Twenty one of 477 HCG cases (4.6%) were associated with epithelial cell abnormalities (ECA). 18 of 21 women with HCG-associated ECA were below 40 years of age; only 3 were =/> 40 years (Figure ​(Figure7).7). The final abnormal cytological interpretations/results in these abnormal cases were as follows: ASCUS (6/21 – 28%), ASC-H (2/21 – 9.5%), LSIL (12/21 – 57%), and HSIL (3/21 – 14%). The association of HCG with ECA was statistically significant (p = 0.0174, chi-square test). In the group with ECA, follow-up cervical biopsy results were available in 10 cases. One LSIL case with non-lesional benign HCG was found subsequently on follow-up cervical biopsy to harbor a high grade squamous intraepithelial lesion (CIN II) (Table ​(Table2).2). A total of four biopsy proven CIN2/3 cases were associated with HCG (Figure ​(Figure6).6). Among these four cases, HCG in the smear actually represented the atypical lesional cells in three cases (one HSIL cytology and two ASC-H cytology cases). Although the total number of HCG-associated abnormal cases in this series is small, an interesting and unexpected finding was that in none of the 124 cases with satisfactory sampling of transformation zone but without HCG showed any epithelial cell abnormality.

Endocervical sampling has been significantly enhanced with the advent of the endocervical brush [8], even as coincidentally increased sampling of the lower uterine segment (LUS) [9,10], endocervical tubal metaplasia (TME) [11,12], brush-induced atypia (brush effect) [13], reparative endocervical changes [14], cone artifact [15], cervical endometriosis [16], and microglandular hyperplasia [17,18] have added to the complex list of benign endocervical glandular HCG that must be distinguished from neoplastic lesions. The detection of TME, approximately 31% in cone or hysterectomy specimens [19], has undoubtedly risen in recent decades as a result of the improved ability of the cytobrush to sample the upper endocervix. TME may present as flat sheets or 3D HCG with a finely granular chromatin, dark nuclei, absent nucleoli. The presence of cilia and peg cells are very helpful. In the absence of ciliated cells, the reporting of atypical glandular cells on the Pap tests is a common outcome [11,12].

Benign endometrial cells often present in the Pap tests as HCG. The cells are small, and crowded; the nuclei may be degenerate and hyperchromatic. Mitotic figures are not typically seen in exfoliated cells but may be seen in directly sampled cells [3]. Benign endometrial cell HCG spontaneously shed in menstrual smears may especially show concerning degenerative chromatin changes, but the cell groups typically lack features commonly described in neoplasia [20], especially the more pronounced nuclear atypia seen at least in higher grade endometrial cancers [21]. Tubal metaplasia of the endometrium and other forms of endometrial metaplasia, also termed epithelial cytoplasmic change [22], have been much more frequently recognized in endometrial surgical pathology specimens than in cytology specimens. The relatively few cytologic descriptions suggest that they may present as HCG and that their accurate recognition can be a significant diagnostic challenge [23,24].

One of the most important abnormal glandular causes of HCG is endocervical neoplasia, including both adenocarcinoma in situ (AIS) and invasive adenocarcinoma [1,3]. Detection rates of AIS on Pap tests have risen with the widespread use of endocervical brush sampling [25]; however, there are many benign conditions which can mimic AIS [26]. Additional features such as marked hyperchromasia, altered nuclear polarity, increased N/C ratio, and associated single cells have been described as helpful diagnostic features [26,27]. An increasingly common endometrioid variant of AIS has a distinct small cell pattern and may be an especially common cause of false negative interpretations as it can be difficult to distinguish from cells sampled from LUS [28]. Presence of endometrial glands and stroma in the cells derived from LUS are helpful cytologic features to distinguish LUS from AIS. Endometrial adenocarcinoma can also present as HCG. Nuclear enlargement and presence of nucleoli are helpful cytomorphologic features (28). Some studies have reported enhanced accurate recognition with liquid-based cytology [30-33].

Parabasal cells associated with an atrophic cellular pattern in postmenopausal or postpartum specimens are often seen as syncytial aggregates of cells with small closely packed parabasal cells with dark nuclei and scant cytoplasm. These may on occasion be difficult to distinguish from CIS [34]. Maturation of the cells at the periphery of the groups of parabasal cells is one clue which may help to recognize the atrophic nature of the smear. A study by Harris et al to evaluate the dense nuclei of HCG using digital measurements revealed that the atrophic groups had significantly lower nuclear area (mean 0.4 μm) than HSIL (mean 0.9 μm) [6]. In another recent study on the cytomorphologic spectrum of ASC-H from the authors' aboratories, it was noted that parabasal cell HCG often present as small dark nuclei with variable cytoplasm and a moderate N/C ratio. The chromatin is typically dark but not clumped. These groups may be interpreted as ASC-H [35]. Recognition of this distinctive cytomorphologic pattern and HPV DNA testing can be very helpful in establishing the benign character of these cell groups [36].

The other most clinically significant differential diagnosis with HCG is that of a high grade squamous intraepithelial lesion (CIN2,3/CIS) with or without involvement of endocervical glands. The cytomorphologic features of HSIL include small dysplastic cells as compared to larger cells of LSIL. HCG in this study never represented lesional LSIL cells. Cells of HSIL usually occur as small single cells, but when present as syncytial-like aggregates, the HCG may be misinterpreted as other benign HCG. Close scrutiny under high-magnification is needed. Attention must be given to features such as increased nuclear size and hyperchromasia, fine-coarsely granular chromatin, and irregular nuclear contours. Nucleoli are usually absent but may be seen along the periphery of the groups, especially when the dysplasia involves endocervical glands [37]. In Liquid based preparations, it has been observed that dispersed single dysplastic cells are more often seen than sheets or syncytial groups [38]. The study of dense HCG by Harris et al found that HSIL groups contained the highest nuclear area (mean-0.9 μm) as compared to invasive squamous cell carcinoma (mean -0.8 μm) or endometrial adenocarcinoma (mean – 0.64 μm) [6]. Interpretation of HCG of HSIL may be challenging in conventional as well as liquid based preparations. In a recent study from the College of American Pathologists PAP Program, poorly performing conventional smear and Thin-Prep HSIL cases were compared. HCG were observed in both the preparations. The HSIL groups were often interpreted as glandular lesions when there was rounding up of cells with small nuclei and smooth contoured borders. On the other hand, these cell groups were interpreted as squamous lesions when elongated and large nuclei were present [38,39]. The distinction of CIS involving the endocervical glands from AIS may impact management decisions. HCG may be seen in both the groups. The cells of AIS show spindling in the center with round to oval nuclei, peripheral palisading, and pseudostratification at the edge [40].

(RMA): Faculty, drafting of manuscript, manuscript writing.