Gynecologic cytopathology

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. 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 ~ slightly smaller than a neutrophil.
2. Parabasal cells:
   • Blue-grey.
   • Assoc. with atrophy.
3. Basal cells:
   • Small cells.
   • Rarely seen.
4. Superficial cell:
   • Nucleus smaller than for intermediate cell.
   • Cytoplasm red.
     • Orange staining superficial cells are hypermature - suggests (abnormal) keratinization.

Glandular cells:

• 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:

• 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.

Abnormal non-malignant cells

1. Clue cells:
   • Purple squamous cell; squamous cell covered with bacteria.
   • Associated with bacterial vaginosis - which is caused by Gardnerella vaginalis.
     • Gardnerella vaginalis is a rod gram variable.
2. Squamous metaplasia:
   • "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:
   • Sheet with well-defined border that is bilayered, i.e. clump of epithelioid cells surrounded by spindle cells.
   • 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.
     • 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 - 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. Atrophy:
   • 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'.
   • Note:
     • 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.

Images:

• Clue cell (atsu.edu).
• Endocervical cells and LSIL (WC).
• Endometrial cells - double contour (techriver.net).
• Endometrial cells with "exodus" pattern (techriver.net).

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	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	DDx: HSIL
Radiation change	Single cells/groups	Well-circumscribed	vacuolated, usu. abundant	Normal NC ratio, enlarged nucleus, no nuclear membrane irregularies	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 - 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).
• 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	Pale-grey fluffy cytoplasm with well-defined nucleus, approx. 30 micrometres.	Acute inflammation (PMNs)	Sexually transmitted		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	?	Should prompt removal of IUD, if present.		Actinomycetes (upenn.edu)
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:

• >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 squamous cells/slide.

Transformation zone (TZ)

The presence of the TZ should be commented on:

• 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

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:

• Candida on Pap test (flickr.com).
• Candida on Pap test - example 1 (WC).
• Candida on Pap test - example 2 (WC).

Trichomoniasis

General

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

Cytopathology

Features:

• Low power: grey blob with a nucleus.
  • Size: approximately 30 micrometres.
  • 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.

Images:

• T. vaginalis - Pap stain (WC).
• T. Vaginalis - Pap stain (WC).
• Trichomonas vaginalis - Giemsa stain (WC).
• T. vaginalis - Giemsa stain, high magnification (bioweb.uncc.edu).

Herpes simplex virus

Features:

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.
• Gram-positive bacteria.
• Microorganism part of the large Actinobacteria group.

Cytopathology

Features:

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

Notes:

• Mycete = fungus.

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:

• LSIL ? (flickr.com).
• LSIL with HPV effect (WC).

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:

• HSIL (WC).
• HSIL (WC).

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:

• HSIL with features suggestive of invasion (WC)

Atypical squamous cells of undetermined significance

• Abbreviated ASC-US.

General

• This is a waffle category that should be used very rarely.

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.

Cytology

Features:

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

DDx:

• HSIL.
• NILM.

Adenocarcinoma

Adenocarcinoma on Pap test is classically divided into:

• Endocervical.
• Uterine.
• Extra-uterine.

Adenocarcinoma vs. squamous carcinoma

• "Feathering" - seen in adenocarcinoma more commonly on smears.
• "Birdtails" - seen on liquid preparations.

Images:

• Feathering in adenocarcinoma (cytology-asc.com).
• AIS (edupathonline.com).

Adenocarcinoma of the endocervix

• 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:

• AIS endocervix (wikimedia.org).

See also

• Cytopathology.
• Basics.
• Cervix.
• Uterus.

External links

• Collection of usual benign findings on pap tests (cytology-asc.com).
• Bethesda system atlas (techriver.net).