Overestimation of Human Immunodeficiency Virus Type 1 Load Caused by the Presence of Cells in Plasma from Plasma Preparation Tubes

The human immunodeficiency virus type 1 (HIV-1) load is an important marker of disease progression and treatment efficacy in patients with HIV-1 infection. In recent years, an increase in the number of samples with detectable HIV-1 RNA has been reported among patients with previously suppressed viral loads, affecting clinical patient care and leading to repeat measurements of viral load and drug resistance. This rise seems to have coincided with the increased use of plasma preparation tubes (PPTs) for sample collection, and we have aimed to explain why PPTs might yield elevated HIV-1 RNA levels. The impacts of different sample-processing procedures on HIV-1 RNA levels were compared retrospectively. Prospectively, the presence of different cells and cell-associated HIV-1 nucleic acids in paired plasma samples from PPTs centrifuged before (PPT1) and after (PPT2) transportation to the laboratory was compared. A retrospective analysis of 4,049 patient samples with <1,000 HIV-1 RNA copies/ml showed elevated HIV-1 RNA levels in plasma from PPT1 compared with the levels from PPT2 and standard EDTA-containing tubes. Prospective data revealed cell-associated HIV-1 nucleic acids and abundant blood cells in plasma from PPT1 but not from the corresponding PPT2. The levels of HIV-1 RNA correlated with the lymphocyte counts in plasma in PPT1. Cells could be removed by the recentrifugation of PPT1 before analysis. In conclusion, the transportation of PPTs after centrifugation may render cells in the plasma fraction containing cell-associated HIV-1 nucleic acids that contribute significantly to the HIV-1 RNA copy numbers in patients with low viral loads.Quantification of human immunodeficiency virus type 1 (HIV-1) RNA in plasma is an essential tool in the clinical management of patients with HIV-1 infection and is used to monitor HIV-1 disease progression and the response to antiretroviral therapy (ART) (9, 12, 14, 15). It is also an important end point in most HIV-1 treatment clinical trials.Although intermittent low-level viremia is often reported among patients receiving seemingly effective ART (4, 13, 16, 23), the unexpected detection of HIV-1 RNA might have a clinical impact and lead to repeat quantification of HIV-1 RNA and testing for drug resistance. In recent years, several HIV-1 clinics and laboratories have reported an increased number of cases of detectable HIV-1 RNA in patients with previous suppressed viral loads, raising concerns about drug resistance and virologic failure (8, 17, 20). At the Oslo University Hospital, Ullevål, we have also experienced an unusual increase in the proportion of plasma samples with HIV-1 RNA levels in the range of 40 to 1,000 copies/ml. This seems to have coincided with a change in the routines for plasma sample collection, and here we present data providing an explanation for this phenomenon.It is recommended that blood samples for HIV-1 RNA quantification be collected in tubes with EDTA as an anticoagulant (3, 12). Standard EDTA-containing tubes require transfer of the plasma to a secondary tube within 6 h after sample collection to reduce the risk of RNA degradation (3). This is often inconvenient in a patient clinic, and as an alternative, plasma preparation tubes (PPTs; Becton Dickinson [BD], Franklin Lakes, NJ) are increasingly often used. Upon centrifugation of the PPTs, a gel barrier separates the plasma from most of the cellular elements, theoretically allowing transportation of the sample in the primary tube.The collection of samples in PPTs from patients receiving effective ART is reported to yield increased levels of HIV-1 RNA compared with the levels in plasma collected in standard EDTA-containing tubes (5, 6, 8, 17, 18, 20, 21). However, the reason for this discrepancy has remained unclear. Our data confirm the previous findings that PPTs generate significantly higher HIV-1 RNA levels in samples from patients with low-level viremia. Additionally, we have shown that the source of this overestimation is cell-associated HIV-1 nucleic acids. These findings necessitate a reconsideration of low-level viral load results in plasma obtained from PPTs not handled with care after centrifugation. Furthermore, we present a simple procedure that will circumvent the problem.