Nonneoplastic Cervical Cytology

Figure 2 shows histology of multilayered squamous epithelium (hematoxylin and eosin, 40×)

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Figure 2:

Histology of multilayered squamous epithelium (hematoxylin and eosin, 40×)

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1. The squamous epithelium is supported by basement membrane and is divided into basal/regenerative layer, the mid-zone forming the major thickness, and the superficial zone

2. The germinal/basal layer is composed of a single row of small regular cells adhering to a basement membrane and showing signs of active growth

3. The mid-zone comprises parabasal and intermediate cells. Parabasal cells are immature and crowded, lying just above the basal layer. The parabasal cell layer is two to three cells deep

4. These cells mature into an intermediate layer of variable thickness in which the cells have more cytoplasm, the nuclei still show a recognizable chromatin pattern and the cells are bound to each other by intercellular cytoplasmic bridges

5. In fully mature cervical squamous mucosa, there is a superficial layer consisting of cells that do not normally mature any further. Intercellular bridges are not highly developed at this level so that the cell bonds are loosely attached. Superficial cells are actually dead or dying and exfoliate spontaneously. The nuclei of the superficial cells are pyknotic.

Mucosal thickness depends on hormonal status as the parabasal, intermediate, and superficial layers are all hormonally responsive. Under the influence of estrogen, a superficial layer develops in about 4 days.

Basal cells

Basal cells are small, undifferentiated cells that measure around 10–12 µm in diameter. These cells have central round to oval nucleus, with fine chromatin and occasional chromocenters and small delicate amount of cytoplasm. They resemble small histiocytes. Isolated basal cells are rarely seen in a Pap smear unless there is severe atrophy. In severe atrophy, they are present as syncytial aggregates or hyperchromatic crowded groups (HCGs).

Basal cells have following functions: They anchor the epithelium to the basement membrane by hemidesmosomes, they form the regenerative epithelium, they help in the formation of basement membrane by sending signals to the stroma. This basement membrane helps to separate the epithelium form the underlying mesenchyme.

Parabasal cells

Moderately large ranging from 15 µm to 30 µm in diameter. They have round to oval outlines with fairly dense green cytoplasm. Nuclei occupy about one half of the cell with a size slightly larger than the red blood cells (RBCs) nucleus. The N/C ratio is approximately 20% but it can be variable. The nuclei are round to oval with finely granular chromatin and occasional chromocenters. Nucleoli are inconspicuous. Immature parabasal cells lie in sheets while more mature cells are usually dissociated. Parabasal cells may predominate in atrophic smears from postmenopausal women. In younger women, postnatal atrophy or high-dose progesterone oral contraception produces similar changes [Figure 3].

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Figure 3:

(a) Liquid-based cytology preparation – smear in atrophy showing parabasal cells (40×). (b) Conventional smear showing parabasal cells and few basal cells, karyorrhexis is also seen in lower half of the field (40×).

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Intermediate cells

Intermediate cells are large polygonal ranging from 35 µm to 40 µm in diameter, depending on their level of maturation. Low intermediate cells are about the size of parabasal cells while high intermediate cells are about the size of superficial cells. These cells are having ample pale green filmy cytoplasm often folded at periphery. Nucleus is round or ovoid, centrally located and the size ranges from 7 µm to 8 µm in diameter [Figure 4]. The chromatin pattern is fine and vesicular. Barr body is easily seen at the nuclear membrane. The N/C ratio is very low (about 3–5%). The nucleus of intermediate cells is a benchmark. It is used to gauge nuclear size, intensity of chromatin staining, and chromatin texture. The intermediate cell nucleus is key reference in evaluation of dysplasia. A dysplastic cell has 3–5 times the area of an intermediate nucleus. Cytoplasmic disintegration (cytolysis) is essentially a feature of intermediate cell.

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Figure 4:

Liquid-based cytology preparation – smear showing intermediate squamous cells with translucent cytoplasm and vesicular nucleus (40×).

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Superficial cells

During child bearing age group of normal women, the bulk of cells seen in cervical cytology is of superficial squamous cells. They usually occur singly and rarely in clusters. These cells are large polygonal with flat delicate, transparent cytoplasm, and measure about 45–50 µm in diameter [Figure 5]. In Papanicolaou stain, the cytoplasm of the majority of superficial cells stains predominantly a delicate pink and has keratohyalin granules [Figure 6] in their cytoplasm. The superficial cells have a centrally placed small pyknotic nucleus. There may be a small clear halo around the pyknotic nucleus as a reminder of its former size. The size of nucleus is less than that of the RBC. If it is larger think of dysplasia. The N/C ratio is about 2–3%. The key distinguishing feature between intermediate and superficial cells is the vesicular chromatin of the earlier and pyknotic of the latter.

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Figure 5:

Liquid-based cytology preparation: Smear shows superficial squamous cells with pyknotic nuclei (40×).

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Figure 6:

Conventional Pap smear showing intermediate and superficial squamous cells with plenty keratohyalin granules. These keratin granules are blue-black, variable sized granules within the cytoplasm (40×).

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Anucleate squames

They are mature superficial squamous cells with loss of nuclei, they stain orange or yellow with eosin [Figure 7].

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Figure 7:

Liquid-based cytology preparation showing plenty anucleate squames (10×).

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Anucleate squames are often present with granular cells, indicating completion of keratinization. Less mature cells from the deeper layers may also become anucleate, usually in association with degenerative changes. Cakes or stacks of anucleate squames may be seen when the cervix or vagina harbors a leukoplakia. It may also represent the hyperkeratotic surface of a benign wart due to human papillomavirus 6, 11 and is a non-classic finding of low-grade squamous intraepithelial lesion.

Endocervical cells

Endocervix is a series of grooves or clefts formed by deep infoldings of mucosa that may tunnel into endocervical stroma forming crypts. A single layer of endocervical columnar cells lines these mucosal folds. They are tall and columnar most are secretory but few are ciliated. Endocervical cells can occur singly, in strips or in sheets. Dissociation is more commonly seen with the LBC [Figure 8a]. Viewed from above, sheets have a honeycomb appearance. Sheets of endocervical cells when viewed from side line up like “picket-fence” palisades with basal nuclei and columnar cytoplasm [Figure 8b]. From above, nuclei appear rounded, from the side, the nuclei are oval of endocervical cells [Figure 8c]. They tend to round up or fold, particularly in a three-dimensional clusters or HCGs. Fine chromatin pattern, one or more small nucleoli are often seen near the nuclear membrane. Nuclei are usually single but occasionally binucleation or multinucleation occurs, particularly in reactive conditions. The nuclear membrane is usually smooth but sometimes there is a nipple-like protrusion seen in cases with estrogen excess. Bare nuclei may accompany groups of degenerate endocervical cells.

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Figure 8:

(a) Liquid-based cytology (LBC) preparation showing endocervical columnar cells singly and in loose clusters. This is an effect of randomization of the specimen during vortexing of LBC vial (40×). (b) Conventional Pap smear showing strips of normal endocervical glandular cells (c) shows flat honeycomb sheets (40×).

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Secretory endocervical cells

It has generous cytoplasm that contains fine mucin vacuole/s. The cells can be plump in the luteal phase, pregnancy, in response to OCP’S, hormone therapy, and endocervical polyp [Figure 9].

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Figure 9:

(a) Conventional Pap smear showing endocervical glandular cells distended with mucin (goblet cells). The mucin takes a light pink color. (b) Smear showing strip of same cells (40×).

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Ciliated endocervical cells

These cells show denser cytoplasm than secretory cells. They look like ciliated respiratory bronchiolar cells. These cells increase in the first phase of menstrual cycle [Figure 10 a and b].

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Figure 10:

(a) Liquid-based cytology preparation and (b) conventional smear showing ciliated columnar endocervical cells (40×).

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High endocervical cells

It is seen high in the endocervix resembling more like endometrial cells. They can occur in crowded groups, high N/C ratio with scant cytoplasm but the nuclei are round and smooth with denser cytoplasm [Figure 11 a and b].

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Figure 11:

Liquid-based cytology preparation: Smear is showing slightly smaller glandular cells in compact group (a) and flat sheet (b) (40×). These may represent high endocervical cells or cells from lower uterine segment collected by the tip of any brush that reaches high in the endocervical canal.

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Endometrial cells

Endometrial cells which are spontaneously shed are usually in a 3-dimensional cluster with or without a central stromal core. Double contour cell balls or wreath with darkly staining stroma inside and lighter staining epithelium wrapped outside are the characteristic arrangement of endometrial cells [Figure 12 a]. It is generally seen in the first 6–10 days of menstrual cycle and known as “exodus” (meaning exit). Factors influencing their presence beyond this may reflect underlying endometrial pathology in a woman over 40 years of age or could be due to exogenous hormonal manipulation such as hormone replacement therapy or oral contraceptive use; tamoxifen use, intrauterine device carriage, and dysfunctional bleeding are further potential sources of endometrial cells being shed outside the allowed phase of the cycle. Endometrial stromal histiocytes commonly accompany the endometrial wreaths during exodus [Figure 12 b and c].

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Figure 12:

(a) Exodus or wreath-like appearance of endometrial cells with central dark core formed by endometrial stromal cells surrounded by endometrial glandular cells (40×). (b and c) Endometrial cell balls in dirty background of histiocytes (red arrows) and inflammatory cells (b: 10×, c: 40×).

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Endometrial cells also occur in 3-dimensional clusters without central stroma. The cells are small and crowded, and the nuclei usually are degenerated and hyperchromatic, appearing as HCG. These cells are generally seen in the second half of menstrual cycle. Distinguishing endometrial cells from endocervical cells is a common problem in Pap test interpretation [Table 1].^[1]

Endometrial stromal cells: They are of two types, superficial stromal cells and deep stromal cells

Superficial stromal cells appear like histiocytes and tend to be loose clusters and are most prominent in the 6^th–10^th day of menstrual cycle along with exodus. Deep stromal cells are typically spindle or stellate shaped with scant cytoplasm having small oval nuclei and scant cytoplasm.

Abnormal shedding of the normal endometrial cells

Normal endometrial cells shed during the first half of the menstrual cycle. It is physiologic exfoliation. Exfoliation other than this time of the endometrial glandular or stromal cells is abnormal, known as “Out-of-phase shedding.” Postmenopausal (above 40 years of age) shedding is always abnormal. The Bethesda system 2001 recommends reporting the presence of normal appearing endometrial cells in women >40 years of age, regardless of the menstrual status.

The causes of abnormal shedding of endometrial cells are as follows: Endometriosis, endometritis, postpartum, abortion, intrauterine device, instrumentation, hormonal therapy, dysfunctional uterine bleeding, submucosal myoma, endometrial polyp, endometrial hyperplasia, and endometrial carcinoma.

The predominant cells seen during menstrual cycle, pregnancy, and menopause

During the 1^st–5^th days of menstrual cycle, there is predominance of intermediate cells but few superficial cells, endometrial glandular and stromal cells, blood, inflammatory cells, and debris are also seen. Endocervical cells are sparse. During proliferative phase, follicular phase, or estrogenic phase days 6^th–14^th, the maturation pattern shifts from intermediate predominant to superficial predominant pattern. During exodus days, that is, 6^th–10^th, there are many histiocytes present. The endometrial cells decrease in number by the 6^th day and normally disappear by the 10^th–12^th day.

At ovulation, that is, approximately 14^th day of the cycle, superficial cells predominate, endocervical cells become secretory, leukocytes are rare, and background becomes clean. Ferning of endocervical mucus may be seen. During the secretory phase or the luteal phase or the pro-gestational state, that is, 14^th–28^th day of the cycle, there is shift from the superficial cells to intermediate cells and after ovulation few navicular cells are also seen. Toward the end of the cycle, there is cytolysis of the cells along with increase in the number of bacteria and leukocytes giving a dirty background [Figure 13a and b].

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Figure 13:

(a) Conventional Pap smear showing predominance of intermediate cells in smear taken during pregnancy (40×). (b) Conventional smear showing cytolysis in the pre-menstrual phase along with commensal Candida group of organisms (40×).

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Just before menstruation, there is shift of cells to superficial type.

Menopause

In early menopause, there is typically an intermediate cell predominance [Figure 14]. This remains till women are sexually active. With time, there is a shift in maturation pattern to parabasal predominance. Cells from all the three layers may be present at times but rarely. Finally, in longstanding atrophy, the superficial and intermediate cells are completely replaced by syncytial aggregates of parabasal and basal cells [Figure 15]. Endocervical cells are usually sparse.

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Figure 14:

Conventional smear showing predominance of intermediate cells that are also forming giant cells in early atrophy (10×).

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Figure 15:

Conventional Pap smear showing sheets of parabasal cells in an atrophic smear (40×).

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MI

It expresses the percentile relationship of parabasal cells to intermediate cells to superficial squamous cells. Cells in cell clusters are not counted, count is performed on single cells. In normal menstruating women, MI is 0:35:65 (indicates zero parabasal cells, 35% of intermediate cells, and 65% of superficial cells). MI of 90:10:0 indicates predominance of parabasal cells.

• This shift to left of MI is seen in physiological conditions such as atrophy and postpartum period

• Women taking oral contraceptive pills or hormone replacement therapy may show variation in this index depending on the proportion of estrogens and progesterone and the duration of consumption of such pills

• A shift to left in this index is seen in women taking progestin-only contraceptives.

KI

It expresses the percentile relationship of superficial squamous cells with pyknotic nuclei to all mature squamous cells. 200–400 consecutive cells in three or four different fields on the smear are counted and evaluated. In a normally menstruating woman, the peak of KI (50–85%) usually coincides with the time of ovulation. KI is also high in pathological conditions like estrogen-secreting ovarian tumors [Figure 16].

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Figure 16:

Conventional smear showing mature superficial and intermediate squamous cells. Ratio of number of superficial squamous cells (with pyknotic nuclei) to total number of mature squamous cells counted will give the karyopyknotic index (10×).

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EI

It expresses the percentile relationship of mature squamous cells with eosinophilic cytoplasm to all mature squamous cells, regardless of status of nucleus [Figure 17]. The peak of EI coincides with the KI and may reach maximum at the time of ovulation.

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Figure 17:

Conventional smear showing plenty mature squamous cells with eosinophilic cytoplasm (40×).

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MV

Meisels in 1967 suggested that a specific numerical value be given to the three types of squamous cells (value of 1.0 to superficial cells, value of 0.5 to intermediate cells, and value of 0.0 to parabasal cells). The MV is expressed by multiplying the percentage of each cell category by its assigned value.

For example, MI of normal menstruating women at the time of ovulation 0:35:65

0 × 0 = 00.00

35 × 0.5 = 17.50

65 × 1.0 = 65.00

MV = 82.5

This system gives figure from 0 to 100, which expresses the hormonal status of the patients.

MV of 100 indicates pure population of superficial cells. MV of 0 indicates pure parabasal cell population. For normal menstruating women, MV is in the range of 50–95. Whereas for women with varying degrees of atrophy, MV is below 50.

1. Folded cell index: It expresses relationship of mature superficial cells or intermediate cells with folded cytoplasm to all the mature squamous cells. This index peaks in the secretory phase of menstrual cycle and in pregnancy [Figure 18a and b]

2. Crowded cell index [Figure 19]: It represents relationship of mature squamous cells in clusters of four or more cells to all mature squamous cells. This index peaks in the secretory phase of the cycle and is also seen in early/ crowded menopause

3. Cornification index: This expresses relationship of superficial cells to all other cells. It peaks at ovulation.

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Figure 18:

(a and b) Conventional Pap smear showing intermediate squamous cells with folded cytoplasm (red arrows), feature commonly seen in smears taken during pregnancy (40×).

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Figure 19:

Conventional smear showing mature squamous cells in clusters (40×).

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