Sanne Hoeks

Cytokine assays 87 6 BL 1 2 3 4 Time (Years) 60 70 80 90 100 110 Change from baseline (%) 1 2 3 4 5 Freeze/thaw cylce 60 70 80 90 100 110 * *** ** * FIGURE 4. Influence of long time storage and multiple freeze thawing cycles. Blood samples of healthy individuals were stored at -80°C and measured at baseline and various time points. As shown in the left panel cytokine levels are subject to change even frozen at -80°C after approximately 2 years of storage. Next blood samples were repeatedly thawed and cytokine profiles were assed. After several cycles cytokine levels are subjected to breakdown, resulting in lower levels (right panel). * p< 0.05, ** p<0.01, *** p<0.001. Several long term stored samples used in experiments or housed in biobanks are, in some cases, not originally collected for the experiments they are used in. A prime example of this is the use of dried blood spots (DBS) in neonatal screening. Excess DBS samples from these procedures are often stored in biobanks for use in other research as the Guthrie card on which samples are stored is an easy and appropriate medium for transport and long term storage. 34, 35, 41 The use of these samples in research and particularly inflammatory marker research means that a comprehensive understanding of sample storage, handling and analyte stability is required. Skogstrand et al. performed experiments in which DBS were stored for several days at various temperatures. Analysis of these samples revealed that the levels of inflammatory markers remained stable for up to 7 days when compared to control DBS samples stored at -20 °C. Any changes seen in analyte levels at this stage were smaller than those observed in plasma and serum from whole blood stored for shorter periods of times. 34 After 30 days however, some pro-inflammatory markers showed a significant change in concentration. In addition to this, at RT or 35 °C, there was no specific trend for individual inflammatory markers but rather a trend towards increased analyte levels with protection of samples against humidification at 4 °C having no positive influence. Even though measurable levels of pro-inflammatory markers were lower in DBS, these levels were more stable than those observed for liquid blood samples. Sample storage for several years, however, still results in varied levels of individual compounds. It is suggested in this study that faster drying of DBS and storage at low temperatures would be the optimal method by which to conserve analyte levels. 34 DBS thus seem to be a possible reliable alternative method for whole