Instability of 8E5 calibration standard revealed by digital PCR risks inaccurate quantification of HIV DNA in clinical samples by qPCR

Abstract

Establishing a cure for HIV is hindered by the persistence of latently infected cells which constitute the viral reservoir. Real-time qPCR, used for quantification of this reservoir by measuring HIV DNA, requires external calibration; a common choice of calibrator is the 8E5 cell line, which is assumed to be stable and to contain one HIV provirus per cell. In contrast, digital PCR requires no external calibration and potentially provides 'absolute' quantification. We compared the performance of qPCR and dPCR in quantifying HIV DNA in 18 patient samples. HIV DNA was detected in 18 by qPCR and in 15 by dPCR, the difference being due to the smaller sample volume analysed by dPCR. There was good quantitative correlation (R² = 0.86) between the techniques but on average dPCR values were only 60% of qPCR values. Surprisingly, investigation revealed that this discrepancy was due to loss of HIV DNA from the 8E5 cell calibrant. 8E5 extracts from two other sources were also shown to have significantly less than one HIV DNA copy per cell and progressive loss of HIV from 8E5 cells during culture was demonstrated. We therefore suggest that the copy number of HIV in 8E5 extracts be established by dPCR prior to use as calibrator.

Figure 1

Comparison between dPCR and qPCR results from 18 PBMC samples from HIV-positive patients, expressed as HIV DNA copies per million cells. The three samples in which HIV DNA was not detected by dPCR are not plotted. (a) qPCR quantities calculated assuming one HIV DNA copy per 8E5 cell. (b) qPCR quantities calculated assuming 0.6 HIV DNA copy per 8E5 cell as determined experimentally by dPCR NB. The dashed line represents equivalence.

Figure 2

HIV DNA copies per cell calculated for different 8E5 sources. (a) Comparison of 8E5 Standards 1, 2 and 3 analysed by dPCR. (b) Effect of culture passage on HIV DNA content per cell for 8E5 Standard 3 measured using the RainDrop^® dPCR platform (c) Effect of culture passage on HIV DNA content per cell for 8E5 Standard 3 measured using the QX200™ dPCR platform. Mean values with standard deviations are plotted.

Figure 3

Median HIV DNA copies per million cells (boxplots with interquartile and range) of seven clinical samples assayed in duplicate by qPCR using 8E5 Standards 1, 2 and 3 for calibration. (a) Calculated assuming one HIV DNA copy per 8E5 cell for all three Standards. (b) Calculated using the actual number of HIV DNA copies per 8E5 cell as determined by dPCR for each of the three Standards.