Reproducibility of HPV DNA Testing by Hybrid Capture 2 in a Screening Setting: Intralaboratory and Interlaboratory Quality Control in Seven Laboratories Participating in the Same Clinical Trial

Francesca Maria Carozzi, PhD; Annarosa Del Mistro, MD; Massimo Confortini, PhD; Cristina Sani, MD; Donella Puliti, MD; Rossana Trevisan; Laura De Marco, PhD; Gillio Tos, PhD; Salvatore Girlando, MD; Paolo Dalla Palma, PhD; Pellegrini, PhD; Maria Luisa Schiboni, PhD; Paola Crucitti, MD; Paola Pierotti, MD; Alberta Vignato, MD; Guglielmo Ronco, PhD


Am J Clin Pathol. 2005;124(5):716-721. 

In This Article

Abstract and Introduction

Within a large Italian randomized trial on new technologies for cervical cancer screening involving 7 laboratories with different levels of experience, an intralaboratory and interlaboratory quality control program for human papillomavirus (HPV) DNA testing by Hybrid Capture 2 (HC2; Digene, Gaithersburg, MD) was implemented. To monitor the hybridization and detection steps, target samples containing purified, concentration-defined, HPV DNA were introduced in each test run. Only 3 of 1,024 showed a mistake in a positive vs negative classification with a 1 relative light unit (RLU)/positive control specimen (PC) ratio cutoff. To monitor the preanalytic steps (particularly denaturation), blinded specimens (33 collected in PreservCyt [Cytyc, Boxborough, MA] and 36 in Specimen Transport Medium [STM, Digene]) were centrally prepared, divided into aliquots, and sent to each laboratory. The multiple-rater κ scores for negative (<1 RLU/PC), low-positive (1 to <11 RLU/PC), and high-positive (≥311 RLU/PC) samples, respectively, were 0.91, 0.60, and 0.69 with PreservCyt and 0.93, 0.87, and 0.90 with STM. Our data showed high reliability and reproducibility with HC2, with κ values higher for STM than ThinPrep (Cytyc) samples.

Human papillomavirus (HPV) infection is the major cause of most cervical cancers and cervical intraepithelial neoplasia worldwide.[1,2,3] Persistent HPV infection by high-risk types is a necessary (although not sufficient) cause for the development of cervical cancer. Consequently, there is strong interest in the use of HPV testing for cervical cancer screening.

By using Hybrid Capture (HC) Systems (Digene, Gaithersburg, MD) or different polymerase chain reaction (PCR) protocols, HPV testing has been evaluated in cervical screening as an adjunct to the Papanicolaou smear or alone as the only screening test. By combining the results of several studies,[4] HPV testing has been shown to have higher sensitivity but lower specificity than conventional cytologic testing in detecting high-grade lesions; this is due mainly to the recognition of transient, clinically silent HPV infections as well. The most relevant result of these studies is the high negative predictive value (approaching 100%) attained by the combination of cytologic and HPV testing, whereas the major drawback of this modality is the increased number of women who need to be referred for colposcopy.

The HC2 HPV DNA test (Digene) is a commercially available microplate assay approved by the US Food and Drug Administration for the detection of HPV DNA and consists of a liquid-phase hybridization by the use of 2 RNA probe mixtures. The HC2 probe mix B detects a group of 13 cancer-associated HPV types: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68, whereas probe mix A detects low-risk types. Previous studies have demonstrated that the HC2 assay is sensitive and specific for detecting HPV DNA from cervical specimens,[5] and it has good agreement with PCR assays.[6,7]

HC2 seems robust, but to consider HPV testing for cervical cancer screening programs, issues such as reproducibility between different testing centers need further attention. Indeed, to achieve quality reproducible results, it has been stated[8] that "in the absence of a formal proficiency testing program, the laboratory must develop its own external program (eg, through interlaboratory exchange) or devise some system for internal proficiency testing." Specimen processing includes critical steps, and lack of optimization potentially can give rise to false-positive results; thus, good reproducibility of blinded clinical samples in the laboratories might warrant the high reliability of trial results.

The reproducibility of HC2 depends on factors influencing the sensitivity and specificity of the reaction: collection, storage, transport and processing of biologic samples, target concentration, presence of reaction inhibitors, occurrence of nonspecific hybridization; and lack of optimization in one or more process phases. In particular, samples collected in fixative-containing solutions (which allow the preparation of cytologic slides) require an additional processing phase to be suitable for HC2 testing. This step, known as sample conversion and necessary to eliminate the fixative, is critical for optimal DNA denaturation and has not been evaluated extensively so far.

In the frame of a large, multicenter, randomized, controlled trial on New Technologies for Cervical Cancer (NTCC study) screening conducted in Italy, we implemented an intra-laboratory and interlaboratory quality control program to monitor reproducibility and accuracy in different steps of the assay in the 7 participating laboratories. The NTCC study involves about 100,000 women aged 25 to 60 years, coming for a new screening round within 9 organized screening programs, who were randomized in 2 arms for 2 periods (2 phases in which recruitment lasted about 1 year each).

During the first phase (HPV testing associated with cytologic testing), HPV testing was performed on samples collected in a fixative-containing transport medium, whereas in the second phase (HPV testing alone), the samples were collected in the Specimen Transport Medium (STM) provided by the HC2 manufacturer (Digene). We studied the performance of the HC2 test with both transport media.


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