Residency · Residency · Pathology

Cervical Cytology and the Bethesda System for Pap Tests

Overview

The Papanicolaou (Pap) test remains a cornerstone of cervical cancer screening, though HPV-primary screening is increasingly adopted as the preferred approach. The Bethesda System for Reporting Cervical Cytology (TBS) provides standardized terminology for reporting squamous and glandular abnormalities. Understanding HPV biology, screening algorithms, and cytologic criteria is essential for interpreting results and guiding patient management.

The Bethesda System: Reporting Categories

Specimen Adequacy

A specimen is considered satisfactory for evaluation when it contains a minimum of 5,000 well-preserved squamous cells (for conventional smears) or 5,000 cells (for liquid-based preparations). The presence or absence of an endocervical/transformation zone (EC/TZ) component is noted, defined as at least 10 well-preserved endocervical or squamous metaplastic cells. Absence of the EC/TZ component is documented but does not by itself render the specimen unsatisfactory. A specimen is unsatisfactory when there is insufficient cellularity, obscuring blood or inflammation, or a broken slide. Quality indicators include air-drying artifact, excessively thick smears, and lubricant contamination.

Negative for Intraepithelial Lesion or Malignancy (NILM)

This category encompasses normal squamous and glandular cells without evidence of neoplasia. Specific organisms may be reported when identified, including Trichomonas vaginalis, Candida species, bacterial vaginosis (shift in flora), Actinomyces, and herpes simplex virus. Reactive changes from inflammation, radiation effect, IUD-associated changes, or atrophy may be noted. Endometrial cells in women over age 45 are reported as present because they may indicate underlying endometrial pathology warranting further evaluation.

Squamous Cell Abnormalities

Atypical Squamous Cells of Undetermined Significance (ASC-US)

ASC-US is characterized by cells with nuclear enlargement 2.5 to 3 times the size of a normal intermediate cell nucleus, mild hyperchromasia, and slight nuclear membrane irregularity. It represents the "gray zone" between clearly reactive changes and a definitive low-grade squamous intraepithelial lesion. It is the most common abnormal Pap result, accounting for approximately 4-5% of all Pap tests. Management uses HPV co-test reflex: HPV-positive results warrant colposcopy, while HPV-negative results return to routine screening.

Atypical Squamous Cells, Cannot Exclude HSIL (ASC-H)

ASC-H applies when atypical cells have features raising concern for a high-grade lesion but are quantitatively or qualitatively insufficient for that diagnosis. These cells are typically small with high nuclear-to-cytoplasmic ratios and hyperchromasia, occurring in small groups or as single cells. Common mimickers include immature squamous metaplasia and atrophy. Management is colposcopy regardless of HPV status, as this category carries a significantly higher risk of underlying CIN2+ (approximately 25-40%) compared to ASC-US.

Low-Grade Squamous Intraepithelial Lesion (LSIL)

LSIL is defined by koilocytosis: perinuclear clearing (a well-defined halo) with nuclear enlargement, hyperchromasia, and nuclear membrane irregularity. It represents productive HPV infection and corresponds to CIN 1 on histologic biopsy. The cells are mature (intermediate or superficial type) with a low nuclear-to-cytoplasmic ratio, and binucleation or multinucleation is common. Management for women over age 25 is colposcopy; for women aged 21-24, repeat cytology in one year is acceptable given the high rate of spontaneous regression in this age group.

High-Grade Squamous Intraepithelial Lesion (HSIL)

HSIL arises from basal and parabasal cell layers and shows markedly increased nuclear-to-cytoplasmic ratios (exceeding 50% of cell area), hyperchromatic nuclei with irregular contours, and coarse chromatin. Cells may be small (basal type) or occur in syncytial sheets. It corresponds to CIN 2 or CIN 3 on biopsy and represents a true precancerous lesion requiring intervention. Management is colposcopy with biopsy, followed by excisional treatment (LEEP) for histologically confirmed CIN 2/3.

Squamous Cell Carcinoma

Invasive squamous cell carcinoma on cytology shows marked nuclear pleomorphism, irregular chromatin, macronucleoli, and tumor diathesis (necrotic debris and blood in the background). The keratinizing type demonstrates dense orangeophilic cytoplasm with tadpole and fiber cell shapes. The non-keratinizing type shows basaloid cells in syncytial sheets.

Glandular Cell Abnormalities

Atypical Glandular Cells (AGC)

AGC describes endocervical or endometrial cells showing nuclear enlargement, hyperchromasia, crowding, and loss of the normal honeycomb pattern. It is subcategorized as AGC-NOS (not otherwise specified) or AGC-favor neoplastic. AGC carries higher clinical significance than ASC-US, with 9-38% of cases associated with significant underlying pathology including adenocarcinoma in situ, invasive adenocarcinoma, or endometrial carcinoma. Management requires colposcopy, endocervical curettage, and endometrial biopsy (particularly in women over 35 or those with abnormal bleeding).

Adenocarcinoma In Situ (AIS)

AIS presents as crowded sheets and strips of endocervical cells with nuclear enlargement, hyperchromasia, pseudostratification, mitotic figures, and apoptotic bodies. A characteristic feathering pattern is seen at the edges of cell groups, and rosette formation may be identified. HPV 18 is the most commonly associated high-risk type. Management requires excisional biopsy (LEEP or cold knife cone) with achievement of negative margins being essential for conservative management.

Adenocarcinoma

Adenocarcinoma is identified by tumor diathesis, irregular cell clusters, loss of normal architecture, and macronucleoli. The report should specify whether endocervical or endometrial origin is suspected, as this distinction guides further workup and management.

HPV Biology and Screening

HPV and Cervical Carcinogenesis

Human papillomavirus is a necessary cause of virtually all cervical cancers. High-risk HPV types include 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68. HPV 16 alone causes approximately 55-60% of cervical cancers, while HPV 18 accounts for 10-15%. The viral oncoproteins E6 (which degrades p53) and E7 (which inactivates Rb) are the molecular drivers of carcinogenesis. Most HPV infections are transient and cleared by the immune system within 1-2 years; only persistent infection with high-risk types leads to progressive intraepithelial neoplasia and eventually invasive cancer.

p16 as a Biomarker

Overexpression of p16 (CDKN2A) results from Rb inactivation by the HPV E7 oncoprotein. Strong, diffuse (block-positive) p16 immunohistochemical staining serves as a surrogate marker for high-risk HPV-driven lesions. The LAST Project recommendations endorse its use on cervical biopsies to distinguish true HSIL from morphologic mimickers such as atrophy, immature squamous metaplasia, and tangential sectioning artifacts. It is not used on Pap tests for primary screening purposes.

Screening Guidelines (USPSTF/ACS)

For women aged 21-24, cytology alone every 3 years is recommended, with HPV co-testing not recommended in this age group due to high transient infection rates. For ages 25-29, options include HPV testing alone every 5 years (preferred), cytology alone every 3 years, or co-testing every 5 years. For ages 30-65, HPV primary screening every 5 years is preferred, with co-testing every 5 years or cytology every 3 years as alternatives. Screening may be discontinued after age 65 if there has been adequate prior screening with negative results. Women who have undergone hysterectomy with removal of the cervix and no history of CIN2+ do not require screening. The American Cancer Society 2020 guidelines recommend HPV-primary screening starting at age 25.

HPV-Primary Screening

FDA-approved HPV tests (such as cobas HPV and Aptima) can now be used as the primary screening modality. Positive results are triaged by genotype: HPV 16 or 18 positivity triggers direct referral to colposcopy, while positivity for other high-risk types is triaged by cytology. HPV-primary screening may eventually replace the Pap test entirely, with implications for reduced need for cytotechnologists and shifts in pathology training emphasis.

Liquid-Based Cytology vs. Conventional Pap

Liquid-Based Preparations (ThinPrep, SurePath)

In liquid-based preparations, cells are collected and rinsed into liquid fixative, then processed to produce a thin monolayer on the slide. Advantages include fewer obscuring elements, better cell preservation, and the availability of residual material for HPV testing and ancillary studies. Liquid-based preparations are now standard in most US laboratories and have similar sensitivity and specificity to conventional preparations for HSIL detection.

Conventional Pap Smear

The conventional technique involves direct smearing of collected cells onto a glass slide with immediate spray fixation. Its advantages include lower cost, making it suitable for resource-limited settings. Disadvantages include obscuring elements such as blood and inflammation, random distribution of cells across the slide, and no residual material available for HPV reflex testing.

<image>A medical illustration showing the cytomorphologic spectrum of squamous cell abnormalities in cervical cytology (Papanicolaou stain). Panel A: Normal superficial and intermediate squamous cells with small pyknotic nuclei and abundant transparent cytoplasm. Panel B: ASC-US showing cells with nuclear enlargement approximately 2.5 times normal, mild hyperchromasia, and slight nuclear membrane irregularity. Panel C: LSIL with classic koilocytes -- cells with well-defined perinuclear halos, enlarged hyperchromatic and wrinkled nuclei, and binucleation, with mature cytoplasm. Panel D: HSIL showing small cells with markedly increased nuclear-to-cytoplasmic ratio exceeding 50%, dense hyperchromatic nuclei with irregular contours, and scant cytoplasm. Panel E: Squamous cell carcinoma with pleomorphic keratinized cells, dense orangeophilic cytoplasm (tadpole and fiber shapes), and a dirty necrotic background (tumor diathesis).</image>

<image>A medical illustration depicting glandular cell abnormalities in cervical cytology. Panel A: Normal endocervical cells in a honeycomb sheet with uniform round nuclei, evenly spaced, and basally located. Panel B: Atypical glandular cells (AGC) showing a crowded sheet of endocervical cells with nuclear enlargement, hyperchromasia, and loss of the honeycomb pattern. Panel C: Adenocarcinoma in situ (AIS) displaying crowded strips of pseudostratified columnar cells with elongated hyperchromatic nuclei, feathering at the edges of cell groups (arrow), and mitotic figures. Panel D: Endocervical adenocarcinoma with irregular three-dimensional cell clusters, macronucleoli, and tumor diathesis in the background.</image>

<image>A flowchart illustrating the cervical cancer screening and management algorithm. Starting with age-based screening entry, showing HPV-primary screening versus co-testing versus cytology alone, then branching pathways for each abnormal result: ASC-US with HPV reflex testing, LSIL, HSIL, ASC-H, and AGC, each leading to colposcopy with appropriate biopsy and management based on ASCCP risk-based guidelines with integration of prior screening history.</image>

Clinical Pearls

ASC-US with negative HPV reflex testing has a very low risk of CIN2+ (less than 2%) and returns to routine screening; this is the most common scenario in ASC-US and avoids the vast majority of unnecessary colposcopies. ASC-H carries a much higher risk of CIN2+ than ASC-US (25-40% versus 5-10%) and always warrants colposcopy regardless of HPV status. AGC is the glandular equivalent of a "cannot exclude high-grade" result and requires both endocervical and endometrial sampling, as up to 38% of cases harbor significant pathology including endometrial carcinoma. Atrophy in postmenopausal women is a major mimic of HSIL because parabasal cells can show high nuclear-to-cytoplasmic ratios and hyperchromasia; a short course of vaginal estrogen before repeat Pap testing can resolve equivocal findings. AIS is often multifocal with skip lesions, meaning that negative LEEP margins do not guarantee complete excision; management decisions between close follow-up and hysterectomy depend on fertility desires. The ongoing shift to HPV-primary screening will fundamentally change the cytopathology landscape, and trainees should understand both cytologic and molecular-based approaches to cervical cancer prevention.

References

• Nayar R, Wilbur DC, eds. The Bethesda System for Reporting Cervical Cytology. 3rd ed. Springer; 2015.
• Fontham ETH, et al. Cervical cancer screening for individuals at average risk: 2020 guideline update from the American Cancer Society. CA Cancer J Clin. 2020;70(5):321-346.
• Perkins RB, et al. 2019 ASCCP Risk-Based Management Consensus Guidelines for Abnormal Cervical Cancer Screening Tests and Cancer Precursors. J Low Genit Tract Dis. 2020;24(2):102-131.
• Darragh TM, et al. The Lower Anogenital Squamous Terminology Standardization Project for HPV-Associated Lesions (LAST Project). Arch Pathol Lab Med. 2012;136(10):1266-1297.
• US Preventive Services Task Force. Screening for Cervical Cancer: US Preventive Services Task Force Recommendation Statement. JAMA. 2018;320(7):674-686.

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