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Definition

The Pap test (Pap smear) is the microscopic examination of cells scraped from the both the outer cervix (ectocervix) and the cervical canal (endocervix). It is called the "Pap" test after its developer, Dr. George N. Papanicolaou, who described a procedure for staining vaginal and cervical cells that gives clearly defined detail to the nuclear chromatin. Using the Papanicolaou stain, he developed a classification system for abnormal cervical cells. Before the application of the Pap test in the 1940s, cervical cancer caused approximately 26,000 deaths in the United States each year. The death rate from cervical cancer since the use of the Pap test has become widely accepted has been reduced by 70%.

Purpose

The Pap test is a screening test used to detect abnormal growth of cervical cells at an early stage, so that if necessary, treatment can be started before the cells become cancerous and invasive. The test helps physicians identify women who are at increased risk of cervical dysplasia (abnormal cells) or cervical cancer. Only an examination of the cervix, and samples of cervical tissue (biopsies) can diagnose precancerous and cancerous changes in the cells that line the uterus, called squamous epithelium.

This microscopic analysis of cells can detect cervical cancer, precancerous changes, inflammation (called vaginitis), infections, and some sexually transmitted diseases (STDs). The Pap test can sometimes detect endometrial (uterine) cancer or ovarian cancer, although it was not designed for this purpose.

Women should begin to have Pap tests at the age of 18 years or whenever they start having sex. Young people are more likely to have multiple sex partners, which increases their risk of certain diseases that can cause cancer, such as human papillomavirus (HPV). The American Cancer Society recommends that a Pap test be done annually for two consecutive negative examinations, then repeated once every three years until age 65 for women without symptoms of gynecologic problems. Many other doctors, however, recommend annual Pap tests for all their patients.

Women with certain risk factors should always have yearly tests. Those at highest risk for cervical cancer are women who started having sex before age 18; those with many sex partners (especially if they did not use condoms, which protect against STDs); those who have had STDs such as genital herpes or genital warts; and those who smoke. Women older than 40 also should have the test yearly, especially in the event of bleeding after menopause. Women over age 60 account for 25% of new cases of cervical cancer and 40% of deaths from this disease. Women who have had a hysterectomy (removal of the uterus) may need to have Pap tests, if the surgery was for cancer, or if the cervix was left in place. Pregnant women should have a Pap test as part of their first prenatal examination.

Women who have a positive test result should be retested more frequently. If atypical squamous cells or low-grade lesions are found they should be tested every four to six months until they have three consecutive normal results. The test should be repeated within two to three months if severe inflammation, infection, or post-menoposal atrophy is found. If atypical cells or low-grade lesions persist, or high-grade lesions are found, colposcopy (examination of the cervix with a magnifying lens) should be performed, and treatment initiated as indicated.

Precautions

The Pap smear is a microscopic evaluation of individual cells, a process that requires interpretation. Differentiation of inflammatory, reactive, and atrophied cells from atypical cells is difficult and cannot always be performed with complete certainty. The test is not 100% sensitive and between 5–10% of cervical abnormalities may be missed. Most false negatives result from poor sample collection (insufficient cervical cells) or poor smear preparation. The finding of abnormal cells on a Pap smear does not mean that the cells were present on previous exams.

The Pap test should be performed in the middle of the menstrual cycle to prevent interference from blood. Sexual intercourse, douching, or the use of vaginal suppositories may affect results. Other factors that can affect test results include: water or lubricant on the specimen from the speculum; blood, mucus, or pus on the slide that obstructs the view of epithelial cells; cell damage during collection; and improper slide fixation. An acceptable smear is one that is correctly labeled with the patient's name, age, and last menstrual period; contains squamous cells covering at least 10% of the slide; and demonstrates the presence of cells from the endocervix and transformation zone. The transformation zone is the area where the squamous epithelium of the ectocervix meets the glandular epithelium of the endocervix.

Description

The Pap test is an extremely cost-effective and beneficial test able to detect about 95% of cervical cancer. According to a report published May 16, 2000 in the Annals of Internal Medicine, the widespread use of this diagnostic procedure decreased the number of cervical cancers in the United Sates from 14.2 per 100,000 in 1973, to 7.8 per 100,000 in 1994. However, the disease still ranks as the ninth-leading cause of cancer deaths in U.S. women.

During the pelvic examination, an instrument called a speculum is inserted into the vagina to open it. A spatula, (Ayre spatula) that is flat at one end and curved at the other so that its contour complements the ectocervix is used to collect the sample. The spatula is firmly rotated using a circular motion to scrape the cells off the ecto-cervix. The flat end can be used to pick up cells which have exfoliated from the rear of the vagina. This procedure, called vaginal pool sampling, is recommended for women in menopause and if signs of inflammation are seen. A tiny brush, pointed spatula, or cotton-tipped swab is used to collect cells from the endocervix. These samples can be mixed and spread evenly on a single glass slide, or a slide or slide section can be used for each. The slide should be dipped in 95% alcohol or sprayed with fixative immediately. Though some women find the procedure uncomfortable, it is usually painless and only takes five to 10 minutes.

A new technique called the Thin Prep is being used by some physicians because it is purported to reduce the false negative rate caused by inadequate smear preparation. For the Thin Prep, the sample is placed in a vial containing a preservative solution. The vial is labeled and sent to the laboratory where a processing instrument disrupts the blood cells and mucus and spreads the decontaminated sample in a thin layer over the slide. Unlike the classical procedure, cells are not left on the collection device. This results in a greater yield of epithelial cells to examine. The staining detail is easier to evaluate because the epithelial cells are not obscured by blood cells or mucus.

Smears are stained with the Papanicolaou stain when they reach the lab. The Pap stain begins with rehydrating the cells in water. The cells are stained with Gill hematoxylin, then dehydrated with 95% ethanol. They are stained with OG-6 followed by EA-65, then fully dehydrated with absolute ethanol. In the last step, they are cleared with xylol, and a coverglass is applied. The entire smear is examined under a microscope. In addition to detecting and classifying abnormalities within the squamous epithelium and glandular epithelium, the smears are also examined for the presence of inflammatory cells (polymorphonuclear white cells, lymphocytes, histiocytes), normal vaginal flora (Lactobacilli), coccobacilli (indicative of vaginal infection), trichomonads (vaginal parasites), yeast, and cytopathic effects of viruses in the epithelial cells.

Squamous epithelial cells from the cervix are evaluated for abnormal intracellular changes that indicate a risk of cancerous transformation. Two systems of classification are widely employed, the CIN (Richart) and Bethesda systems. Both describe a progression of cells from normal to low risk, then to high risk, then to malignant

cells. The CIN system uses the term, cervical intraepithelial neoplasia (CIN) to describe premalignant cells. CIN-I is characterized by mild cellular abnormalities (mild dysplasia), CIN-II moderate dysplasia, and CIN-III severe dysplasia. CIN-III includes the presence of immature cells with cancerous features that have not yet invaded the surrounding connective tissue. This is called carcinoma in situ. When such cells are found beyond the transformation zone (within the underlying stroma), the lesion is classified as invasive cancer (squamous carcinoma). The CIN classification system classifies cells that are most likely benign (called squamous atypica) and low-risk precancerous cells in the category of CIN-I. In 1989, the Bethesda system was introduced in order to more clearly define the difference between mild dysplasia that is likely to be benign and that which is pre-cancerous. The former comprise a group called ASCUS which stands for atypical squamous cells of undetermined significance. This distinguishes cells that are often reactive from those of the next group, low-grade intraepithelial neoplasia (LSIL) that show precancerous changes, but are at a low risk of transforming into cancerous cells. ASCUS is reserved for cells that cannot be conclusively called benign. Classification of a smear as ASCUS is based upon judgement and depends upon the quality of the smear and the numbers and appearance of atypical cells present. A pap test in which ASCUS is found should be repeated in three to four months, and if ASCUS is detected the second time, the patient should be evaluated by colposcopy and biopsy. Between 19% and 57% of these patients will be reclassified as SIL on the

basis of biopsy. The LSIL category is the counterpart of the CIN-I category. The final category of the Bethesda system is high-grade intraepithelial neoplasia (HSIL) which comprises both CIN-II and CIN-III groups including carcinoma in situ. Beyond HSIL, the lesion is classified as an invasive squamous cell carcinoma.

In general, cervical cells are classified as ASCUS if the nuclear enlargement is no greater than three-fold the size of the nucleus of a normal intermediate squamous cell, or there is mild hyperchromasia (increased chromatin staining). LSIL cells are superficial or intermediate squamous cells that display a nucleus that is at least three-fold larger than the normal intermediate squamous cell. There is moderate variation in the size and shape of the nucleus. Nuclear hyperchromasia is present either as uniformly granular or smudged chromatin staining. In addition, cells that are associated with infection by HPV have a cytoplasm with hollowed-out cavities. About 80% of cervical cancers are associated with HPV infection. Therefore, these cells, called koilocytes, are classified as LSIL provided that some nuclear abnormality or binucleation is present. HSIL cells are immature squamous cells (smaller cells) with a three-fold or greater nuclear enlargement, an increased nuclear to cytoplasm ratio, severe hyperchromasia with irregular chromatin and nuclear membrane contour. They are usually seen in streaming rows or groups of attached cells.

Preparation

While most women are not routinely advised to make any special preparations for a Pap test, some simple preparations may help to ensure that the results are reliable. Among the measures that may help increase test reliability are:

• abstaining from sexual intercourse 24 hours prior to the test
• not douching 18–72 hours before the test
• avoiding vaginal creams or medications one week before the test

If possible, women may want to ensure that their test is performed by an experienced gynecologist and sent to a certified laboratory. Certification requires successful participation in a proficiency testing program approved by the U.S. Department of Health and Human Services. In such a program every cytotechnologist reading pap smears is tested at least once per year and is required to meet specific performance criteria.

Before the exam, the physician will take a complete sexual history to determine a woman's risk status for cervical cancer. Questions may include date and results of the last Pap test, any history of abnormal Pap tests, date of last menstrual period and any irregularity, use of hormones and birth control, family history of gynecologic disorders, and any vaginal symptoms. These topics are relevant to the interpretation of the Pap test, especially if any abnormalities are detected. Immediately before the Pap test, the woman should empty her bladder to avoid discomfort during the procedure.

Aftercare

Harmless cervical bleeding is possible immediately after the test; women may need to use a sanitary napkin. They should also be sure to comply with their doctor's orders for follow-up visits.

Complications

No appreciable health risks are associated with the Pap test. However, abnormal results (whether valid or due to technical error) can cause significant anxiety. Women may wish to have their sample retested, either by the same laboratory or via computer-assisted screening. Two re-screening programs approved by the Food and Drug Administration are called Papnet and AutoPap QC.

Results

Normal (negative) results from the laboratory exam mean that no atypical cells were detected, and the cervix is normal. It is important to remember that an abnormal (positive) result does not necessarily indicate cancer. Fully 60–70% of abnormal results resolve by themselves, and only 1% of mild abnormalities ever develop into cancer. Between 19% and 57% of patients with ASCUS will be reclassified as having SIL (mostly LSIL) following biopsy. Approximately 57% of LSIL lesions regress on their own (i.e., return to normal); 32% remain LSIL on retesting, 11% progress to HSIL, and 1% may progress to invasive carcinoma. Approximately 43% of HSIL (CINII) lesions regress, 35% remain HSIL on retesting, and 22% progress to CIN-III. Approximately 5% of HSIL (CIN-II) lesions progress to invasive cancer. Approximately 32% of HSIL (CIN-III) lesions regress, up to 55% persist on repeat exam, and more than 12% progress to invasive carcinoma.

Treatment

CHANGES OF UNKNOWN CAUSE (ASCUS OR SQUAMOUS ATYPICA). The most common abnormality (found in 50–60% of abnormal tests) is ASCUS. If squamous atypica is thought to be inflammatory or reactive, this will be noted on the report as well as any evidence of infection (e.g., coccobacilli, yeast, white blood cells) seen on the microscopic exam. These women may be treated for infection and then undergo repeat Pap testing in two to three months. If ASCUS is present without signs of inflammation, re-testing is performed every four to six months for two years or until three consecutive tests are negative. If the lesion persists, or ASCUS is seen twice within a two-year period, colposcopy is recommended.

DYSPLASIA. Typically, dysplasia causes no symptoms, although women may experience abnormal vaginal bleeding. Because dysplasia can be precancerous, it should be treated if it is moderate or severe. Treatment of dysplasia depends on the degree of abnormality. In women with no other risk factors for cervical cancer, mild dysplasia may be simply observed over time with repeat testing, every four to six months as described above. If the lesion persists, colposcopy with biopsy and scraping of the endocervix are often recommended.

The second most common finding (about 30–40% of abnormal tests) is LSIL, which includes mild dysplasia or CIN I and changes caused by HPV. Unlike cancer cells, these cells do not invade normal tissues. Women are most susceptible to mild dysplasia at ages 25–35 years. HSIL (found in 5–10% of abnormal Pap tests) includes moderate to severe dysplasia or carcinoma in situ (CIN II orIII). The frequency of HSIL is highest at ages 30–40. In women with HSIL lesions, colposcopy, biopsy, and treatment (excision or destruction of the lesion) are performed. In addition to surgical resection (removal), several outpatient techniques are available: conization (removal of a cone-shaped piece of tissue), laser surgery, cryotherapy (freezing), electrosurgical cauterization, and radiation. Cure rates are nearly 100% after prompt and appropriate treatment of carcinoma in situ. Of course, frequent checkups are then necessary.

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KEY TERMS

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Carcinoma in situ—Precancerous cells that are present only in the ectocervix (i.e., do not extend beyond the basement membrane). The abnormal cells do not extend beyond the transformation zone.

Cervical intraepithelial neoplasia (CIN)—A term used to categorize degrees of dysplasia arising in the cervical epithelium (outer cervix).

Cervix—The opening between the vagina and the uterus, or womb.

Cytology—The study of cells, their origin, structure, function, and pathology.

Dysplasia—Abnormal changes in cells.

Human papilloma virus (HPV)—The leading STD in the United States. Various types of HPV are known to cause cancer.

Neoplasia—Abnormal growth of cells, which may lead to a neoplasm, or tumor.

Squamous intraepithelial lesion (SIL)—A term used to categorize the severity of abnormal changes arising in the squamous cells of the cerrvix.

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In addition to abnormal squamous epithelium, abnormal glandular cells from the endocervix may be found. Atypical glandular cells of undetermined significance (AGUS) is used to designate cells that cannot be classified with certainty as benign, precancerous, or cancerous. AGUS should be investigated further to determine the risk of endometrial carcinoma. Malignant glandular cells may also be found on the Pap smear and may result from cervical or vaginal adenocarcinoma. This cancer is uncommon in women under 40, and most common in women over 50 who have postmenopausal bleeding. Malignant glandular cells are more often recovered from vaginal pool sampling or aspiration than from cervical scraping, and therefore, vaginal cell smears should be made along with cervical smears for women in menopause. Hysterectomy is recommended for confirmed cases of endometrial adenocarcinoma.

CANCER. Human papilloma virus (HPV), the most common STD in the United States, may be responsible for many cervical cancers. Cancer may be manifested by unusual vaginal bleeding or discharge, bowel and bladder problems, and pain. The peak ages for cervical cancer are between 45 and 55 years. Biopsy is indicated when any abnormal growth is found on the cervix, even if the Pap test is negative.

Invasive cervical cancer is usually treated with surgery or radiation, or both. Most cases of invasive cervical cancer are treated with radical hysterectomy. Chemotherapy may be used if the cancer has spread to lymph nodes or other organs. Survival rates at five years after treatment of early invasive cancer are about 90%; rates are below 60% for more severe invasive cancer. That is why prevention, risk reduction, and frequent Pap tests are the best defense for a woman's gynecologic health.

Health care team roles

The slides are prepared by a gynecologist. Cytotechnologists, laboratory professionals who specialize in the study of cells, read the Pap smears looking for abnormal cells. Abnormal findings may be reviewed by the laboratory's pathologist.

Victoria E. DeMoranville