Evaluation of Effect of Specimen-Handling Parameters for Plasma Preparation Tubes on Viral Load Measurements Obtained by Using the Abbott RealTime HIV-1 Load Assay

HIV-1 viral load testing is essential to the management of HIV-1-infected patients, and proper specimen handling ensures accurate viral load (VL) results. This study was performed to (i) evaluate the effect of freezing plasma in situ in BD Vacutainer plasma preparation tubes (PPT) on the accuracy of HIV-1 viral load results using the Abbott RealTime HIV-1 assay and (ii) evaluate the effect of whole-blood storage in the PPT for 6 h at room temperature prior to centrifugation (PPT6H) rather than 2 h as specified in the PPT product insert. Of the 64 HIV-positive subjects evaluated, 29 had average viral load counts of >40 copies/ml in at least one of the tubes tested and 35 subjects had a result of either “undetected target” or “below the limit of quantification” (LOQ) for all or some of the tubes regardless of handling condition. For the 29 subjects with VLs that were >LOQ, the mean biases between plasma from Vacutainer K₂EDTA tubes and plasma frozen in situ in PPT and between K₂EDTA tube plasma and plasma from PPT6H (log₁₀ copies/ml) were 0.005 and −0.001, respectively, and r² was >0.92 for all correlations. We conclude that VLs determined from plasma frozen in situ in PPT are equivalent to VLs in K₂EDTA tube plasma and can be used for accurate quantification of HIV-1 RNA in the Abbott RealTime HIV-1 assay. Furthermore specimens collected in PPT can be stored for 6 h at room temperature with no effect on viral load results as measured by the Abbott RealTime HIV-1 assay.Accurate quantification of human immunodeficiency virus type 1 (HIV-1), also referred to as HIV-1 viral load (VL) testing, is essential for effective management of HIV-1-infected patients. The BD Vacutainer plasma preparation tube (PPT) (BD Preanalytical Systems, Franklin Lakes, NJ) was developed to facilitate the handling of plasma specimens used for HIV-1 viral load testing. The PPT contains a K₂EDTA additive and a polyester gel which, upon centrifugation, forms a barrier that separates blood cells from plasma, allowing storage, freezing, and shipment of the plasma specimen in situ in PPT. Use of PPT also results in a decrease in the amount of labor required to process specimens, elimination of a potential source of error in specimen labeling, and reduction in the risk of HIV exposure associated with the transfer of separated plasma to secondary tubes for shipping.Earlier studies using PPT for specimen collection demonstrated compatibility of this tube with HIV-1 VL tests (1, 4, 5). Over the past few years, however, investigators have reported that freezing plasma in situ in a PPT produced higher HIV-1 viral load results compared to plasma from K₂EDTA tubes when tested in the Cobas Amplicor HIV-1 monitor system (Roche Molecular Diagnostics, Pleasanton, CA) (2, 3, 11). Elevated levels of HIV-1 VL from PPT frozen in situ after centrifugation were first reported by Squires et al., who showed that PPT yielded higher HIV RNA levels than K₂EDTA tubes. The disparity in quantification, seen in both standard and ultrasensitive assays, was more apparent in specimens with VLs between the limit of quantification (LOQ), 50 copies/ml, and 1,000 copies/ml and more pronounced in specimens close to or below 50 copies/ml (3, 10, 11). In fact, studies that compared plasma aspirated from K₂EDTA tubes with plasma frozen in situ in PPT show that VLs that are clearly below the LOQ in the former are quantifiable in the latter (3, 8, 11). Such discrepant results for plasma collected in different tubes from the same subject could be interpreted as virological failure, and these results, therefore, have therapeutic implications.Additional studies reported that specimens which were aspirated and transferred to another tube after centrifugation did not show patterns of artificially increased viral load (8, 10). Similarly, recentrifugation of specimens transported in PPT at 4°C eliminated the inaccurate quantification of HIV seen in plasmas frozen in situ in PPT (6, 9).Such observations led investigators to associate elevated viral loads in plasma in PPT with the presence of cell-associated nucleic acids released from cells trapped within or adhered to the surface of the gel barrier in PPT (6). Depletion of cellular material by recentrifugation of separated, unfrozen plasma from PPT resulted in lower VLs, indicating that cell-associated nucleic acid contributed to the elevation in VL. This finding was compatible with an earlier study which had shown that the HIV proviral DNA associated with cellular (genomic) DNA was in part responsible for the increase in viral load (13).The aim of this study was to evaluate the performance of PPT with the new automated RealTime HIV-1 assay (Abbott Molecular, Des Plaines, IL) for the quantification of HIV-1 viral load using the Abbott m2000sp and m2000rt systems for sample processing and amplification/detection, respectively. Specifically, we were interested in determining the effect of freezing plasma in situ in PPT on HIV-1 viral load analyzed using the Abbott RealTime HIV-1 assay. In addition, the study evaluated the effect on viral load of whole-blood storage in PPT for 6 h before centrifugation rather than 2 h, as specified in the PPT product insert.