The document provides information on cervical cytology screening and the Bethesda System for reporting results. It describes the Papanicolaou staining technique and liquid-based cytology collection methods. The Bethesda System categories for specimen adequacy, interpretation, and epithelial cell abnormalities are outlined. Normal cervical cell types are defined. Reactive, infectious, and other non-neoplastic findings are described. Atypical squamous cells of undetermined significance, low-grade squamous intraepithelial lesions, and high-grade squamous intraepithelial lesions are defined. Adenocarcinoma in situ and adenocarcinoma are also summarized.

1. CERVIX-CYTOLOGY
BETHESDA SYSTEM
Abdul Quddus BS MLT

2. Papanicolaou (Pap) stain
 Alcohol dried; better for nuclear detail
 Stains ribosomes blue green, particularly in
parabasal cells, mesothelial cells and
metaplastic squamous cells
 Stains metabolically inactive cells pink, such
as superficial cells
 Stains keratinized cells or thick specimens
orange (benign or malignant)

3. Papanicolaou (Pap) stain

4. Liquid based cytology
 Head of spatula, where cells are lodged, is
broken off into small glass vial containing
preservative fluid, or rinsed directly into
preservative fluid
 Sample is sent to lab, then spun and treated
to remove mucus, pus or other obscuring
material
 Random sample of remaining cells is taken
and deposited onto a slide
 Reduces number of inadequate smears and
need for repeat smears

5. Liquid based cytology

6. Diff-Quik stain
 An air dried, Giemsa-type stain
 Better for background material or to
assess adequacy of endocervical
smears to detect C. trachomatis
 Used for fine needle aspirates, not for
cervical smears

7. CERVIX - Normal cells
 Basal cells
 Parabasal cells
 Intermediate cells
 Superficial cells
 Endocervical cells

8. Basal Cells
 Small undifferentiated cells
 Seldom identified in pap smears

9. Parabasal cells
 Cytoplasm is round, dense, basophilic
 Nucleus is vesicular, central, round
and relatively large (50μm²)
 May see naked nuclei
 Higher N/C ratio and smaller size than
intermediate cells

10. Parabasal cells

11. Intermediate Cells
 Cytoplasm is polygonal, transparent,
basophilic
 Nucleus is about the size of a red
blood cell (35 μm²), is vesicular,
round/oval
 Nuclear texture and size is reference
for dysplasia

12. Intermediate Cells

13. Superficial cells
 cytoplasm is polygonal, transparent,
eosinophilic
 Nucleus is pyknotic, round/oval

14. Superficial cells

15. Endocervical cells
 Columnar cells
 Vacuolated or granular cytoplasm
 Prominent cell borders
 Basal nuclei with fine granular
chromatin
 Occasional nucleoli
 Honeycomb appearance
 Ciliated if tubal metaplasia

16. Endocervical cells

17. Squamous Metaplasia
 Single cells, loosely cohesive groups or
sheets
 Variable cell size and shape
 Cell borders are more defined with maturity
 Cytoplasm is variable with occasional
vacuoles
 Nuclei is slightly larger than intermediate
cell nuclei, has finely granular and evenly
distributed chromatin, nuclear membrane is
smooth, no prominent nucleoli

18. Squamous Metaplasia

19. BETHESDA
SYSTEM

20. BETHESDA SYSTEM
 Uniform terminology introduced in
1988 and revised in 1991 and 2001
 To establish uniform terminology and
standardize diagnostic reports.
 In addition, it introduced a
standardized approach for reporting if
an individual specimen is adequate for
evaluation.

21. BETHESDA SYSTEM, 2001
 Specimen adequacy
 General categorization (optional)
 Interpretation/result
 Automated review and ancillary
testing (include if appropriate)
 Educational notes and suggestions
(optional)

22. Adequacy
 Satisfactory for evaluation (describe
presence or absence of
endocervical/transformation zone
component and all other quality
indicators, such as partially obscuring
blood, inflammation, etc.)
 Unsatisfactory for evaluation
(indicate reason)
 Specimen rejected/not processed
(indicate reason)

23. Assessment of adequacy
 Adequate number of squamous cells
(conventional smear should have 8000-12000
cells, liquid-based preparation should have
5000 cells)
 The presence or absence of endocervical
cells should be reported; an adequate number
of endocervical cells (at least 10 well-
preserved endocervical or metaplastic cells)
confirms sampling of transition zone
 Specimen with more than 75% of cells
obscured by inflammation and bacteria is
unsatisfactory (however, should still report
presence of abnormal cells)

24. Unsatisfactory: scant
squamous cellularity

25. Adequate

26. Interpretation/result
 Negative for Intraepithelial Lesion or
Malignancy (NILM)
 Epithelial Cell Abnormality
 Other malignant neoplasm (Specify)

27. Negative for Intraepithelial
Lesion or Malignancy (NILM)
 Organisms
 Trichomonas vaginalis
 Fungal organisms morphologically consistent
with Candida species
 Shift in flora suggestive of bacterial vaginosis
 Bacteria morphologically consistent with
Actinomyces species
 Cellular changes associated with Herpes
simplex virus

28. Trichomonas vaginalis
(pear shaped)

29. Candida (Branching
pseudohyphae and spores)

30. Gardenella Vaginalis
(Clue cells)

31. Actinomyces
(Dust bunnies)

32. Herpes Simplex

33.  Other non-neoplastic findings (optional to
report, list is not inclusive)
 Reactive cellular changes associated with:
• inflammation (includes typical repair)
• irradiation
• Intrauterine contraceptive device (IUD)
 Glandular cells status post hysterectomy
 Atrophy

34. Reactive cellular changes,
Repair
 Flat
monolayer
sheets with
distinct
cytoplasmic
outlines,
streaming
nuclear
polarity,
prominent
nucleolus in
almost every
cell.

35. Reactive cellular changes,
Radiation
 Enlarged nuclei
with abundant
polychromatic
cytoplasm with
vacuolization.
Mild nuclear
hyperchromasia
without coarse
chromatin,
prominent
nucleoli

36. Reactive cellular changes,
IUD
Glandular cells in
small clusters with
increased N/C
ratio and
cytoplasmic
vacuoles. Nuclear
degeneration and
prominent nucleoli
present.

37. Glandular cells status post
hysterectomy
 Goblet cell
metaplasia
and bland
cellular
features.

38. Atrophy
 Sheets of
uniform orderly
parabasal
cells. Some
nuclei may
show grooves,
but chromatin
pattern is fine.
Atrophic cells
may have
nucleoli.

39.  Other
Endometrial cells (in a woman greater
than or equal to 40 years of age;
specify if “negative for squamous
intraepithelial lesion”)

40. Endometrial cells
 Cells occur in small
clusters. Small,
round nuclei similar
in size to a normal
intermediate cell
nucleus.
Inconspicuous or
absent nucleoli.
Cytoplasm is scant,
basophilic and
sometimes
vacuolated. Cell
borders are ill-
defined.

41. Epithelial Cell Abnormality
 SQUAMOUS CELL
 Atypical squamous cells
 - of undetermined significance (ASC-US)
 - cannot exclude HSIL (ASC-H)
 Low grade squamous intraepithelial lesion (LSIL)
 - encompassing HPV/mild dysplasia/CIN I
 High grade squamous intraepithelial lesion (HSIL)
 - encompassing: moderate and severe
dysplasia/CIN2/CIN3/CIS
 - with features suspicious for invasion (if
invasion suspected)
 Squamous cell carcinoma

42. Atypical squamous cells - of
undetermined significance
(ASC-US)
 May be neoplastic (HPV related, LSIL,
HSIL) or reactive
 Nuclear changes more marked than
reactive, less than LSIL; nucleus is 2.5-3x
size of intermediate cell nucleus
 In perimenopausal women (40-55 years),
cells with bland nuclear enlargement (2-3x
size of intermediate cell nuclei), smooth
nuclear membranes and fine chromatin are
likely to be negative for SIL/malignancy

43. Atypical squamous cells - of
undetermined significance
(ASC-US)

44. Atypical squamous cells -
cannot exclude HSIL (ASC-H)

45. Squamous Intraepithelial
Lesions

46. Low grade squamous
intraepithelial lesion (LSIL)
 HPV/mild dysplasia/CIN I
 usually transparent cytoplasm
 enlarged nuclei at least 3x size of nucleus of
intermediate cell, but N/C ratio is less than 1/3
 hyperchromasia (darker than intermediate cell)
 cell may be enlarged (largest atypical cells in
gynecologic cytology are LSIL), but still has
elevated N/C
 no nucleoli
 binucleation is more common than multinucleation
 koilocytes may be present

47. LSIL

48. LSIL

49. HSIL / CIN II / moderate
dysplasia
 N/C ratio is 1/3 to ½
 cell size is same as squamous metaplastic
or parabasal cells
 polygonal shape (like intermediate or
superficial cell)
 denser cytoplasm
 enlarged and hyperchromatic nucleus
 nuclear membranes may be irregular
(crinkled paper)
 no nucleoli

50. HSIL / CIN II / moderate
dysplasia

51. HSIL / CIN II / moderate
dysplasia

52. HSIL / CIN III / severe
dysplasia
 Usually single, small, round/oval cells
 scant cytoplasm
 nuclei same size as dysplastic cells but
increased N/C ratio (greater than 1/2) since
only minimal rim of cytoplasm
 nuclei vary markedly in contour and have
irregular coarsely clumped chromatin
 no macronucleoli
 no tumor diathesis

53. HSIL / CIN III / severe
dysplasia

54. HSIL / CIN III / severe
dysplasia

55. HSIL-Carcinoma in Situ
 Hyperchromatic crowded groups
 Undifferentiated cytoplasm
 Ill-defined cell borders
 Loss of nuclear polarity

56. Carcinoma in situ

57. Carcinoma in situ

58. Squamous cell carcinoma
 Highly irregular shaped cells (tadpole,
caudate)
 Keratinized are orange, often with
squamous pearls
 Nonkeratinized cells have dense, hard,
basophilic cytoplasm; also cannibalism
(tumor cells surround other cells)
 Tumor diathesis in background (necrosis,
hemorrhage, inflammatory cells) is
suggestive of malignancy

59. Squamous cell carcinoma

60. Squamous cell carcinoma

61. EPITHELIAL ABNORMALITIES
- GLANDULAR
 Atypical
 Endocervical cells, NOS or specify in comments
 Endometrial cells, NOS or specify in comments
 Glandular cells, NOS or specify in comments
 Atypical
 Endocervical cells, favor neoplastic
 Glandular cells, favor neoplastic
 Endocervical adenocarcinoma in situ
 Adenocarcinoma
 Endocervical
 Endometrial
 Extrauterine
 Not otherwise specified (NOS)

62. Atypical Glandular Cells -
AGC
 Morphological changes of glandular
cells which are too pronounced for
inflammatory or reactive origin, but
insufficient to diagnose an
adenocarcinoma
 More likely to be neoplastic if
decreased cytoplasm, irregular
nuclear membranes and nucleoli
present

63. Atypical Glandular Cells -
AGC

64. Atypical Glandular Cells -
AGC

65. Adenocarcinoma in situ
(AIS)
 Tightly crowded sheets of malignant
cells with architectural disarray, often
with short strips of pseudostratified
columnar cells near edges
 Nuclei may be partially denuded,
causing a feathered appearance
 Nuclei are enlarged, usually oval, and
hyperchromatic
 Often prominent nucleoli
 No tumor diathesis

66. Adenocarcinoma in situ
(AIS)

67. Adenocarcinoma in situ
(AIS)

68. Adenocarcinoma
 Multilayering
 May form glandular structures with central
lumina or acinar formations with peripheral
nuclei
 Cells are large or small with pleomorphism;
have fluffy cytoplasm, cytoplasmic
vacuolization, loss of nuclear polarity, true
nuclear crowding, nuclei with clumped
chromatin, marked variation of nucleoli,
occasional mitotic figures; invasion is often
characterized by heavy blood with abundant
glandular material

69. Adenocarcinoma

70. Adenocarcinoma

71. ANCILLARY TESTING
 Describe briefly the test method(s)
and report the result so that it is easily
understood by the clinician

72. AUTOMATED REVIEW
 If case is examined by automated
device, specify the device and result

73. EDUCATIONAL
NOTES/SUGGESTIONS
 If provided, should be concise and
consistent with clinical guidelines
published by professional
organizations

74. THANK YOU