Biobanking represents a new and valuable way in which translational research can be performed. It is critical that biobanks conform to the highest standards and provide researchers with materials of consistently high quality. The IDB utilizes standardized operating procedures based upon EEC standards (ISO guideline 34, No. 17025: 2005), works within the UNE-EN-ISO 9001:2000 guidelines, and is a member of the International Society for Biological and Environmental Repositories to maintain high standards and to facilitate future interbiobank networking capabilities. Preanalytical variation is a major source of experimental error,14
and an important consideration for clinical archives is ensuring that materials do not become degraded between collection and delivery to researchers. Thus, IDB PVB samples for the HIRD study were processed within a venepuncture-to-freezer time of <4.5
h for all samples. As part of ongoing reviews of IDB standards, we constantly monitor available quality-control data generated by studies using IDB samples.
Overall, the RNA integrity of the PBMNC preparations for the HIRD study was high, with all having RIN values well above the minimum recommended for microarray analyses. There were no significant differences between mean RIN values for PBMNCs collected at the 4 different visits or between those before and after H1N1 vaccination. Nevertheless, there was a range of RIN values observed and the potential source(s) of variation were therefore investigated.
Initially, we determined whether there was an association between ischemic times as this could affect the integrity of RNA as well as even the types of mRNA expressed.15
However, there was no association between these variables (P
>0.05) or between the individual subcomponents of this delay. Others have also noted that small delays in ischemic procurement times (up to 1
h) do not significantly decrease RNA quality from pancreatic cancers.16
Similarly, in this report, delays of up to 3
h had no detectable influence on PBMNC RNA quality. The main contributor to ischemic time for the IDB samples was that they were dispatched from the clinic in batches of 4 donations and the delay was due to the time involved in consenting, questioning and bleeding subsequent donors.
The quality of PBMNC preparations was inferred from 2 proxy indicators, RNA and PBMNC yields; however, neither was associated with RIN values. Another possibility was that samples had become degraded in a time-dependent manner during storage. Had this been the case, those stored longest should have had lowest RIN scores, but this was not observed. There was no variation in procedures or reagent degradation. Additionally, there was no evidence of freezer malfunction during the study (these were alarmed to the mobile telephones of key workers and also monitored daily for temperature fluctuations, which are logged). The fact that the IDB was processing between 1.5 and 2.9
L of blood/week over this time means that there was a high turnover of all reagents and also makes the explanation of reagent degradation unlikely. The polynomial line of best fit indicated that RNA integrity was lower during spring and autumn, yet higher during the summer. Despite this analysis, the reason for this variation could not be explained by technical variations or intervolunteer characteristics. Similarly, the specimens were received frozen in Germany (and shipping had a negligible effect upon RNA yield or quality17
) and no differences in the different RNA preparations by Miltenyi Ltd. were detected.
There are some caveats with the present observations. First, the time of storage between collection and analyses was relatively short (8 months) and biobanks may need to store samples for many years, especially if medical information associated with disease outcomes is required. Second, although RIN values are a good measure of RNA quality, it would be useful in future studies to additionally compare RIN to polymerase chain reaction performance on a target gene to see whether this is comparable. Finally, it would also be interesting to determine whether different technicians have similar RIN scores.
There are physiological factors beyond the IDB's control that might explain the present observations. For example, no differences were observed between baseline differential blood counts for those volunteers with RIN values of 8.5 or less, but others have reported nadirs in PVB subsets of CD4+, CD8+, and NK cells,18
as well as a zenith in vitamin D levels,19
in the summer. Both of these factors may affect the overall quality of PBMNC RNA. However, to determine the true nature of this seasonal variation, dedicated studies need to be performed.
Many others have considered the viability of archived samples, although most studies have been restricted to the analyses of labile serum components such as enzymes20
or recording the number of freeze–thaw cycles that individual samples have undergone.21
The IDB has a policy of not reissuing plasmas or sera that have undergone a freeze–thaw process and has checked the virological viability of plasmas collected from other cohorts by successfully isolating and sequencing human immunodeficiency virus nucleic acids from around 33% of its archived stocks. Here, the IDB had a unique opportunity to directly assess the quality of stored RNA and, by inference, also that of the PBMNC preparations. The samples themselves had only been archived for a relatively short time period (maximum 8 months) and it is entirely possible that a greater range of (lower) RIN values would have been observed upon prolonged storage.
In conclusion, although the IDB was able to provide high-quality PBMNC RNA preparations sufficient for transcriptome analyses to researchers, there was some variation in RNA quality albeit minimal. The reason for these differences could not be ascertained from recorded technical variables other than the date of sample processing, which implied a seasonal variation rather than degradation.