Sanne Hoeks

Chapter 6 86 time as patient samples should also be used as comparative controls. Samples should also be collected at the same time of day as, it is known that analyte levels are influenced by circadian rhythms. This is exemplified in Rheumatoid arthritis where proinflammatory cytokines (TNF and IL-6) released at night contribute to morning disease related symptoms. 38 If all these steps can be taken then artificial analyte changes can be circumvented. 4 8 24 48 Time after sampling (h) -20 0 20 40 60 80 100 Change form baseline at 20ºC (%) 4 8 24 48 -20 0 20 40 60 80 100 Change form baseline at 4ºC (%) Citrate NaHep EDTA Serum *** *** *** ** ** ** FIGURE 3. Influence of time and temperature after blood draw. Blood was collected from healthy donors in EDTA (EDTA), SodiumHeparin (NaHep), Sodium Citrate (Citrate) and clotting (serum) tubes. Subsequent to centrifugation, cell free plasma or serum was collected and stored for 4, 8, 24 and 48 hours at room temperature or at 4°C. All the plasma samples where then analyzed by multiplex immunoassay. as described elsewhere. 15 The figure illustrates the level of inflammatory cytokines for various tube types when samples were left at room temperature (left) and 4 degrees (right). *p<0.05, ** p<0.01, *** p<0.001. Long term stability and repetitive use Previous work of our group has shown that most cytokines remain stable within a two year period however degradation of some cytokines can already be seen within one year of storage at -80 °C. 26 In general a decrease over time is seen losing approximately 10–20% each year after 2 years of storage (Fig. 4). Eventually, a five- fold increase or decrease in different cytokine levels can occur after 5 years as a result of cross-reactivity between protein epitopes. 39 In addition to this storage effect, repetitive freeze thawing cycles also influence cytokine recovery. The levels of these molecules can either be stable, increased or decreased after multiple freeze– thawing cycles, depending on each cytokine. In general, cumulative levels significantly decrease (Fig. 3). Thus, in order to optimally measure cytokine levels, samples should be analyzed within 2 years and undergo minimal freeze–thawing procedures so as to maintain stable cytokine levels. 26, 39, 40 If the two year time period is however, not conducive to the experimental set up, individual changes in cytokine/chemokine levels should be assessed before conducting these long term storage studies, or biobanking.