcinogenesis
 Relative conservative treatment for premalignant lesions
 Cost effectiveness

15. SIGNIFICANCE
 Detects precancerous & invasive cancer cervix cases in early stages.
 Positive screeners can be selected for selective tests and
management .
 With treatment, progression of disease is halted. Thus morbidity
associated with advanced cancer decreases.
 Mortality reduces by 20-60 %.
 Helps us to study natural history of disease.

16. WHEN TO SCREEN ?
 Start within 3 years of onset of sexual activity or by age of
21(ACOG) whichever is first.
 WHO recommends screening after the age of 30.
 Risk factors for cervical dysplasia
 Early onset of sexual activity
 Multiple sexual partners
 Tobacco
 Oral contraceptives

17. SCREENING FREQUENCY
 Yearly ,until three consecutive pap smears are normal and
then every three yearly.
 Annual screening for high-risk women is highly
recommended.

18. WHEN TO STOP SCREENING
 Age 65 and “adequate recent screening”
 Three consecutive normal pap smears
 No abnormal pap smears in last 10 years
 No history of cervical or uterine cancer
 Hysterectomy for benign disease
 Hysterectomy for invasive cervical cancer

19. WHAT DOES A PAP TEST INVOLVE?
 Performing a vaginal speculum exam during which a health care
provider takes a sample of cells from a woman’s cervix using a small
flat spatula or brush.
 Smearing and fixing cells onto a glass slide.
 Sending the slide to a cytology laboratory where it is stained and
examined under a microscope to determine cell classification.
 Transmitting the results back to the provider and then to the woman.

20. PROCEDURE
COLLECTION OF SPECIMEN
Prerequisites for collection of specimen
 Women should be tested two weeks after the first day of their last
menstrual period.(Day 14 of cycle is optimal).
 Proper technique is very important
 More problems are due to improper sampling than screening
 Not to be collected during menses
 Avoid vaginal contraceptives, vaginal medications for at least 48
hrs before taking smear
 Abstinence from sex for 24 hrs
 Postpartum smear should be taken only after 6 - 8 weeks of
delivery

21. PRECAUTIONS TO BE FOLLOWED
• Specimens should be obtained after a nonlubricated
speculum (moistened only with warm water
if needed) is inserted.
• Excess mucus or other discharge should be
removed gently with ring forceps holding a folded
gauze pad.
• The sample should be obtained before the application
of acetic acid or Lugol iodine.
• An optimal sample includes cells from the ectocervix
and endocervix.

22. INFRASTRUCTURE REQUIRED
 Private exam area
 Examination table
 Trained health professionals
 Sterile vaginal speculum
 Supplies and equipment for preparing and interpreting the Pap smears (e.g.,
spatulas, glass slides, fixative, stains, microscopes)
 Marker/pencil/glass writer/labels
 Cytology requisition forms
 Record books or sheets
 Cytology laboratories with skilled personnel to interpret results
 Pathologists
 Transportation of slides to the laboratory and back
 Information systems to ensure follow-up contact with clients
 Quality assurance system to maximize accuracy

23. CONVENTIONAL PAP SMEAR
Method
 Patient in dorsal position
 Good illumination is necessary
 Cusco’s speculum is inserted to visualise & fix the cervix
 Inspection of cervix done & findings are noted
 Ayres spatula is inserted first. It is placed at cervical os so that
longer end goes into cervical canal and smaller end rests on
ectocervix
 Spatula is rotated through 360 degrees maintaining contact with
ectocervix
 Do not use too much force [bleeding /pain]
 Do not use too less force [inadequate sample]
 Sample is smeared evenly on the slide and fixed immediately
 Both sides of spatula are to be smeared

26. FIXATION
COMMON FIXATIVES
 95% Ethanol.
 95% Rectified Spirit.
 100% Methanol.
 80% Isopropanol or Propanol.
 Ether/95% Ethanol ( 1: 1).
 Spray fixatives contains isopropanol and propylene glycol.

27. FIXATION METHODS
There are two options for smear fixation.
1.Coating fixatives contain alcohol and polyethylene glycol and
are applied by pump sprays, by droppers from dropper
bottles, or by pouring from an individual envelope
included as part of a slide preparation kit.
2. The smear can be immersed directly into a container
filled with 95% ethanol.

28.  Slide should be labeled properly with patients name, identification
no. and details
 Detailed history and clinical examination findings are to be
mentioned
 Patient details and clinical findings are to be maintained in a
register
 Advice is given regarding further follow up and treatment

29. STAINING
Stains for Pap:
1.Harris Hematoxylin
2.OG 6 Stain
Orange Green 6, 0.5 solution in 95% ROH 100 ml
Phosphotungstic acid 0.015gm
3. EA 50
light green SF, yellowish 0.1% solution in 95% ROH 45ml
Bismarck brown 0.5 in 95% ROH 10 ml
Eosin Y, 0.5% in 95% ROH 45 ml
Phosphotungstic acid 0.2 gm
Lithium Carbonate. Sat. aq. Solution 1 drop

30. PROCEDURE FOR STAININING
 Fix in ether-ROH and pass thru 80% ROH, 40% ROH and
distilled H2O.
 Stain in Harris Hematoxylin for 4-5 minutes.
 Wash with H2O.
 Pass thru 0.25% HCl in 50% ROH.
 Immerse in 1.5% NH4OH in 70% ROH for 1 minute.
 Rinse in 70% ROH and pass thru 80% and 95% ROH.
 Stain with OG 6 for 1.5-2 minutes.

31. Contd..
 Pass thru 3 changes of 95% ROH.
 Stain with EA 65 or EA 50 for 3 minutes.
 Pass thru 3 changes of 95% ROH.
 Dehydrate and clear in:
a. absolute ROH,
b. equal parts of ether and absolute ROH,
c. 2 changes of xylol
 Mount in Canada Balsam.

32. PAP KIT
This complete kit contains a Medline speculum, cervical scraper
and two 6" polyester-tipped applicators. There's also a pair of latex
gloves, lubricating jelly, two frosted end slides, slide holder and
mailer.

33. AUTOMATED PAP STAINING

34. LIQUID BASED CYTOLOGY
 It means cytology (study of cells )through a liquid medium.
 Cells are collected from cervix(any other site) are placed
directly into liquid preservative, rather than transferring to a slide.
 Sample is processed and resultant thin smear easy to screen.

35. ADVANTAGES
 Simplifies the collection process for the smear taken
 Reduced inadequate rate
 Cells better preserved and not obscured by blood,mucus, or
inflammatory cells
 Infectious organisms retained and better preserved
 Quicker to screen and report
 Multiple slides can be produced allowing further testing.
 Residual material in the vial can be made available for further tests
eg; HPV tests
 Facilitates computer assisted screening which can be available in
future(automated cytology)

36. DISADVANTAGES
 More costly than conventional pap smears
 Preparation is more laborious ,intensive than Conventional
 Loss of background material
 Some differences in architecture and morphology
 Requires training for both the screeners and the smear takers

37. LIQUID BASED CYTOLOGY TECHNIQUES
 MANUAL METHOD
 THINPREP PAP TEST
 SUREPATH PAP TEST
 MONOPREP PAP TEST

38. MANUAL METHOD
LBC FIXATIVE FOR PAP SMEAR
 Alcohol- 50ml
 10% formalin – 50ml
 Sodium chloride -0.5gm
 Sodium citrate- 0.5gm

39. POLYMER PREPARATION
 Agarose- 2gm
 Polyethylene glycol- 10ml
 Poly-L-lysine- 2ml
 Distilled water – 88ml
 (Filter before use to avoid precipitation) (Solution kept in refrigerator)
PHOSPHATE BUFFER PREPARATION
 Solution A- Sodium hydrogen orthophosphate – 9.45gm
 Distilled water – 1lt.
 Solution B- Potassium di hydrogen orthophosphate – 9.8gm
 Distilled water – 1lt.
FOR 100ML BUFFER
 Solution A- 49.6ml
 Solution B – 50.4ml
 (Solution kept in Refrigerator)

40. 1. Receive the sample from the ward uniquely labelled.
2. Mix sample properly before transferring to the test tube.
3. Take clear test tube and transfer the sample to the test tube.
4. Centrifuge at 2000 RPM for 5 minutes
5. Decant supernatant and mix properly.
6. Add1-2ml of polymer reagent to the deposit
7. Centrifuge at 2000 RPM for 5 minutes.

41. 8. Decant supernatant mix deposit properly.
9. Add1-2ml of cold phosphate buffer to the deposit.
10 Centrifuge at 2000 RPM for 5 minutes.
11 Decant supernatant mix properly.
12 Take a clear glass slide.
13. Pipette deposit on the slide in circular
14. Prepared slides can be dried at room temperature, in the hot air
over at 40-50’c for 5 to 10minutes.
15. The slide can be further fixed by dipping in 95% alcohol for 2
hours
16. Stained with Pap stain.

42. THIN PREP
 The practitioner obtains the ThinPrep Pap sample with either a
broom-type device or a plastic spatula/endocervical brush
combination.
 The sampling device is swirled or rinsed in a METHANOL-BASED
preservative solution (PreservCyt) for transport to the cytology
laboratory and then discarded.
 Red blood cells are lysed by the transport medium.
 The vials are placed one at a time on the ThinPrep 2000 instrument.
 The entire procedure takes about 70 seconds per slide and results in
a thin deposit of cells in a circle 20 mm in diameter (contrast with
cytospin diameter=6mm)

43. 1. The sample vial sits on a stage and a hollow plastic cylinder with a 20-mm diameter
polycarbonate filter bonded to its lower surface is inserted into the vial. A rotor spins
the cylinder for a few seconds, homogeneously dispersing the cells.
2. A vacuum is applied to the cylinder, trapping cells on the filter. The instrument
monitors cell density.
3. With continued application of vacuum, the cylinder (with cells attached to the filter)
is inverted 180 degrees, and the filter is pressed against a glass slide. The slide is
immediately dropped into an alcohol bath.

45. SURE PATH
 The practitioner snips off the tip of the collection device and
includes it in the sample vial.
 The equipment to prepare slides includes a Hettich centrifuge and a
PrepStain robotic sample processer with computer and monitor.
 The PrepMate™ is an optional accessory that automates mixing the
sample and dispensing it onto the density reagent.
 Red blood cells and some leukocytes are eliminated by density
centrifugation.
 In addition to preparing an evenly distributed deposit of cells in a
circle 13 mm in diameter, the method incorporates a final staining
step that discretely stains each individual slide.
 Media is primarily ETHANOL BASED

46. 1. TThThe sample is quickly vortexed.
2. A proprietary device, the Cyringe™, disaggregates large clusters by syringing the sample through a
small orifice.
3. The sample is poured into a centrifuge tube filled with a density gradient reagent.
4. Sedimentation is performed in a centrifuge. A pellet is obtained and resuspended, and the
sedimentation is repeated.
5. The tubes are transferred to the PrepStain instrument, where a robotic arm transfers the fluid into a
cylinder. Cells settle by gravity onto a cationic polyelectrolyte-coated slide.The same robotic arm
also dispenses sequential stains to individual cylinders.

47. MONOPREP PAP TEST
 The practitioner obtains the MonoPrep sample with standard collection
devices that are swirled or rinsed in a preservative-filled collection
vial, after which the sampling device is discarded.
 Red blood cells are lysed by the transport medium.
 The vials are delivered to the laboratory where slides are prepared
using the MonoPrep Processor, a fully automated, batch processing
instrument capable of processing 40 samples per hour, with a
throughput capacity of 324 samples per 8-hour run.
 MonoPrep provides a significant reduction in unsatisfactory slides.

48. 1. An integrated stirrer mixes the specimen briefly to disperse mucus and aggregates.
2. The specimen is aspirated into the hollow stirrer and dual-flotechnology captures
a representative sample on a frit-backed filter.
3. The filter is pressed against the slide to transfer the cells onto a 20-mm diameter
circular area.
4. After cell transfer, the instrument applies a premeasured amount of alcohol
fixative directly onto the slide.

49. THIN PREP SURE PATH

50. CONVENTIONAL PAP VS LBC
Conventional Pap Smear
 Majority of cells not captured
 Non-representative transfer of
cells
 Clumping and overlapping of
cells
 Obscuring material
Liquid-based Cytology
 Virtually all cells of sample are
collected
 Randomized, representative
transfer of cells
 Even distribution of cells
 Minimizes obscuring material

51. METHODS OF SCREENING SMEARS

52. CERVICAL HISTOLOGY

53. The uterine cervix protrudes into the upper
vagina and contains the endocervical canal,
linking the uterine cavity with the vagina.
The endocervical canal EC is lined by a single
layer of tall columnar mucus-secreting
epithelial cells.
The ectocervix V is lined by thick stratified
squamous epithelium.
The junction J between the ecto- and
endocervical epithelium is quite abrupt and is
normally located at theexternal os, the point
at which the endocervical canal opens into the
vagina.

54. SQUAMOUS EPITHELIUM
The squamous epithelium covering the
exocervix is normally noncornified, and it
grows, matures, and accumulates glycogen
in its upper layers in response to circulating
estrogens.

55. ENDOCERVICAL EPITHELIUM
The endocervix is lined by a single
layer of mucin secreting epithelium
composed of cells with small, often
basilar, nuclei above which is mucin-
filled cytoplasm which imparts a
‘picket fence’ appearance .

56. TRANSFORMATION ZONE
Transformation zone is the zone between the original squamo columnar junction
and the new squamo columnar junction.

57. REPORTING SYSTEMS
 George N Papanicolaou (1954)
5 classifications based on certainty of finding malignant cells
 Descriptive system – WHO - (1968)
based on morphologic criteria – included mild, moderate,
severe dysplasia and Ca In Situ
 Richart – CIN –based on histologic diagnosis
 Bethesda system – TBS (1988)
National cancer institute revised in 1991 ,2001 and 2014

58. NORMAL CELLULAR ELEMENTS
A.SQUAMOUS CELLS
1.SUPERFICIAL SQUAMOUS
CCELLCELLS  The superficial cells have smaller
condensed (pyknotic) nuclei. Light
brown glycogen is present in the
cytoplasm.
 Cells are polygonal in shape with
keratohyaline granules in the
cytoplasm.

59. 2.INTERMEDIATE CELLS
 A typical intermediate cell with a
polygonal cytoplasmic profile. The
nucleus possesses finely granular
chromatin with longitudinal
groove.
 The cross-sectional area of the
intermediate nucleus is
approximately 35 μm 2.

60. 3.PARABASAL CELLS
 The parabasal cell exhibits typical
features with an oval nucleus, fine
chromatin, and a cross sectional
area of approximately 50 μm 2 .
 The cytoplasm is dense and heaped
up.

61. B.GLANDULAR CELLS
1.ENDOCERVICAL CELLS
Endocervical cells when
viewed from the side
present in a “picket-fence”
configuration
There is normal nuclear
polarity and ample
evidence of apical
mucin in these columnar
cells.

62. 2.ENDOMETRIAL CELLS
 A tight cluster of endometrial
glandular cells with nuclei having
cross-sectional areas slightly
smaller than the 35 μm 2 of
intermediate cells.
 Endometrial cell nuclear to
cytoplasmic ratios are high and the
cells tend to form three
dimensional groups.

63. The 2014 BETHESDA SYSTEM FOR REPORTING
CERVICAL CYTOLOGY
SPECIMEN TYPE:
Indicate conventional smear (Pap smear) vs. liquid-based preparation vs. other
SPECIMEN ADEQUACY
• Satisfactory for evaluation ( presence or absence of endocervical/transformation
zone component and any other quality indicators, e.g., partially
obscuring blood, infl ammation, etc. )
• Unsatisfactory for evaluation
– Specimen rejected/not processed
– Specimen processed and examined, but unsatisfactory for evaluation of epithelial
abnormality(reason)

64. GENERAL CATEGORIZATION
• Negative for Intraepithelial Lesion or Malignancy
• Others :Endometrial cells ( in a woman ≥45 years of age )
• Epithelial Cell Abnormalities
INTERPRETATION/RESULT
 NEGATIVE FOR INTRAEPITHELIAL LESION OR MALIGNANCY
(When there is no cellular evidence of neoplasia, state this in the
GeneralCategorization above and/or in the Interpretation/Result section of the
report -whether or not there are organisms or other non-neoplastic findings)
 OTHER
Endometrial cells ( in a woman ≥45 years of age )( Specify if “negative for
squamous intraepithelial lesion” )
 EPITHELIAL CELL ABNORMALITIES
 OTHER MALIGNANT NEOPLASMS

65. ADJUNCTIVE TESTING
Brief description of the test method(s) and report the result so that it is
easily understood by the clinician.
COMPUTER-ASSISTED INTERPRETATION OF CERVICAL CYTOLOGY
If case examined by an automated device, specify device and result.
EDUCATIONAL NOTES AND COMMENTS APPENDED TO CYTOLOGY
REPORTS ( optional )
Suggestions should be concise and consistent with clinical follow-up
guidelinespublished by professional organizations (references to relevant
publications may be included).

66. ADEQUACY
An adequate pap smear is one that includes a sampling of both the exocervix and
endocervix.
An adequate conventional smear has an estimated minimum of approximately
8,000-12,000 well visualized and preserved squamous cells.
An adequate liquid based preparation should have an estimated minimum of
5,000 well visualized/preserved squamous cells

67. “SATISFACOTRY FOR EVALUATION”
 Approriate labelling and identifying information.
 Relevant clinical information.
 Adequate number of well preserved and well visualized squamous
epithelial cells.

68. Satisfactory, but borderline squamous cellularity (LBP, SurePath). At 40×,
there were approximately 11 cells per field when ten microscopic fields
along a diameter were evaluated for squamous cellularity; this would give
an estimated total cell count between 5,000 and 10,000

69. UNSATISFACTORY FOR EVALUATION
 Lack of patient identification on specimen.
 Slide that is broken and cannot be repaired, or cellular material that is
inadequately preserved.
 Scant squamous epithelial component(well preserved and well visualized
squamous epithelial cells covering less than 10% of the slide surface)
 Obscuring that precludes interpretation of approximately 75% or more of
epithelial cells.

70. Unsatisfactory due to scant squamous cellularity.
Endocervicalcells are seen in a honeycomb arrangement (LBP,
ThinPrep at 10× magnification)

71. CONDITIONS WITH ABNORMAL PAP
 INFECTIONS
 NON NEOPLASTIC CONDITIONS
 EPITHELIAL CELL ABNORMALITIES

72. INFECTIONS

73. LACTOBACILLI/DODERLEIN’S BACILLI
 Lactobacilli are gram-positive
facultative anaerobic rod- shaped
bacteria observed in about 50% of
normal healthy adult female
population.
 These bacilli release enzymes
causing extensive cytolysis of
glycogen containing cells.
 Mainly affect intermediate and
superficial cells.
 Parabasal cells are generally
spared.
Bacteria: lactobacilli and cytolysis ( A:CP ),(B:LBP- Thin
Prep ) shows the presence of a cytolytic background with
cell debris and numerous stripped nuclei of intermediate
cells.
A B

74. BACTERIAL VAGINOSIS
Bacteria – coccobacilli ( CP :A)(LBP-SUREPATH-B). Shift in flora suggestive of bacterial
vaginosis. “Clue cell” and filmy background due to large numbers of coccobacilli.
Relatively cleaner background is seen in LBP.
A B

75. HERPES SIMPLEX
 Cellular changes consistent with
herpes simplex virus ( CP ).
 The eosinophilic intranuclear
“Cowdry A-type” inclusions are seen.
 The “ground-glass” appearance of
the nuclei is due to accumulation of
viral particles leading to peripheral
margination of chromatin.
 The inset shows a SurePath liquid-
based preparation with a typical
multinucleated herpetic cell showing
“ ground- glass” appearance of the
nuclei

76. ACTINOMYCES
 Actinomyces is often associated
with intrauterine device (IUD)
usage.
 Tangled clumps of filamentous
organisms, often with acute angle
branching, are recognizable as
“cotton ball” clusters on low
power .
 Filaments sometimes have a radial
distribution or have an irregular
“woolly body”appearance.
 Masses of leukocytes adherent to
microcolonies of the organism with
swollen filaments or “clubs” at the
periphery may be identified.

77. CANDIDA ALBICANS
LBP , ThinPrep : pseudohyphae (left) “spearing” or a “shish kebab” appearance
of squamous cells (right). This feature is readily appreciated at low power, even
when the pseudohyphae are not prominent.

78. TRICHOMONAS
Image A shows CP and B shows LBP ;THIN PREP(polyball appearance of neutrophills)
Trichomonas is a pear-shaped, oval to round, cyanophilic organism that ranges in size from 15-30 microns.The
nucleus is pale, vesicular and eccentrically located. Eosinophilic granules are often visible in the cytoplasm.
A B

79. CYTOMEGALOVIRUS
 The cytopathic effect of
cytomegalovirus (CMV) affects
mostly the endocervical glandular
cells but can also be present in
stromal cells
 Cellular and nuclear enlargement.
 Large eosinophilic intranuclear
viral inclusions with a prominent
halo.
 Small cytoplasmic, basophilic
inclusions can also be present.

80. NON NEOPLASTIC CONDITIONS

81. Includes the following
 Squamous metaplasia
 Keratotic changes
 Typical parakeratosis
 Hyperkeratosis
 Tubal metaplasia
 Pregnancy related cellular changes
 Atrophy
 Reactive /reparative cellular changes
 Associated with inflammation
 Lymphocytic cervicitis
 Associated with radiation
 Associated with IUD usage

82. SQUAMOUS METAPLASIA
 ( LBP , SurePath ):A characteristic
metaplastic cell is found in the
center of the field.
 The nucleus is round to oval with fi
ne, evenly distributed chromatin.
 The nuclear to cytoplasmic ratio is
variable, and in this instance, it
approaches one to one.
 The mean nuclear area is larger than
that of the intermediate cell and
similar to the parabasal cell at 50
μm 2 .

83. KERATOTIC CHANGES
TYPICAL PARAKERATOSIS
 Both are examples of “typical
parakeratosis” showing miniature
squamous cells with small bland,
pyknotic nuclei.
 “A” shows the “squamous pearl”
formation from a 49-year-old
woman being followed up after
treatment for SIL.
 ‘B’ shows small cluster of miniature
squamous cells.
A B

84. HYPERKERATOSIS
 A- LBP , ThinPrep shows a group
of anucleate squamous at low
power.
 B- LBP , ThinPrep shows anucleate,
mature polygonal squamous cells
with ghostlike “nuclear holes”
A B

85. TUBAL METAPLASIA
CP :Ciliated cells derived from tubal
metaplasia. Terminal bar
and cilia at left edge ( arrow)
LBP , Thin Prep:A linear array of
cells showing tubal metaplasia

86. PREGNANCY RELATED CHANGES
 Hormonal changes
 Decidual changes
 Synctiotrophoblastic changes
 Cytotrophoblastic changes
 Arias stella reaction

87. HORMONAL CHANGES
NAVICULAR CELLS In pregnant women, squamous cells become laden
with glycogen, and have a vaguely “boatlike” shape referred to
as“navicular”cells {A: LBP - ThinPrep , and B : LBP - SurePath}
A
B

88. ATROPHY
Atrophy ( LBP , ThinPrep ): flat, monolayer
sheet of parabasal-type cells, with preserved
nuclear polarity
Atrophy with infl ammation ( CP ):shows
classic finding of granular debris in
background, degenerating parabasal cells and
polymorphonuclear leukocytes.

89. REACTIVES CHANGES ASSOCIATED WITH
INFLAMMATION
 ( CP ) A 26-year-old woman, day 14
of menstrual cycle with mild
vaginal discharge.
 Squamous cells show mild nuclear
enlargement with nuclear
hypochromasia, perinuclear halos,
and cytoplasmic polychromasia
resulting in a “moth-eaten”
appearance.
 Trichomonads are seen in the
background.

90. LYMPHOCYTIC CERVICITIS
( CP )Abundant lymphoid cells with a tingible
body macrophage located centrally
LBP-THIN PREP

91. RADIATION EFFECT
(CP)A;shows irregularly shaped abundant cytoplasm and the streaming or
“windblown” edges of the. Nuclei are typically enlarged and may be pale or become
hyperchromatic as nuclear material condenses. Nucleoli are typically seen along with
numerous polymorphonuclear leukocytes in the background.
B:(LBP , ThinPrep ) radiated cells in liquid-based preparations do not tend to show the
streaming and the cytoplasm is typically more dense. Nuclear degeneration and
cytoplasmic vacuolization are common in both preparation types
A B

92. IUD EFFECT
( CP ) shows small cluster of glandular
cells with cytoplasmic vacuoles
displacing nuclei.
LBP , Thin Prep :cellular groups tend to be
tighter.

93. EPITHELIAL CELL ABNORMALITIES

94. ASC-US
 LBP-SUREPATH
 Single atypical squamous cell with
ill-defined cytoplasmic halo in a
background of inflammation.
 Nuclei are approximately two and
one half to three times the area of
the nucleus of a normal
intermediate squamous cell
(approximately 35 mm 2 ) or twice
the size of a squamous metaplastic
cell nucleus (approximately 50 μm
2 )

95. ASC-US ( CP ): Cells with multinucleation, nuclear enlargement.

96. ASC-H
 ASC-H ( LBP, SurePath ): Routine cytology for a 30-year-old woman.
 Rare metaplastic cells with dense cytoplasm and nuclear enlargement
with hyperchromasia are present in a background of scattered acute
inflammation

97. ATYPICAL GLANDULAR CELLS
ATYPICAL ENDOCERVICAL CELLS, (CP):
Sheet of glandular cells showing nuclear
enlargement, marked variation in
nuclear size, prominent nucleoli,
and distinct cell borders.
ATYPICAL ENDOMETRIAL CELLS ( LBP ,
ThinPrep ). Three-dimensional grouping of
small cells with crowded round or oval
nuclei.

98. L-SIL
LBP-THIN PREP
LBP-THIN PREP:Nuclear enlargement and
hyperchromasia of sufficient degree seen for
the interpretation of LSIL

99. LBP-THIN PREP
LSIL on cytology is characterized by mature squamous cells with
enlarged nuclei with variable chromatin and nuclear membranes.
Koilocytosis or perinuclear cavitation in the cytoplasm,
a characteristic of HPV cytopathic effect is present.

100.  Nuclei are generally hyperchromatic but may be normochromatic.
 Nuclei show variable size (anisonucleosis).
 Chromatin is uniformly distributed and ranges from coarsely granular to
smudgy or densely opaque .
 Contour of nuclear membranes is variable ranging from smooth to very
irregular with notches .
 Binucleation and multinucleation are common .
 Nucleoli are generally absent or inconspicuous if present.
 Koilocytosis or perinuclear cavitation consisting of a broad, sharply
delineated clear perinuclear zone and a peripheral rim of densely stained
cytoplasm is a characteristic viral cytopathic feature but is not required for
the interpretation of LSIL
 Cells may show increased keratinization with dense, eosinophilic cytoplasm
with little or no evidence of koilocytosis.

101. MIMICS OF L-SIL
PSEUDOKOILOCYTES
( LBP , ThinPrep ): Glycogen in squamous
cells can give the appearance of
“pseudokoilocytosis” . The halos associated
with glycogen often have a yellow refractile
appearance.
HERPES
( LBP , ThinPrep ): A 25-year-old woman.
Endocervical cell and intermediate cells
showing herpes virus cytopathic effect with
clearing of chromatin.

102. H-SIL
( CP ): The dysplastic cells are seen here
in a syncytial cluster or hyperchromatic
crowded group
CP:There is variation in nuclear size and
shape, and the cells have delicate
cytoplasm

103.  The cells of HSIL are smaller and show less cytoplasmic maturity than cells of
LSIL.
 Cells occur singly, in sheets, or in syncytial-like aggregates .
 Syncytial aggregates of dysplastic cells may result in hyperchromatic crowded
groups.
 Nuclear to cytoplasmic ratio is higher in HSIL compared to LSIL.
 Contour of the nuclear membrane is quite irregular and frequently
demonstrates prominent indentations.
 Nuclei are generally hyperchromatic but may be normochromatic or even
hypochromatic.

104.  Chromatin may be fine or coarsely granular and is evenly distributed.
 Nucleoli are generally absent, but may occasionally be seen, particularly
when HSIL extends into endocervical gland spaces or in the background of
reactive or reparative change.
 Appearance of the cytoplasm is variable; it can appear “immature,” lacy, and
delicate or densely metaplastic occasionally, the cytoplasm is“mature” and
densely keratinized (keratinizing HSIL)

105. SQUAMOUS CELL CARCINOMA
SQUAMOUS CELL CARCINOMA,
KERATINIZING ( LBP , SurePath ):The
malignant cells in variable shapes and
sizes with some keratinized “tadpole
cells.”
( CP ): There is markedpleomorphism
of cell size and shape, cytoplasmic
keratinization, and tumor diathesis
in the background

106.  Presents predominantly as isolated, single cells and less commonly in cellular
aggregates.
 Marked variation in cellular size and shape is typical, with caudate and spindle
cells that frequently contain dense orangeophilic cytoplasm.
 Nuclei vary markedly in area, nuclear membranes may be irregular, and numerous
dense opaque nuclei are often present.
 Chromatin pattern, when discernible, is coarsely granular and irregularly
distributed with chromatin clearing.
 Macronucleoli may be seen .
 Associated keratotic changes (hyperkeratosis or parakeratosis) may be present
but are not suffi cient for the interpretation of carcinoma in the absence of
nuclear abnormalities.
 A tumor diathesis may be present.

107. ADENOCARCINOMA
ADENOCARCINOMA, ENDOCERVICAL ( CP ):Nuclei are enlarged and
pleomorphic with irregular chromatin distribution and prominent or
macronucleoli. Cytoplasm is finely vacuolated with prominent blood
-filled background

108.  Abundant abnormal cells, typically with columnar confi guration.
 Single cells, two-dimensional sheets or three-dimensional clusters, and
syncytial aggregates .
 Enlarged, pleomorphic nuclei demonstrate irregular chromatin distribution,
chromatin clearing, and nuclear membrane irregularities .
 Macronucleoli present
 Cytoplasm is usually fi nely vacuolated.
 Necrotic tumor diathesis is common.

109. ADJUNCTIVE TESTING
HIGH RISK HPV TESTING
 There are four hrHPV tests that are FDA approved for performance
inassociation with cervical cytology. Three are DNA based and one is RNA
based.
 Triage of an abnormal cytology result by a hrHPV test effectively improves
the balance of sensitivity vs. specifi city for colposcopic referral and
prevalent disease detection.
 HPV genotyping refers to the selective reporting of individual HPV types in
conjunction(HPV 16 nd 18) with a positive pooled high-risk test
IMMUNOCHEMICAL ASSAYS
 The best-studied biomarkers are p16, ProExC, and Ki67
 p16 and ProExC are both markers of an aberrant cell cycle which has been
affected by the oncogenic effects of HPV.
 Ki67 is a marker of cellular proliferation.
 p16 stains both the nucleus and cytoplasm; ProExC and Ki67 stain the
nucleus

110. SAMPLE REPORT
SPECIMEN TYPE:
Conventional pap smear
SPECIMEN ADEQUACY:
Satisfactory for evaluation; endocervical/transformation zone present.
INTERPRETATION:
Negative for intraepithelial lesion or malignancy
NOTE
High-risk HPV typing is NEGATIVE
COMMENT
As Per the 2012 ASCCP management guidelines, a negative result for HPV testing
when associated with an NILM cytology means the patient has signifi -
cantly less than a 1 % chance of HSIL (CIN3) lesion and repeat testing at
decreased intervals is not warranted

111. HORMONAL CYTOLOGY
 Maturation of vaginal squamous cells from one cell to another is hormone
dependent.
 The quantitative ratio between the different cell types can reflect the index
of the hormonal status of the female.
 For hormonal assesment ideal site is lateral vaginal wall.
 Estrogens have proven action on maturation of squamous epithelium of
vagina.
 Its excess causes enhancement of maturation and the smear contains more of
superficial cells, on the other hand its lack causes lower degree of
maturation or the atrophy of squamous epithelium,the same effect could be
reflected due to antagonistic action of the excess of progesterone.

112. CELLULAR INDICES FOR HORMONAL
ASSESSMENT
KARYOPYKNOTIC INDEX
EOSINOPHILIC INDEX
FOLDED CELL INDEX
CROWDED CELL INDEX
MATURATION INDEX

113. KARYOPYKNOTIC INDEX
Ratio between the superficial squamous cells with pyknotic nuclei to all mature
squamous cells irrespective of staining character.
Peak of KPI usually coincides with the time of ovulation and may reach 50-85.
EOSINOPHILIC INDEX
Ratio of mature squamous cells with eosinophilic cytoplasm to all mature
squamous cells irrespective of size of nucleus.
Peak value is 50-75 during ovulation.
MATURATION INDEX
It is count of the parabasal cells,intermediate and superficial cells (P : I : S)
In a normal menstruating woman during ovulation the menstruation index will
be 0/35/65.
In postmenopausal it will be 85/15/0.

114. OTHER INDICES
FOLDED CELL INDEX
Ratio of mature squamous cells with folded margins to all mature squamous
cells irrespective of staining chararcter.
Folding is usually observed in cells containing glycogen.
CROWDED CELL INDEX
Represents the relationship of mature squamous cells lying in clusters of four or
more cells, to all mature squamous cells.

115. THE MATURATION VALUE
Meisels (1967) suggested that a specific numerical value be attached to the
three principal subgroups of the squamous cells—a value of 1.0 to superficial
squamous cells, a value of 0.5 to intermediate cells, and a value of 0.0 to
parabasal cells.
The maturation value (MV) in Patient with MI 0:35:65 (normal menstruating
woman at time of ovulation)
0 X 0 = 00
35 X 0.5 = 17.5
65 X 1 = 65.O
TOTAL = 82.5
An MV of 100 indicates a pure population of superficial squamous cells, MV of
zero indicates a pure population of parabasal cells.
For menstruating normal women, the MV is between 50 and 95; for women with
varying degrees of atrophy of squamous epithelium, the MV is below 50.

116. TAKE HOME MESSAGE FOR ALL WOMEN:
CERVICAL CANCER IS A PREVENTABLE DISEASE
"No woman has to die from cervical cancer," says Ault, professor of obstetrics
and gynecology at Emory. "It’s almost entirely preventable with recommended
Pap guidelines. If everyone got a regular Pap test and the HPV (human
papillomavirus) vaccine we could reduce the number of women diagnosed with
cervical cancer by more than 75 percent."

117. REFERENCES
 Koss Diagnostic Cytology and Its Histopathologic Bases, 5th Edition
 The Bethesda System for Reporting Cervical Cytology Definitions, Criteria,
and Explanatory Notes Third Edition
 CYTOLOGY: DIAGNOSTIC PRINCIPLES AND ISBN: 978-1-4160-5329-3 CLINICAL
CORRELATES Copyright © 2009 by Saunders, an imprint of Elsevier Inc.
 HISTOLOGY FOR PATHOLOGISTS BY STACEY E MILLS.
 Robbins and Cotran Pathologic Basis of Disease
 Wheater’s Functional Histology A Text and Colour Atlas

118. THANK YOU