To assess the effects of long-term storage and freeze-thaw cycles among samples collected in MoBa we designed a quality control study to mimic our study conditions. The MoBa study collects whole blood with EDTA as anti-coagulant from pregnant women throughout Norway [4
]. After collection, the specimens for plasma are spun within 4 hours of collection and shipped overnight at room temperature to a central Biobank in Oslo [4
]. At receipt in the Biobank, samples are aliquoted and placed in long-term freezer storage. A majority of plasma samples are placed on 96-well plates, with up to 95 individuals on one plate; thus specimens may undergo many freeze-thaw cycles until a specific aliquot is pulled.
For our quality assurance specimens, we created an EDTA plasma pool by collecting blood from 40 healthy adult donors in Oslo. Both males and non-pregnant females were included. The blood was collected venously in EDTA tubes (BD Vacutainer). Our goal was to obtain sufficient sample volume to allow repeated measures over time without the need for a new quality assurance pool. To simulate methods used in the MoBa study, the blood stood on the bench for up to 2 hours before plasma was centrifuged at 1800g for 10 minutes at room temperature, and plasma separated. The samples were pooled and stored at 4 °C overnight before aliquoting onto microtitre plates (AB Gene, Epsom, UK), 300 μl per well and sealed with Easy pierce heat sealing foils (AB gene, Epsom, UK). After aliquoting, all plasma samples were stored in the same manner as other MoBa samples, in electric freezers at −80 °C.
To evaluate the impact of freeze-thaw events on specimen quality, we thawed plates weekly for up to 100 weeks. For each time point, three samples, representing three different plates, were sent to the laboratory and were analyzed as three independent samples. shows the schedule of analyte testing following multiple freeze-thaw cycles over time. The EDTA plasma was thawed once a week. The thawing was done at room temperature (1 hour). The samples were retrieved using a pipette to pierce through the foil. When the retrieval of all selected wells on a plate was completed, the holes in the foil were re-sealed with small pieces of a re-sealing heat foil (AB gene, Epsom, UK). For freeze-thaw cycles where no retrievals were done, the samples were homogenized and left on the bench for another 1 hour before freezing again to mimic the approximate retrieval time. We drew an aliquot for chemical analysis after 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 75 and 100 freeze-thaw cycles; t1,t2,…,t100. A time zero sample (t0) was analyzed on fresh plasma 24 hours after collection and simultaneously with freezing the rest of the plasma.
Experimental design. The figure shows the timeline for the analysis
To assess that possible change in marker concentrations were the result of the freeze-thaw process and not storage time in the freezer, we also sent samples that had never been thawed before to analysis after 12 and 24 months, t12m, t24m.
The EDTA plasma was analysed for sodium, cholesterol, triglycerides, free fatty acids, vitamin E and AST and are presented in . We chose this suite of analytes to represent different aspects of sample integrity and as surrogates for other chemical agents which might behave similarly. Sodium was chosen as a marker of volume; if the sample was evaporating, the sodium concentration would be anticipated to increase [6
]. We chose cholesterol and triglycerides as measures of lipid degradation as other investigators have reported changes over time in these analytes [7
]. For sample analysis, we chose laboratories where the analyses were conducted on routine basis. When possible we also chose clinical laboratories where the analyses were ISO 17025 accredited. The samples for sodium, cholesterol, triglycerides, vitamin E and AST were sent at room temperature and analyzed the same day. Samples for free fatty acids, were sent frozen (−80 °C) to analysis and have therefore undergone an additional freeze-thaw cycle.
For each time period and each analyte, we calculated the mean and standard deviation and compared the means after each time point. We compared all sample results to the values reported in the baseline sample (t1
). The t0
sample provides a “fresh” level, but because all specimens in the MoBa will undergo one freeze-thaw cycle at a minimum, this sample was not used to explore changes as a result of freeze-thaw events. Only one of the 360 results was excluded as an outlier using Grubb’s test [11
]. We calculated the percent change from baseline (t1) as follows: ((tx
)*100. Changes in mean component concentrations were evaluated using paired t-tests. All statistical analyses were conducted in SPSS (version 11, Chicago, Illinois). We used p<0.05 as a measure of statistical significance.