NEWS & EVENTS

NEWS ARCHIVE | EDUCATIONAL RESOURCES

LIQUID BASED CERVICAL SCREENING

The conventional Pap smear is the traditional cervical screening test that has been used over the past fifty years to identify precancerous or cancerous abnormalities. Over 50 million are performed in the United States each year. The Pap smear, followed with careful clinical evaluation and biopsy, has helped reduce cervical cancer from one of the leading causes of death in U.S. women. Approximately 13,000 new cases and 4,500 deaths occur each year. While all recognize the strengths of the Pap smear in identifying cervical abnormalities, there are also inherent weaknesses. The average conventional slide contains 50,000-300,000 cells to review. Variables making review more difficult include air-drying artifact, thick smears, excessive blood, inflammation, bacteria, and low cellularity. Sampling is also a significant factor. Much of the cellular sample on the collection device is discarded and does not make it to the slide. Also, the part of the sample that is smeared on the slide may not be representative of the entire specimen. It is estimated that only 6%-60% of cells collected actually end up on a conventional smear.

In recent years, new technologies have been developed to increase the sensitivity of the Pap smear and thereby decrease the false negative rate. One technical improvement is the evolution of liquid based thin-layer preparations. This method offers ease of performance and improves the technical quality of the specimen. Improvements include reduction in the number of unsatisfactory specimens and limited specimens caused by obscuring blood, inflammation, and debris, poor fixation, inconsistent technical slide preparation, and increased sensitivity for the detection of LSIL AND HSIL. Since the majority of the cells are transferred to the liquid vial, extra sample material is available, limiting the need to recall the patient if additional testing is required.

Two liquid based technologies are currently available, the ThinPrep Pap Test by Cytyc Corporation and SurePath (previously known as AutoCyte) by TriPath Imaging Inc. Both improve the quality of the cervical/endocervical sample through improved slide preparation. This is achieved by rinsing the collection device(s) in a preservative fluid to generate a suspension of cells that is processed to deposit a monolayer of cells on a microscope slide. The FDA approved the ThinPrep Pap test in 1996, and it has been offered at Physicians Reference Laboratory (PRL) since September 1999. Utilizing this technique, the cervical/endocervical sample is collected using a broom-type or cytobrush/spatula cervical sampling device. These collection devices are similar to those used for conventional smears but must be made of plastic. The sampling device is rinsed into a ThinPrep vial containing PreservCyt transport medium, which immediately fixes the sample. The vial is capped, properly labeled, and transported to PRL with a completed requisition for processing. During processing, red cells, white cells, and mucus threads that commonly obscure the conventional Pap smear are almost entirely removed. Below are representative images of each specimen type from the same patient.

Conventional Pap Smear	ThinPrep Pap Test
With the conventional Pap smear method, cells can be obscured by blood, mucus, and inflammation.	The ThinPrep Pap Test method preserves the cells and minimizes cell overlap, blood, mucus, and inflammation.

Several published studies on the ThinPrep Pap Test have shown an improved rate of detecting LSIL over conventional Pap smears, an improved rate of detecting HSIL, and a decrease in the rate of ASCUS reporting. There is no evidence of superiority in diagnosing adenocarcinomas or other glandular lesions, but diagnostic difficulties also exist for glandular lesions with the conventional Pap smear. The percentage of specimens reported as "satisfactory but limited by" and "unsatisfactory" decrease due to the elimination of obscuring entities and air-drying artifact. Studies have also been published concluding there is no statistically significant difference in the sensitivity and specificity between conventional pap smears and liquid based preparations. Some of these researchers suggest the improved detection of cervical abnormalities is not due to the liquid based preparation but rather a consequence of a better sampling technique. Currently, the majority opinion in the literature is that the ThinPrep Pap Test is more effective than the conventional smear for the detection of precancerous cervical lesions in a variety of patient populations. In our practice, ThinPrep Paps currently make up approximately 30%-35% of the total Pap smear volume. We have documented a slight decrease in the percentage of cases reported as ASCUS, a slight increase in the percentage of LSIL, and no change in the percentage of HSIL. LSIL is easier to recognize on ThinPrep Paps, but HSIL can be more difficult.

The ASCUS/LSIL Triage Study (ALTS) was a clinical trial sponsored by the National Cancer Institute to find the best way to manage ASCUS and LSIL Pap results. For women with an ASCUS result, HPV testing was found to be highly sensitive in detecting the underlying abnormalities that need immediate attention. If the HPV test is negative, it is unlikely that a high-grade lesion is present. If positive, colposcopy or other immediate follow-up is recommended. Based on these findings, experts at two recent meetings have clarified the terminology used to report abnormal results on Pap tests and developed new consensus guidelines for managing patients with abnormal cervical cytology. The 2001 Bethesda system workshop recommendations for reporting cervical/vaginal cytologic diagnoses include an optional section to report ancillary HPV DNA testing results. The Consensus Conference for the Management of Cytological Abnormalities and Cervical Cancer Precursors, sponsored by the American Society for Colposcopy and Cervical Pathology (ASCCP), include the option of DNA testing for high-risk types of HPV in the management of patients with ASCUS or for postmenopausal women or adolescents with LSIL.

Currently, the only FDA approved commercially available in vitro diagnostic test for HPV DNA is the Hybrid Capture 2 (hc2) HPV DNA Assay manufactured by Digene. The FDA has approved the use of the PreservCyt collection medium used for ThinPrep Paps as a specimen collection medium for Digene’s hc2 HPV DNA Test. It is the only liquid based Pap collection kit with such approval at this time. A proposal has been submitted to the FDA for the SurePath Pap test, and it is currently pending. Submission of a specimen in this fashion permits both Pap screening and HPV testing following a single patient visit. The Pap can be ordered by the clinician with “reflex HPV testing”, i.e. if the Pap is interpreted as ASCUS, the sample vial is automatically referred for HPV testing. The FDA has also approved testing for chlamydia and gonorrhea on the ThinPrep Pap vial. This enables the clinician to perform up to 4 separate tests on one patient sample from one office visit, and patient management can be based on a treatment algorithm using the results of these combined tests. Chlamydia trachomatis and Neisseria gonorrhea testing are performed by PRL. At this time, HPV DNA testing is submitted to the Mayo Medical Laboratories in Rochester, Minnesota.

In conclusion, the conventional Pap smear is one of the great success stories in medicine and has saved many lives. It continues to perform well, particularly when the most preventable causes of suboptimal smear preparation are addressed. Suggested tips for preparing and submitting high quality smears for evaluation include:

• Spray-fix the smear immediately.
• Remove excess mucus, exudate, and blood from the cervix prior to sampling.
• Use the spatula first, then the brush.
• Do not take smears while the patient is menstruating.
• Fill out the requisition completely with the appropriate clinical information.
• Cytohistologic correlation is very important, so always try to send the follow-up biopsy to the same laboratory that reported the Pap smear.

While liquid based Pap smears demonstrate distinct advantages in slide quality and adjunctive HPV testing, there is no consensus recommendation in favor of abandoning conventional Pap smears. Clinicians are encouraged to familiarize themselves with these new techniques, as well as the 2001 Bethesda system workgroup recommendations and ASCCP management guidelines, and make choices based on the medical benefit to the patient.

COLLECTION AND PROCESSING PROTOCOL

OR	Step 1 A gynecologic sample is collected using a broom-type or cytobrush/spatula cervical sampling device.
	Step 2 Instead of smearing the cells on a slide, the sampling device is rinsed into a ThinPrep vial containing PreservCyt® transport medium by vigorously swirling in the vial 10 times. The device is then discarded.
	Step 3 The sample vial is capped, labeled, and sent to the laboratory for slide preparation.
	Step 4 At the laboratory, the vial is placed into the ThinPrep 2000 Processor. First, a gentle dispersion step breaks up blood, mucous, non-diagnostic debris, and then thoroughly mixes the sample. A negative pressure pulse is generated which draws fluid through a TransCyt® Filter that collects a thin, even layer of diagnostic cellular material. The ThinPrep 2000 Processor constantly monitors the rate of flow through the TransCyt Filter during the collection process to prevent the cellular presentation from being too scant or too dense. The cellular material is then transferred to a glass slide and fixed.

Amy J. Owen, MD
Technical Director, Cytology
June 2002