Optimization of specimen-handling procedures for accurate quantitation of levels of human immunodeficiency virus RNA in plasma by reverse transcriptase PCR

Abstract

Human immunodeficiency virus type 1 (HIV-1) RNA levels in plasma are currently widely used clinically for prognostication and in monitoring antiretroviral therapy. Accurate and reproducible results are critical for patient management. To determine the effects of specimen collection and handling procedures on quantitative measurement of HIV-1 RNA, we compared anticoagulants and sample processing times. Whole blood was collected from 20 HIV-1-infected patients in EDTA, acid citrate dextrose (ACD), and heparin tubes, aliquoted, and stored at room temperature. Plasma was separated from whole-blood aliquots prepared at < or =1, 3, 6, 24, and 48 h postcollection and then stored at -70 degrees C until use. HIV-1 RNA levels were determined by the AMPLICOR HIV-1 MONITOR assay. Heparinized plasma samples, which were pretreated with heparinase prior to analysis, had the lowest baseline HIV-1 RNA levels. In the first 6 h, HIV-1 RNA levels decreased by 10, 20, and 31% in EDTA, ACD, and heparin tubes, respectively. From 6 to 48 h postcollection, HIV-1 RNA levels decreased in all anticoagulants, albeit at a slower, more consistent rate. Our results indicate that EDTA should be the anticoagulant of choice for plasma HIV-1 RNA measurement by reverse transcriptase PCR, but ACD tubes are acceptable if the plasma is separated within 6 h of blood collection. Caution must be applied in the interpretation of absolute HIV-1 RNA copy number values obtained with suboptimal specimen collection and processing procedures.

FIG. 1

(A) Baseline (≤1 h) HIV-1 RNA copy numbers in plasma collected from infected individuals with three anticoagulants. HIV-1 RNA levels are expressed as the numbers of HIV-1 RNA copies per milliliter of plasma. Symbols: ○, patients who were being treated with ZDV at the time of specimen collection; •, patients who were not receiving ZDV at the time of specimen collection. Horizontal bars indicate the means of the measured variables. (B) Comparison of HIV-1 RNA copy numbers, as determined by RT-PCR, of EDTA-, ACD-, and heparin-treated plasma at baseline. Results are expressed as percentages of the mean EDTA baseline HIV-1 RNA copy number. Horizontal bars indicate SD. P values were determined by the Kruskal-Wallis test.

FIG. 2

(A) Stability of HIV-1 RNA in whole blood stored at room temperature. Results are expressed as mean percentages of baseline copy numbers for EDTA (top curve), ACD (middle curve), and heparinized (bottom curve) plasma. Horizontal bars represent SD. (B) Stability of HIV-1 RNA in whole blood stored at room temperature in comparison to mean baseline (≤1 h) EDTA-treated specimens. Results are expressed as mean percentages of EDTA baseline copy numbers for EDTA (top curve), ACD (middle curve), and heparinized (bottom curve) plasma. Horizontal bars represent SD.