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.


METHODS
PCR was performed on cellular digests of archival, Papanicolaou-stained cervical smears and unstained cervical smears using primers to the human beta-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.


CONCLUSIONS
The hematoxylin and aluminum sulfate within Papanicolaou stain inhibit PCR but can be removed from Papanicolaou-stained cytologic specimens using a simple destaining procedure.
Chen
J T
JT
Department of Pathology, Johns Hopkins School of Medicine, Baltimore, Maryland 21287-6940, USA.
Lane
M A
MA
Clark
D P
DP
D000534:Alum Compounds / Q000494:pharmacology*; D004396:Coloring Agents; D004247:DNA / Q000032:analysis; D005260:Female; D005914:Globins / Q000235:genetics; D006416:Hematoxylin / Q000494:pharmacology*; D006801:Humans; D065006:Papanicolaou Test; D016133:Polymerase Chain Reaction / Q000187:drug effects / Q000379:methods*; D013194:Staining and Labeling / Q000379:methods*; D014626:Vaginal Smears
D000534:Alum Compounds; D004396:Coloring Agents; C041524:aluminum sulfate; D005914:Globins; D004247:DNA; D006416:Hematoxylin