THE PAPANICOLAOU SMEAR

AAFP Scientific Assembly - 1994

E.J. Mayeaux, Jr., M.D.
Assistant Professor of Family Medicine
Clinical Assistant Professor of Obstetrics and Gynecology
Louisiana State University Medical Center - Shreveport, Louisiana

Goals & Objectives

• Understand the various past systems of reporting pap smears.
• Gain familiarity with the new Bethesda system.
• Identify the lesions likely to be missed on pap smears.
• Learn the various techniques for obtaining a pap smear.

Background

• History of Cervical Cancer Screening Techniques
• 1925 - Hans Hanselman introduces colposcope.
• 1926 - Aureli Babes publishes on cytologic screening. Little publicity.
• 1928 - Papanicolaou presents findings on cytologic screening.
• 1941 - Papanicolaou publishes on cytologic screening. Widely read and accepted.
• 1955 - Scheffey introduces colposcopy to US.

• Epidemiologic Proof for Cervical Ca Screening - Why we do it...
• MacGregor (1976):
  Screened women - invasive cancer rate = 30-50/100,000
  Unscreened women - invasive cancer rate = 310/100,000
• Fidler (1968):
  Screened women - invasive cancer rate = 5/100,000
  Unscreened women - invasive cancer rate = 29/100,000
• Walton (1976):
  Strong correlation between screening intensity and cervical cancer mortality .
• Adami (1994):
  "Cytologic screening reduces mortality from cervical cancer by earlier diagnosis of invasive disease" Cancer 1994; 73:140-7.

"... a majority of women diagnosed with invasive carcinoma of the uterine cervix do not receive routine pap smears..." NY State J of Med

• Epidemiology of Cervical Cancer
• 20,000 new cases per year
• 7,600 deaths per year
• 3.5% of all female deaths per year
• One death every 53 minutes

It is the clinician's responsibility to understand the implications of the cytology report, no matter the classification used, and monitor the quality of the service.

Pap Smear Terminology: Previous and Current Systems

* CIN = cervical intraepithelial neoplasia.

B. Pap Classes Are Out Because:

• Do not reflect current understanding of pathology
• Classes not transferrable to histology terms
• No classes for non-cancerous entities
• No longer uniform
• Years of experience have demonstrated a lack of reproducibility in assessing cervical lesions to mild, moderate, or severe dysplasia

The 1991 (revised) BETHESDA SYSTEM

Important Changes Over Old Systems:

• Pap considered a Medical Consult
• Pathologist responsible for diagnosis
• Referring physician provides history
• Must have a statement of adequacy
• Recommendations regarding follow-up should be made by pathologist

The Bethesda System Report Includes:

• Whether the pap is an adequate sample.
• If there is infection.
• Low grade lesions (Condyloma and CIN I).
• High grade lesions (CIN II, III, and CIS).
• Cancer.

Only Two New Classification Terms:

1. "Low-grade squamous lntraepithelial lesion" (low-grade SIL)
   Cellular changes associated with HPV
   Mild (slight) dysplasia/CIN 1
2. "High-grade squamous intraepithelial lesion" (high-grade SIL)"
   Moderate dysplasia/CIN II
   Severe dysplasia/CIN III
   carcinoma in situ/CIN III

Endocervical cells

1. Used to determine the adequacy of a pap smear.
2. Currently considered an important factor in assessing adequacy of pap smear.
3. Statistically significant relationship between patient age/fertility state and yield of endocervical cells.
4. No significant association between parity or phase of menstrual cycle and yield.
5. Significant association of these cells with documented presence of red blood cells on the smear.
6. No support to the hypothesis that inflammation is linked to the presence or absence of these cells.

Koilocytes - Cellular evidence of HPV infection - findings:

• Perinuclear halo
• Denser than normal chromatin
• Altered nuclear - cytoplasmic ratio
• Abnormal mitotic figures

Miscellaneous

1. "Atypia" is used only if undetermined significance and should include a recommendation for follow-up.
2. Descriptive diagnosis should be given to common problems (changes due to trich, yeast, etc.)
3. Metaplasia - The physiologic conversion of columnar endocervical cells to flat exocervical squamous cells. Norman finding - no special follow-up needed.
4. "Parakeratosis" is a term for the persistence of the nuclei of the keratinocytes into the stratum corneum (horny layer) of the skin. Parakeratosis is normal in the epithelium of true mucus membranes of the mouth and vagina.
5. "Dyskeratosis" is a term for abnormal, premature, or imperfect characterization of the keratinocytes.
6. Hyperkeratosis implies increased keratin in the sample and should be followed-up closely since there may be an increased risk of cancer. J Fam Pract 1993; 33:354-8.

POINTS ABOUT THE PAP

Highly effective for screening only.

1. Not diagnostic
2. Identifies those at risk
3. 20-25% false negative rate
4. No patient with an HPV induced lesion detected on cytology may be assumed not to have a lesion greater than that.
5. Therefore, all positive paps need colposcopy.

HPV found in 10% of women between 15 and 50 years of age with normal cervical cytology. Lancet 1987 2:703.

Whatever explanations for a false negative rate, It is now accepted that the (pap) smear does not reflect... histology, ...and follow-up with cytology alone of patients with abnormal smears, can be most unreliable. Chanen 1990, AUST NZ J Obstet Gyn

Inadequacies in Pap Smear Screening

1. False negative Paps - 5-50% with 10 - 20% average.
2. Failure to identify high risk patient at entry.
3. Inaccurate or incomplete reports from the lab to clinic to patient.
4. Lack of adequate tracking and follow-up.
5. Poor patient compliance.

Summary of lesions missed by Pap:

1. Occur outside of a large eversion.
2. Small lesions.
3. Advanced invasive lesions since they have infection and necrotic tissue, which can obscure the true cytology. Koss, JAMA, February 1989
4. Rapidly progressive lesions.
5. Lesions deep in the cervical canal.

Factors That Diminish the Accuracy of Pap Smears

Clinician Related Factors

• Contamination with blood or lubricants
• Mislabeled or unlabeled slides
• Inadequate clinical history
• Inadequate sampling of the transformation zone
• Slide material too thick or insufficient
• Performing pap in spite of obvious infection

Laboratory Related Factors

• Confusing smears or names
• Failure to identify dysplastic cells
• Misinterpretation of diagnostic cells
• Poorly controlled technical process

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Guidelines for Obtaining a Pap Smear

• Visual inspection of the lower genital tract and cervix through the speculum is a prerequisite to optimal sample collection.
• The location and appearance of the transformation zone is variable depending on such factors as vaginal pH, pregnancy, hormonal mileu, prior therapy, and individual anatomy.
• An optimal cervical specimen includes sampling of the squamous and columnar epithelium, encompassing in particular the transformation zone, where the majority of cervical neoplasias arise.

Collecting the Pap Smear

• Label the frosted end of the glass slide with the patient's name prior to sample collection.
• Insert the speculum, which may be slightly moistened with water or saline if necessary.
• No other lubricants should be used.
• Visually inspect the cervix for abnormalities.
• Identify the transformation zone, if visible, and direct sampling efforts to encompass this area.
• The endocervical limit of the transformation zone is dynamic, defined by the leading edge of the migrating squamo-columnar junction.
• In post menopausal women, it is often high in the endocervical canal and no longer visible.

Collecting the Pap Smear - Spatula

• Choose the contoured end of the spatula which best conforms to the cervix and the transformation zone.
• Rotate the spatula 360o about the circumference of the cervix, while maintaining firm contact with the epithelial surface.
• With a clockwise rotation beginning and ending at 9 o'clock (or counter-clockwise rotation from 3 o'clock to 3 o'clock), the collected material is retained on the upper horizontal surface as the instrument is removed.
• Do not smear the sample at this time unless you immediately fix the specimen.
• Hold the spatula between the fingers of the non-sampling hand (or rest it on the glass slide with the specimen face-up), while the cervical brush material is collected.
• Spread the material collected on the spatula evenly over the slide with a single smooth stroke motion.

Collecting the Pap Smear - Cervix Brush

• These brushes have circumferential bristles that come into contact with the entire os surface on insertion.
• The brush need only be turned 1/4 turn.
• Roll the brush across the slide by twirling the handle.
• Not currently recommended for pregnancy.

Collecting the Pap Smear

• The object is to quickly but evenly spread the cellular material in a monolayer on the slide.
• Thin out large clumps of material as much as possible, while avoiding excessive manipulation which can damage cells.
• Transfer material from both sampling instruments to the slide within a few seconds and fix immediately in order to avoid air-drying artifact.
• Immediately fix the specimen by either immersing the slide in 95% ethanol or coating the slide with a surface fixative.

H. Spatula & Brush - 3 Options for Transferring Material:

Option #1

• Smear the spatula on the upper half of the slide.
• Roll the brush across the lower half of the slide.
• Immediately fix slide.

Option #2

• Smear the spatula sample across the slide
• roll the brush material directly over the previously spread sample.
• Immediately fix slide.

Option #3

• Smear the spatula sample over the left-hand side of the slide
• cover the right-hand side with cardboard and immediately spray fix.
• Roll the brush material onto the right-hand side of the slide and spray fix.

Collecting the Pap Smear - Plastic "Broom"

• Another collection instrument, a plastic "broom-like" brush (Cervex-brush or Papette), simultaneously samples the endocervix and ectocervix.
• To use the "broom," insert the long central bristles into the os until the lateral bristles bend against the ectocervix.
• Rotate 3-5 times in both directions.
• To transfer material, stroke both sides of the "broom" across the glass slide.
• Place second stroke exactly over the first stroke.

Collecting the Pap Smear - Cotton Swab

Except in pregnancy, the use of a cotton tip applicator is not recommended. It usually provides less cellular samples, possibly because of trapping of material in the cotton fibers.

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Screening Intervals

A. Screening Intervals vs Cervical Cancer Incidence

• Q 1 year = 1.00
• Q 2 years = 1.01
• Q 3 years = 3.90 Obstet Gynecol 1989; 74:838

ACOG, AAFP, and ACS Recommendations (1988)

"...women who are, or have been sexually active, or have reached age 18 years, have an annual Pap test and pelvic examination. After a women has had three or more consecutive satisfactory normal annual examinations, the Pap test may be performed less frequently at the discretion of her physician."

RISK FACTORS FOR DEVELOPING CERVICAL CANCER

• Three or more lifetime sexual partners...... Some say more than one in a lifetime...
• First sexual intercourse before age of 18
• Genital warts (papilloma virus) or other venereal diseases
• A sexual partner with condyloma or a history of genital warts
• A sexual partner who has had intercourse with a woman who developed cervical cancer, abnormal Paps, or had genital warts
• Smoking
• Maternal DES (diethylstilbesterol) exposure during pregnancy
• A previous abnormal Pap smear

RISK FACTORS - Conclusions

Only a small percentage of women are classified as low risk. The vast majority of women have one or more risk factors and are considered at greater risk for developing cervical cancer. Due to this trend, most women should be screened at least once a year, as a general rule, to ensure early diagnosis of the disease.

Rarely do I ever recommend going longer than 1 year between examinations. We now know the pap can miss up to 25% of lesions, even when done correctly. Jack Pfenninger

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The 1991 Bethesda System

Adequacy of the specimen
Satisfactory for evaluation
Satisfactory for evaluation but limited by . . . (specify reason)
Unsatisfactory for evaluation . . . (specify reason)

General categorization (optional)
Within normal limits
Benign cellular changes (see descriptive diagnosis)
Epithelial cell abnormality (see descriptive diagnosis)
Descriptive diagnoses
Benign cellular changes

Infection
Trichomonas vaginalis
Fungal organisms morphologically consistent with Candida species
Predominance of coccobacilli consistent with shift in vaginal flora
Bacteria morphologically consistent with Actinomyces species
Cellular changes associated with Herpes simplex virus
Other

Reactive changes
Reactive cellular changes associated with inflammation (includes typical repair), atrophy with inflammation ("atrophic vaginitis"), radiation, intrauterine contraceptive device, other

Epithelial cell abnormalities
Squamous cell
Atypical squamous cells of undetermined significance (qualify)
Low-grade squamous intraepithelial lesion encompassing: HPV mild dysplasia / CIN **
High-grade squamous intraepithelial lesion encompassing: moderate and severe dysplasia, CIS/ CIN 2 and ClN 3
Squamous cell carcinoma
Glandular cell
Endometrial cells, cytologically benign, in a postmenopausal woman
Atypical glandular cells of undetermined significance (qualify)
Endocervical adenocarcinoma
Endometrial adenocarcinoma
Extrauterine adenocarcinoma
Adenocarcinoma (not otherwise specified)
Other malignant neoplasms: specify

Hormonal evaluation (applies to vaginal smears only)
Hormonal patten compatible with age and history
Hormonal pattern incompatible with age and history (specify)
Hormonal evaluation not possible due to (specify)

HPV = human papillomavirus
CIN = cervical intraepithelial neoplasia
CIS = carcinoma in situ.
* -Atypical squamous or glandular cells of undetermined significance should be further qualified as to whether a reactive or a premalignant / malignant process is favored.
**-Cellular changes of HPV-previously termed koilocytosis, koilocytotic atypia or condylomatous atypia are included in the category of low-grade squamous intraepithelial lesion.

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