Gynecologic cytopathology

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Gynecologic cytopathology is a subset of cytopathology. Gynecologic usually refers to Pap test specimens, i.e. uterine cervix, vaginal vault; other gynecologic specimens are considered non-gynecologic.

This article deals only with cervical cytopathology. An introduction to cytopathology is in the cytopathology article.

Preparation

The standard for Pap test is the Papanicolaou stain. It is described in the staining article and discussed in the context of cytopathology in the cytopathology article.

Slide marking conventions

Conventions are important for facilitating communication between various team members. They are discussed in the cytopathology article.

Normal cells

Squamous cell types:[1]

  1. Intermediate cells:
    • In clusters or single.
    • 30-50 micrometres in diameter.
    • Associated with progesterone - (light) blue.
    • This is the cell of reference in Pap test, i.e. other cells are measured against this cell when assessing a Pap test.
      • Nucleus ~ 7-8 micrometers.
  2. Parabasal cells:
    • Blue-grey.
    • Assoc. with atrophy.
  3. Basal cells:
    • Small cells.
    • Rarely seen.
  4. Superficial cell:[2]
    • Nucleus smaller than for intermediate cell.
    • Cytoplasm red.
      • Orange staining superficial cells are hypermature - suggests (abnormal) keratinization.

Glandular cells:[3]

  • Sheets of cells with regular spacing.
  • Relatively high NC ratio (when compared to intermediate cells).
  • Nucleoli (like most glandular cells).
  • Nucleus approximately the size of an intermediate cell nucleus.

Mix of cells

The mix of cells is dependent on age and hormones:[4]

  • Progesterone - makes the Pap test blue... more intermediate cells.
  • Yonger patients have a mix of cells.
  • Menopausal patients... more parabasal cells.
  • Older patients... more estrogen, glycogen.

Unusual non-malignant cells

  1. Clue cells:
    • Purple squamous cell; squamous cell covered with bacteria.
    • Associated with bacterial vaginosis - which is caused by Gardnerella vaginalis.[5]
      • Gardnerella vaginalis is a rod gram variable.[6]
  2. Squamous metaplastic cells:
    • "Dense" cytoplasm.
    • Nucleus ~2X the size of an intermediate cell nucleus.
    • Nucleoli.
    • Note:
      • Squamous metaplastic cells have a similar appearance to parabasal cells; they cannot be differentiated on morphologic grounds.
      • Squamous metaplastic cells have a high NC ratio - they are differentiated from HSIL via nuclear features (dark staining + irregular nuclear contour = HSIL).
      • Slight nuc. contour irregularies are accepted, may be darker staining.
  3. Endometrial cells:[7]
    • Cluster of cells with a well-defined border that is bilayered, i.e. a clump of (epithelioid) stromal cells surrounded by (flatted) glandular cells. Classically described as a cluster with a double contour; known as exodus pattern.[8]
    • Scant cytoplasm.
    • Chromatin clumping.
    • Raisin-like nuclei - approximately the size of an intermediate cell nucleus.
      • Nuclei can be considered normal if nucleus less than 2X the size of an intermediate cell nucleus.
    • Notes:
      • Endometrial cells may appear irregular in the context of an intrauterine device (IUD); abnormalities in the context of an IUD are often ignored.
        • Classic change: cytoplasmic vacuolization.
      • The presence of endometrial cells on a Pap test on a woman >=40 years old (per Bethesda guidelines) should be noted in the pathology report[9] - this prompts an endometrial biopsy.
        • In my humble opinion, reporting benign endometrial cells in premenopausal women is not evidence-based practise; the practise is driven by lawsuit-paranoia in the USA.
  4. Atrophic cells:[10]
    • Cells smaller.
    • Cytoplasm grey/blue.
    • No "dancing"/"sparkling" chromatin.
    • +/-"Dirty" background - degenerated cells, inflammatory cells (neutrophils, histiocytes).
      • May mimic "dirty" background of tumour, i.e. 'tumour diathesis'.
    • Notes:
      • Usually older women.
      • Main DDx is HSIL which has chromatin changes.
  5. Tingible body macrophages:
    • Abundant cytoplasm with vacuolization.
    • May be seen in the context of chlamydia.
  6. Navicular cells:
    • Intermediate cells with:
      1. Folded edges.
      2. Abundant cytoplasmic glycogen.
    • Notes:
      • Cells often described as boat-shaped.
      • May resemble the lenses of coke bottle glasses.
      • Associated with: pregnancy, contraception with medroxyprogesterone acetate (Depo-provera).[11]

Images:

Glycogen halos versus HPV effect

HPV effect (koilocyte) Glycogen halo
Discolouration of halo Clear Yellow
Nuclear changes Associated with nuc. changes Normal nuclei
Cell-to-cell variability No - all clear Yes - some yellow some clear

Gynecologic pathology in tables

Normal cells

Cell Architecture Cell borders Cytoplasm DNA DDx
Intermediate cell (IC) Single cells Irregular Blue, abundant Small nucleus (~ size of PMN), no nucleolus -
Superficial cell (SC) Single cells Irregular Red, abundant Small nucleus, 1/2 size of IC nucleus, no nucleolus -
Squamous metaplastic cell Single cells/clumps of cells Smooth/oviod shape Dense, dark blue 2X IC nucleus, nucleolus, no membrane irreg., no chromatin changes DDx: HSIL, basal cell
Endometrial cell Well-circumscribed clump/ball of cells with squamoid covering cells; referred to as "exodus" pattern[12] Indistinct within cluster Blue, small/very scant Small, dark, nuclear moulding, degenerative changes (chromatin clumping) DDx: HSIL, basal cell.
Glandular (endocervical) cell Sheets of cells with regular spacing, columnar morphology may be apparent, +/-palisading at edge of clump Often distict Blue, scant-to-moderate Nucleus ~ size of an IC nucleus, no membrane irreg., no chromatin changes DDx: endometrial cell
Atrophy Single cells/groups Well-circumscribed Grey/blue dense, may be scant Large NC ratio, nuc. membrane irregularities, NO chromatin clumping[13] DDx: HSIL
Radiation changes Single cells/groups Well-circumscribed vacuolated, usu. abundant Normal NC ratio, enlarged nucleus, no nuclear membrane irregularies, +/-multinucleation DDx: LSIL, vitamin B12 def.

Note:

  • If only normal cells are present the diagnosis is negative for intraepithelial lesion and malignancy (NILM).

Abnormal cells

Cell Architecture Cell borders Cytoplasm DNA Other
Low-grade squamous intraepithelial lesion (LSIL) Single cells/groups Irregular or moderately-circumscribed Blue, abundant - NC ratio ~ 1:3 Large nucleus (3-4X IC nuc. - see Note 1), perinuclear clearing, nuc. membrane irregularities, chromatin clumping DDx: HSIL, reactive changes
High-grade squamous intraepithelial lesion (HSIL) Often single cells, may be groups Well-circumscribed Dark blue, scant - NC ratio ~ 1:2 Large nucleus (3-4X IC nuc. - see Note 1), nuc. membrane irregularities, clumping of coarse chromatin, dark nuc. staining, +/- small nucleoli DDx: squamous metaplasia, atrophy with atypia, superficial endometrial cells
Atypical squamous cells of undetermined significance (ASC-US) Single cells/groups Irregular or moderately-circumscribed Blue, abundant cytoplasm Moderately enlarged nucleus (~2.5-3.0X IC nuc.), minimal changes in nuclear membrane and chromatin DDx: LSIL, reactive changes
Atypical squamous cells, cannot exclude HSIL (ASC-H) Often single cells, may be groups Irregular or moderately-circumscribed Blue, moderate-to-scant cytoplasm Moderately enlarged nucleus (~1.5-2.0X IC nuc.), minimal changes in nuclear membrane and chromatin DDx: HSIL, AIS
Atypical glandular cells (AGC) Usu. groups of cells Usually well-circumscribed (?) Dark blue dense, scant Moderately enlarged nucleus (~2X IC nuc.), nuc. membrane irregularities, chromatin clumping, dark nuc. staining, nucleoli DDx: AIS, HSIL
Adenocarinoma in situ (AIS) groups; rosette formation Usually well-circumscribed Dark blue dense, scant Large nucleus (>=2X IC nuc.), nuc. membrane irregularities, chromatin clumping, dark nuc. staining, nucleoli (very common), pseudostratification (as in endocervical AIS) DDx: AGC, HSIL
Features of SCC (see Note 2) Large clusters of cells with irreg. edge and "streaming", +/-blood, necrotic debris Poorly seen Dark blue dense, scant Large NC ratio, nucleolus, nuc. membrane irregularities, chromatin clumping DDx: HSIL

Note 1:

  • LSIL/HSIL nucleus - at least 3X IC nucleus.
    • ASCUS nucleus - at least 2.5X IC nucleus.
      • 3X is not an absolute requirement to call SIL, i.e. SIL may be called with a smaller nucleus in circumstances where other nuclear features are at the extremus of malignant.
  • Large nuclear size, membrane irregularities, "clumpy" chromatin and dark nuc. staining - are the key features.
    • Perinuclear clearing is quite subjective.
      • The best perinuclear halos have a sharp punched-out edge.

Note 2:

  • By definition, it is not possible to diagnose squamous cell carcinoma (SCC) on a pap test as one cannot demonstrate stromal invasion.

HSIL versus LSIL

HSIL LSIL
NC ratio[14] - see Note 1 ~1:2 ~1:3
Nuclear membrane irregularities Marked - distinct notches Moderate
Chromatin granularity Coarse, clumped, +/-nucleolus (red) Coarse, no nucleolus
Cytoplasmic staining Dark Light
Perinuclear clearing Usually absent Often present
Binucleation Uncommon May be present
Maturity of squamous cell Normal maturity Hypermature (orangeophilic cell present)
Image (example) HSIL (WC) LSIL (WC), LSIL & endoCx (WC)

Note 1:

  • The single most useful feature is NC ratio but it is not definitive; NC ratio should be evaluated in the context of nuclear irregularities (nuclear membrane smoothness, chromatin pattern, presence of nucleolus).[14]
  • It may be easier to think in terms of cell size - approximate values are:
    • HSIL cells: < 1/2 size of IC.
    • LSIL cells: classically the size of IC.

Infectious organisms

Disease Organism Group Dx features Associated features Clinical Reference Image
Trichomoniasis Trichomonas vaginalis Protozoan Pear-shaped pale-grey fluffy cytoplasm with well-defined nucleus, approx. 30 μm. Acute inflammation (PMNs), may be seen with Leptothrix (hair-like appearance ~0.5 x 20 μm) Sexually transmitted [15] T. vaginalis - Pap stain (WC), Trichomonas - Pap stain (WC)
Candidiasis Candida albicans Fungi Branching hyphae ~= 1/2 the dia. of IC nucleus, red PMNs ? ? Candida on Pap test (WC)
Herpes Herpes simplex virus (HSV 1 - less commonly, HSV 2 - more commonly) Virus Large ground glass nuclei then multinucleation with moulding & inclusions with clear halo ? Sexually transmitted ? HSV (WC),HSV (WC), Herpes simplex virus - surgical (virology.org)
Actinomycetes Actinomycetes Gram-positive bacteria Clusters of cocci in chains - hyphae-like appearance low power: pom-pom or fuzzy ball-like appearance Should prompt removal of IUD, if present. [15] Actinomycetes (gfmer.ch), Actinomycetes (quizlet.com)
Bacterial vaginosis (see Note 1) Gardnerella vaginalis Gram-variable rod "Clue cell": bacterial clusters attached to a purple squamous cell ? Assoc. Fishy smell ? Bacterial vaginosis (WC), Clue cell (atsu.edu)

Note 1:

  • Usually not reported.

Adequacy of specimens

There is a generally accepted standard for cervical (liquid-based) cytology specimens:[16]

  • >5000 squamous cells/slide, if no abnormality is present.
    • If abnormal cells are present, any number of cells is acceptable.
      • This works-out to approx. 4 cells/HPF.
        • Where: HPF = area seen at 400X with an eye piece diameter is ~22 mm.
      • 10 HPFs are counted and a table is used to see whether the sample is adequate.

Note:

  • The standard for conventional pap smears is: 8000-12000 (well-visualized) squamous cells.[17]

Transformation zone (TZ)

The presence of the TZ should be commented on:[18]

  • An adequate TZ is 10 cells - endocervical cells or squamous metaplastic cells (per Bethesda).

Difficulties in obtaining a TZ may arise in the following populations:

  • Pregnant (endocervical canal not sampled).
  • Menopausal.
  • Young nulliparous.

Candida

Main article: Candidiasis

General

  • Common.
  • May be asymptomatic.
  • Usually Candida albicans.

Cytology

Features:

  • Typically in clusters - lead to darkened clusters of squamous cells (at low power).
  • May appear to "shish kabob" the cell; may appear to puncture the cell membrane (as they overlie it).
  • Red staining hyphae; width of hyphae ~= 1/2 the diameter of an intermediate cell nucleus; branches.

Notes:

  • Presence should be noted in the pathology report.

Images:

Trichomoniasis

General

  • Caused by Trichomonas vaginalis - a protozoa.
  • Sexually transmitted.
  • Common.

Cytopathology

Features:

  • Low power: grey blob with a nucleus, may be pear-shaped:
    • Size: approximately 30 micrometres.[15]
    • Shape: usually oval, may have teardrop-shaped.
    • Flagellum - hair-thin locomotive stucture, usu. barely visible at 200X - diagnostic feature.

Cytopathological associations:

  • Acute inflammation (neutrophils), often marked - key feature at low power.
  • Reactive squamous cells with:
    • Nucleoli,
    • Perinuclear halos, and
    • Moth-eaten cytoplasm; cytoplasm that has multiple vacuoles with star-like spaces.

Notes:

  • Trichomonas is tricky - it is easy to miss if one is not suspicious, in the context of inflammation.
  • May vaguely resemble a neutrophil:
    • Flagellum useful to differentiate.
    • Neutrophil has multiple lobulations of the nucleus.
  • May be seen in association of Leptothrix.
    • Appearance: long, hair-like.
    • Size: ~0.5 x 20 micrometres.

Images:

Herpes simplex virus

Main article: Herpes simplex virus

General

  • May be HSV1 or HSV2.
    • Classically HSV2 based on epidemiology and location.

Cytology

Features:[15]

  1. Early: Large "ground-glass" nuclei - nuclei with hazy & uniformly dull appearance.
  2. Late: multi-nucleation with moulding of nuclei and nuclear inclusions surrounded by a clear halo.

Actinomycetes

Main article: Actinomycetes

General

  • Presence should prompt removal of intrauterine device (IUD), if present.[15]
  • Gram-positive bacteria.
  • Microorganism part of the large Actinobacteria group.

Cytopathology

Features:[15]

  • Clusters of filamentous bacteria.
    • Hyphae-like appearance/"filamentous".

Notes:

  • Mycete = fungus.[19]

Squamous intraepithelial lesion (SIL)

General:

  • The nucleus makes it SIL.
  • The cytoplasm determines the grade (LSIL vs. HSIL).

Management (in short):

  • LSIL = repeat Pap test in 6 months.
  • HSIL = referal for coloposcopy.

Low-grade squamous intraepithelial lesion

  • Abbreviated LSIL.

General

  • Usually regress, i.e. will disappear on their own.

Cytopathology

Features:

  • Nuclei 3x size of intermediate cell.
  • Irreg. nuclear border.
  • Perinuclear 'cavity' (clearing).
    • The best perinuclear halos have a sharp punched-out edge.

Images:

High-grade intraepithelial lesion

  • Abbreviated HSIL.

General

  • Often progress to cervical cancer.

Cytopathology

Features:

  • Often single cells.
  • Blue cells - nucleus and cytoplasm.
  • Increased NC ratio - key feature.
  • Irregular nuclear border.
  • Chromatin clumping.

Image:

Squamous cell carcinoma

  • Abbreviated SCC.
Main article: Squamous cell carcinoma
  • Some believe that one can diagnosis SCC on a pap test.
    • This is nonsense, as SCC implies invasion which cannot be seen on a pap test.

Features suggestive of invasion:

  • Nucleoli.
  • Blood.
  • Necrotic debris.
  • Clumps of large cells.

Image:

Atypical squamous cells of undetermined significance

  • Abbreviated ASC-US or ASCUS.

General

  • This is a waffle category that should be used very rarely.
  • May be an indication for HPV testing.

Cytology

Features:

  • Nuclear size >2.5X IC nucleus, but <3X IC nucleus.

Atypical squamous cells, cannot exclude high-grade squamous intraepithelial lesion

  • Abbreviated ASC-H.

General

  • This is a waffle category that should be used very rarely.
  • Higher HPV positivity vs. ASC-US.[20]

Cytology

Features:

  • Atypia that falls short of diagnosing HSIL:
    • Increased NC ratio.

DDx:

Atypical glandular cells

  • Abbreviated AGC.
  • Previously atypical glandular cells of undetermined significance, abbreviated AGUS.

General

Microscopic

Features:

  • Atypical glandular cells:
    • Cell cluster with cells with a diameter <= 2x intermediate cell nucleus.
    • Some features of nuclear atypia, e.g. irregular nuclear membrane, granular chromatin, nuclear hyperchromasia, nuclear enlargement.

DDx:

  • Adenocarcinoma in situ.

Adenocarcinoma in situ

  • Abbreviated AIS.

Adenocarcinoma in situ on Pap test is classically divided into:

  • Endocervical.
  • Uterine.
  • Extra-uterine.

Adenocarcinoma vs. squamous carcinoma

  • "Feathering" - seen in adenocarcinoma[21] more commonly on smears.[22]
  • "Birdtails" - seen on liquid preparations.

Images:

Adenocarcinoma of the endocervix

General

  • Associated with HPV.

Cytopathology

Features:

  • Cluster of small cells.
    • Cells approximately the size of a lymphocyte ~ 10 micrometres.
  • Nucleoli - key feature (may be subtle).

Negatives:

  • Lack cilia.
    • Cilia on cells is a feature of benignancy and should sway the pathologist away from adenocarcinoma.

Image:

Carpet beetle larval parts

General

  • Uncommon distinctive contaminant.[23]
  • Fragment of a beetle.
  • Benign.

Cytology

Features:

  • Slender long structure - fern-like.

Image:

Radiation changes in cervical cytology

General

  • Radiation is used to treat cervical cancer.

Cytology

Features:[24]

  • Architecture: single cells/groups.
  • Cell borders: well-circumscribed.
  • Cytoplasm: vacuolated, usually abundant.
  • Nucleus:
    • Enlarged nucleus - but normal NC ratio.
    • No nuclear membrane irregularies.
    • Chromatin: "smudgy".
    • +/-Multinucleation.

DDx:

  • LSIL.
  • Vitamin B12 deficiency.

Images:

See also

References

  1. ↑ Half-day. 10 November 2008.
  2. ↑ SM. 14 January 2010.
  3. ↑ SM. 14 January 2010.
  4. ↑ GR. 4 February 2010.
  5. ↑ Scott TG, Smyth CJ, Keane CT (February 1987). "In vitro adhesiveness and biotype of Gardnerella vaginalis strains in relation to the occurrence of clue cells in vaginal discharges". Genitourinary medicine 63 (1): 47–53. PMC 1194007. PMID 3493202. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1194007/.
  6. ↑ Taylor-Robinson D (1984). "The bacteriology of Gardnerella vaginalis". Scand J Urol Nephrol Suppl 86: 41–55. PMID 6399409.
  7. ↑ SM. 14 January 2010.
  8. ↑ URL: http://nih.techriver.net/view.php?patientId=78. Accessed on: 31 March 2012.
  9. ↑ Thrall MJ, Kjeldahl KS, Savik K, Gulbahce HE, Pambuccian SE (August 2005). "Significance of benign endometrial cells in papanicolaou tests from women aged >=40 years". Cancer 105 (4): 207-16. doi:10.1002/cncr.21156. PMID 15900572.
  10. ↑ DeMay, RM. The Art & Science of Cytopathology: Exfoliative Cytology. 1996. ISBN 0-89189-322-9. PP.116-7.
  11. ↑ Volk, EE.; Jax, JM.; Kuntzman, TJ. (Sep 2000). "Cytologic findings in cervical smears in patients using intramuscular medroxyprogesterone acetate (Depo-provera) for contraception.". Diagn Cytopathol 23 (3): 161-4. PMID 10945902.
  12. ↑ URL: http://nih.techriver.net/view.php?patientId=221. Accessed on: 26 November 2011.
  13. ↑ DeMay, RM. The Art & Science of Cytopathology: Exfoliative Cytology. 1996. ISBN 0-89189-322-9. PP.116-7.
  14. ↑ 14.0 14.1 Slater, DN.; Rice, S.; Stewart, R.; Melling, SE.; Hewer, EM.; Smith, JH. (Aug 2005). "Proposed Sheffield quantitative criteria in cervical cytology to assist the diagnosis and grading of squamous intra-epithelial lesions, as some Bethesda system definitions require amendment.". Cytopathology 16 (4): 168-78. doi:10.1111/j.1365-2303.2005.00264.x. PMID 16048503. http://www3.interscience.wiley.com/journal/118661591/abstract?CRETRY=1&SRETRY=0.
  15. ↑ 15.0 15.1 15.2 15.3 15.4 15.5 Humphrey, Peter A; Dehner, Louis P; Pfeifer, John D (2008). The Washington Manual of Surgical Pathology (1st ed.). Lippincott Williams & Wilkins. pp. 446. ISBN 978-0781765275.
  16. ↑ UHN PCY50001.08 P.10.
  17. ↑ Sheffield, MV.; Simsir, A.; Talley, L.; Roberson, AJ.; Elgert, PA.; Chhieng, DC. (Mar 2003). "Interobserver variability in assessing adequacy of the squamous component in conventional cervicovaginal smears.". Am J Clin Pathol 119 (3): 367-73. PMID 12645338.
  18. ↑ GR. 4 February 2010.
  19. ↑ URL: http://en.wiktionary.org/wiki/-mycete#English. Accessed on: 14 September 2011.
  20. ↑ Srodon, M.; Parry Dilworth, H.; Ronnett, BM. (Feb 2006). "Atypical squamous cells, cannot exclude high-grade squamous intraepithelial lesion: diagnostic performance, human papillomavirus testing, and follow-up results.". Cancer 108 (1): 32-8. doi:10.1002/cncr.21388. PMID 16136595.
  21. ↑ URL: http://www.cytology-asc.com/cec/endocx/. Accessed on: 13 September 2011.
  22. ↑ Belsley, NA.; Tambouret, RH.; Misdraji, J.; Muzikansky, A.; Russell, DK.; Wilbur, DC. (Apr 2008). "Cytologic features of endocervical glandular lesions: comparison of SurePath, ThinPrep, and conventional smear specimen preparations.". Diagn Cytopathol 36 (4): 232-7. doi:10.1002/dc.20782. PMID 18335553.
  23. ↑ Bechtold, E.; Staunton, CE.; Katz, SS.. "Carpet beetle larval parts in cervical cytology specimens.". Acta Cytol 29 (3): 345-52. PMID 3859134.
  24. ↑ Gupta, S.; Mukherjee, K.; Gupta, YN.; Kumar, M. (Aug 1987). "Sequential radiation changes in cytology of vaginal smears in carcinoma of cervix uteri during radiotherapy.". Int J Gynaecol Obstet 25 (4): 303-8. PMID 2887465.

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