After the introduction of the Bethesda system for reporting cervical smear abnormalities, a binary format of cytological diagnosis has been recommended that distinguishes low from high grade squamous intraepithelial lesions. Abnormalities in the low grade category combine cellular changes previously interpreted as koilocytic atypia with those of mild dysplasia. The reasons for this are the difficulty in reproducibly separating koilocytic atypia from mild dysplasia and the presumption that their natural history and associated HPV types are similar (9). It has been shown that LGSIL's are likely to be infected with HPV6, 11, 42, 43 and 44 (6,10).
The Bethesda system categorises miniature polygonal squamous cells with dense, orangeophilic or eosinophilic cytoplasm and small pyknotic nuclei that demonstrate cellular pleomorphism (caudate or elongate shapes) and/or increased nuclear size or hyperchromasia as atypical squamous cells of uncertain significance (ASCUS), or squamous intraepithelial lesions (SIL) depending on the degree of the cellular abnormalities (8).
Atypical squamous cells are those cells with cytomorphological changes that are more serious than those of reactive/reparative changes, but donot fulfil the criteria for SIL. Specifically the criteria include: nuclear enlargement, hyperchromasia or mildly coarse but regularly distributed chromatin, mild nuclear membrane irregularities, nuclear halo without significant nuclear atypicality, and atypical degenerated or air-dried nuclei. Hyperchromasia can be defined as smudged or opaque nuclei, whereas coarse chromatin was defined as finely granular and regularly distributed chromatin (11).
Huddock etal (5) defines squamous atypia as nuclear enlargement to approximately three times that of a normal intermediate squamous cell with a normal to mildly hyperchromatic chromatin pattern.
Hyperkeratosis is defined as the presence of anucleated squames (5).
Parakeratosis is defined as the presence of orangeophilic squamous cells with pyknotic nuclei (5). These cells have a low N/C ratio and smoothly contoured, oval or round, pyknotic (ink dot-like) nuclei with dense, uniform chromatin (8).
Atypical parakeratotic cells were isolated or clustered. They were small cells, 2-3 times the diameter of a neutrophil. The dense cytoplasm was orangeophilic or eosinophilic with a variable, often irregular outline. They had a high N/C ratio, irregular nuclear contour, and dark, often irregularly distributed chromatin, Nucleoli are usually not present. The most striking features were elevated N/C and an irregular, angulated ("raisinoid") or triangular nuclear contour (8).
Voytek etal (8) found that atypical parakeratosis was an important marker for SIL, in contrast to typical parakeratosis which had no associated increased incidence of cervical dysplasia. The presence of a clean smear background without extensive inflammation and without degenerated cells and a history of dysplasia or at high risk as well as abundant well-preserved atypical parakeratotic cells would favour a dysplasia associated atypical parakeratosis (8).
Abu-Jawdeh etal (11) demonstrated that coarse chromatin was the cytological parameter most predictive of SIL. 46% of cases of squamous atypia that had coarse chromatin showed SIL on histology compared with 22% of those without coarse chromatin. Atypical squamous cells with coarse chromatin without nucleoli are more likely to be dysplastic than reactive (11).
Paavonen etal (12) suggest the following classification for the evaluation of epithelial cell abnormalities:
Superficial squamous cell atypia was defined as minimal to moderate nuclear and cytoplasmic changes, including a slight increase in nuclear size(10-13um), usually regular nuclear contour, mild hyperchromasia, frequent binucleation, frequent dyskeratosis and the absence of a distinct perinuclear halo.
Metaplastic cell atypia was defined as atypical changes seen in metaplastic cells, including slight nuclear enlargement (10-12um), slight hyperchromasia and chromocentres or nucleoli present, and a regular or slightly irregular nuclear contour.
Koilocytic squamous cell atypia was defined as the presence of minimal nuclear changes in squamous cells, ie, mild hyperchromasia, frequent binucleation or multinucleation but usually a regular nuclear contour, and a distinct perinuclear halo with a dense outer rim of cytoplasm.
Paavonen etal (12) found that patients with benign atypias (as defined above) often had CIN present on their cervix and that metaplastic cell atypia combined with squamous cell atypia had a significantly higher progression rate than any other type of atypia.
HPV causes changes in the nucleus and cytoplasm of infected cells including koilocytosis, parakeratosis, hyperkeratosis and various nuclear changes including enlargement and irregularity of the nuclear membrane. The chromatin may appear smudgy or even mildly hyperchromatic but usually is normochromic and evenly distributed. The nucleus may be pyknotic (5).
The definitive diagnosis of HPV on smears required the presence of koilocytes. These are squamous cells with paranuclear halos and peripheral condensation of the cytoplasm in combination with enlarged nuclei, often with nuclear membrane irregularity or binucleation (5).
It seems that HPV requires a certain degree of cellular maturity for the induction of cytopathic effect, so that as the degree of CIN increases the prevalence of koilocytosis decreases. Several investigators have found that progression from borderline changes to high grade dysplasia was more likely if cytological evidence of HPV was absent (7).
Cecchini etal (2) studied five nonclassic cytologic signs of HPV (mild koilocytosis, mild dyskeratosis, binucleation or multinucleation, cleared cytoplasm and nuclear hyperchromatism) and found that they were significantly but not independently associated with HPV infections.
Hudock etal (5) have shown that parakeratosis and/or hyperkeratosis alone are not indicative of HPV infection but keratinisation and squamous atypia (see above) without inflammation does correlate with an increased incidence of HPV on follow-up.
A moderate degree of nuclear atypia is frequently seen in condylomatous lesions. There is a group of condylomata, however, in which the degree of atypia is so marked that an erroneous cytological diagnoses of dysplasia, CIS or even invasive keratinising carcinoma could easily be made on the cellular sample (1). Parabasal cells from atypical condyloma may have amphophilic cytoplasm, karyorrhexis and smudging of chromatin, where as cells from moderate dysplasia (with which it may be confused) will tend to have cyanophilic cytoplasm and nuclei with a sharply defined chromatin pattern (1). Even more confusing is a smear patten consisting of very pleomorphic mature squamous cells, which occur singly or in thick aggregates. There is macrocytosis with dense orangeophilic or amphophilic cytoplasm. Perinuclear clearing may be entirely absent. the nucleus is very hyperchromatic, almost pyknotic, much enlarged and irregular in shape. The chromatin is smudged. These cells may look very similar to those shed from an invasive SCC, however there is no tumour diathesis and the nuclei do not have the sharply defined chromatin structure found in carcinoma. The N/C remains within normal limits (1).
CIN 1 or mildly dysplastic cells have hyperchromatic sometimes irregular nuclei at least four times larger than those of an intermediate cell with slight to moderate chromatin coarsening. The cytoplasm in such cells was usually that of a superficial cell and either did or did not show evidence of a HPV effect (9).
The cell shape is usually polyhedral and there may be some moderate cellular pleomorphism. The cells will be either single or occasionally clumped (10).
Jovanovic etal (4) have documented a group of post menopausal epithelial alterations that may mimic low grade precursor lesions with features similar to those of koilocytic atypia. Cytoplasmic halos, nuclear enlargement and multinucleation may occur in post menopausal squamous atypia, but certain features distinguish this from HPV. There is generally lesser nuclear enlargement (2 fold or less versus 3 fold or more), less variable staining intensity and finely and evenly distributed nuclear chromatin, in contrast to the denser and coarser chromatin pattern in koilocytic atypia. Moreover, post menopausal squamous atypia has greater uniformity of nuclear halo contour, in some cases having a characteristic "fried egg" appearance (4).
Post partum smears may contain atypical cells that can be mistaken for dysplastic or malignant squamous cells (Arias-Stella cells, decidual cells and trophoblastic cells) (3). The recognition of these cells of maternal-fetal origin is facilitated by the presence of syncytiotrophoblastic and endometrial cells or a mixture of cell types suggesting endometrial shedding. Mononucleated trophoblastic cells have poorly defined cytoplasmic borders, abundant eosinophilic-to-amphophilic cytoplasm that is often vacuolated and variably-sized nuclei with thick nuclear membranes, fine chromatin and occasional nucleoli. Where trophoblastic cells are associated with the hyalinised matrix of a placental implantation site, the cells are widely spaced rather than contiguous, as would be seen in an invasive neoplasm (3).