Gyenocologic Pap Test Collection Procedure


University of Virginia Health System

Department of Medical Laboratories

Division of Cytopathology

  Revised October 2010

Revised September 2012




A Pap test is a screening test that can also be a diagnostic test. Its primary purpose is to identify patients who have cellular changes that place them at risk for the development of cervical cancer. The Pap test is a highly complex laboratory test requiring careful patient preparation, skill in clinical collection, highly skilled and controlled laboratory processing, and professional interpretation.  Like all laboratory tests, the Pap test is not perfect. A Pap test is best viewed as a moderately sensitive, highly specific test with an established false negative rate of no less that 5%.

In cases of routine screening for cervical cancer and its precursors, cellular material can be obtained from the uterine cervix using a variety of sampling devices. Most clinics at the University of Virginia Health Sciences Center use the spatula and/or an endocervical brush. The cervix and the area adjacent to the cervix (fornices) must be fully visible.  The ecto- and endocervix should be sampled separately. The material obtained can be rinsed into liquid medium (ThinPrep Pap test), our preferred method, or, only exceptionally, spread on one or two glass slide slides (conventional Pap smear).

ThinPrep Pap test supplies are available from the Hospital Storeroom: Item ID 92189 tray of 25 Thinprep Pap test vials and Item ID 92190 bag of 25 collector sets (spatulas and brushes). Cervex broom collectors and conventional Pap test supplies are available through Laboratory Customer Service at 924-LABS. Carefully read the manufacturer's instructions for proper applications and all contraindications prior to using the cytobrush.



1. The patient should NOT make an appointment for her Pap test during her menstrual period. The preferred time for the examination is two weeks after the first day of her last menstrual period.

2. The patient should be instructed NOT to use vaginal medications, vaginal contraceptives, lubricants, or douches for 48 hours before her appointment. Intercourse is not recommended the night before the examination.


1. The physician should not use any lubricant during his examination prior to collecting the cell sample.

2. Every effort is made to identify and sample the squamo-columnar junction and/or transformation zone since the majority of squamous lesions arise within this area.

3. Excess blood, mucus, or inflammatory exudate may be gently blotted away with a gauze pad. Do not scrape or wash this material away since such actions may adversely affect the subsequent cellular sample.

4. The Pap test should always be taken prior to other testing (e.g. cultures, tissue sampling, application of acetic acid, etc.)

The ThinPrep Pap test requires unique specimen collectors and a special collection fluid. Some physician training in proper collection techniques is also needed. To arrange training, contact Laboratory Customer Service at 924-LABS. In addition, an instructional video on ThinPrep Pap test collection is available from the Cytology Laboratory (924-2770) and on-line from Hologic Corporation (see #9 below).


1.  Assemble collection devices and ThinPrep Pap test vial.

2.  Obtain an adequate sampling of the ectocervix using a plastic spatula. (A wooden spatula is not suitable as the cellular material is more difficult to rinse into the collection fluid.)

3.  Rinse the spatula into the solution vial by swirling the spatula vigorously in the vial 10 times. Discard the spatula.

4.  Obtain an adequate sampling from the endocervix using an endocervical brush device. Insert the brush into the endocervical canal until only the bottom-most fibers are exposed. Slowly rotate ¼ or ½ turn in one direction. DO NOT OVER ROTATE.

5.   Rinse the brush in the solution vial by rotating the device in the solution 10 times while pushing against the vial wall. Swirl the brush vigorously to further release material. Discard the brush.

6.   Tighten the cap so that the torque line on the cap passes the torque line on the vial.

7.   Record the patient’s name and history number on the vial or attach a printed patient identification label. Complete a cytopathology test request form (in preprinted paper form or test order printed from Epic).

8.   Send labeled specimen vial and completed request form to the laboratory for processing. No refrigeration of the specimen is necessary.

9.   On-line video available:



NOTE: Currently 99.9% of Pap tests are ThinPrep, which is the expected sample type.

1. Using a lead pencil, write the patient's name and/or medical record number on the frosted end of each glass slide to be used. Use a diamond point pencil to label plain glass slides. All slides submitted to the laboratory must be clearly labeled with the patient identification information at the time of specimen collection. Paper labels affixed to the slides are NOT an acceptable means of slide identification. Federal regulations require that unlabeled slides must be returned to the sender for identification and correction. Laboratory personnel cannot correct this error.

2. Have spray fixative nearby to allow for immediate fixation of the prepared slides. Review and follow the manufacturer's instructions for correct application. 

3. Under direct vision, sample the ectocervix and endocervix separately using the appropriate sampling device. Prepare one combined slide or two separate slides.

a.        To prepare a single combined smear, sample the endocervix first using the endocervical brush.  Insert the brush into the external os and gently rotate the device using ¼ or ½ turn to obtain the cell sample (over rotation may cause bleeding).  Do not smear, but allow the material to remain on the brush.  Scrape the ectocervix with the spatula and spread the material rapidly onto the upper end of the slide.  Quickly roll the endocervical brush through the ectocervical material to the end of the slide.  Perform this technique as rapidly as possible to prevent drying artifacts.  Immediately spray fix by thoroughly soaking the cellular sample while holding the spray fixative container about 6-8 inches from the slide. Allow spray fixative to evaporate.

b.         To prepare separate slides, rotate the spatula with pressure over the entire ectocervix. Spread the cellular material evenly and rapidly across the glass slide being careful to avoid the labeled area. Immediately spray fix by thoroughly soaking the cellular sample while holding the spray fixative container about 6-8 inches from the slide. Allow spray fixative to evaporate.

 For the second slide, insert the endocervical brush into the external os and obtain the cell sample as previously described.  Remove the brush and roll it across the slide to remove the cellular material. Rubbing or scraping the brush across the slide creates artifacts that may hamper evaluation. Immediately spray fix by thoroughly soaking the cellular sample while holding the spray fixative container about 6-8 inches from the slide. Allow spray fixative to evaporate.

4. Place fixed slides in an appropriate container designed to prevent breakage for transport to the laboratory. Complete a cytopathology test request form (in preprinted paper form or test order printed from Epic).

5.  Deliver specimen slides and request form to the laboratory at the convenience of the submitting physician or department.


IN PATIENTS AT RISK FOR ENDOMETRIAL DISEASE, CERVICAL SAMPLES ARE USUALLY INADEQUATE TO SENSITIVELY DETECT ENDOMETRIAL LESIONS. Aspiration of material from the vaginal pool may provide some information; however, more direct sampling is preferred. There are several procedures for obtaining endometrial material, all of which require specialized skills and equipment. Consultation with clinicians in the Department of Obstetrics and Gynecology who are familiar with such procedures is recommended prior to specimen collection.



A cytopathology test request form (in preprinted paper form or test order printed from Epic) containing the following required information must accompany each specimen:

  1. Patient name, patient history number (medical record number), and date of birth (age is not sufficient as a means of patient identification).
  2. Name of the ATTENDING physician or authorized nurse practitioner requesting the test. A resident may be listed only if accompanied by the name of a staff physician.
  3. Clinic, floor, or service generating the specimen.
  4. Date of specimen collection.
  5. ICD-9 code indicating the medical necessity for the test. The code should be included in the space/field provided. Medicare regulations for ICD-9 coding are written on the back of the multipart preprinted test request form.
  6. Type or source of specimen (check box on form or use drop down box in Epic).
  7. Test order: check the appropriate box on the paper form or use the on-line dropdown list in Epic to order Pap test screening alone or Pap test with HPV testing. Explanations for HPV test ordering) are on the back of the top copy of the paper request form.
  8. All pertinent clinical history/findings or other data relating to the requested evaluation.

For preprinted versions of the Cytopathology Request form, computer generated registration labels are the preferred method for providing patient identification information (i.e. name, history number, date of birth) for outpatients.

Clinical history or findings include the information listed below. This information is important for accurate interpretation of smears and should correlate with the assigned ICD-9 code provided on the request form.

1. All previous disease processes (e.g. dysplasia, cancer, etc.) and/or therapy (e.g. radiotherapy, cryo/laser therapy, estrogen replacement therapy, etc.) that could influence the cytologic interpretation of the Pap test. Case numbers from previous cytology or surgical specimens are helpful, but should NOT replace written descriptions of previous findings.

2.  Current patient symptoms or complaints.

3.  Abnormal findings discovered during the physical examination.

4.  Menstrual history 

  •  a. Indicate menstrual status by checking the appropriate box on the request form.
  •  b. The most recent date for normal menses or expected bleeding is written on the “LMP” date line. 
  •  c. For post-menopausal patients on estrogen replacement therapy, the date for normal withdrawal bleeding is given on the LMP date line. 
  • d. All ABNORMAL bleeding in post-menopausal patients should be indicated in descriptive terms in the clinical history/findings section of the request form or by checking the appropriate box. Do not use the LMP date line for this information.

All of the information discussed above is essential for the efficient processing of the specimen and the timely reporting of results. Missing vital data may delay completion of the case.



The University of Virginia Cytology Laboratory uses the 2014 Bethesda System for reporting the cytologic interpretations of gynecologic Pap specimens.  A description of this system is given in the following pages.

Results are usually available within five to seven working days after receipt of the specimen in the laboratory.  Inpatient and stat cases are read as quickly as possible, usually the next working day after receipt in the laboratory.   



In 1988 the Division of Cancer Prevention and Control at the National Cancer Institute convened a workshop of experts and consultants to review existing terminology for the reporting of cervical/vaginal diagnoses and to recommend effective methods for standardizing diagnostic reporting.  As a result of this workshop, the Bethesda System (TBS) was published as a guideline for uniform terminology and reporting of cervical/vaginal diagnoses.  Under this system the final report consists of three major components 1) a statement of adequacy, 2) a general categorization/ descriptive diagnosis, and 3) recommendations for follow-up procedures, if necessary.  A second workshop was held in 1991 at the National Cancer Institute for further discussion and review of TBS.  Some minor changes and clarifications of TBS resulted from that meeting.

The 2001 Bethesda Conference provided a review of the application of TBS over the previous 10 years. After much discussion, significant revision of reporting terminology was made. The most significant change was the combination of the “within normal limits” category and the “benign cellular changes” category under the heading of “Negative for Intraepithelial Lesion or Malignancy.” Infectious organisms are reported directly with the “Negative” interpretation. Benign findings are considered optional and are reported as additional findings under the “Negative” heading. The revised version was published as a reporting system for cervical cytology.

The UVA Cytology Laboratory uses a slightly modified version of TBS for reporting cervical interpretations. The modifications are generally in specific wording and in the inclusion of additional descriptive diagnosis in order to facilitate patient management by clinicians at our institution.  The intent, content, and scope of TBS remain intact.


Satisfactory for Interpretation:

Satisfactory gynecologic specimens are defined as specimens having sufficient numbers of well-preserved, well-stained, unobscured epithelial cells such that a cytologic interpretation can be rendered.  No conditions exist (e.g. excessive inflammation or blood, poor preservation, etc.) that prevent the cytologic interpretation. 

Relevant clinical information and/or history are considered necessary for accurate evaluation of Pap smears.  These data may clarify otherwise uncertain cytologic findings.  Therefore, the provision of pertinent patient information that may influence the interpretation of the smears is necessary for a "satisfactory" specimen.

Some specimens may have conditions that hamper but do NOT prevent a reasonable cytologic interpretation. The cellular material remains sufficient to render an interpretation although that interpretation may be somewhat limited in scope or specificity. Reasons for the limitations are stated in the report under the heading “Statement of Adequacy.”

Unsatisfactory for Interpretation:

Unsatisfactory specimens are defined as specimens in which conditions exist (e.g. too few epithelial cells, excessive inflammation or blood, poor preservation, etc.) that prevent a reasonable cytologic interpretation.  Rendering a cytologic interpretation on the specimens would not meet basic standards of care. In this category the statement of adequacy or lack thereof, becomes the descriptive interpretation.  All identifiable limiting factors for the unsatisfactory results are stated in the report.  No cytologic interpretation is given.

If abnormal cells are detected, regardless of other factors, the specimen is NEVER categorized as unsatisfactory.

The laboratory has procedures in place to avoid reporting unsatisfactory results whenever possible. Prior to recording a specimen as unsatisfactory, the residual material in the specimen vial is evaluated for physical findings that may indicate that additional processing could potentially improve sample adequacy. If merited, additional slides are made. In addition, all specimens in which basic criteria for adequacy are not met are sent for a second cytotechnologist review and sign out. If the two cytotechnologists agree, the unsatisfactory results are reported. If the two reviewers do not agree, the case is referred to the pathologist for final interpretation.


The interpretation element of the report is largely self-explanatory.  The descriptive terminology used in our laboratory is listed on pages 8-10.  TBS limits the term "atypical cells" to those cases in which the cytologic findings are of undetermined significance.  Whenever possible, the use of the term should be further clarified when a high-grade lesion cannot be excluded. The old numerical Papanicolaou classification system is not considered acceptable in the modern practice of diagnostic cytopathology.


Suggestions for follow-up are optional.  When included in the report, they should be concise and consistent with clinical follow-up guidelines published by professional organizations (e.g., ASCCP). TBS does not contain guidelines for patient management.


The results of any ancillary testing performed on the specimen will be given either in the final report or as an addendum to that report. The results will provide a brief description of the test method and the test outcome so that clinicians can easily understand them. Refer to the section on HPV testing pages 10-11.



I.          Statement of adequacy

Satisfactory for interpretation – limiting factors listed as needed

Unsatisfactory specimen - requires explanation, give all limiting factors


Limiting factors for satisfactory specimens:

Scant numbers of epithelial cells

Endocervical/transformation zone component absent/scant

Poor fixation or preservation

Partially obscuring inflammatory exudate

Partially obscuring blood

Partially obscuring bacterial overgrowth

Mechanical distortion

Cellular degeneration

Pertinent clinical information that may affect cytologic interpretation was not provided.

Explanations for unsatisfactory specimens:

Insufficient numbers of epithelial cells for adequate cytologic interpretation

Completely obscuring inflammatory exudate

Completely obscuring blood

Completely obscuring bacterial overgrowth

Poor fixation or preservation of the epithelial cells prevents adequate cytologic evaluation

Mechanical distortion of the epithelial cells prevents adequate cytologic evaluation

Cellular degeneration prevents adequate evaluation of the epithelial cells

II.        Descriptive interpretation/results categories

Negative for Intraepithelial Lesion or Malignancy

Infections (reported with the Negative interpretation):

Fungal organisms consistent with Candida species

Bacteria morphologically consistent with Actinomyces species

Trichomonas vaginalis infection

Cellular changes indicative of cytomegalovirus

Cellular changes indicative of Herpes simplex virus


Additional cytologic findings (reporting optional):

Benign cellular changes associated with an inflammatory process

Atrophic cervicitis/vaginitis

Benign cellular changes associated with ionizing radiation

Benign cellular changes associated with a reactive/reparative process

Benign cellular changes associated with atrophy

Benign cellular changes, associated cause not identified

Benign cellular changes associated with presence of intrauterine contraceptive device

Tubal metaplasia present

Effects of nonsteroidal estrogen exposure

Parakeratosis (reported only when present in quantity)

NOTE:  This entity may occur alone in response to various stimuli, or may be                             associated with squamous neoplasia.

Anucleated squamous cells present (reported only when present in quantity)

NOTE:  This entity may occur alone in response to various stimuli, or may be                             associated with squamous neoplasia.

Endometrial material present in postmenopausal woman

Endometrial material present, recommend clinical correlation

NOTE:  This is reported in women 40 years or older when not accounted for by patient history.

Pregnancy elements present

Endocervical-type cells present in vaginal smear


Epithelial cells abnormalities:

Atypical squamous cells present

Atypical squamous cells, cannot exclude a high-grade intraepithelial lesion

Low-grade squamous intraepithelial lesion (mild dysplasia)

High-grade squamous intraepithelial lesion (moderate dysplasia)

High-grade squamous intraepithelial lesion (severe dysplasia)

Squamous cell carcinoma (statement of grade and type may be made)

Atypical endocervical cells present

Atypical endometrial cells present

Atypical glandular cells present

Atypical endocervical cells present, favor neoplastic

Atypical glandular cells present, favor neoplastic

Endocervical adenocarcinoma-in-situ

Suspicious for malignancy

Squamous cell carcinoma

Adenocarcinoma (if possible site of origin suggested)

Adenosquamous carcinoma

Small cell undifferentiated or neuroendocrine carcinoma

Malignant mixed mullerian tumor     


Metastatic carcinoma

Other malignancies (identify type if possible)


Other diagnoses (free text)

Comments for interpretations:

Paucity of atypical cells prevents definitive diagnosis

Predominance of cocci/bacilli consistent with shift in vaginal flora

III.       Suggestions for follow-up

Based on cytologic findings, recommend repeat Pap test           

Recommend clearing of inflammation followed by repeat Pap test

Recommend treatment for the infection followed by repeat Pap test

Recommend repeat Pap test at mid-cycle

Recommend treatment with topical estrogen followed by repeat Pap test

Other recommendations (free text)

IV.       Hormonal Evaluation

(Performed only on lateral vaginal wall specimens, requires specific request from ordering physician/nurse practitioner)

MI   /  /    (interpretation required)

hormonal evaluation not possible - cervical specimen or inflammation present or insufficient patient history


1.      Gilbert FE, Hicklin MD, Inhorn SL, et al.  Standards of Adequacy of Cytologic Examination of the Female Genital Tract.  Am J Clin Path 1974;61:285-286.

2.      Lundbery GD, ed.  Quality Assurance in Cervical Cytology - The Papanicolaou Smear.  JAMA 1989;262:1672-1679.

3.      Greening SE:  The Adequate Papanicolaou Smear Revisited.  Diagn Cytopathol 1985;1:55-56.

4.      Vooys PG, Elias A, van der Graaf Y, et al.  Relationships Between the Diagnosis of Epithelial Abnormalities and the Composition of Cervical Smears.   Acta Cytol 1985;29:323-328.

5.      Lundberg GD, ed.  The 1988 Bethesda System for Reporting Cervical/Vaginal Cytologic Diagnoses.  JAMA 1989;262:931-934.

6.      Broder S. The Bethesda System for Reporting Cervical/Vaginal Cytologic Diagnoses - Report of the 1991 Bethesda Workshop.  JAMA 1992;267:1982.

7.   The Bethesda System for Reporting Cervical/Vaginal Cytologic Diagnoses. Acta Cytol 1993;37:115-124.

8.      Solomon D, Davey D, Kurman R, et al. The 2001 Bethesda System – terminology for reporting results of cervical cytology. JAMA 2002;287:2114-2119.

9.      Solomon D, Nayar R (editors). The Bethesda System for Reporting Cervical Cytology (second edition). Springer, New York, 2004.



The Molecular Diagnostics Laboratory, in conjunction with the Cytopathology Laboratory, offers HPV High-Risk DNA with genotyping on the ThinPrep Pap Test.  This test is designed to identify women infected with any of 14 high-risk HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68) that may be associated with cervical cancer or its precursors, including information on high risk HPV genotypes.  The Roche Cobas 4800 provides clinicians with a result that differentiates HPV 16 and HPV 18 from the 12 other HPV high-risk types.  HPV testing via the Roche Cobas 4800 has been found to be more sensitive and more specific than cytology alone in detecting persistent HGSIL or cancer in women with 1 of the 14 high-risk genotypes.  Physicians ordering the ThinPrep Pap Test have the option of selecting ancillary HPV testing to be performed on the original Pap test specimen submitted for a patient.  HPV testing on ThinPrep samples should be ordered within three weeks from the date of specimen collection. Because of storage limitations within the laboratory, specimen vials are not kept beyond the three week time period.

The testing is performed in the Molecular Diagnostics division of the Medical Laboratories on material from the ThinPrep Pap test submitted to the Medical Laboratories for cervical cancer screening.


The most recent version of the preprinted cytopathology test request form and Epic on-line test order provide specific items for ordering HPV testing.  In addition, information on test ordering is provided on the back of the pre-printed form.

Epic HPV Test Orders

At the time the ThinPrep sample is submitted for cytologic evaluation, HPV testing can be ordered from the dropdown list for Epic on-line ordering.

1.      HPV Primary Screening (means the Primary Screening FDA algorithm)

     o   If HPV PRIMARY SCREENING TEST is selected, the following algorithm will be used:

·         If POSITIVE for HPV 16 or HPV 18: No further testing will be performed.  Per the ASCCP guidelines, patients testing positive for HPV 16 and/or HPV 18 should be considered for immediate colposcopy, regardless of the concurrent cytology result.

·         If NEGATIVE for all HR HPV types: No further testing will be performed.  Patients testing negative for HR HPV types have a <1% chance of HSIL lesion.

·         If HR HPV POS, NOT HPV 16 or HPV 18: Samples will be automatically reflexed for PAP smear.  Clinicians will not need to order reflex testing or request a separate PAP smear.  Per ASCCP guidelines, patients positive for HPV types other than 16 and 18 should be considered for immediate colposcopy if concurrent cytology is abnormal at a threshold of ASCUS or above


2.      HPV-CoTest with Pap: Refer immediately for HPV testing once the Pap test has been run.

3.      HPV-reflex for ASCUS: Perform HPV testing only if the cytologic interpretation is ASCUS or AGUS.

4.      Pap Test only, NO HPV performed.

5.      HPV-Non-cervical/vaginal specimens.


Pre-printed Cytopathology Request forms HPV Test Orders

At the time the ThinPrep sample is submitted for cytologic evaluation, HPV testing can be ordered HPV testing can be ordered by checking the appropriate box.

     o  ThinPrep Pap with Reflex HPV Test: Perform HPV testing only if the cytologic interpretation is ASCUS or AGUS.

     o  ThinPrep Pap with HPV Test Regardless of Interpretation: Perform HPV testing even if the interpretation is “negative” (usually in patients with a prior history of abnormal findings on previous Pap tests or as part of a follow-up for previously treated intraepithelial lesions).

     o  ThinPrep Pap with HPV Screening for Women 30 and older: Refer immediately for HPV testing once the Pap test has been run. This order is totally independent of the cytology results of the Pap test.

Pap tests with unsatisfactory interpretations are not appropriate for HPV testing.

The cytopathology report will indicate in the “notes” section whether or not HPV has been ordered and if sufficient and/or acceptable specimen exists to perform HPV testing.  Note:  If you have ordered HPV testing and a comment regarding that order is NOT on the final report, please contact the Cytology Laboratory immediately to verify that the test was ordered.  If the order was missed by the laboratory staff, it can be ordered immediately.

If HPV testing has not been ordered on the original specimen request or the clinician wants to change the type of HPV test order, an add-on request can be made.  A written order signed by the requesting physician/nurse for the request can be faxed to the Cytology Laboratory (434-924-0217) or an Epic on-line order can be faxed so that the HPV request can be initiated.  Such a request should be made within 5 days of the sign-out date of the cytopathology final report.  In the event that sufficient and/or appropriate sample is not available, the laboratory will notify the requestor.  The person taking the request will not be able to give you this information at the time the request is made.


Final results of HPV testing, regardless of algorithm requested, will be available in “Cytology, GYN” under the “Pathology” tab in EPIC.  Clients without access to EPIC will receive HPV results via method used to receive all other laboratory test results.  

Please contact the Clinical Microbiology Director, Dr. Melinda Poulter, at PIC 3677 with questions.  Dr. Mark Stoler is available at PIC 4316 to discuss algorithmic or medical implementation questions.


Wright, T., Stoler, M., Sharma, A., Zhang, G., Behrens, C., Wright, T.L., Evaluation of HPV-16 and HPV-18 Genotyping for the Triage of Women with High-Risk HPV+ Cytology, Negative Results. AM J Clin Patholo 2011;136:578-596.