Though it is written on the sign

“ DO NO PLUCK THE FLOWER”

It is useless against the wind who can’t read. (By an unknown Japanese poet)

Introduction.

• Pap-test is the procedure used for early detection of intraepithelial lesions and carcinoma of cervix.

• Pap-test is named after George Papanicolaou as he developed the procedure in the 1930’s

Cervix

• Cervix is lower portion of uterus, roughly cylindrical and measuring 3cm in all directions.

• The stroma is consist mainly of fibrous tissue and smooth muscle fibres with branches of uterine vessels.

Cervical lining epithelium.

• Vaginal aspects of cervix is lined with squamous epithelium that turns back towards the posterior vaginal wall, forming fornices.

• Endocervical canal is lined with mucosecreting, monolayered columnar epithelium. Nuclei are vesicular and often contain a small nucleoli and X chromatin body affixed to nuclear membrane.

• The two layers meet at the squamocolumnar junction which is situated at external os.

• Squamocolumnar epithelium is not fixed and moves upward or downwards at different times.

Histology of cervix

• At birth and infancy SC junction is situated at exocervix.

• After menopause it recedes into the endocervical canal.

• At puberty columnar epithelium of the exocervix is gradually replaced by squamous metaplasia. Hence squamous metaplasia is a physiological process and need not be mentioned in diagnosis.

• (Red marked area is SC junction)

Transformation zone

• The area of squamous metaplasia that covers preexisting endocervical glands is called transformation zone.

Squamous epithelium

• Squamous epithelium consists of three layers.• - Parabasal cell layers consists of immature

cells• - Intermediate cells• - Superficial cells containing most mature

cells.• Germinal or basal cells lie directly on

basement membrane and these are small columnar shaped cells.

Parabasal cells

Intermediate and superficial epithelial cells.

Hormonal influence

• Thickness and maturation of the squamous epithelium depends on the hormonal stimulation:

• Estrogen causes maturation.

• Progesteron on the other hand, opposes complete maturation, while causing thickening of the intermediate layer.

Endocervix

• Endocervical lining epithelium is of tall columnar mucous secreting type with basal nuclei, often contain a small nucleolus and the X-chromatin body affixed to the nuclear membrane, which is smooth and regular. When irritated, the columnar cells may display cilia.

• Underneath the columnar layer are few reserve cells which are not easily observed in histology. These reserve cells can multiply and differentiate to produce metaplasia.

Endocervical cells.

Endometrial cells

• Endometrial cells are observed on the smear during first ten days of the cycle. Those are smaller than endocervical cells with scant, sometimes vacuolated, cyonophilic cytoplasm and grouped in tight three dimensional clusters. Nuclei are vesicular with fine chromatin pattern. From day 6th to day 10th of the cycle EGC form tight balls surrounded with pale eosinophilic cytoplasm, originating in endometrial stroma. These has been called `exodus’ by Papanicolaou.

Endometrial cells

Histiocytes

• Small histiocytes are observed at the end of the cycle and during first 10 days of the proliferative phase. Thought to be originated in the endometrial stroma. Equal to the size of parabasal cells, cyaonophilic, foamy cytoplasm. Nuclei are notched, or kidney or bean shaped and there may be 2 or more nuceli. When nuceli are numerous and histiocytes become large they are known as multinucleated giant cells. In absence of inflammation they are scavangers, removes debris and blood after menstruation.

Granulocytes

• Granulocytes are observed at the beginning and end of the menstrual cycle and do not indicate the presence of inflammation.

• Lymphocytes and plasma cells are rarely observed on normal smears.

• Psammoma like concretions are occasionally observed and probably represents inspissated mucus.

When to collect sample :

• The sample to be collected 10-14 days after the 1st day of the last menstrual period.

• Things to be noted before collection

Vaginal medications, lubricants, vaginal contraceptives, or vaginal douches to be avoided 48 hours before sample collection.

Collection Procedure(Conventional Pap smear)

• Supplies: Vaginal speculum, plastic extended-tip or wooden

spatula, cytobrush, fixative or (spray fixative or bottle of 95% ethyl alcohol), clean glass slides.

PROCEDURE:• Label one slide with the details of the patient, and

specimen source • Place the patient in the lithotomy position. • Examine the patient with a dry of slightly water

moistened speculum to give adequate exposure of the entire cervix and upper vagina.

• Excessive mucous from the cervical lips and vaginal canal to be cleaned.

TYPES OF SPATULA

1. AYRE’S SPATULA

2. WOODEN

SPATULA

3. ENDOCERVICAL

BRUSH

Technique

• In V-C-E technique:• Full visualiztion of cervix and upper part of vagina

is necessary• First sample obtained from the posterior fronix

with a wooden tongue depressor or an Ayre spatula. The material is kept on the instrument.

• Then a scraping with a second spatula is made from the exocervix, and special care is taken to sample all of the transformation zone. Again material is kept on the spatula.

• Lastly, the endocervical sample is secured with a cotton tipped applicator or with an endocervical brush.

• Now all these three samples are quickly transferred to a slide.

Technique

• In V-C-E technique:• - First sample

obtained from the posterior fronix with a wooden tongue depressor or an Ayre spatula. The material is kept on the instrument.

Technique

• Then a scraping with a second spatula is made from the exocervix, and special care is taken to sample all of the transformation zone. Again material is kept on the spatula.

Technique

• Lastly, the endocervical sample is secured with a cotton tipped applicator or with an endocervical brush.

Preparation of smears.

• All these three samples are quickly transferred to a slide.

• Endocervical sample is smeared from edge to edge near the end, farthest from the label.

• Exocervical sample is spread in the middle of the slide.

• The posterior vaginal vault material is placed nearest to the label.

V-C-E smears.

• Now all these three samples are quickly transferred to a slide.

• The V-C-E smear provides three distinct samples from the female genital tract, thus reducing the chances of missing a significant lesion.

• At the same time it is economical and practical :• -Only one glass slide is to be stained, screened and

stored for each patient.• Once the smear has been collected and adequately

fixed, it must be sent to cytopreparatory laboratory at earliest, with completed requisition form.

V-C-E smear.

• The sample next to the label is from posterior vaginal vault (V).

• The middle sample is from scraping of the transfromation zone.

• Endocervical sample is farthest from the label.

Specimen Adequacy Satisfactory for evaluation" indicates that the specimen has all of the following:• Appropriate labeling and identifying information• Relevant clinical information• Adequate numbers of well-preserved and well-visualized squamous epithelial cells.• An adequate endocervical / transformation zone component (from a patient with a cervix).• Quality of the Pap smear will still be noted when:• Less than 10 well preserved endocervical or metaplatic cells are seen• Blood or inflammation moderately obscuring the Pap smear

"Unsatisfactory for evaluation"

It designates that the specimen is unreliable for the detection of cervical epithelial cell abnormalities. This term is used if any of the following apply:• A broken slide that cannot be repaired.• Scanty squamous epithelial component (less than 8,000 well preserved and well visualized cells on conventional slides or

less than 5,000 well preserved and well visualized cells on

liquid-based preparations)• Obscuring blood, inflammation, thick areas, poor fixation,

air-drying artifact, contaminant, etc. that precludes interpretation of approximately 75% of the epithelial cells.

Contradictions to perform a Pap Smear

1. Menstruation. 2. If a douche or vaginal medication has

been used in preceding 48 hours. 3. Cryosurgery or cautery within the

previous 3 months.

Some causes of false positive Pap Smears:

• 1. Recent cryocautery(within 3 months). 2. Folic acid deficiency. 3. Corticosteroid medication. 4. Digitalis. 5. Recent (3-6 months) pelvic irradiation.

6. Inflammation or repair related to

infection or recent therapy.

High risk factors related cervical neoplasia:

1. Early age at first coitus. 2. Multiple partners, especially in the teens. 3. First pregnancy under age 20. 4. Multiple early pregnancies. 5. History of sexually transmitted diseases, e.g.

HPV, gonorrhea, syphilis, Herpes Type II, Trichomonas, Chlamydia, Condylomata and Haemophilus.

High risk male exposure.

A. Occupation group of husband is important. Mannual labours have three times

greater risk as compared with professionals.

B. Genital cancers found in other sites, i.e. penile or prostratic malignancies.

C. Sexual promiscuity with or without history of venereal disease.

Factors unrelated to cervical neoplasia:

1. Family history. 2. Menstrual history. 3. Parity per se. 4. Diet. 5. Chronic non-specific cervicitis. 6. Mechanical irritation. 7. Exposure to external carcinogen, e.g., coal tar

products, such as douches or vaginal applications.

How often  should be Pap Smear to be done.

• After first sexual intercourse or at the age of 21 years,  any woman has to undergo Pap-test within 3 years. 

• Annual Pap-test  recommended for the age group of 21-30 years old. After 30 years woman who test negative for three years consecutively,  can have the test after every 3 years.

• High risk patients, with dysplastic and CIN findings, should be followed more closely, at least semi-annually.

Liquid Based Pap smear

• Instead of "smearing“ the cells (as in conventional type) onto a slide, the collected cells are placed in a vial of preservative solution and the vial is sent to the laboratory. The collected cells are placed onto a slide in the laboratory using technology that clears obscuring debris and prepares a thin layer of cells for the cytotechnologist to review under a microscope.

Endocervical cells

• Naked nuclei of endocervical cells in mucus streaks.

Endocervical cells.

• Endocervical cells with anisokaryosis,

Thank you