Stable hepatitis C virus RNA detection by RT-PCR during four days storage

Abstract

Background: Suboptimal specimen processing and storage conditions of samples which contain hepatitis C virus (HCV) RNA may result in a decline of HCV RNA concentration or false-negative results in the detection of HCV RNA in serum. We evaluated the stability of HCV RNA in serum and clotted blood samples stored at room temperature or at 4 degrees C for 4 days with the aim of optimizing the standard procedures of processing and storage of samples.

Methods: Blood from five HCV RNA positive patients was collected in tubes with and without separator gel, centrifuged 1 or 6 hours after collection. Samples were then left 6, 24, 48, 72 or 96 h at room temperature (21.5 - 25.4 degrees C) or at 4 degrees C before determining their HCV RNA level using the COBAS AMPLICOR HCV MONITOR Test, vs 2.0 (Roche Diagnostic Systems).

Results: The logarithm of the HCV RNA level measurements remained within a 0.3 value of the means for 4 days at both temperatures (room temperature or 4 degrees C).

Conclusions: We conclude that blood samples may be collected and aliquoted within 6 h of collection and can be stored at 4 degrees C for 72 hours as proposed by the manufacturer without significant differences in measured HCV RNA level. Our results indicate that lapses in this scheme may still yield reliable results.

Figure 1

Viral load measurements over time in patient 1 in IU/ml on a log scale. SS-TA: tube without serum separator at room temperature; SS-4°C: tube with serum separator at 4° C; AS-1H-TA: tube with separator, centrifuged within the first hour and kept at room temperature; AS-1H-4°C: tube with separator, centrifuged within the first hour and kept at 4°C; AS-6H-TA: tube with separator, centrifuged after 6 hours and kept at room temperature; AS-6H-4°C: tube with separator, centrifuged after 6 hours and kept at 4°C. The mean of measurements at each point of time is given in open triangles linked by a thick line.

Figure 2

Viral load measurements over time in patient 2 in IU/ml on a log scale. SS-TA: tube without serum separator at room temperature; SS-4°C: tube with serum separator at 4°C; AS-1H-TA: tube with separator, centrifuged within the first hour and kept at room temperature; AS-1H-4°C: tube with separator, centrifuged within the first hour and kept at 4°C; AS-6H-TA: tube with separator, centrifuged after 6 hours and kept at room temperature; AS-6H-4°C: tube with separator, centrifuged after 6 hours and kept at 4°C. The mean of measurements at each point of time is given in open triangles linked by a thick line.

Figure 3

Viral load measurements over time in patient 3 in IU/ml on a log scale. SS-TA: tube without serum separator at room temperature; SS-4°C: tube with serum separator at 4°C; AS-1H-TA: tube with separator, centrifuged within the first hour and kept at room temperature; AS-1H-4°C: tube with separator, centrifuged within the first hour and kept at 4°C; AS-6H-TA: tube with separator, centrifuged after 6 hours and kept at room temperature; AS-6H-4°C: tube with separator, centrifuged after 6 hours and kept at 4°C. The mean of measurements at each point of time is given in open triangles linked by a thick line.

Figure 4

Viral load measurements over time in patient 4 in IU/ml on a log scale. SS-TA: tube without serum separator at room temperature; SS-4°C: tube with serum separator at 4°C; AS-1H-TA: tube with separator, centrifuged within the first hour and kept at room temperature; AS-1H-4°C: tube with separator, centrifuged within the first hour and kept at 4°C; AS-6H-TA: tube with separator, centrifuged after 6 hours and kept at room temperature; AS-6H-4°C: tube with separator, centrifuged after 6 hours and kept at 4°C. The mean of measurements at each point of time is given in open triangles linked by a thick line.

Figure 5

Viral load measurements over time in patient 5 in IU/ml on a log scale. SS-TA: tube without serum separator at room temperature; SS-4°C: tube with serum separator at 4°C; AS-1H-TA: tube with separator, centrifuged within the first hour and kept at room temperature; AS-1H-4°C: tube with separator, centrifuged within the first hour and kept at 4°C; AS-6H-TA: tube with separator, centrifuged after 6 hours and kept at room temperature; AS-6H-4°C: tube with separator, centrifuged after 6 hours and kept at 4°C. The mean of measurements at each point of time is given in open triangles linked by a thick line.