Cervical and Vaginal Cytology

Screening Guidelines

Cervical cancer screening guidelines differ around the world. In the United States, recommendations are issued by the American College of Obstetricians and Gynecologists, the US Preventive Services Task Force, and a consortium of the American Cancer Society, the American Society for Colposcopy and Cervical Pathology (ASCCP), and the American Society for Clinical Pathology. Their guidelines differ in minor ways, but there is general agreement on the larger points, including longer screening intervals and a later age to start screening (age 21) than had been recommended in the past ( Table 1.1 ). For women 21 to 29 years of age, they recommend a Pap test every 3 years. For women between ages 30 to 65, there are several options: a Pap test alone every 3 years; a test for high-risk types of the human papillomavirus (HPV) every 5 years; or a “cotest” (a Pap plus an HPV test) every 5 years. The guidelines address women with an average risk for cervical cancer. Women at higher risk—those with a history of cervical cancer, in utero diethylstilbestrol (DES) exposure, and immunocompromise (due to organ transplantation, chemotherapy, long-term corticosteroid treatment, or infection with the human immunodeficiency virus [HIV])—may benefit from more frequent screening. Because women with human immunodeficiency virus/acquired immunodeficiency syndrome have higher rates of cervical cancer than the general population, it is recommended that HIV-seropositive women have a Pap test twice during the first year after diagnosis of HIV infection and, if the results are normal, annually thereafter. Adherence to screening guidelines is critical for cervical cancer prevention: in Sweden, for example, women who had not had a Pap test within the recommended screening interval had a higher risk of cervical cancer than those who had been screened (odds ratio 2.52).

Because of the strong association between HPV and cervical neoplasia (see “Squamous Intraepithelial Lesions” below), a test for high-risk HPV types can be used by itself as an initial screen (so-called “primary HPV screening”). If positive, partial genotyping (for HPVs 16 and 18) and Pap cytologic study are performed; a referral to colposcopy is recommended if genotyping shows HPV 16 or 18, if the Pap cytology result is abnormal, or if a subsequent HPV test shows persistence of virus. There is evidence that primary HPV screening is equivalent or superior to screening algorithms based on Pap cytologic study, and the US Food and Drug Administration (FDA) has approved the Roche cobas HPV Test and the BD Onclarity HPV Assay for primary HPV screening. The International Agency for Research on Cancer and the World Health Organization have endorsed primary HPV testing, and in 2017 the Netherlands was the first country to implement it nationwide.

Guidelines for Managing Women with Abnormal Pap Results

In 2012, the ASCCP revised its guidelines for the management of women with abnormal cervical cytologic study, HPV, and histopathologic results. These guidelines, mentioned throughout this chapter in the relevant sections, apply only to women whose abnormalities are detected during screening. Management is individualized for women with postcoital or unexplained abnormal vaginal bleeding, pelvic pain, abnormal discharge, or a visible cervical lesion.

HPV Vaccination

The development of prophylactic HPV vaccines has opened a new opportunity for cervical cancer prevention. The vaccines consist of empty protein shells called virus-like particles that are made up of the major HPV capsid protein L1; they contain no DNA and are not infectious. Two of the vaccines are Gardasil and Gardasil 9 (Merck & Co., Inc.). Gardasil is a quadrivalent vaccine against HPV types 6, 11, 16, and 18, and Gardasil 9 is a nine-valent vaccine against HPV types 6, 11, 16, 18, 31, 33, 45, 52, and 58. A third vaccine—Cervarix (GlaxoSmithKline)—withdrew from the US market in 2016 due to low market demand. They have all shown extraordinary efficacy in preventing type-specific histologic CIN 2,3 lesions, with no difference in serious adverse effects compared with placebo. The American Cancer Society recommends routine HPV vaccination for girls and boys ages 11 and 12 years. It also recommends the vaccine to males and females ages 13 to 26 to “catch up,” having missed the opportunity to be vaccinated at the recommended age. In 2016, it was estimated that 65% of girls and 56% of boys aged 13 to 17 years in the United States had initiated HPV vaccination. Of note, screening guidelines are no different for the vaccinated population as compared with those not vaccinated. Continued screening, even for the vaccinated population, remains important because (1) these vaccines do not protect against all cervical cancers; (2) the duration of protection is unknown; (3) they are not effective in treating prevalent HPV infections; and (4) the cost of the vaccines might limit their use in some populations.

Smears

Smears are often prepared using the combination of a spatula and brush. The spatula is used first. Although a wooden or plastic spatula is acceptable, the plastic spatula is recommended because wooden fibers trap diagnostic material. The spatula is rotated at least 360 degrees. The sample can be smeared on half of a slide and spray fixed (the other half should be covered to avoid coating it with fixative before the endocervical sample is applied). Alternatively, one may set aside the spatula sample momentarily while the endocervical brush sample is obtained.

After the brush is inserted in the endocervical canal, some bristles should still be visible. If inserted too far, there may be inadvertent sampling of the lower uterine segment (LUS), which causes diagnostic difficulties because its epithelium resembles a high-grade squamous intraepithelial lesion (HSIL) and adenocarcinoma in situ (AIS). The brush should be rotated gently only one-quarter turn. A larger rotation is unnecessary because the circumferential bristles are in contact with the entire surface the moment the brush is inserted.

The spatula sample, if not already applied and fixed, should be applied to the slide, then the brush sample rolled over the slide, followed by immediate fixation. The two samples can be placed in quick succession on two separate halves of the slide, or the endocervical sample can be rolled directly over the spatula sample, both covering the entire slide. Immediate fixation (within seconds) is critical to prevent air-drying artifact, which distorts the cells and hinders interpretation.

The broomlike brush (“broom”) has a flat array of plastic strips contoured to conform to the cervix, with longer strips in the middle. This design allows simultaneous sampling of the endocervix and ectocervix. The long middle strips are inserted into the os until the shorter outer strips bend against the ectocervix. The broom is rotated three to five times. To transfer the material, each side of the broom is stroked once across the slide in a painting motion.

The cotton swab moistened with saline solution is no longer recommended because its fibers trap cells, reducing the efficiency of cell transfer onto slides.

There are two options for smear fixation. Coating fixatives contain alcohol and polyethylene glycol and are applied by pump sprays, by droppers from dropper bottles, or by pouring from an individual envelope included as part of a slide preparation kit. Alternatively, the smear can be immersed directly into a container filled with 95% ethanol.

Samples for liquid-based cytologic study are obtained as described above except that, instead of smearing the cells on a slide, the collection device is rinsed in a vial containing a liquid fixative. In the United States, the liquid-based Pap test is more common than the smear.

Liquid-Based Cytologic Study

In 1996, the US (FDA) approved the ThinPrep as an alternative to the cervicovaginal smear. This was followed 3 years later by approval of the AutoCyte Prep (now SurePath; BD TriPath, Burlington, NC). Liquid-based cytologic study (LBC) was an important step in the development of automated Pap screening devices—an improved preparation was needed to minimize cell overlap so that automated instruments would perform better in identifying abnormal cells. But LBC performed so well in clinical trials against smears that it found a market independent of automated screening. Although a number of studies showed an increased detection of cytologic low-grade squamous intraepithelial lesions (LSIL) or HSIL with LBC, subsequent meta-analyses and prospective randomized trials failed to demonstrate a significant difference between smears and LBC in the detection of histologic CIN2,3. Nevertheless, LBC offers several advantages over smears: the opportunity to prepare duplicate slides and even cell block preparations from the residual sample ; the option of “out-of-vial” aliquoting for HPV, chlamydia, and gonorrhea testing; an improved substrate for automated screening devices; and a thinner cell preparation that most pathologists and cytotechnologists find less tiring to review than smears.

ThinPrep Pap Test

The practitioner obtains the ThinPrep Pap sample with either a broom-type device or a plastic spatula/endocervical brush combination. The sampling device is swirled/rinsed in a methanol-based preservative solution (PreservCyt) for transport to the cytology laboratory and then discarded. Red blood cells are lysed by the solution. The vials are placed one at a time in the ThinPrep 2000 instrument or in batches in the ThinPrep 5000 ( Fig. 1.1A ). This results in a thin deposit of cells in a circle 20 mm in diameter (contrast with Cytospin: diameter = 6 mm). In most cases, only a fraction of the sample is used to prepare the slide used for diagnosis. If needed, the residual sample is available for additional ThinPrep slide preparation, cell block preparation, or molecular diagnostic testing (e.g., high-risk HPV, Chlamydia , gonorrhea).

A multicenter, split-sample study found that the ThinPrep detected 18% more cytologic cases of LSIL and more serious lesions as compared with smears, with no significant difference in the detection of organisms. A number of studies have shown significant increases in the detection of cytologic HSIL after the implementation of the ThinPrep. Subsequent meta-analyses and a prospective randomized trial, however, failed to demonstrate a significant difference between smears and ThinPrep in the detection of histologic CIN2,3. Data suggest that the ThinPrep is equivalent to the smear in the detection of endocervical adenocarcinoma in situ and endometrial disease. The ThinPrep collection vial has been approved by the FDA for testing for HPV, useful for primary screening alongside the pap (“cotesting”) and for managing women whose Pap test results show atypical squamous cells (ASC).

Historical Overview

Automated cytology screening devices have been under development since the 1950s. The first computerized screening system was developed in the United States by Airborne Instruments Inc. and was called the Cytoanalyzer. In preclinical trials it did not perform as well as expected, and the project was discontinued. The difficulty of the task was soon appreciated, especially the inherent problems with analyzing smears prepared in the conventional manner. Despite setbacks, research into cervical cytology screening continued throughout the following decades, with the development of the TI-CAS, Quantimet, BIOPEPR, CERVIFIP, CYBEST, DIASCANNER, FAZYTAN, and LEYTAS. Some of these instruments are now in museums, but others have served as prototypes for systems that are now commercially available.

In the 1990s, researchers in the United States and Canada established private enterprises supported by venture capital to develop a commercial automated screening instrument. Foremost in the field were AutoCyte (formerly Roche Image Analysis Systems), Cytyc, Neopath, and Neuromedical Systems. A three-way merger took place in 1999, when AutoCyte, after purchasing the intellectual property of Neuromedical Systems, merged with Neopath to form a new company called TriPath Imaging, acquired in 2006 by Becton Dickinson. In 2007, Cytyc Corporation, developer of the ThinPrep Pap Test and ThinPrep Imaging System (TIS), merged with Hologic Inc. and became a wholly-owned subsidiary of Hologic.

In 1998, the FDA approved the AutoPap System (now called the FocalPoint Slide Profiler; BD TriPath Imaging, Burlington, NC) as a primary screener for cervicovaginal smears, followed by approval in 2002 for use with SurePath slides. In 2003, the FDA approved the TIS (Hologic, Marlborough, MA) as a primary screener for ThinPrep Pap slides, and in 2008 it approved the FocalPoint Guided Screening (GS) Imaging System. Neither is approved in the United States for automated screening of nongynecologic cytology specimens.

ThinPrep Imaging System

The TIS uses the principle of location-guided screening to aid the cytotechnologist in reviewing a ThinPrep Pap slide. The TIS consists of two components, the Image Processor (”imager”) and the Review Scope ( Fig. 1.2A–B ). Stained and coverslipped ThinPrep slides are placed in a cartridge (each cartridge holding 25 slides), and up to 10 cartridges are loaded onto the bench-top imager. The imager has the capacity to screen more than 300 slides per day. It scans the slides and identifies 22 fields of view (FOV) on each slide that, based on optical density measurements and other features, are the most likely to harbor abnormal cells. The x- and y-coordinates of the 22 FOVs are stored in a database and retrieved at a later time. The server is electronically linked to one or more Review Scopes in the laboratory. A Review Scope resembles a standard microscope but is augmented with an automated stage, a pod that controls the stage and objectives, and a keypad. The scope also has a camera that reads the slide identifier when the slide is loaded onto the stage. When a valid slide identifier is recognized, the server sends its coordinate information to the scope, permitting the cytotechnologist to navigate to the 22 FOVs using the pod. Navigation to each FOV is done geographically, that is, using the shortest distance from one FOV to the next. The cytotechnologist uses the pod to advance forward or return back through the FOVs, changing objectives as needed. If no abnormal cells are found in any of the FOVs, the case has been completed and can be reported as negative. If any abnormal cells are found in any of the FOVs, a review of the entire slide must be performed. This can be done using the “Autoscan” function on the Review Scope, with preset, customized user screening preferences. The Review Scope has both electronic and physical slide dotting capabilities.

The accuracy of the TIS was evaluated in a clinical trial at four laboratories. ThinPrep slides were first screened manually and the results recorded. They were then rescreened using the TIS. Truth adjudication was performed by expert review of all abnormal cases and a proportion of negative slides. The TIS detected significantly more abnormal slides (ASC-US or greater) than manual review (82% versus 76%). A later split-sample study comparing smears versus the TIS for ThinPrep slides showed a significantly higher detection rate of histologic HSIL (CIN2,3) with the TIS. Because 22 FOVs represent approximately 25% of the ThinPrep cell spot, implementation of the TIS enhances productivity.

Implementing the TIS requires adopting the proprietary ThinPrep Pap stain, to which some adjustment is necessary because it yields darker nuclear staining of metaplastic and endocervical cell clusters than most traditional Pap stains. The TIS does not eliminate false-negative results, which are still encountered, albeit less frequently than in the absence of imaging. A number of postapproval studies have shown significant increases in the detection of cytologic LSIL and HSIL after implementation of the TIS.

Specimen Adequacy

One of the most important advances of the Bethesda System is its recommendation that each Pap report include an explicit statement of adequacy. In 1988, the Bethesda System proposed three categories for specimen adequacy: “satisfactory,” “less than optimal” (renamed “satisfactory but limited by ….” in 1991), and “unsatisfactory.” The 2001 Bethesda System eliminated the middle category because it was confusing to clinicians and prompted unnecessary repeat Pap tests. Nevertheless, the 2014 Bethesda System advocates mentioning the presence or absence of a transformation zone component and permits comments on obscuring elements.

It is easy to determine whether a specimen is adequate or unsatisfactory in most cases. Slides received without patient identification or broken beyond repair should be rejected as unsatisfactory. An appropriately labeled smear with an adequate complement of well-preserved squamous and endocervical cells is clearly satisfactory. About 1% or less of Pap test results are interpreted as unsatisfactory. Unsatisfactory Pap test results can be finalized by a cytotechnologist and need not be reviewed by a cytopathologist (see Chapter 19 ). One of the components of an adequate Pap test result is an adequate squamous component , defined as a minimum estimated number of well-preserved, well-visualized squamous cells, the minimum being different for smears versus liquid-based preparations:

The minimum number of 5000 squamous cells for an adequate LBC Pap test result was based on correlations made between the false-negative rate and squamous cell cellularity. Because LBCs likely represent a more homogeneous representation of the material obtained by the collection device, a more stringent squamous cellularity requirement was imposed on smears.

The cellularity of the squamous cell component is estimated; laboratories are not expected to count individual cells. With experience, an adequate squamous cell component is apparent in most cases. In borderline cases, techniques are available for estimating adequacy: reference images for smears and a spot-counting procedure for liquid-based preparations.

A spot-counting method is used to evaluate LBCs with borderline squamous cellularity. A minimum of ten fields are counted along a diameter that includes the center of the slide ( Fig. 1.3A ). If the cell circle has blank spots, these should be represented in the fields counted ( Fig. 1.3B ). The average number of squamous cells is then compared against tables that take into account the objective, the eyepiece field number, and the diameter of the circle that contains cellular material. For example, with an Field Number 20 (FN 20)) eyepiece and a ×40 objective, the sample is adequate if the average number of squamous cells counted is greater than 3.1 for a ThinPrep slide.

An additional slide can be prepared when the initial slide has insufficient cellularity, in the hopes that the new slide will be more cellular. The addition of a washing step with 10% glacial acetic acid increases the percentage of satisfactory ThinPrep Pap test results, uncovering occasional cases of SIL and invasive cancer. Adequacy should be assessed individually, however, not cumulatively; two unsatisfactory slides do not add up to a satisfactory slide.

Squamous cellularity is sometimes difficult to estimate, for example, when there is marked cell clustering or cytolysis. In certain clinical settings, particularly in women with atrophy and after radiation therapy to the pelvis, a lower number may be adequate. In these situations, cytologists are expected to use their judgment when evaluating adequacy rather than rigidly applying the numerical criteria.

For women with an unsatisfactory Pap test result, repeat cytologic study in 2 to 4 months is recommended. In women with an unsatisfactory Pap test result and a positive HPV test result, either a repeat Pap test in 2 to 4 months or colposcopy is acceptable.

In the 2014 Bethesda System, the presence or absence of an endocervical/transformation zone component is noted on the report. An endocervical component is considered present if 10 or more endocervical or squamous metaplastic cells, either isolated or in groups, are present. The data on the endocervical component as a measure of sample adequacy are contradictory. The importance of endocervical cells was first suggested by cross-sectional studies, which showed that smears are more likely to contain SIL when endocervical cells are present. Data from retrospective case–control studies, however, do not support this: investigators have found no association between false-negative Pap test results and the absence of endocervical cells. Retrospective cohort studies have shown that women whose initial smears lack endocervical cells do not develop more lesions on follow-up than women whose smears do have an endocervical component, implying that an endocervical component is not essential. Currently, a smear without endocervical cells is not considered unsatisfactory, although the absence of an endocervical/transformation zone component is mentioned as a “quality indicator.” A repeat Pap test is not necessary.

General Categorization

The general categorization is an optional component of the Bethesda System.

“Other” includes benign-appearing endometrial cells in women over 45 years of age. Specimens are categorized according to the most significant abnormality identified.

Interpretation and Results

Recommended terminology for reporting findings is listed in Table 1.2 . Mentioning nonneoplastic findings, other than organisms, is optional, given that many clinicians desire the Pap test report to be as concise as possible. Findings of no clinical consequence, if mentioned, may result in confusion and even unnecessary repeat testing. Nevertheless, many cytologists believe it is important to document that certain findings were interpreted as benign, particularly those that can mimic a neoplasm.

Squamous Cells

The ectocervix is lined by a stratified squamous epithelium that matures under the influence of estrogen. The most mature squamous cells are called superficial cells . They have a small, pyknotic nucleus that is 5 to 6 μm in diameter. Intermediate cells have a larger nucleus measuring 8 μm in diameter, which is not pyknotic but instead has a finely granular texture. Intermediate cells are occasionally binucleated and even multinucleated. Both superficial and intermediate cells are large polygonal cells with transparent pink or green cytoplasm ( Fig. 1.4 ). Superficial and intermediate cells are the predominant cells in cytologic samples from women of reproductive age.

Immature squamous cells are called parabasal cells and basal cells . Because a Pap test does not usually scrape off the entire thickness of the epithelium but only the upper few layers, immature cells near the base of a mature epithelium are not usually sampled. An immature epithelium, however, is composed throughout its thickness by parabasal-type or basal-type cells. Immature epithelium is common at the transformation zone, where it is called squamous metaplasia, and whenever there is squamous epithelial atrophy due to a low estrogen state. Thus so-called “parabasal” and “basal” cells are typically obtained from squamous metaplasia or atrophic epithelium. Squamous atrophy is encountered in a variety of clinical settings associated with a low estrogen state.

Parabasal cells are round or oval rather than polygonal and have a variably sized nucleus that is usually larger than that of an intermediate cell. Basal cells are even smaller and have very scant cytoplasm ( Fig. 1.5 ).

Basal and parabasal cells are the hallmark of atrophy. With a deeply atrophic cervical epithelium, no superficial or intermediate cells are seen, only parabasal and basal cells. In addition, atrophic epithelium, particularly in postmenopausal women, is prone to injury and inflammation and often shows a spectrum of changes that should be recognized as normal and not confused with a significant lesion. Sheets of immature squamous cells are crowded and syncytium-like, mimicking the crowded cells of an HSIL ( Fig. 1.6A ). Nevertheless, the chromatin texture in atrophy is finely granular and evenly distributed, nuclear contours remain mostly smooth and thin, and mitoses are generally absent. A curious variant, transitional cell metaplasia , is notable for prominent longitudinal nuclear grooves (“coffee-bean nuclei”), wrinkled nuclei, and small perinuclear halos ( Fig. 1.6B ). Cellular degeneration is seen in some cases of atrophy ( Fig. 1.7A ). Dark blue, rounded, amorphous masses known as “blue blobs,” thought to represent either condensed mucus or degenerated bare nuclei, are sometimes seen ( Fig. 1.7B ), as is a granular background (see Fig. 1.7A ) that resembles the necrosis associated with invasive cancers.

Parabasal-type cells are also the constituents of squamous metaplasia of the endocervix. Squamous metaplasia is a common morphologic alteration of the endocervical epithelium usually limited to the transformation zone in women who otherwise have good squamous maturation. It is identified cytologically as flat sheets of immature squamous cells, often arranged in an interlocking fashion like paving stones ( Fig. 1.8 ). They may show mild variation in nuclear size, with slightly irregular contours and slight hyperchromasia.

Squamous metaplasia, as defined cytologically, is always composed of immature squamous cells. So-called mature squamous metaplasia , a histologic term describing mature squamous epithelium overlying endocervical glands, is not recognized as such on cytologic preparations.

Other normal changes of squamous cells are hyperkeratosis and parakeratosis. Hyperkeratosis is a benign response of stratified squamous epithelium due to chronic mucosal irritation, as in uterine prolapse. Anucleate, mature, polygonal squamous cells appear as isolated cells or plaques of tightly adherent cells ( Fig. 1.9A ). Such cells are benign and should not be considered abnormal. This cytologic picture is mimicked by contamination of the slide by squamous cells of the vulva or skin from the fingers of the persons handling the slide.

Parakeratosis , a benign reactive change also caused by chronic irritation, is characterized by small, heavily keratinized squamous cells with dense orangeophilic cytoplasm and small, pyknotic nuclei ( Fig. 1.9B ). When such densely keratinized cells show nuclear atypia in the form of enlargement and membrane irregularity, they are called “atypical parakeratosis” and should be categorized as an epithelial cell abnormality (see further on).

Endocervical Cells

The endocervix is lined by mucin-producing columnar cells that have abundant vacuolated cytoplasm and a basally placed nucleus with finely granular chromatin. Nucleoli are inconspicuous but become prominent in reactive conditions such as cervicitis. Endocervical cells are often identified in strips or sheets rather than as isolated cells ( Fig. 1.10 ). When arranged as strips, the cells have the appearance of a picket fence. When in sheets, they resemble a honeycomb because of the well-defined cell borders and uniform cell arrangement. Rarely, mitoses are identified. They should not raise suspicion of a neoplasm if the cells are otherwise normal in appearance. Tubal metaplasia is a benign alteration of the endocervical epithelium found in about 30% of cone biopsy and hysterectomy specimens ( Fig. 1.11 ).

Exfoliated Endometrial Cells

Spontaneously exfoliated, menstrual endometrial cells are seen if the Pap test specimen is taken during the first 12 days of the menstrual cycle.

Exfoliated endometrial cells are most easily recognized when they are arranged in spherical clusters ( Fig. 1.12A ). They are small, with a dark nucleus and (usually) scant cytoplasm; occasional cells may have more abundant, clear cytoplasm. Clusters have a scalloped contour due to the slight protrusion of individual cells. Apoptosis is common. Isolated endometrial cells are also seen, but they are less conspicuous because of their small size.

Occasionally, endometrial cell clusters consist of an obvious dual cell population, with small, dark stromal cells (in the center) and larger glandular cells (around the edges) ( Fig. 1.12B ). Most endometrial cell clusters, however, do not have this dual population; clusters like that in Fig. 1.12A might be of endometrial glandular or stromal origin or a mix of both and are best referred to simply as “exfoliated endometrial cells.”

Shedding endometrial cells after day 12 (“out of phase”) is associated with endometritis, endometrial polyps, and intrauterine devices (IUDs). In a young woman, abnormal shedding is almost never due to endometrial adenocarcinoma. For this reason, endometrial cells need not be mentioned in the report for women under 45 years of age. Sometimes a pathologist might do so anyway, to document that the cells were identified and interpreted as benign endometrial cells. Endometrial cells are notorious for their ability to cause diagnostic difficulty because a variety of neoplastic cells resemble endometrial cells. In a woman 45 years of age or older, benign-appearing endometrial cells are reported because of the small risk of endometrial neoplasia.

The differential diagnosis includes a number of very significant lesions that mimic endometrial cells and thus are sometimes mistakenly interpreted as normal, particularly if the woman is in the first 12 days of her menstrual cycle. Attention to certain cytologic details can help avoid some if not all of these misattributions. A minority of HSILs are comprised of relatively small cells. Like endometrial cells, their nuclei are dark, and they have scant cytoplasm ( Fig. 1.13A ). HSIL cells, even when small, are usually bigger than endometrial cells, vary more in size, and have denser cytoplasm. HSIL clusters are usually less well circumscribed and not as spherical as endometrial cell balls. Some poorly differentiated squamous cell carcinomas are comprised of small dark cells that mimic endometrial cells to perfection ( Fig. 1.13B ). In such cases, suspicious clinical findings (e.g., postcoital bleeding) might be the only clue to the correct interpretation. Most AIS have a columnar cell structure, but a minority are made up of smaller and rounder cells ( Fig. 1.13C ), particularly with liquid-based preparations. Careful examination for focal columnar differentiation and mitoses can be very helpful. The rare small cell carcinoma of the cervix may display crush artifact ( Fig. 1.13D ), which is rarely seen with endometrial cells.

Abraded Endometrial Cells and Lower Uterine Segment

The endocervical sampling device occasionally inadvertently samples the LUS or endometrium. This is especially likely when the endocervical canal is abnormally shortened, such as after a cone biopsy or trachelectomy.

The characteristic feature is the combination of glands and stroma, often in large fragments ( Fig. 1.14A–C ), either together or separated. Glandular cells of the LUS resemble endocervical cells, but have a higher nuclear-to-cytoplasmic ratio, are more hyperchromatic, and can be mitotically active. Because of their very high nuclear-to-cytoplasmic ratio, there’s a risk of confusion with a significant squamous or glandular lesion.

Trophoblastic Cells and Decidual Cells

Syncytiotrophoblastic cells from placental tissue are seen very rarely, perhaps in about 0.1% of Pap test results from pregnant women. The cells are large, with abundant blue or pink cytoplasm. They have multiple nuclei that have a granular chromatin texture and slightly irregular contours. Trophoblastic cells can be distinguished from multinucleated histiocytes because their nuclei are darker and more irregular in contour ( Fig. 1.15 ). They do not show the prominent molding and ground-glass appearance of nuclei of herpes simplex infection. Immunostains for human chorionic gonadotropin and human placental lactogen can be used to confirm their identity as trophoblastic cells. The presence of syncytiotrophoblastic cells is not a reliable predictor of an impending abortion.

Decidual cells may be seen during pregnancy and the postpartum period, but they are often difficult to recognize and identify as such. They are usually isolated cells with abundant granular cytoplasm, a large vesicular nucleus, and a prominent nucleolus. They often show degenerative changes.

Inflammatory Cells

Neutrophils are seen in all Pap samples and do not necessarily indicate infection, but they are present in increased numbers after injury or infection. Lymphocytes and plasma cells are rare, but occasionally—most often in older women—they are numerous ( Fig. 1.16A–B ). This pattern is called follicular cervicitis because biopsy specimens show lymphoid follicle formation. The lymphocytes of follicular cervicitis can be confused with HSIL cells, endometrial cells, and lymphoma. Histiocytes are associated with a myriad of conditions (e.g., menses, pregnancy, foreign bodies, radiotherapy, and endometrial hyperplasia and carcinoma) ( Fig. 1.17 ), but by themselves are a nonspecific finding of no clinical significance.

Lactobacilli

The vagina is colonized by Gram-positive rod-shaped bacteria of the genus Lactobacillus . They are beneficial because they produce lactic acid, which reduces the ambient pH and possibly protects from infection by Candida and other pathogens. Lactobacilli metabolize the glycogen contained within exfoliated squamous cells. The resulting cellular pattern, commonly seen during the second (luteal) phase of the menstrual cycle, is known as cytolysis —bare intermediate cell nuclei, fragments of squamous cytoplasm, and abundant bacterial rods ( Fig. 1.18 ). Cytolysis can interfere with one’s ability to evaluate the nuclear-to-cytoplasmic ratio, an important criterion in grading SILs.

Artifacts and Contaminants

Artifacts and specimen contaminants such as trichomes are illustrated in Fig. 1.19 . For laboratories that use the ThinPrep method, one of the most commonly encountered artifacts happens when the sample is contaminated by a lubricant, gel, or vaginal medication containing carbomers or Carbopol polymers ( Fig. 1.19B ). These interfere with cellular deposition during slide preparation, resulting in an unsatisfactory slide. A note in the report can be helpful, for example:

NOTE: This sample appears to be unsatisfactory due to the presence of an obscuring material, possibly lubricant. Nonapproved lubricants, and some gels or medications, may contain ingredients that interfere with Pap slide preparation (notably carbomers and Carbopol polymers). Of note, disposable specula may be precoated with interfering lubricants. In all cases, lubricant should be used sparingly when obtaining a Pap. For more information, please contact the Cytology Laboratory.

“String of pearls” is the descriptive name given to an uncommon but striking artifact seen with ThinPrep Pap tests: a long string with bulbous excrescences ( Fig. 1.19C ). The cause is unclear, but it might represent a tight coil of mucus. Although originally described in a case report from a postmenopausal woman, they can be seen at any age.