The cost-effectiveness of cytologic screening for vaginal neoplasia after removal of the cervix for benign disease has not been demonstrated. Nonetheless, periodic cytologic evaluation of the vagina in such cases, based on the above risk factors is warranted.

Methods of Collection:

In the recent past, adaptation in the collection and preparation methods, new methods of interpretation, and the introduction of adjunctive tests have been shown to improve the sensitivity of the Pap test without markedly decreasing its specificity. Originally, cervical cells samples were obtained from the posterior vaginal pool. It has been shown repeatedly that this is a poor source of cells and that the sample should be collected only from the portio and endocervix. Cervical cytology samples should be obtained using either a spatula and endocervical brush or an instrument that can collect cells from both the ectocervix and endocervix.

Slide Preparation:

The Traditional Method: the conventional method of preparation for the laboratory is to spread the collected material on a glass slide and to fix it immediately either by spraying the slide or by immersing it in vial of preservative. To avoid air-drying errors in the interpretation the slide should be fixed right away. Sometimes the smears placed on the glass slide are thick and difficult to analyze.

The ThinPrep: the liquid-based system uses a proprietary fixative into which the clinician places the collection devices. The devices are agitated for a prescribed period, and the vial containing the fixative is sealed, labeled, and forwarded to the laboratory where the slide actually is prepared. The U.S. Food and Drug Administration (FDA) allows the ThinPrep technique to be marketed as better able to detect both low-grade and high-grade squamous intraepithelial lesions (LSIL and HSIL, respectively) than the conventional method of preparation (3).

The SurePath System: it also uses a proprietary preservative solution into which a supplied collection device is placed. The liquid and the collection device are sent to the laboratory. The FDA has approved this technique for use and allows it to be marketed as equivalent to the conventional Pap test. Several other methods of cell collection and preparation currently are in development. None of these had yet received FDA approval.

Automated Slide Interpretation:

Because the interpretation of cervical cytology is labor intensive, there has been great interest in the development of computer-based systems that can read cervical cytology accurately. The FocalPoint slide profiler has been approved by the FDA for primary screening of cervical cytology. This device identifies up to 25% of slides as negative for CIN for which no human review is required (4). Because computerized interpretation may decrease turn-around time and can potentially be cost saving, a number of corporations are developing slide interpretation products. The Bethesda 2001 Terminology Committee suggested that whenever a slide is prepared or interpreted using any form of automation, that fact should be mentioned in the report that is returned to the clinician.

Various Diagnostic Terminologies:

Traditional Nomenclature	CIN Nomenclature	Bethesda System Nomenclature (SIL)
Squamous atypia	Squamous atypia	ASCUS
Condyloma	Condyloma	Low-grade SIL
Mild dysplasia	CIN I	Low-grade SIL
Moderate dysplasia	CIN II	High-grade SIL
Severe dysplasia	CIN III	High-grade SIL
Carcinoma-in-situ (CIS)	CIN III	High-grade SIL

I. Cytology of Squamous Epithelium:

During normal maturation, the squamous epithelium of the uterine cervix can be conceptualized as differentiating from basal/reserve cells to parabasal cells to intermediate cells to superficial cells. These four cells are the keys to the most common daily diagnostic problem in cytology. The cytoplasm provides information about the origin and functional differentiation of a cell. For this reason, cytoplasmic features are used to determine the degree of squamous differentiation. The cytoplasmic hallmarks of squamous differentiation are distinct cell boundaries and the accumulation of dense cytoplasm. The nucleus provides information about the health of the cell (whether it is normal, inflamed, hyperplastic, or neoplastic). Nuclear features determine where in the continuum of neoplastic transformation, or carcinogenesis, the cell may be. Changes in nuclear size, configuration, and chromatin (hyperchromasia, coarsening, and eventually irregular distribution) and the appearance of visible nucleoli are the main nuclear features of ensuing carcinogenesis.

Matrix of Diagnostic Categories click to enlarge

Inflammatory Changes and Specific Infections:

Pruritis and vaginal discharge are among the most common reasons that a woman seeks medical advice from her gynecologist. Although Pap smear can be useful in identifying specific infectious agents, it should not be used in lieu of more effective diagnostic tests. Many things can cause inflammation; the mere presence of inflammatory change on Pap smear is a poor indicator of the presence of infection. Inflammatory change can mimic dysplasia. Patients with persistent inflammatory change are at high-risk for a bona fide squamous abnormality (cervical intraepithelial neoplasia/ squamous intraepithelial lesion). Differential Diagnosis of Dysplasia vs Inflammatory Change: Inflamed nuclei are big, but not dark; or dark, but not big. Red nuclei indicate inflammation. Dysplastic nuclei are big and dark. Blue nuclei indicate true dysplasia. Dysplasia vs inflammation: matter of degree: more pleomorphic, larger nuclei; more irregular nuclear outline; more abnormal chromatin crisp and distinct; more cellular disorder. Halos: inflammatory vs kilocytotic (5).
Trichomonas Vaginalis: this is an oval or pear-shaped organism that varies from 8 to 30 micro m. The trichomonad nucleus (thin, elliptical) must be identified to diagnose this infection. Red granules in cytoplasm may be seen. Slightly enlarged, dark nuclei and perinuclear halos are common, mimicking low grade dysplasia.
Leptothrix: it is mixed lactobacilli. The organisms are long, thin (less than half as thick as Candida) and flexible. If leptothrix is present, Trichomonas is usually present, but reverse is not true.
Candida Species: it is associated with a change in vaginal glycogen flora or pH. For example: Pregnancy, late luteal phase of cycle, diabetes mellitus, immunosuppression, debilitating disease, steroids, birth control pills, broad spectrum antibiotics, chemotherapy are associated with candida infection. Pseudohyphae (sticks) and yeast (stones) are seen.
Actinomyces: it is associated with IUD use; rarely is associated with other foreign objects (tampons or pessaries). The patient may be asymptomatic or have pelvic pain. Cytologic findings are colonies of variably gram-positive, long, thin, filamentous bacteria that are reddish, branch are irregularly beaded, and radiate from central area.
Herpes: it can be asymptomatic or present as blisters, which can ulcerate and be painful. Herpes simplex types I and II are morphologically indistinguishable. The most characteristic cells are multinucleated and the nuclei mold each other. The nuclei are enlarged and the chromatin marginates, resulting in a ground glass appearance. There may be red nuclear inclusions. Late changes are characteristic and diagnostic; early changes can mimic CIN III. Patients with herpes infection are in high-risk group for CIN/SIL.
Gardnerella vaginalis (Bacterial Vaginosis): it is a gram-negative, comma-shaped coccobacillus. The bacteria tend to agglomerate onto squamous cells (clue cells). Smears usually show a characteristic granular blue background of small coccobacilli, but the background is otherwise clean, often accompanied by slight parakeratosis.
Chlamydia trachomatis: it is an obligate intracellular bacterium that is associated with granular cytoplasmic inclusions. It is the most common cause of non-gonococcal urethritis/ cervicitis. It may cause 20 to 25% of cases of pelvic inflammatory disease, which can result in infertility and ectopic pregnancy. Chlamydia trachomatis is frequently asymptomatic. The value of Pap smear in diagnosing Chlamydia is uncertain. Fine vacuolization of metaplastic cells (having a "moth eaten" appearance) may correlate with high risk of infection. Nebular bodies though rare and difficult to see, may be more specific. Chlamydia changes can mimic low-grade dysplasia.

II. Cytology of the Glandular Epithelium:

The glandular epithelium of the female genital tract includes the lining of the endocervix, endometrium, and fallopian tube. Pap smear is not nearly as good a screening test for glandular lesions as it is for squamous lesions. Endocervical cells are tall and columnar, and can be secretory or ciliated. Endocervical cells can be seen singly or in strips or sheets. See pictures below:

click to enlarge

Endometrial cells can also form three-dimensional clusters of glandular cells, without central stroma. The cells are small and crowded, and the nuclei are usually degenerated and hyperchromatic can mimic carcinoma in situ. A common everyday problem in Pap smear diagnosis is distinguishing endometrial cells from endocervical cells. This can be important, since shedding of endometrial cells is abnormal in the second half of the menstrual cycle (especially past 40 years of age) or any time in postmenopausal women. Abnormal shedding of endometrial cells carries with it an increased risk of endometrial hyperplasia or neoplasia. See picture below:

click to enlarge

Summary:

Although the Pap smear can fail at many levels, ironically, the single most important error is failure of women to get Pap smears in the first place. Cervical carcinoma is preceded by a long prodrome of preinvasive disease that can be detected and treated, which should make it possible to prevent this cancer. The Pap smear is only a screening test for cervical cancer; it has a low, but significant diagnostic error rate. A "negative" report does not guarantee the absence of cervical cancer. Close surveillance of high-risk patients, including those with multiple infections and heavy inflammation, is important. All abnormal Pap smear results should be followed up, and of great importance suspicious lesions should be biopsied and suspicious symptoms investigated, even when the Pap is negative.

References:

1. ACOG Technology Assessment in Obstetrics and Gynecology. No 2, December 2002.
2. ACOG Committee opinion. No 152, March 1995.
3. Berstein SJ et al. Liquid-based cervical cytologic smear study and conventional Papanicolaou smears: a meta-analysis of prospective studies comparing cytologic diagnosis and sample adequacy. Am J Obstet Gynecol 2001;185:308-17.
4. Alosia LM et al. Performance of the AutoPap primary screening system in the education of the AutoPap primary screening system in the detection of high-risk cases in cervico-vaginal smears. Acta Cytol 2001;45:704-8.
5. The Art & Science of Cytopathology by Richard M DeMay.

All images: The Art & Science of Cytopathology by Richard M DeMay.