Medical history has consistently shown that change is hard and breakthroughs often unwelcome. Papanikolaou’s work was met with skepticism and resistance from the scientific community. It took over a decade to validate the diagnostic potential of the vaginal smear. In 1943 Papanikolaou and gynecologist Dr. Herbert Traut published their findings and conclusions in the famous monograph Diagnosis of Uterine Cancer by the Vaginal Smear. This diagnostic procedure was named the Pap test [2].
With 528,000 new cases per year, cervical cancer is now the fourth most common cancer affecting women worldwide after breast, colorectal, and lung cancer. Eighty percent of the global burden falls in areas with low-income countries especially of sub-Saharan Africa. Over a fifth of newly diagnosed cases are from India [3].
In sub-Saharan Africa, 34.8 new cases of cervical cancers are diagnosed per 100,000 women annually, and 22.5 per 100,000 women die from the disease, while in North America these figures are as low as 6.6 and 2.5 per 100,000 women, respectively. The drastic reduction in the incidence of cervical cancer in North America and Oceania over the past 50 years is attributed to widespread use of the Pap test for screening [3].
4.2 Screening for Cervical Cancer
Most of the premalignant and malignant lesions of the cervix are of the squamous type. Human papillomavirus (HPV) types 16 and 18 are the dominant oncotypes in squamous lesions, but type 18 is relatively more important in glandular lesions. HPV infections occur in the transformation zone, an area which undergoes physiological metaplasia from glandular to squamous epithelium at the onset of adolescence. Persistent infection produces perturbation of the cell-cycle controls, resulting in cervical intraepithelial neoplasia (CIN). At their mildest (CIN 1), these lesions are generally no more than manifestations of HPV infection, but at their most severe (CIN 3), the risk of progression to cancer is high. The risk in CIN 1 lesions is about 1 %, while it is 5 % and 12 % among those with CIN 2 and CIN 3, respectively [4]. The progression to invasive carcinoma is a slow process, giving a wide opportunity for detection by exfoliative cytology [5–8].
4.3 Cervical Cytology
Papanikolaou devised a class system for reporting cervical smears, intended to convey degree of suspicion that the patient had cancer: class I, the absence of atypical or abnormal cells; class II, atypical cytology but no evidence of malignancy; class III, cytology suggestive of but not conclusive for malignancy; class IV, cytology strongly suggestive of malignancy; and class V, cytology conclusive for malignancy. Later descriptive terms borrowed from histologic classifications of preinvasive squamous lesions were used (Fig. 4.2).
Fig. 4.2
Classifications for cervical cytology: The graded shades of colors emphasize a morphologic continuum of cytologic findings with indiscrete transitions, blue negative, green equivocal, yellow low-grade intraepithelial abnormalities, orange high-grade intraepithelial abnormalities, and red carcinoma. The categories of atypical squamous cells of undetermined significance (ASC-US) and atypical squamous cells that cannot exclude high-grade squamous intraepithelial lesion (ASC-H) are represented by dots. NILM negative for intraepithelial lesion, HSIL high-grade intraepithelial lesion, CA cancer, NOS not otherwise specified, KA koilocytic atypia, CIN cervical intraepithelial neoplasia, CIS carcinoma in situ [9]
The dysplasia nomenclature divided lesions into carcinoma in situ; high-risk lesion of immature, undifferentiated atypical cells; and dysplasia, considered to be a low-risk lesion. In the 1960s, Dr. Ralph Richart challenged the duality of dysplasia/carcinoma in situ and proposed a new term, cervical intraepithelial neoplasia (CIN), which was graded from 1 to 3. Due to lack of universal application, a uniform system was needed for grouping in studies and evolving treatment protocols. Developed in 1988 and revised in 2001, the Bethesda system for reporting cervical cytology accomplished these goals. The name for preinvasive squamous lesions was changed to squamous intraepithelial lesion, subdivided into only two grades, low and high. This paradigm shift from the CIN concept was based on HPV biology. Low-grade lesions are transient infections that carry little risk for oncogenesis, whereas most high-grade lesions are associated with viral persistence and a significant potential for progression to invasive cancer [10].
4.4 The Bethesda System (TBS) [11, 12]
The Bethesda system is a standardized framework for reporting cervical cytology that includes a descriptive diagnosis and an evaluation of specimen adequacy. It provides a clear guidance for clinical management.
4.4.1 Satisfactory or Unsatisfactory Smear
Smears are reported as “satisfactory for evaluation” or “unsatisfactory for evaluation.” Minimal squamous cellularity requirements for a specimen to qualify as “satisfactory” are 8,000 to 12,000 well-visualized squamous cells for conventional smears and 5,000 squamous cells for liquid-based preparations. A comment on the presence or absence of endocervical/transformation zone component is also included. There should be at least ten well-preserved endocervical or squamous metaplastic cells (Fig. 4.3).
Fig. 4.3
Clusters of endocervical cells. A minimum of ten well-preserved endocervical or squamous metaplastic cells are an indicator of transformation zone or endocervical sampling (Courtesy Dr. Satish Phatak)
Samples that cannot be accessioned, for example, broken or unlabelled slide, are labeled as unsatisfactory. Certain smears are designated as unsatisfactory following microscopic evaluation. Specimens with more than 75 % of epithelial cells obscured by blood or inflammation are “unsatisfactory.” A specimen is considered “partially obscured” when 50–75 % of the epithelial cells cannot be visualized. Evaluation of specimen adequacy is considered to be the single most important quality assurance component of the Bethesda system (Fig. 4.4).
Fig. 4.4
Squamous cells obscured by polymorphs (a) and blood (b) (Courtesy Dr. Satish Phatak)
4.4.2 General Categorization
This is an optional component of the Bethesda system, to allow clinicians to triage reports readily. Smears with no abnormality or reactive changes are categorized as “negative for intraepithelial lesion or malignancy.” Categories for epithelial abnormalities and other morphologic findings that may indicate some increased risk like benign-appearing endometrial cells in a woman >40 years of age are included.
4.4.3 Interpretation/Result
4.4.3.1 Negative for Intraepithelial Lesion or Malignancy
Specimens for which no epithelial abnormality is identified are reported as “negative for intraepithelial lesion or malignancy.” Organisms like Trichomonas vaginalis, Candida species, shift in flora suggestive of bacterial vaginosis, Actinomyces species, and herpes simplex virus are also reported. Reporting nonneoplastic findings like reactive cellular changes associated with inflammation, radiation, intrauterine contraceptive device, or atrophy are optional.
4.4.3.2 Epithelial Cell Abnormalities: Squamous Cells
The first category is atypical squamous cells to designate “cellular abnormalities that are more marked than those attributable to reactive changes but that quantitatively or qualitatively fall short of a definitive diagnosis of ‘squamous intraepithelial lesion’ (SIL).” This class is further subdivided into two atypical squamous cells of undetermined significance (ASC-US) and atypical squamous cells that cannot exclude high-grade squamous intraepithelial lesion (ASC-H) (Fig. 4.5). The qualifier “undetermined significance” indicates that some cases of ASC-US are associated with underlying CIN 2 or 3. ASC-H includes approximately 5–10 % of ASC cases and reflects a mixture of true HSIL and its mimics. Estimates suggest that 10–20 % of women with ASC have underlying CIN 2 or 3 and that 1 in 1000 may have invasive cancer [13].
Fig. 4.5
Atypical squamous cells of undetermined significance: designate cellular abnormalities that are more marked than reactive changes but fall short of a definitive diagnosis of squamous intraepithelial lesion (Courtesy Dr. Satish Phatak)
Low-grade squamous intraepithelial lesions (LSILs) affect intermediate or superficial cells, which show nuclear enlargement, moderate variation in nuclear size, and slight irregularities in nuclear shape and contour. LSIL encompasses lesions previously described as koilocytosis and mild dysplasia (CIN 1). Classic koilocytes have large, sharply defined perinuclear cytoplasmic cavities surrounded by dense rims of cytoplasm (Fig. 4.6). LSILs are low-risk intraepithelial lesions majority of which regress spontaneously.
Fig. 4.6
Koilocyte: HPV-infected, intermediate, or superficial cells with perinuclear cytoplasmic cavities (Courtesy Dr. Satish Phatak)
High-grade squamous intraepithelial lesions (HSILs) carry a significant risk for progression to cervical cancer. HSIL is usually a lesion of smaller, less mature squamous cells. Nuclear-to-cytoplasmic ratio, hyperchromasia, irregular chromatin distribution, and membrane contour irregularity are all more severe than in LSIL. HSIL encompasses lesions previously described as moderate dysplasia (CIN 2) and as severe dysplasia/carcinoma in situ (CIN 3) (Fig. 4.7).
Fig. 4.7
High-grade squamous intraepithelial lesions (HSIL): high nuclear-to-cytoplasmic ratio, hyperchromasia, irregular chromatin distribution, and membrane contour irregularity (Courtesy Dr. Satish Phatak)
Classic squamous cell carcinoma shows abundant necrotic debris: a granular, amorphous precipitate with nuclear debris and red blood cells called “tumor diathesis.” This is associated with hyperchromatic crowded groups of atypical cells or abundant atypical keratinized cells with unusual shapes (“tadpoles,” “fiber cells”) (Fig. 4.8).
Fig. 4.8
Squamous cell carcinoma: atypical keratinized cells with abnormal shapes (Courtesy Dr. Satish Phatak)
4.4.3.3 Epithelial Cell Abnormalities: Glandular Cells
Similar to the ASC, atypical glandular cells include glandular cells that demonstrate changes beyond benign reactive processes but are insufficient to be labeled as in situ or invasive adenocarcinoma. These are further classified to indicate their origin as endocervical or endometrial origin whenever possible.
Endocervical adenocarcinomas, in situ and invasive, are high-grade lesions that demonstrate nuclear enlargement, hyperchromasia, stratification, and mitotic activity. Invasive carcinoma shows additional features of invasion, including prominent nucleoli and tumor diathesis.
Endometrial adenocarcinoma yields fewer cells than endocervical lesions, which are directly sampled. The cytologic features vary according to the grade of tumor. Tumor diathesis is often difficult to appreciate.
An intermediate category of atypical endocervical cells that favor neoplastic includes smears with atypical features not amounting to an interpretation of AIS but conveys a significant level of concern.
4.4.4 Others
Additional findings like endometrial cells if seen in a woman 40 years of age or older are noted, regardless of the date of the last menstrual period. Although usually benign in nature, the presence of endometrial cells, if not associated with menses or after menopause, may indicate risk for an endometrial abnormality.
4.4.5 Automated Review and Ancillary Testing
Instrumentation and automated review result are included in cytology report if the slide is scanned by automated computer systems. The assay and result of an ancillary molecular test performed are also added when appropriate.
4.4.6 Educational Notes and Suggestions
These are optional in a report; format and style vary according to preferences of the laboratory and clinicians.
4.5 Sample Collection [14]
Adequate sample collection and proper submission to the laboratory with appropriate clinical information play a pivotal role in cervical cytology reporting. Majority of false negatives are attributed to improper sample collection or lack of skill and knowledge of the individual who obtains the specimen [15–17].
4.5.1 Patient Preparation
Collection of sample is planned approximately 2 weeks (10–18 days) after the first day of menstrual period. The woman is instructed against the use of douche, tampons, birth control foams, jellies, or other vaginal creams or vaginal medications for 48 h prior to the test. Also, she is advised to refrain from intercourse 48 h prior to the test.
4.5.2 Test Requisition
The requisition form must include patient details including name, age, menstrual status (LMP, hysterectomy, pregnant, postpartum, hormone therapy), and previous abnormal cervical cytology result and previous treatment, biopsy, or surgical procedure. The source of specimen, e.g., cervical and vaginal, should be mentioned along with the history of the use of hormone/contraceptive use and relevant clinical findings (abnormal bleeding, grossly visible lesion, etc.).
4.5.3 Labeling the Sample
The glass slide or specimen vial must be labeled with patient’s first and last names at the time of the collection of sample. For glass slides a solvent-resistant pen or pencil can be used on the frosted end of the slide. For liquid-based samples, the information is affixed to the vial.
4.5.4 Visualization of the Cervix for the Collection of an Adequate Sample
The smear is collected before bimanual examination and application of acetic acid or Lugol’s iodine. A sterile bivalve speculum of appropriate size is inserted into the vagina without lubrication in the dorsolithotomy position. Warm water or water-soluble jelly may be used to facilitate insertion. The jelly should not be applied on the tip of the speculum. Excess mucus or other discharge should be removed gently with ring forceps holding a folded gauze pad. A well-placed speculum would allow visualization of the os and ectocervix. The transformation zone may be easily visualized in some patients, while it may be high in the endocervical canal in others. The position of transformation zone varies in an individual over time as well. Various factors influence the position of transformation zone including changes in vaginal pH; hormonal changes including pregnancy, childbirth, and menopausal status; and hormonal therapy. Cervical stenosis in postmenopausal patients or postradiation therapy may prevent visualization of the transformation zone. It is important to sample the endocervix in these patients. A sample from the vaginal cuff is sufficient in a post-hysterectomy setting.
4.5.5 Collection Devices
Several devices including endocervical brushes, wooden and plastic spatulas, and plastic “broom-type” samplers are available for sampling. Plastic spatulas are preferred over wooden as they retain cellular material [18]. Also the use of moistened cotton-tipped swab results in incomplete sampling as cells adhere to the cotton and do not transfer well to the glass slide. Overall, the cytobrush and spatula together provide the best specimen quality for cervical cytology [19].
4.5.6 Collection of Samples for Conventional Smear Preparation Using the Spatula and Endocervical Brush
The ectocervix is sampled first using a plastic spatula. The notched end of the spatula that corresponds to the contour of the cervix is rotated 360° around the circumference, retaining the sample on the upper surface. The endocervical brush is inserted in the endocervical canal so that some bristles are still visible, rotated 45°–90°, and removed. Now the sample on the spatula is spread evenly and thinly lengthwise by single uniform motion on one half of glass slide. The endocervical brush is rolled along the remaining half. Forceful smearing in multiple directions is avoided. The entire slide is then rapidly fixed in alcohol and the collection devices are discarded. The vaginal fornix and ectocervix should always be sampled before the endocervix/transformation zone. If the order is reversed, bleeding secondary to abrasion from the brush may obscure the cellular material (Fig. 4.9).
Fig. 4.9
(a) Endocervical brush and (b) Ayres spatula
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