Pap cytology: commonly used in cervical cancer screening
Well-established and widely used, the Pap test continues to play a major role in reducing the incidence and mortality of cervical cancer.1
Use the illustration to explore the main steps in Pap cytologic testing.2-4

Obtain cells from the cervix

Prepare cells for evaluation

Evaluation
Obtain cells from the cervix
Cervical cells are collected by gently scraping surfaces of the cervix with a brush or spatula. Cells are collected from both the ectocervix and the endocervix. The procedure is completed within a few minutes and may cause mild crampy discomfort for the patient.2,3
Prepare cells for evaluation
In the traditional Pap smear, cervical cells are spread across a slide and an alcohol solution is immediately sprayed on to preserve the cells. Alternately, liquid-based cytology (LBC) yields a cervical specimen that can be used to perform multiple tests or additional reflex testing, if necessary. This may eliminate the need for a woman to return to have another sample collected.4
Evaluation
Samples are stained and cells are evaluated through a microscope. Some labs use automated initial evaluations with a computer-guided microscope, which can assist in identifying samples that are most likely to have abnormalities. Slides with potentially suspicious cells are then evaluated by cytologists or pathologists.2
Interpretation of cytology results
Different categories of cellular abnormality have evolved over time, and different systems are used in different countries. However, the most commonly used system of categorizing cytologic abnormalities is the Bethesda system. These consensus guidelines continue to be updated, with the most recent version proffered in 2001.5
Explore the Bethesda classification system
Bethesda system for reporting cervical cytology results5,6
Normal cells
CATEGORY | CYTOLOGIC PRESENTATION (40x) | FREQUENCY7,8 |
Negative for intraepithelial lesion or malignancy (NILM) | ![]() |
≥90% |
Squamous cell abnormalities
CATEGORY | CYTOLOGIC PRESENTATION (40x) | FREQUENCY7,8 |
Atypical squamous cells of undetermined significance (ASC-US) | ![]() |
≥90% |
Atypical squamous cells, cannot exclude high-grade squamous intraepithelial lesion (ASC-H) | ![]() |
<1% |
Low-grade squamous intraepithelial lesion (LSIL) Encompassing human papillomavirus (HPV), mild dysplasia and cervical intraepithelial neoplasia (CIN) 1 |
![]() |
1% to 2% |
High-grade squamous intraepithelial lesion (HSIL) Encompassing: moderate and severe dysplasia, carcinoma in situ, CIN 2, and CIN 3 |
![]() |
<1% |
Squamous cell carcinoma | ![]() |
<0.5% |
Glandular cell abnormalities
Note: One reason that atypical glandular cells are found less frequently is that they are located in the endocervix and are not as easily accessible for sampling.3
CATEGORY | FREQUENCY7,8 |
Atypical glandular cells (AGC) Specify endocervical, endometrial, orglandular cells not otherwise specified |
<1% |
AGC, favor neoplastic Specify endocervical or not otherwise specified |
<1% |
Endocervical adenocarcinoma in situ (AIS) | <1% |
Adenocarcinoma | <1% |
ASC-US or LSIL Pap cytology almost always trigger more frequent and intensive monitoring. New analytical approaches may be able to reduce some of the uncertainty associated with these results so that additional testing can be better focused on patients who are at the highest risk.
Key categories of cervical cytologic abnormality
Although some Pap cytology results such as high-grade squamous epithelial lesion (HSIL) typically call for immediate excision or other intervention, many much more common results are equivocal and require further monitoring and assessment in order for sound clinical decisions to be made.
Atypical squamous cells
The category atypical squamous cells (ASC) is used to describe squamous cells that do not look entirely normal, but also cannot be clearly identified as neoplastic. Because of this ambiguity, the ASC designation typically triggers additional testing, such as HPV testing, colposcopy, or biopsy, in order to more definitively identify the cells.1,9
Atypical squamous cells of undetermined significance (ASC-US). Criteria for this category include10:
- Cells resemble superficial or intermediate squamous cells in size and configuration
- Nuclei are two to three times the size of a normal intermediate cell
- Nuclei are round to oval with minimal irregularities
- Nuclei are normochromatic to slightly hyperchromatic
Atypical squamous cells, cannot exclude high-grade squamous intraepithelial lesion (ASC-H). ASC-H samples contain some cells that are suggestive of a high-grade lesion, but the finding is not definitive. Criteria include10:
- Nuclei often have uneven chromatin and are hyperchromatic
- Nuclear contour is often irregular
Further testing done on women with this cytologic finding has revealed that more than 60% typically test positive for high-risk HPV (hrHPV).6
Studies have shown that a woman with a result interpreted as ASC has a 5% to 17% chance of having CIN 2/3 confirmed by biopsy,while ASC-H cases reveal CIN 2/3 in 12% to 50% of cases.7,11
Read More
Low-grade squamous intraepithelial lesion
In low-grade squamous intraepithelial lesion (LSIL), the nuclei of cells display a number of abnormalities, including bi- and multi-nucleation. Some nuclei are surrounded by a “halo” (referred to as koilocytosis) and, in association with degeneration of nuclei, cells have darker nuclei (referred to as hyperchromatism).12
Read More
Atypical glandular cells
Atypical glandular cells (AGC) are seen in fewer than 1% of Pap tests, and even these results are not reproducible with further testing. Still, an AGC Pap cytology finding is associated with greater risk of pre-cancer or cancer than any Pap category, other than HSIL.6,1
Pap cytology: concerns and caveats
Although Pap testing has proven to be a useful screening tool, some drawbacks have also been noted. These include:
Limited sensitivity
Pap cytology is a specific technique but has limited sensitivity. When used without adjunct testing, disease may be missed. In fact, up to a third of cervical cancers occur in screened women with normal Pap cytology.1,6,15,16
Subjective interpretation
Interpretations of samples are based on individual judgments of pathologists or technicians, which can compromise reproducibility. Guidelines are provided, but no definitive objective standard exists.6,17
Interpretation errors
False-negative reports may occur when pathologists or technicians fail to detect abnormal cells. False-positive results may also occur when normal cells are incorrectly categorized as abnormal.6
Sample quality
When samples are prepared as a smear, quality may not be sufficient to enable acceptable analysis.5
These issues can lead to over- or underestimation of risk.1,14
- Overestimation of risk may lead to unnecessary tests or treatments. This can be of particular concern to younger women, who have many false-positive tests and for whom treatment for suspicious lesions may have long-term consequences for fertility and pregnancy
- Underestimation of risk can result in delays or absence of action that might prevent or compromise treatment