Specimen Collection, Adequacy & Requisition


Cervical Cytology Practice Guideline

Approved by the ASC Executive Board November 10, 2000



III.   Specimen Collection and Submission

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.394041  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.

III.A.   Patient Preparation

To optimize collection conditions, a woman should:42  

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

III.B.   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.  The requisition should request the following information as required by CLIA ’88.43

  1. Patient’s name (any name change in the past 5 years should be noted.)
  2. Age and/or date of birth.
  3. Menstrual status (LMP, hysterectomy, pregnant, postpartum, hormone therapy.)
  4. Previous abnormal cervical cytology result, previous treatment, biopsy or surgical procedure.
  5. Patient’s risk status for developing cervical cancer, e.g. “high risk”.   The clinician should expect that the laboratory would rely upon the information provided on the current requisition in arriving at an assessment of risk status.  (See section IIB.)
  6. Source of specimen, e.g. cervical, vaginal.

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

  1. Hormone/contraceptive use.
  2. Relevant clinical findings (abnormal bleeding, grossly visible lesion, etc.)

III.C.  Labeling the Sample

The glass slide or specimen vial 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. Individual laboratories may require a second identifier such as date of birth, medical record number, social security number or collection date. The lab must have a written procedure that specifies the requirements for proper specimen identification.  For glass slides, the required information is written in solvent resistant pen or pencil on the frosted end of the slide.   For liquid based samples, the required information must be affixed to the vial.

III.D.  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 may be 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.44   The transformation zone may be easily visualized or may be 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 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.

III.E.  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.45   The use of a cotton-tipped swab is NOT recommended, even if the swab is moistened.41  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.46   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 III.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.41   Liquid based methods require the use of collection devices that have been approved by the FDA for use with the particular specimen preparation instrument.

III.F.  Techniques for Sample Collection


III.F.1.  Collection of cervical/vaginal specimens for conventional smear preparation using the 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 360º around the circumference of the cervical os, retaining the 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 45-90º 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.41,47   The entire slide is then rapidly fixed by immersion or spray and the collection devices are discarded.  Note: use of the endocervical brush may be contraindicated in pregnant patients. Refer to the package insert.  If the above-described sampling order is reversed, bleeding secondary to abrasion from the brush may obscure the cellular material.

III.F.2.  Collection of cervical/vaginal specimens for liquid-based preparations using the spatula and endocervical brush

For liquid based preparations, the ectocervix should be sampled using the same procedure as for conventional Pap smears.  However, the spatula with the cellular material is rinsed in the specimen vial and then discarded.  The endocervical specimen is collected using the same technique as for conventional Pap smears.  However, the endocervical brush is rinsed in the vial and then discarded.  Manufacturers’ directions must be followed.48

III.F.3.  Collection of cervical/vaginal specimens for conventional smear preparation using the “broom-like” device

The ectocervix and endocervix are collected simultaneously with the “broom-like” device.  The central bristles of the broom are inserted into the endocervical canal until the lateral bristles bend fully against the ectocervix.  The sampling device is rotated 360º in the same direction five (5) times while maintaining gentle pressure.  The broom is removed and with a single paint stroke motion the cellular sample is transferred down the long axis of the labeled surface of the slide.  The broom is turned over and the paint stroke motion is repeated over the same area.  The slide is rapidly fixed either by immersion or spray and the device is then discarded.

III.F.4.  Collection of cervical/vaginal specimens for liquid-based preparations using the “broom-like” device

The ectocervical and endocervical specimens are collected with the “broom-like” device simultaneously.  The central bristles of the device are inserted into the endocervical canal until the lateral bristles fully bend against the ectocervix.  Maintaining gentle pressure, the broom is rotated in a clockwise direction 360º for a total of five (5) times.  The broom is then rinsed in the specimen vial. Manufacturers’ directions vary and must be referred to and followed.48,49  

III.G.  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 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.50    The immersion technique requires use of a separate container for each specimen and changing or filtering the alcohol between specimens.

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.41

III.H.  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 the specimen has been collected. Manufacturers’ 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 for sampling of the endocervix.  The “broom-like” device is also available.  Clinical judgment is required to determine the appropriate device, as there is no single sampling device that is optimal for all clinical circumstances. 

There is variation in placement of the vaginal, ectocervical and endocervical samples on the glass slide.    For VCE slides, the vaginal sample is collected first and placed on the slide near the frosted end within the section labeled “V”.   The ectocervical specimen is then collected and smeared within the section of the slide labeled “C”.  The endocervical specimen is collected last, and smeared within the section of the slide labeled “E”. The slide is then rapidly fixed.  Another option is to mix a 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.5152

While this section discusses the consensus of the cytologic community regarding the most appropriate and effective 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 patients. 

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39  McGoogan E, Colgan TJ, Ramzy I et al.  Cell preparation methods and criteria for sample adequacy: IAC Task Force Summary. Acta Cytol 1998; 42:25-32.

40  Vooijs GP, Elias A, Van der Graaf Y, Poelen-van de Berg M.  The influence of sample takers on the cellular composition of cervical smears. Acta Cytol 1986; 35:251-257.

41  Thompson D.  Adequate “Pap” Smears: A guide for sampling techniques in screening for abnormalities of the uterine cervix.  Laboratory Proficiency Testing Program of Canada, 1989.

42  American Cancer Society, Atlanta, Georgia.  Web site: http:\www.cancer.org

43  Clinical Laboratory Improvement Amendments of 1988; Final Rule.  Federal Register.  Feb. 28, 1992; Vol. 57:  493.1105.

44  NCCLS.  Papanicolaou Technique; Approved Guideline.  NCCLS Document 15-A (ISBN 1-56238-238-1) Villanova, Pennsylvania:  NCCLS; 1994; 14:  no. 8.

45  Rubio CA.  The false negative smear. Obstet and Gynecol 97; 49:576-580. 

46  Boon ME, Guilloud JC, Rietverd WJ.  Analysis of five sampling methods for the preparation of cervical smears.  Acta Cytol 1989; 33: 843-848.

47  Luzzatto R, Boon ME.  Contribution of the endocervical cytobrush sample to the diagnosis of cervical lesions.  Acta Cytol 1996; 40:1143-1147.

48  Cytyc Corporation, 85 Swanson Road, Boxborough, Massachusetts, 01719

49  TriPath Imaging, Inc. 700 Plantation Drive, Burlington, North Carolina, 27215

50  Somrak TM, Sorensen K, Abdul-Karim F.  Pap smear: Collection, handling and quality assurance.   Chicago: ASCP Press; 1990.

51  Saitas VL, Hawthorne C, Cater J, Bibbo M.  Single slide versus double slide: A comparative study. Diagn Cytopathol 1995; 12:317-320.

52  Quakenbush S R. Single slide Pap smear: An acceptable alternative to the double-slide Pap smear.  Diagn Cytopathol 1999:20:317-320.