False-positive results of plasma PCR for cytomegalovirus DNA due to delayed sample preparation

Positive results by cytomegalovirus (CMV) PCR of plasma are considered predictive of active CMV infection in kidney allograft recipients. To assess whether contamination with leukocyte-derived CMV DNA can distort the results, aliquots of whole-blood samples from 60 CMV immunoglobulin G-positive patients with leukocyte CMV DNAemia were stored for up to 24 h at room temperature (RT) and at 4 degrees C before plasma preparation. Native and ultrafiltered plasma samples were tested by CMV and beta-globin PCRs. Among 30 latently infected patients (negative for CMV pp65 antigens), low baseline rates (10%) and levels (median number of copies, 10 [per 10 microl]) of CMV plasma DNAemia in native plasma samples increased significantly over time (after 4 h at RT, 37% [P < 0.001]; median number of copies, 45 [P < 0.001]). Similar effects were found during storage at 4 degrees C. Ultrafiltration reduced the levels of CMV plasma DNAemia, but by 6 h of storage the levels were significantly elevated as well. CMV and beta-globin DNA kinetics in plasma were parallel. In contrast, 30 actively infected patients (pp65 positive) had high baseline rates (87% in native samples) and levels (median number of copies, 75) of CMV plasma DNAemia. No significant effects of storage or ultrafiltration and no concordance with beta-globin DNA kinetics were seen. In conclusion, delayed preparation of plasma samples bears a significant risk of false-positive CMV PCR results, probably due to leukocyte lysis. This has important implications in the clinical setting and for PCR standardization.