Fig. 5.1
Nuclear area (LBP, ThinPrep). The nuclear area of an intermediate squamous cell is approximately 35 μm2. This is used as a reference to measure abnormal squamous cells such as ASC-US (approximately 100 μm2) and LSIL (approximately 150–175 μm2)
Fig. 5.2
Low-grade squamous intraepithelial lesion (LSIL) (a, left: LBP, ThinPrep and b, right cervix, H&E stain). Nuclear enlargement and hyperchromasia are of sufficient degree for the interpretation of LSIL (a & b). HPV-associated cytoplasmic changes are not a prerequisite for LSIL
Fig. 5.3
LSIL (LBP, ThinPrep). A 32-year-old woman, day 15, routine cervical cytology screening. Note the overall large cell size, “smudged” nuclear chromatin, well-defined cytoplasm, and multinucleation
Fig. 5.4
LSIL (LBP, ThinPrep). Routine screen from a 32-year-old woman. Nuclear abnormalities are required to make an interpretation of LSIL. HPV cytopathic effect manifested by perinuclear cavitation often accompanies the nuclear abnormalities but is not required for an interpretation of LSIL
Fig. 5.5
LSIL (LBP, SurePath). Cells with diagnostic koilocytic features of LSIL have a sharply defined perinuclear cavity, condensation of cytoplasm around the periphery, and abnormal nuclear features including enlargement and nuclear membrane irregularity. In liquid-based samples, nuclear hyperchromasia may be less evident
Fig. 5.6
LSIL (LBP, ThinPrep). A 28-year-old woman with a history of ASC-US and positive hrHPV testing. LSIL on cytology is characterized by mature squamous cells with enlarged nuclei with variable chromatin and nuclear membranes. Koilocytosis or perinuclear cavitation in the cytoplasm, a characteristic of HPV cytopathic effect is present, however it is not required for an interpretation of LSIL
Fig. 5.7
Pseudokoilocytes (LBP, ThinPrep). Glycogen in squamous cells can give the appearance of “pseudokoilocytosis” (a). The halos associated with glycogen often have a yellow refractile appearance (b). The nuclear abnormalities required for an interpretation of LSIL are absent. Follow-up in both cases was NILM
Fig. 5.8
ASC-US versus LSIL (a left CP, b Right LBP, ThinPrep). Atypical squamous cells with orangeophilic cytoplasm (“atypical parakeratosis”). These cells have some features of SIL; however, such keratinized lesions may be difficult to grade. hrHPV triage is helpful in determining follow-up
Fig. 5.9
ASC-US versus LSIL (LBP, ThinPrep). A 32-year-old woman. Clusters of squamous cells may be seen in “spikelike” aggregates; such clusters should be classified based on the degree of nuclear abnormalities. This patient had an LSIL interpretation on a conventional smear 2 months before this cytology which was interpreted as ASC-US. hrHPV test was positive
Fig. 5.10
ASC-US versus LSIL (CP). Nuclear features are borderline between those required for ASC-US and LSIL. Cases such as this will no doubt have poor interobserver reproducibility as demonstrated in various studies including the Bethesda 2001 BIRST project
Fig. 5.11
ASC-US versus LSIL (LBP, ThinPrep). Abnormal nuclear enlargement without concomitant HPV cytopathic change is identified in this Pap test from a 32-year-old woman. The hallmark of LSIL is an enlarged nucleus, often as much as four to six times the area of a normal intermediate cell nucleus. The N/C ratio is low and hyperchromasia varies, especially in liquid-based preparations
Fig. 5.12
Herpes (LBP, ThinPrep). Routine cervical cytology. A 25-year-old woman. Endocervical cell (a) and intermediate cells (b) showing herpes virus cytopathic effect with clearing of chromatin. These cells can be mistaken for ASC-US or LSIL (b) or occasionally HSIL (a) when obvious nuclear changes associated with herpes virus infection are not seen. Looking elsewhere on the same slide will usually clarify that the changes are due to herpes cytopathic effect
Fig. 5.13
Radiation change versus squamous cell carcinoma (CP). (a) A 61-year-old woman with a history of squamous cell carcinoma and radiation. Mature squamous cell showing cytomegaly, low N/C ratios, intracytoplasmic vacuoles with neutrophils. The mild enlargement of the nucleus should not be mistaken for LSIL. (b) Patients radiated for squamous cell carcinoma may also show tumor cells with radiation effect. These changes should be distinguished from radiation changes in benign cells (a)
Squamous cell changes associated with HPV infection encompass “mild dysplasia” and “CIN 1.” Several studies have demonstrated that the morphologic criteria for distinguishing “koilocytosis” from mild dysplasia or CIN I vary among investigators and lack clinical significance. In addition, both lesions share similar HPV types, and their biologic behavior and clinical management are similar, thus supporting a common designation of LSIL [20–22].
5.3.1 Criteria
Cells occur singly, in clusters, and in sheets.
Cytologic changes are usually confined to squamous cells with “mature” intermediate or superficial squamous cell-type cytoplasm.
Overall cell size is large, with fairly abundant “mature” well-defined cytoplasm.
Nuclear enlargement more than three times the area of normal intermediate nuclei results in a low but slightly increased nuclear to cytoplasmic ratio (Fig. 5.1).
Nuclei are generally hyperchromatic but may be normochromatic.
Nuclei show variable size (anisonucleosis).
Chromatin is uniformly distributed and ranges from coarsely granular to smudgy or densely opaque (Fig. 5.2).
Contour of nuclear membranes is variable ranging from smooth to very irregular with notches (Fig. 5.2).
Binucleation and multinucleation are common (Fig. 5.3).
Nucleoli are generally absent or inconspicuous if present.
Koilocytosis or perinuclear cavitation consisting of a broad, sharply delineated clear perinuclear zone and a peripheral rim of densely stained cytoplasm is a characteristic viral cytopathic feature but is not required for the interpretation of LSIL (Figs. 5.4 and 5.6).
Cells may show increased keratinization with dense, eosinophilic cytoplasm with little or no evidence of koilocytosis.
Cells with koilocytosis or dense orangeophilia must also show nuclear abnormalities to be diagnostic of LSIL (Figs. 5.4–5.6); perinuclear halos or clearing in the absence of nuclear abnormalities does not qualify for the interpretation of LSIL (Fig. 5.7; see Fig. 2.36).
Preparation-Specific Criteria
In LSIL, there are minimal differences between conventional preparations and liquid-based preparations.
The nuclei may show less hyperchromasia on LBPs, but overall the morphology of the cells is the same as in conventional preparations.
5.4 Problematic Patterns in LSIL
An interpretation of LSIL should be based on strict criteria to avoid unnecessary follow-up of women for nonspecific morphologic changes. By and large, the interobserver reproducibility of LSIL on cytology is far greater than LSIL (CIN 1) on histology [23]. A few pitfalls and gray areas should be kept in mind.
5.4.1 Keratinized Squamous Cells (Fig. 5.8)
Parakeratosis, as represented by miniature squamous cells with round to oval small, pyknotic nuclei and low nuclear to cytoplasmic ratios, is by itself not an HPV-related entity (see Chap. 2). However, parakeratosis may be found as a background pattern in HPV-associated lesions and as such should elicit a careful search for classic HPV-related cytologic changes (see Figs. 2.15 and 2.16). Keratinized cells showing nuclear abnormalities and low N/C ratios should be categorized as “atypical squamous cells–undetermined significance” (ASC-US) (see Figs. 4.15 and 4.16) or higher, based on the degree of nuclear abnormality (Figs. 5.8 and 5.9).
5.5 Mimics of LSIL
5.5.1 Pseudokoilocytosis (Fig. 5.7)
Cytoplasmic perinuclear clearing without accompanying atypical nuclear features should not be considered as LSIL (Fig. 5.7a). Small indistinct perinuclear halos are often seen in Trichomonas infections or in other reactive processes (see Figs. 2.36 and 2.52). Cytoplasmic vacuolization due to glycogen often takes on a yellow refractile, “cracked” appearance (Fig. 5.7b).
5.5.2 Herpes Cytopathic Effect (Fig. 5.12)
Classical herpes cytopathic effect, with multinucleated cells showing nuclear molding, margination of chromatin, and clear, ground glass nuclei, does not typically pose a differential diagnostic problem in comparison to LSIL. However, early herpes cytopathic effect may lack diagnostic nuclear features. Given the nuclear enlargement and degenerative chromatin, which may be hyperchromatic, such cases may be mistaken for LSIL (Fig. 5.12b). These cells lack the other changes of HPV cytopathic effect such as koilocytosis, and often other cells in the preparation will show more classic diagnostic changes of herpes. Occasionally, herpetic changes may also mimic HSIL (Fig. 5.12a).
5.5.3 Radiation Changes (Fig. 5.13)
Cells showing the effects of ionizing radiation have a low nuclear to cytoplasmic ratio with large nuclei which are often the same size as those seen in LSIL. The cytoplasm of these cells is usually quite distinctive with a two-toned, vacuolated appearance that lacks the perinuclear clearing and peripheral condensation present in a typical koilocyte (Fig. 5.13a; see Fig. 2.43). Patients radiated for squamous cell carcinoma may also show tumor cells with radiation effect (Fig. 5.13b), and these changes should be distinguished from radiation changes in benign cells.
5.6 Management of LSIL
In the data from the ASCUS-LSIL Triage Study (ALTS), hrHPV types were detected in 85 % of LSIL cases, with the conclusion being that HPV testing is not a useful triage strategy for cytologic LSIL, particularly in young women because of the high prevalence of HPV infection in this age group [24]. On the contrary, reflex HPV testing is acceptable for LSIL in postmenopausal women due to higher specificity in this population.
With the advent of HPV co-testing in women over the age of 30, many women with an interpretation of LSIL will have concurrent HPV testing. Thus, the 2012 ASCCP management guidelines recommend that women under the age of 25 with a cytologic interpretation of LSIL be followed up with cytology at 12 months. Women 25 years and older can be cotested in 3 years if they are HPV negative, but colposcopic examination is recommended if HPV positive. Women of unknown HPV status should have a repeat cytology in 12 months [18].
5.7 High-Grade Squamous Intraepithelial Lesion (HSIL) (Figs. 5.14–5.48)
Fig. 5.14
High-grade squamous intraepithelial lesion (HSIL) (LBP, ThinPrep). There is a mixture of dysplastic cells here, one large LSIL cell, and four adjacent, small, high N/C ratio cells with nuclear features consistent with HSIL
Fig. 5.15
High-grade squamous intraepithelial lesion (HSIL) (CP). The dysplastic cells are seen here in a syncytial cluster or hyperchromatic crowded group
Fig. 5.16
HSIL-syncytial cluster (LBP, SurePath). As in conventional smears, crowded hyperchromatic cell groups should be examined with care. If a squamous abnormality is suspected, a thorough search for single dysplastic cells in the background is warranted. Follow-up showed HSIL (CIN 3) with endocervical gland involvement
Fig. 5.17
HSIL (CP). A 58-year-old postmenopausal woman on hormone replacement therapy. Hyperchromatic crowded groups seen at low power require careful examination at higher magnification. Flattening at the edge of the cell cluster and whorling in the center are suggestive of HSIL over a glandular abnormality. Follow-up showed HSIL (CIN 3) with endocervical gland involvement
Fig. 5.18
HSIL (CP). Nuclear changes are HSIL; however, the nuclear/cytoplasmic (N/C) ratio is on the low end for HSIL
Fig. 5.19
HSIL (CP). There is variation in nuclear size and shape, and the cells have delicate cytoplasm
Fig. 5.20
HSIL (CP). HSIL with “metaplastic” or dense cytoplasm, in contrast to that seen in the syncytial groups of HSIL (Fig. 5.19)
Fig. 5.21
HSIL (CP). HSIL cells with some variation in cell size and N/C ratios. A cluster such as this may be misinterpreted as squamous metaplastic cells if examined only under lower magnification. Follow-up showed HSIL (CIN 3)
Fig. 5.22
HSIL (a, b LBP, ThinPrep). HSIL that is markedly hypochromatic. A diligent search may reveal more classic cells elsewhere on the same slide. (a) On the left side, note syncytial arrangement and nuclear grooves. (b) On the right side, abnormal naked nuclei and a hyperchromatic, high N/C ratio single HSIL cell are seen
Fig. 5.23
HSIL (a, b LBP, SurePath). Note the nuclear envelope irregularities and abnormal chromatin. As seen here in LBPs, hyperchromasia may not be as prominent as in conventional smears
Fig. 5.24
HSIL (LBP, ThinPrep). Cells showing variably sized, ovoid nuclei with prominent nuclear grooves. In this case, the chromatin is not particularly hyperchromatic, and cytoplasm has ill-defined borders
Fig. 5.25
HSIL (CP). A 42-year-old woman. Although uncommon, nucleoli may be seen in HSIL, especially with extension into endocervical gland spaces. The chromatin may appear less coarsely granular
Fig. 5.26
Fig. 5.27
HSIL (a, b: LBP, ThinPrep). A 29-year-old woman from a high-risk clinic. Close attention to isolated cells is required when screening LBPs because the abnormal isolated cells may not be as apparent as clusters of HSIL cells and may lie between benign cell clusters or in “empty spaces” on the preparation. When the criteria for HSIL are met, such cells should be interpreted as HSIL and not ASC-H. Both images (a and b) demonstrate such cells. Follow-up showed HSIL (CIN 3)
Fig. 5.28
HSIL (LBP, ThinPrep). Isolated single abnormal cells (arrow) are more often seen in LBPs. These small cells may be seen in the spaces between cells as seen here and may be easily missed on screening. The inset magnifies the cell indicated by the arrow, which shows abnormal features including a large hyperchromatic nucleus with irregular nuclear membranes and increased N/C ratio
Fig. 5.29
HSIL (LBP, ThinPrep). A 32-year-old woman with a history of abnormal Pap tests and positive hrHPV testing. A syncytial cluster of cells with overlapping of hypochromatic nuclei are seen. The nuclei are often less hyperchromatic in liquid-based preparations. Follow-up cone biopsy revealed HSIL (CIN 3)
Fig. 5.30
HSIL (CIN 3) ( cervix, H&E stain). The histology of HSIL (CIN 3) reflects the findings seen in clusters of HSIL seen on cytology. The abnormal immature cells show minimal maturation from the base of the epithelium to the surface with nuclear size and shape variation
Fig. 5.31
HSIL with extension into endocervical gland space (LBP, SurePath). Note flattening of cells at the edge of the cluster, a feature that favors HSIL over a glandular lesion
Fig. 5.32
HSIL (CIN 3) with extension into endocervical glands (cervix, H&E stain). Squamous dysplasia, especially high-grade lesions, often extends into endocervical glands replacing the normal endocervical glandular cells
Fig. 5.33
HSIL (CP). A 30-year-old woman with atypical glandular cells on a prior Pap test. When HSIL lesions involve endocervical glands, they may show features that overlap with those of adenocarcinoma in situ (AIS). Note normal columnar cells with residual mucin at the right upper edge of the cell cluster (arrow). Follow-up showed CIN with endocervical gland involvement
Fig. 5.34
HSIL (LBP, SurePath). A 44-year-old woman. Syncytial cluster of HSIL cells with features of endocervical gland extension. Such “hyperchromatic crowded groups” may raise a wide differential diagnosis under low magnification; attention to architectural pattern and cellular detail are necessary for correct interpretation. Follow-up showed HSIL (CIN 3) with endocervical gland involvement
Fig. 5.35
HSIL (a and b LBP, SurePath). This rare example of HSIL (a) shows a loosely aggregated group of dysplastic cells having a spindled appearance reminiscent of endometrial stromal cells. The cells at the margins of the group show tapered cytoplasmic ends. The nuclei show atypical chromatin and irregular nuclear contours that are more in keeping with the high-grade squamous lesion. Compare the cytologic features with shed endometrium (b)
Fig. 5.36
HSIL (LBP, SurePath). HSIL can present in three-dimensional groups that closely mimic shed endometrial cells. In this example, the nuclei are smaller that might be expected for the typical HSIL; however, they do show atypical chromatin and irregular contours. Apoptotic debris is present within the groups, a feature that is commonly present in shed endometrium
Fig. 5.37
HSIL (LBP, SurePath). In some cases of HSIL, more voluminous amounts of cytoplasm with cytoplasmic appendage formation reminiscent of repair can be present. Note also the presence of intermixed inflammatory cells within the group, another feature that overlaps with reparative changes. Such samples should be interpreted cautiously, with an attempt to find more typical HSIL cells
Fig. 5.38
HSIL (LBP, ThinPrep). Abnormal, large stripped nuclei are seen that are considerably bigger than the intermediate cell nuclei. Such cells should elicit a search for classic, intact HSIL cells elsewhere on the same preparation. These stripped nuclei should be distinguished from endometrial cells or the stripped clusters of atrophic nuclei that are often seen in LBPs in the background of atrophy
Fig. 5.39
HSIL–stripped nucleus pattern (CP). A 38-year-old woman with a history of LSIL. These abnormal stripped nuclei are often a useful diagnostic clue that other abnormal cells may be identified on the same slide. They should be distinguished from the bare intermediate cell nuclei seen in cytolysis (Fig. 2.62) and from “small blue cells” (see Fig. 3.7)
Fig. 5.41
NILM; endocervical microglandular hyperplasia (a LBP, ThinPrep, b CP). A 34-year-old woman on day 19 of menstrual cycle. Degenerated endocervical cells, seen in a streaming pattern along with thick mucus, is a pattern that has been associated with microglandular hyperplasia (b). The appearance is more subtle in liquid-based preparations (a). When identified, it is typically during the second half of the menstrual cycle, often in women taking oral contraceptives, and may mimic HSIL at low magnification. Follow-up cytology showed NILM
Fig. 5.42
HSIL (CP). Classification of atypical keratinized cells depends on the degree of nuclear abnormality, the N/C ratio, and to some extent on the pleomorphism of the abnormal cells. This image shows a range of cells from the LSIL cells seen in the center to the HSIL cells seen around the periphery. The high-grade cells have an increased N/C ratio as well as more marked variability in cytoplasmic shape (see also Figs. 5.8 and 5.26)
Fig. 5.43
HSIL (LBP, ThinPrep). These cells demonstrate marked pleomorphism of the nuclei and keratinized cytoplasm. The marked variation in shape and the presence of cells with a high N/C ratio is consistent with an interpretation of HSIL
Fig. 5.44
HSIL (LBP, ThinPrep). A 42-year-old woman. Keratinized dysplastic cells with nucleoli and angulated or “carrot”-shaped nuclei that may raise suspicion for invasion and qualify for an interpretation of HSIL cannot rule out invasion. Follow-up showed only HSIL (CIN 3) that was keratinizing
Fig. 5.45
HSIL (LBP, SurePath). HSIL in atrophy may be difficult to distinguish from clusters of benign atrophic squamous cells. In HSIL, as seen here, the cells show a syncytial arrangement, and looking at these clusters by focusing in different planes allows one to better distinguish them from the parabasal cells in the background
Fig. 5.46
HSIL (CP). Clusters of parabasal cells are commonly identified in the background of HSIL in atrophy. The HSIL illustrated here shows a sheet-like arrangement, a pattern commonly seen in HSIL, with significant nuclear size variation and a loss of polarity with overlapping of the nuclei. HSIL in the background of atrophy can be a diagnostic challenge
Fig. 5.47
LSIL with some cells suggesting the possibility of a concurrent HSIL (CP). Routine screen from a 28-year-old woman. Most of these cells qualify as LSIL; however, there are three atypical metaplastic cells at the top center (arrow) that raise the possibility of a high-grade lesion. Cases such as this are may be interpreted as LSIL with a comment explaining the possibility of HSIL or as LSIL with an additional interpretation of ASC-H. The presence of a few diagnostic HSIL cells in the background of a predominant LSIL pattern should be interpreted as HSIL. Follow-up in this case showed HSIL (CIN 2)
Fig. 5.48
HSIL (LBP, ThinPrep). In this case, diagnostic HSIL cells are present. Even if these cells are seen in the background of a majority of LSIL elsewhere on the slide, the final interpretation should be HSIL
5.7.1 Criteria
The cells of HSIL are smaller and show less cytoplasmic maturity than cells of LSIL (Fig. 5.14).
Syncytial aggregates of dysplastic cells may result in hyperchromatic crowded groups. (HCG) of immature cells which should always be carefully assessed for nuclear abnormalities (Fig. 5.15, 5.16, and 5.17).
While overall cell size is variable, in general, the cells of HSIL are smaller than those of LSIL. Higher-grade lesions often contain quite small basal-type cells (Figs. 5.28, 5.40, and 5.45).
Degree of nuclear enlargement is more variable than that seen in LSIL. Some HSIL cells have the same degree of nuclear enlargement as in LSIL, but the cytoplasmic area is decreased, leading to a marked increase in the nuclear to cytoplasmic ratio (Figs. 5.18 and 5.19). Other cells have very high nuclear/cytoplasmic ratios, but the actual size of the nuclei may be considerably smaller than that of LSIL, at times even as small as a normal intermediate cell nucleus (Fig. 5.21).
Nuclear to cytoplasmic ratio is higher in HSIL compared to LSIL.
Nuclei are generally hyperchromatic but may be normochromatic or even hypochromatic (Fig. 5.22).
Chromatin may be fine or coarsely granular and is evenly distributed.
Contour of the nuclear membrane is quite irregular and frequently demonstrates prominent indentations (Figs. 5.20 and 5.23) or grooves (Fig. 5.24).
Nucleoli are generally absent, but may occasionally be seen, particularly when HSIL extends into endocervical gland spaces or in the background of reactive or reparative change (Fig. 5.25).
Appearance of the cytoplasm is variable; it can appear “immature,” lacy, and delicate (Fig. 5.19) or densely metaplastic (Fig. 5.20); occasionally, the cytoplasm is “mature” and densely keratinized (keratinizing HSIL) (Figs. 5.26 and 5.43).
Preparation-Specific Criteria
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