Detection of human papillomavirus in cervical carcinoma: Comparison of peroxidase, nanogold, and catalyzed reporter deposition (CARD)-Nanogold in situ hybridization

We compared three in situ hybridization (ISH) methods for their applicabili ty and sensitivity in detecting human papillomavirus (HPV) in 61 cases (1 G rade 1, 18 Grade 2, 42 Grade 3) of routinely processed squamous cell cervic al carcinoma. A commercially available biotinylated probe for HPV-16/18 was applied to serial sections and detected by conventional streptavidin-bioti n-peroxidase ISH, streptavidin-Nanogold-silver ISH, and catalyzed reporter deposition (CARD)-Nanogold-gold ISH, The latter method involved signal ampl ification by peroxidase-catalyzed deposition of biotinylated tyramides at t he hybridization sites, followed by detection of accumulated biotin by stre ptavidin-Nanogold made visible by autometallography, The HPV-16/18 detectio n rates for the three methods were 39.3, 44.3, and 65.6%, respectively. In all of the three ISH methods, a punctate staining pattern (single or multip le intranuclear spots of variable size), presumably indicating viral integr ation, was highly predominant among the positive cases. Two of the cases id entified as positive by streptavidin-biotin-peroxidase ISH were rated negat ive with streptavidin-Nanogold-silver ISH, whereas six cases that were clea rly negative with streptavidin-biotin-peroxidase ISH became positively stai ned with streptavidin-Nanogold ISH, All of these discordant cases were posi tive by the highly sensitive CARD-Nanogold-gold ISH, In addition, the high detection sensitivity of CARD-Nanogold-gold ISH was confirmed by its abilit y to detect single copies of HPV-16 in SiHa cells, In general, we found tha t the intense black reaction product from Nanogold autometallography gave s uperior contrast to that obtained with the peroxidase system. After tyramid e signal amplification, the staining was so clearly visible that preparatio ns could be readily screened under low magnification. Our findings precisel y demonstrated the need for improved sensitivity in the in situ detection o f HPV, The CARD-Nanogold-gold technology looks promising as a highly sensit ive method for routine ISH in molecular pathology.