This slide presentation summarizes a cytology case involving a 38-year-old female patient. A cervical smear shows atypical cells with dense, scanty cytoplasm and enlarged hyperchromatic nuclei throughout the smear. Background shows sheets of neutrophils, coccobacilli and hemorrhage. The specimen is categorized as an epithelial cell abnormality and interpreted as a high-grade squamous intra-epithelial lesion. Colposcopic examination is suggested. The presentation provides further details on cytology techniques, classifications of abnormal cervical cells, screening guidelines, and characteristics of low and high-grade squamous lesions.

1. SLIDE PRESENTATION
CYTOLOGY
Sansar Babu Tiwari, MBBS, PGY I
Department of Pathology
TUTH
1st August 2019
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2. Single Pap-stained Cervical Smear of 38 years female
(Conventional cytology)
Smear shows superficial, intermediate, and parabasal
cells along with few endocervical cells.
Atypical cells are found arranged singly, in sheets and
in clusters throughout the smear.
These cells are round to oval (parabasal) in shape with
dense, scanty cytoplasm, enlarged hyperchromatic
nuclei, coarse chromatin and irregular nuclear rim
with high N : C ratio and inconspicuous nucleoli.
SLIDE PRESENTATION
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3. Background shows sheets of neutrophils and
coccobacilli along with hemorrhage.
Necrosis and mitosis is not seen
SLIDE PRESENTATION
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4. Specimen: Cervical smear
Adequacy: Satisfactory for evaluation; endocervical component
present
General Categorization*: Epithelial cell abnormality, squamous
Interpretation: High-grade squamous intra-epithelial lesion (HSIL)
Adjunctive Testing*: Not done in smear
Note*: Suggest colposcopic examination
* Optional to mention in Report.
SLIDE PRESENTATION
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5. Olympus, 4x
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6. Olympus, 4x
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7. Olympus, 20x
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8. Olympus, 20x
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9. Olympus, 40x
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10. Olympus, 40x
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11. Columnar lining changes into
squamous in TZ
TZ is a dynamic point, keep on
changing. Move out towards
ectocervix during puberty,
pregnancy and pills. Move
inwards when hormonal
influences are absent like after
menopause.
CERVIX
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12. BURDEN OF CERVICAL CANCER
WHO:
Cervical cancer is the second most common cancer in women living
in less developed regions with an estimated 570 000 new cases in
2018 (84% of the new cases worldwide).
In 2018, approximately 311 000 women died from cervical cancer;
more than 85% of these deaths occurring in low- and middle-
income countries.
High risk HPV: 16, 18, 31, 33, 35,39, 45, 51, 52, 56, 58, 59, 68, 69, 82
Low risk HPV: 6, 11, 40, 42, 43, 44, 54, 61, 72, 81
On average, 50% of HPV infections are cleared within 8 months and
90% are cleared within 2 years.
CERVICAL CANCER OVERALL BURDEN
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13. CERVICAL NEOPLASIA CLASSIFICATION
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14. HPV and cervical cancer
14
• Although HPV has been
firmly established as a
common cause of
cervical cancer, it is not
sufficient to cause
cancer.
• Host immune and co-
carcinogen exposure
influences the fate of
HPV and progression to
cancer.

15. Gardenella and (pre)neoplasia
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16. Incidence of cervical cytology
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17. United States Preventive Service Task Force
(USPSTF), 2018 recommends:
• Age to initiate: 21 years
• Age to discontinue: 65 years
• Between 21-29 years: Pap test every 3 years
• After 30 years: One of these methods:
• Pap test every 3 years
• hrHPV testing alone every 5 years
• Co-testing (Pap test and HPV testing) every 5 years
Routine Screening of patients
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18. OBTAINING PAP SMEAR
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19. CONVENTIONAL VS LBP
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20. SUREPATH VS THINPREP
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21. SUREPATH VS THINPREP
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22. SUREPATH VS THINPREP
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23. TYPES OF SQUAMOUS CELLS
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24. OTHER CELL TYPES
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25. SUPERFICIAL CELL
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26. INTERMEDIATE CELL
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27. PARABASAL CELL
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28. ENDOCERVICAL CELL
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29. METAPLASTIC CELL
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30. 30

31. 31

32. 32
Proliferative Phase

33. 33
Ovulatory Phase (14-16 days)

34. 34
Luteal Phase

35. LSIL cervical cytologic specimens that contain a few cells that
are suspicious for but not diagnostic of HSIL are reported as
atypical squamous cells, cannot exclude a high-grade
squamous intraepithelial lesion(ASC-H). There was previously
no recommendation regarding how to report these.
Benign-appearing endometrial cells are reported only in
women ≥45 years. This is a change from Bethesda 2001, which
used an age threshold of ≥40 years.
Pages: 191  324
Images: 186  370
Bethesda 2014 vs 2001
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36. 1. Description of specimen type and test requested –
cervical or vaginal sample, conventional Pap smear,
liquid-based cytology, and/or reflex human
papillomavirus (HPV) test.
2. A description of specimen adequacy
3. A general categorization (optional) – Negative,
epithelial cell abnormality, or other
4. An interpretation/result – Either the specimen is NILM
(although organisms and reactive changes may be
present) or there is an epithelial cell abnormality as
defined, or there is another finding(increased risk like
endometrial cells in women ≥45 years).
Bethesda 2014 Overview of Report
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37. 5. A description of any ancillary testing or automated
review that was performed (eg. HPV, AutoPap)
6. Educational notes and suggestions by the pathologist
(optional)
Bethesda 2014 Overview of Report
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38. 38

39. 39

40. 1. Cellularity >5000 (LBP), 8000-12000 (CP)
>2000: Low cellularity but satisfactory: Post-radiation, Post-TH vaginal)
2. Endocervical component
EC/TZ is not necessary for adequate specimen, only squamous
cellularity is needed
Adequate TZ requires at least 10 well preserved EC or squamous
metaplastic cells
No relation with further diagnosis with SIL
3. Obscuring factors (Blood, lubricants)
>75% of sq cells (not the slide area) : unsatisfactory
4. HPV testing on unsatisfactory specimen
HPV test could be falsely negative in unsatisfactory specimen
However, if HPV is positive in these specimen, follow up is still needed
ADEQUACY
40

41. ASC refers to cytologic changes suggestive of SIL, which
are qualitatively and quantitatively insufficient for
definite interpretation
Three essential features
1. Squamous differentiation
2. Increased ratio of nuclear to cytoplasmic area
3. Minimal nuclear hyperchromasia, chromatin
clumping, irregularity, smudging or multinucleation
ATYPICAL SQUAMOUS CELL
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42. • Definition: cytological changes suggestive of LSIL that are
quantitatively and qualitatively insufficient for a definitive diagnosis
• CRITERIA
– Cells resemble superficial and intermediate squamous cells in size
and configuration
– Nuclei are approximately
2 and half to three times the area of the nucleus of a normal intermediate
squamous cell or twice the size of a squamous metaplastic cell nucleus.
– Slighlty increased N:C ratio
– Minimal nuclear hyperchromasia and irregularity of chromatin
distribution or nuclear shape
– Nuclear abnormalities associated with dense orangeophilic
cytoplasm (atypical parakeratosis)
ATYPICAL SQUAMOUS CELL -UNDETERMINED
SIGNIFICANCE (ASC-US)
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43. ATYPICAL SQUAMOUS CELL -UNDETERMINED
SIGNIFICANCE (ASC-US)
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44. ATYPICAL SQUAMOUS CELL -UNDETERMINED
SIGNIFICANCE (ASC-US)
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45. ATYPICAL SQUAMOUS CELL -UNDETERMINED
SIGNIFICANCE (ASC-US)
45

46. • Definition: cytological changes suggestive of HSIL that are
quantitatively and qualitatively insufficient for a definitive diagnosis
• Patterns:
A. Small cells with high N:C Ratios (Atypical immature Metaplasia)
• Cells usually occur singly or in small fragments of less than 10
cells, occasionally in conventional smears, cells may stream in
mucus.
• Cells are size of metaplastic cells with nuclei that are about 1.5
to 2.5 times larger than normal
• N/C ratio may approximate that of HSIL
ASC-H Vs HSIL: Nuclear abnormalities such as hyperchromasia,
chromatin irregularity and abnormal nuclear shapes with focal
irregularity favor an interpretation of HSIL.
ATYPICAL SQUAMOUS CELL –CANNOT EXCLUDE HSIL
(ASC-H)
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47. B. Crowded Sheet Pattern:
• Dense cytoplasm, polygonal cell shape and fragments with
sharp linear edges generally favor squamous over
glandular (endocervical) differentiation.
C. ASC-H Mimics:
• Isolated endocervical cells
• Degenerated endometrial cells
• Macrophages
• IUD: may shed rare cells with high N:C ratio
• Pregnancy and post-partum: atypical appearing
decidualized stromal cells
ATYPICAL SQUAMOUS CELL –CANNOT EXCLUDE HSIL
(ASC-H)
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48. ATYPICAL SQUAMOUS CELL –CANNOT EXCLUDE HSIL
(ASC-H)
48

49. • CRITERIA
– Cells occur singly, in clusters, and in sheets
– Cytological changes are usually confined to
squamous cells with ‘mature’ intermediate or
superficial squamous cell- type cytoplasm
– Overall cell size is large, with fairly abundant
‘mature’ well-defined cytoplasm
– Nuclear enlargement more than 3 times the area of
normal intermediate nuclei results in a low but
slightly increased N:C ratio
Low-grade Squamous Intraepithelial Lesion (LSIL)
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50. • CRITERIA
– 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
Low-grade Squamous Intraepithelial Lesion (LSIL)
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51. • CRITERIA
– Koilocytosis or perinuclear cavitation consisting of a
broad, sharply delineated clear peri-nuclear 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
– Cells with koilocytosis or dense orangeophilia must also
show nuclear abnoramalities to be diagnostic of LSIL,
perinuclear halos in the absence of nuclear
abnormalities does not qualify for the interpretation of
LSIL
Low-grade Squamous Intraepithelial Lesion (LSIL)
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52. Low-grade Squamous Intraepithelial Lesion (LSIL)
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53. • Pseudokoilocytosis
• Herpes Cytopathic
Effects
• Radiation Changes
MIMICS OF LSIL
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54. L
S
I
L
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55. FOLLOW-UP OF LSIL
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56. FOLLOW-UP OF LSIL
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57. High-grade Squamous Intraepithelial Lesion (HSIL)
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58. • CRITERIA
– Cells occur singly, in sheets, or in syncytial aggregates
– Smaller and show less cytoplasmic maturity than cells of
LSIL
– Syncytial aggregates of dysplastic cells may result in
hyperchromatic crowded groups
– Overall cell size is variable, in general, the cells of HSIL
are smaller than those of LSIL. Higher-grade lesions often
contain quite small basal-type cells
– Degree of nuclear enlargement is more variable than
that seen in LSIL. Even if nucleus size is same as that of
LSIL, cytoplasmic area is decreased leading to marked
increase in N:C ratio.
High-grade Squamous Intraepithelial Lesion (HSIL)
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59. • CRITERIA
– Nuclei are generally hyperchromatic but may be
normochromatic or even hypochromatic
– Chromatin may be fine or coarsely granular and is evenly
distributed
– Contour of the nuclear membrane is quite irregular and
frequently demonstrates prominent indentations or
grooves
– 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 changes
– Cytoplasm: variable; immature, lacy or delicate or
densely metaplastic ; mature and densely keratinized as
in keratinizing HSIL.
High-grade Squamous Intraepithelial Lesion (HSIL)
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60. High-grade Squamous Intraepithelial Lesion (HSIL)
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61. • Syncytial Aggregates/ Hyperchromatic Crowded
Groups:
• SIL with Endocervical Gland Involvement
• Endometrial cells or Squamous Repair
• Keratinizing High Grade Lesion
• HSIL in Atrophy
• LSIL with some features suggestive of concurrent
HSIL
Problematic Pattern in HSIL
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62. Note the flattening of cells at the edge of cluster.
SIL with EC gland involvement
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63. • Isolated cells
Reserve cells
Parabasal cells
Immature sq. met. cells
• Histiocytes or Lymphocytes
Kidney shaped
Nuclear membrane
notching and irregularity
is absent
• Decidualized Stromal cells
History of pregnancy, more
granular but less dense
cytoplasm, prominent
nucleoli.
Mimics of HSIL
63

64. H
S
I
L
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65. Follow-up of HSIL
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66. Follow-up of HSIL
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67. • Definition:
–WHO, 2014: SCC is an invasive epithelial
tumor composed of squamous cells of
varying degrees of differentiation
Keratinizing vs Non-keratinizing SCC
The Bethesda system doesnot subdivide
squamous cell carcinoma per se.
Squamous Cell Carcinoma
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68. • CRITERIA:
– 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
Squamous Cell Carcinoma KERATINIZING
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69. • CRITERIA:
– Macronucleoli may be seen but are less common in
NKSCC
– As keratotic changes (hyper or para) may be present
but are not sufficient for the interpretation of
carcinoma in the absence of nuclear abnormalities
– A tumor diasthesis may be present but is usually less
than that seen in NKSCC
Squamous Cell Carcinoma KERATINIZING
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70. Squamous Cell Carcinoma KERATINIZING
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71. • CRITERIA:
– Cells occur singly or in syncytial aggregates with
poorly defined cell borders
– Cells may be somewhat smaller than those of many
HSIL, but display most of the features of HSIL
– Nuclei demonstrate markedly irregular distribution
of coarsely clumped chromatin with chromatin
clearing
– Nucleoli may be prominent
– A tumor diasthesis consisting of necrotic debris and
broken-down elements is often present
Squamous Cell Carcinoma NON-KERATINIZING
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72. Squamous Cell Carcinoma NON-KERATINIZING
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73. 1. The Bethesda System of Reporting Cervical
Cytopathology, 3rd edition, 2014
2. The Bethesda System and Beyond, BD
Surepath
3. Robbins and Cotran Pathological Basis of
Disease, 9th edition, 2015
4. Uptodate, 2019
5. www.ncbi.nlm.nih.gov/pubmed
REFERENCES:
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