Analytical performance of an automated assay quantifying HIV-1 from dried blood spots.

BACKGROUND: Performance of an automated sample preparation and viral load quantification for HIV-positive dried blood spots (DBS) on the Siemens VERSANT(®) kPCR Molecular System has been previously demonstrated with clinical samples. OBJECTIVES: Evaluation of the analytical performance of the automated assay using HIV-positive DBS prepared from a dilution series. STUDY DESIGN: Over 300 DBS of HIV-1 nucleic acids from a dilution series in lysed whole blood (322 copies/mL to over 1.6 × 10(7)copies/mL) were spotted onto Whatman 903 cards and analyzed to evaluate analytical performance. Cross contamination was examined with a checkerboard pattern of 82 alternating negative and 1 × 10(6)copies/mL samples. RESULTS: Analytical sensitivity evaluation with a single 50 µL spot demonstrated a limit of detection (LoD) of 866 copies/mL. Above the LoD, linearity (difference between linearized and observed mean values) was within ± 0.10 log, and accuracy (difference between expected and observed mean values) was within ±0.18log. Imprecision for dilution series levels more than two-fold above the LoD was measured as 20% to 27% CV of quantification. No cross contamination was observed. CONCLUSIONS: The HIV-1 DBS Assay performed similarly to the VERSANT HIV-1 RNA 1.0 Assay (kPCR) in assay linearity, accuracy, and imprecision. The assay was sensitive enough to run single 50µL spots and used an unmodified VERSANT(®) SP Module with only a 30 min incubation prior to automated sample preparation. DBS-specific assay calibrators and controls, expressly formulated for easy frozen storage and identical processing to samples, were employed.

A sensitive branched DNA HIV-1 signal amplification viral load assay with single day turnaround.

Branched DNA (bDNA) is a signal amplification technology used in clinical and research laboratories to quantitatively detect nucleic acids. An overnight incubation is a significant drawback of highly sensitive bDNA assays. The VERSANT® HIV-1 RNA 3.0 Assay (bDNA) ("Versant Assay") currently used in clinical laboratories was modified to allow shorter target incubation, enabling the viral load assay to be run in a single day. To dramatically reduce the target incubation from 16-18 h to 2.5 h, composition of only the "Lysis Diluent" solution was modified. Nucleic acid probes in the assay were unchanged. Performance of the modified assay (assay in development; not commercially available) was evaluated and compared to the Versant Assay. Dilution series replicates (>950 results) were used to demonstrate that analytical sensitivity, linearity, accuracy, and precision for the shorter modified assay are comparable to the Versant Assay. HIV RNA-positive clinical specimens (n = 135) showed no significant difference in quantification between the modified assay and the Versant Assay. Equivalent relative quantification of samples of eight genotypes was demonstrated for the two assays. Elevated levels of several potentially interfering endogenous substances had no effect on quantification or specificity of the modified assay. The modified assay with drastically improved turnaround time demonstrates the viability of signal-amplifying technology, such as bDNA, as an alternative to the PCR-based assays dominating viral load monitoring in clinical laboratories. Highly sensitive bDNA assays with a single day turnaround may be ideal for laboratories with especially stringent cost, contamination, or reliability requirements.