College of American Pathologists
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cap today

October 2005

Richard A. Savage, MD, Editor

Q. We have started to use SurePath liquid-based cytology for our Pap tests. Can human papillomavirus testing be done on these samples collected in SurePath vials? We are looking for a way to test our ASC-US and above cases for HPV.

A.Testing for high-risk types of human papillomavirus, or HPV, has gained clinical acceptance in the United States for supplemental testing of women for whom the results of primary cervical cytologic screening are atypical (ASC-US). More recently, HPV DNA co-testing along with cervical cytology has been approved as a sensitive modality for primary screening in women over the age of 30. Testing for HPV provides guidance as to how these patients should be managed.

According to 2001 American Society for Colposcopy and Cervical Pathology guidelines, testing for high-risk HPV DNA is preferred for triage management, and HPV DNA testing should be performed using a sensitive molecular technique. Available methods for testing for HPV DNA include Hybrid Capture 2 (HC2) DNA Assay, chromogenic in situ hybridization methodology, such as Inform, or PCR techniques. The only FDA-approved in vitro diagnostic test for HPV DNA is the HC2 DNA Assay, manufactured by Digene. Previous tests, including ViraPap, ViraType, and HPV Profile, are no longer manufactured or distributed.

HPV testing is a high-complexity test with certain requirements for quality assurance/quality control, proficiency testing, and personnel. Approved cervical samples for the Digene HC2 test include using the Digene specimen collection kit (cervical brush plus specimen transport media) or the Cytyc PreservCyt cervical cytology cell-collection fluid.

Laboratories that wish to use the TriPath SurePath Test Pack as a specimen-collection medium for the Digene HC2 test should realize that it is not FDA-cleared as an approved specimen source. Federal (that is, CMS) and some state regulations stipulate that validation is required for in-house (that is, home brew), non-FDA cleared methods, or modifications of the manufacturer’s procedure. This is the current route that laboratories can use to begin HPV DNA testing on SurePath samples, either from the residual vial contents or processed cell buttons. A minimum of 2 mL of residual SurePath cell collection fluid is required for testing with Digene HC2.

According to the Centers for Medicare and Medicaid Services, a validation study must establish performance specifications as well as calibration and control procedures and define ongoing QA, including proficiency testing and method comparison. The validation study data should establish precision, sensitivity, specificity, interfering substances, reportable range (linearity), and reference range of the specimen. Laboratories should refer to CLSI (formerly NCCLS) guidelines for more information regarding molecular tests. In-house validation must determine any method interferences with the HPV assay. Improper collection, sampling devices, and collection solutions can cause method interference. Optimum SurePath specimen retention should also be established. For example, HC2 HPV testing can be performed on Cytyc PreservCyt cell samples for up to three weeks after collection. Finally, false-negative results can be due to insufficient samples (unsatisfactory Pap).

A laboratory that is deliberating HPV testing should remember that there is no single answer regarding the decision to send it out or perform it in-house. Workflow and potential testing volumes should be considered, as well as local clinical practice and expectations. Payer mix and local payments for HPV DNA testing (CPT 87621) vary. Most labs need many samples per run to break even using HC2 testing. Costs and reimbursement for other methods vary remarkably.

Joel S. Bentz, MD
University of Utah
Health Sciences Center
Salt Lake City
Member, CAP
Cytopathology Committee

Q. Earlier this year, “Q&A” editor Richard A. Savage, MD, invited readers to answer the question, Can you recommend containers for transporting breast biopsy specimens in fixative? Here are four responses.

A. Our practice uses the Trans Spec container (Ezem Inc., Westbury, NY). It is a circular clear plastic device that compresses the specimen, allowing a specimen radiograph. The holder has holes in it, and the entire container is immersed in formalin for transport. Our transport always occurs within 24 hours. The specimen in formalin is refrigerated until transport because the compression device prevents direct contact with formalin on two sides of the specimen. This device was the most effective one that we tested.

A. We receive breast biopsy specimens with a plastic grid that overlays the specimen, holding it in place. The container, called TransSpec, is made specifically for breast biopsy specimens. The inner clear plastic grid has small holes so you can ink the area of radiographic interest before removing the grid. This container can be x-rayed so the specimen can be viewed on the radiography and the area of interest localized with coordinates from one to 15 on one side of the plastic grid and A to O on the other side. The client can wrap Parafilm around the container’s snap lid to prevent leakage during transport.

A. We have the radiology staff put a straight pin, though some use hypodermic needles, through the grid holes at the areas of interest while in the compression container. They then remove the specimen from the radiologic compression container and send it in an appropriate-sized container with formalin.

They also need to send the specimen mammogram because the needles or pins may fall out or they may forget that we do not want the entire specimen to be blocked. Just having the specimen mammogram, without pins, but with the needle locator wire, is immensely helpful. With a biopsy for a nodule, I can find the nodule without pins, wires, or x-rays.

A. After the surgeon has excised the tissue containing the localization wire, the radiologist inserts a hypodermic needle into the specimen and then circles the lesion on the specimen mammogram. With two landmarks, localization wire and hypodermic needle, it is easy to use the specimen mammogram to re-orient the specimen in 3D and sample the suspicious area. The specimen is then shipped in a standard sealed container.

More than 6,000 laboratories are accredited by the CAP, and approximately 23,000 laboratories are enrolled in the College’s proficiency testing programs.

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