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Studies have demonstrated that plasma samples collected and stored frozen using vacutainer plasma preparation tubes (PPT) may result in falsely elevated viral load (VL) values with the Roche COBAS TaqMan HIV-1 v1.0 test. At the University of Connecticut Health Center, a total of 349 samples from HIV-1-infected patients on HAART were collected and stored frozen in PPT. Viral load (VL) results were obtained using the Roche COBAS TaqMan HIV-1 v2.0 test (CTM v2.0) and Abbott RealTime HIV-1 assay (RealTime HIV-1). Of the 349 samples, 260 (74.5%) had VL values that differed by >0.5log10copies/mL; 64 of these were quantified by both assays. The remaining 196 samples were detected by CTM v2.0 but not detected in RealTime HIV-1: 62 of the most discordant samples in this category (CTM v2.0 detected/RealTime HIV-1 not detected) were further analyzed using two nested RT-PCR assays targeting pol integrase: full-length (864nt) and a highly conserved subregion (134nt). No HIV-1 RNA was detected in the discordant samples, confirming RealTime HIV-1 results. The increase in VL reactivity with the CTM v2.0 assay was presumably due to proviral DNA captured by the CTM total nucleic acid extraction chemistry but not the RNA-specific extraction procedure used in RealTime HIV-1. These results suggest that using CTM v2.0 with samples frozen in PPT could have significant clinical implications for HIV-1 patient management.