Anatomic Pathology:

Specimen Collection for Cervico-Vaginal Smears (Please contact Delta Lab before sending the sample):


The importance of proper specimen collection and submission cannot be overemphasized. At least one half to two thirds of false negatives are the result of patient conditions present at the time of sample collection and submission and the skill and knowledge of the individual who obtains the specimen. The clinical community is responsible for training health care personnel to assure that adequate cervical cytology samples are collected and submitted to the laboratory with appropriate clinical information. The laboratory provides feedback on sample adequacy via individual reports, and may elect to provide summary information regarding patient sampling to its clients.

1- Patient Preparation:

To optimize collection conditions, a woman should:

  • Schedule an appointment approximately two weeks (10-18 days) after the first day of her last menstrual period
  • Not douche 48 hours prior to the test
  • Not use tampons, birth control foams, jellies or other vaginal creams or vaginal medications for 48 hours prior to test
  • Refrain from intercourse 48 hours prior to test
2- Test Requisition:

Under the supervision and guidance of the physician, a laboratory requisition must be legibly and accurately filled out before obtaining the cellular sample. The laboratory requisition is the main communication link between the physician and the laboratory (pathologist). The requisition should request the following information as a minimum. All entries must be in English and date of last menstrual period should preferably in Gregorian and not Arabic calendar.

  • The patient’s name
  • Age and/or date of birth
  • Menstrual status (LMP, hysterectomy, pregnant, postpartum, hormone therapy)
  • Previous abnormal cervical cytology result, previous treatment, biopsy or surgical procedure.
  • Patient’s risk status for developing cervical cancer (if clinically relevant).
  • Source of specimen, e.g. cervical, vaginal

Appropriate clinical history provided by the physician on the requisition should include:

  • Hormone/contraceptive use.
  • Relevant clinical findings (abnormal bleeding, grossly visible lesion, etc.)
3- Labelling Samples:

The glass slide (except specimen for automated machine) must be labeled with a unique identifier, usually the patient’s first and last names, at the time of the collection of the cellular sample. The required information is written in solvent resistant pen or pencil on the frosted end of the slide.

4. Visualization of the Cervix for Collection of an Adequate Sample:

Collection of a cervical cytology specimen is usually performed with the patient in the dorsolithotomy position. A sterile, or single-use bivalve speculum of appropriate size is inserted into the vagina without lubrication. Warm water maybe used to facilitate insertion of the speculum. The position of the speculum should allow for complete visualization of the os and ectocervix.

The transformation zone is the site of origin for most cervical neoplasia and should be the focus of cytology specimen collection. The transformation zone may be easily visualized or maybe high in the endocervical canal. Location varies not only from patient to patient, but in an individual over time. Factors producing variation include changes in the vaginal pH, hormonal changes including pregnancy, childbirth, and menopausal status, and hormonal therapy. In postmenopausal patients or women who have received radiation therapy, cervical stenosis may prevent visualization of the transformation zone. It remains important to sample the Endocervix in these patients. This may require more extensive clinical procedures. If a patient has had a hysterectomy, a vaginal sample is sufficient, with particular attention to sampling the vaginal cuff.

5- Collection Devices:

There are a variety of collection devices available for sampling the endocervix, transformation zone and ectocervix. They include endocervical brushes, wooden and plastic spatulas, and plastic “broom-type” samplers. Plastic spatulas are preferred over wooden since the wooden spatulas retain cellular material. The use of a cotton-tipped swab is NOT recommended, even if the swab is moistened. Cells adhere to the cotton and do not transfer well to the glass slide, which results in an incomplete specimen. Analysis of different sampling methods has shown that overall, the cytobrush and spatula together provide the best specimen for cervical cytology. However, the choice of a particular device is dependent on variations in the size and shape of the cervix and the clinical situation. As stated in D, age, parity, and hormonal status of the patient can affect the exposure of the transformation zone. Previous therapy, such as conization, laser therapy or cryotherapy, can also change the features of the cervix. The clinician ought to consider these factors when choosing a collection device. (Liquid-based methods require the use of collection devices that have been approved by FDA for use with the particular specimen preparation instrument).

6- Techniques for Sample Collection:
– Collection of cervical/vaginal specimens for conventional smear preparation using spatula and endocervical brush.

The vaginal fornix and ectocervix should be sampled before the endocervix/transformation zone. First, a sample of the ectocervix is taken using a plastic (or wooden) spatula. The notched end of the spatula that corresponds to the contour of the cervix is rotated 360 degrees around the circumference of the cervical os, retaining sample on the upper surface of the spatula. Grossly visible lesions, including irregular, discolored or friable areas should be directly sampled and can be placed on a separate slide, especially if the lesion is distant from other collection areas. The spatula is held with the specimen face up while the endocervical sample is collected.

Sampling of the endocervix requires insertion of the endocervical brush into the endocervical canal until only the bristles closest to the hand are visible. The brush is rotated 45-90 degrees and removed. At this time, the sample on the spatula is spread evenly and thinly lengthwise down one half of the labeled slide surface, using a single uniform motion. The endocervical brush is then rolled along the remaining half of the labeled slide surface by turning the brush handle and slightly bending the bristles with gentle pressure. The brush should not be smeared with force or in multiple directions. The entire slide is then rapidly fixed by immersion or spray and the collection devices are discarded.

Note: use of the endocervical brush maybe contraindicated in pregnant patients. Refer to package insert. If the above-described sampling order is reversed, bleeding secondary to abrasion from the brush may obscure the cellular material

– Cell Fixation for Conventional Cervical Cytology

Immediate fixation of the cellular sample, within seconds of specimen collection, is necessary to prevent air-drying. Air-drying obscures cellular detail and compromises specimen evaluation. Immersing the slide in alcohol or spraying with fixative can prevent air-drying artifact.

If the specimen is immersed in alcohol, it may remain in the alcohol (absolute ethyl alcohol) for transport to the laboratory. Alternatively, the specimen can be immersed in alcohol for 20-30 minutes, removed and allowed to air dry, then placed in a container /mailer for transport to the laboratory. The immersion technique requires use of a separate container for each specimen and changing or filtering the alcohol between specimens. Isopropyl or rubbing alcohol is not recommended to be used as fixative.

If a specimen is spray-fixed, only quality controlled cytology fixatives should be used. Hair spray should NOT be used. Whether using a pump spray, aerosol fixative or single application packet, the manufacturer’s instructions on the container and package insert should be followed. Generally, spray fixatives should be 6-10 inches (15-25 cm) from the glass slide when applied.

– Variability in Specimen Collection and Submission Practices

Variations in specimen collection include the use of conventional Pap smear collection on a glass slide/slides or collection in a liquid fixative. Additional variation is encountered in rinsing the collection devices and handling of the devices after specimen has been collected. Manufacturer’s instructions and/or package inserts should be consulted and recommendations followed.

Other variations include the use of different collection devices. The plastic spatula is preferred to the wooden spatula. The endocervical brush is preferred fro sampling the endocervix. Clinical judgment is required to determine the appropriate device, as there is no single sampling device

Another option is to mix the vaginal pool sample with the cervical specimen. This somewhat protects the cellular material from air-drying prior to fixation. Yet another option is to smear the ectocervical specimen on the slide, and then directly roll the endocervical brush on top followed by fixation.

No consensus has been reached on the clinical benefit of one slide versus two slides for cervical cytology. Several comparative studies have been performed and concluded that the single slide method is an acceptable alternative to the double slide method. The single slide method decreases the number of slides screened in the laboratory reduces costs for glass slides, and requires less space for storage.

While this section discusses the consensus of the cytologic community regarding the most appropriate methods of specimen collection and submission, it is not intended to supplant or establish the gynecologic community’s standard of care and practice regarding these issues. Nor is this Guideline intended to diminish the responsibility of clinicians to be aware of and apply the standards applicable to their medical specialty and their individual