In situ hybridization is the only DNA—or RNA-based molecular biology based test that allows for the direct correlation of the results with the histologic and cytologic features of the sample. The DNA/RNA extraction that precedes filter hybridization (hybrid capture or Southern blot) hybridization and the polymerase chain reaction (PCR) precludes this type of analysis. In order for the in situ hybridization to detect a given DNA or RNA target, there must be at least 10–20 copies of the target per cell. In comparison, Southern blot hybridization and the hybrid capture assay can detect one DNA or RNA target per 100 cells. PCR is even more sensitive if the hot start maneuver is employed; under these conditions, it can detect one DNA or RNA target per 100,000 cells. It is evident that in situ hybridization is a relatively insensitive test. A reflection of this relative insensitivity is seen in occult or latent infection by a virus where the copy number is low. In such situations, the virus is rarely detected by in situ hybridization even though it was detected by either PCR or filter hybridization (1–6). As stressed in this chapter, this is actually an advantage of in situ hybridization over the more sensitive assays. In situ hybridization detects only productive infection by human papillomavirus (HPV). It will not detect the low copy subclinical or “incidental” infection of HPV in the setting of a normal Pap smear (Fig. 1). When one realizes that incidental HPV infection can occur in 15–20% of normal Papanicolaou (Pap) smears, as determined by the hybrid capture or PCR assays, it is evident that it is advantageous for in situ hybridization NOT to detect the virus, given the realization that most concur there is no reason to clinically treat HPV in the absence of any pathology.

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