Inhibitors of the polymerase chain reaction in papanicolaou stain: Removal with a simple destaining procedure

OBJECTIVE: To identify inhibitors of the polymerase chain reaction (PCR) within Papanicolaou stain and to develop a method of removal. STUDY DESIGN: PCR was performed on cellular digests of archival, Papanicolaou-stained cervical smears and unstained cervical smears using primers to the human b-globin gene to determine if Papanicolaou stain inhibited the reaction. Next, in order to identify the specific reagents responsible for inhibition, components of the Papanicolaou stain were added to PCRs performed on purified human genomic DNA. Finally, PCR was performed on cellular digests of cervical smears that had been destained using 1% HCl to determine if the inhibitors of PCR were removed. RESULTS: Cellular digests from Papanicolaou-stained cervical smears did not yield products from PCR, whereas cellular digests from unstained cervical smears always yielded PCR products. Analysis of individual Papanicolaou stain reagents revealed inhibition of PCR by hematoxylin and aluminum sulfate. These inhibitors could be removed from Papanicolaou-stained cervical smears by destaining the slides with 1% HCl. CONCLUSION: The hematoxylin and aluminum sulfate within Papanicolaou stain inhibit PCR but can be removed from Papanicolaou-stained cytologic specimens using a simple destaining procedure.