Effects of anticoagulant, processing delay, and assay method (branched DNAversus reverse transcriptase PCR) on measurement of human immunodeficiencyvirus type 1 RNA levels in plasma

We conducted two studies to determine the potential influence of delays in blood processing, type of anticoagulant, and assay method on human immunode ficiency virus type 1 (HIV-1) RNA levels in plasma. The first was an experi mental study in which heparin- and EDTA-anticoagulated blood samples were c ollected from 101 HIV-positive individuals and processed to plasma after de lays of 2, 6, and 18 h. HIV-1 RNA levels in each sample were then measured by both branched-DNA (bDNA) and reverse transcriptase PCR (RT PCR) assays. Compared to samples processed within 2 h, the loss (decay) of HIV-1 RNA in heparinized blood was significant (P < 0.05) but small after 6 h (bDNA assa y, -0.12 log(10) copies/ml; RT-PCR, -0.05 log(10) copies/ml) and after 18 h (bDNA assay, -0.27 log(10) copies/ml; RT-PCR, -0.15 log(10) copies/ml). De cay in EDTA-anticoagulated blood was not significant after 6 h (bDNA assay, -0.002 log(10) copies/ml; RT-PCR, -0.02 log(10) copies/ml), but it was aft er 18 h (bDNA assay, -0.09 log(10) copies/ml; RT-PCR, -0.09 log(10) copies/ ml). Only 4% of samples processed after 6 h lost more than 50% (greater tha n or equal to 0.3 log(10) copies/ml) of the HIV-1 RNA, regardless of the an ticoagulant or the assay that was used. The second study compared HIV-1 RNA levels in samples from the Multicenter AIDS Cohort Study (MACS; samples we re collected in heparin-containing tubes in 1985, had a 6-h average process ing delay, and were assayed by bDNA assay) and the British Columbia Drug Tr eatment Program (BCDTP) (collected in EDTA- or acid citrate dextrose-contai ning tubes in 1996 and 1997, had a 2-h maximum processing delay, and were a ssayed by RT-PCR). HIV-1 RNA levels in samples from the two cohorts were no t significantly different after adjusting for CD4(+)-cell count and convert ing bDNA assay values to those corresponding to the RT-PCR results. In summ ary, the decay of HIV-1 RNA measured in heparinized blood after 6 h was sma ll (-0.05 to -0.12 log(10) copies/ml), and the minor impact of this decay o n HIV-1 RNA concentrations in archived plasma samples of the MACS was confi rmed by the similarity of CD4+-cell counts and assay-adjusted HIV-1 RNA con centrations in the MACS and BCDTP.