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

Abstract

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.

Coamplification of HIV-1 Proviral DNA and Viral RNA in Assays Used for Quantification of HIV-1 RNA

“…Artificially elevated HIV-1 RNA levels in PPTs are suggestive of HIV-1 viremia and consequently associated with clinical failure to HAART (1, 3, 4, 13). Recent reports have determined that elevated VLs in PPTs, linked to transient viremia or blips (14), are due to technical aberrations caused by cell-associated HIV nucleic acids (5,6,8). Our data clearly suggest that this artificial elevation in VL is due to the presence of HIV-1 proviral DNA from residual cells present in the plasma specimen.…”

“…In a recent prospective study, Kran et al (5) observed a direct correlation between the number of cells present in the plasma, measured in the Advia 60 cell counter, and the amount of HIV-1 that was quantified using the Roche COBAS AmpliPrep-COBAS TaqMan (CAP/TAQ) HIV-1 test. Using paired specimens that were collected and centrifuged in PPTs, they found lower cell counts and reduced HIV-1 VL in the PPTs that were recentrifuged prior to HIV-1 quantification versus the PPTs that were centrifuged once, after phlebotomy.…”

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

“…Discrepancies in VL results obtained after freezing the plasma in situ in PPT are attributed to the presence and amplification of cellular proviral DNA in the extracted RNA that is used for testing (6), although some investigators believe that platelets associated with the virus could play a role in the process (9). In fact our own studies (13) identified the presence of integrated proviral DNA in the plasmas frozen in situ in PPT, suggesting that this proviral DNA, upon amplification in the Roche Cobas monitor assay, yielded quantifiable VL results.…”

“…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.…”