The detection of viral nucleic acids in intraocular fluids and tissues by PCR has become increasingly important in clinical ophthalmology. While much attention has been directed toward minimizing false-positive reactions resulting from specimen contamination or amplicon carryover, relatively little attention has been given to the causes of false-negative PCRs. This report describes a PCR inhibitor in normal aqueous and vitreous fluids that can produce false-negative PCR results. As little as 0.5 l of vitreous fluid and 20 l of aqueous fluid can completely inhibit DNA amplification in a 100-l PCR mixture. This inhibition was not primer specific, nor was it due to chelation of Mg 2؉ ions or DNase activity in the ocular fluid. The inhibitor was completely resistant to boiling for 15 min. However, the inhibitory effects were completely removed by a single chloroform-isoamyl alcohol (24:1) extraction. The extent of PCR inhibition depended upon the type of thermostable DNA polymerase used in the reaction. Taq DNA polymerase was very sensitive to the inhibitor, while thermostable DNA polymerases from Thermus thermophilus HB-8 (Tth) and Thermus flavus (Tfl) were completely resistant. Thus, the inhibitory effects of intraocular fluids on PCRs can be removed by diluting the specimen, by chloroform extraction, or by using Tth or Tfl DNA polymerases.

The prevalence of herpesvirus DNA detectable by PCR techniques in ocular fluids appears to be quite low despite the high proportion of patients who tested positive for herpesvirus antibodies. Therefore, a positive result obtained in a patient presenting with vitreoretinal inflammation should be regarded as significant.

The use of molecular diagnostic testing is increasing in the clinical setting; therefore, data regarding DNA stability in clinical specimens are essential for correct test performance and interpretation. This study was designed to determine DNA stability in peripheral blood and solid tissue under different storage conditions. DNA quality and yield were assayed by spectrophotometric absorbance, gel electrophoresis, and suitability for Southern hybridization and polymerase chain reaction (PCR), the most widely employed clinical DNA analyses. A second goal of the study was to evaluate DNA stability during storage at 4 degrees C for 1 month to 3 years. The data show that freezing or refrigeration of separated leukocytes is preferable for short- to intermediate-term storage and freezing is preferable for solid tissue. DNA degradation varying from slight to severe is seen inconsistently with such specimens, probably due to sampling of unevenly frozen-tissue areas. Depending on the degree of DNA degradation, analysis may still be possible by PCR and in some cases even by Southern hybridization. Once isolated, DNA was stable at 4 degrees C for at least 3 years. These results suggest a more flexible approach to specimen requirements for molecular pathology, as some samples that would routinely be rejected gave interpretable results.

This finding shows that benign epithelial cells can be mobilized during breast surgery; this effect of surgical manipulation warrants caution in the interpretation of RT-PCR positivity for cytokeratin mRNA in the peripheral blood of patients undergoing surgery for breast cancer.

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