Sanne Hoeks

Cytokine assays 85 6 blood at 4 °C before the removal of plasma was sufficient to maintain cytokine levels when compared to control plasma samples (plasma immediately isolated and store at frozen until analysis). After 4h at 4 °C however, cytokine levels already began to increase. 34 Samples stored at RT before separation showed a greater increase in cytokine levels than those seen at 4 °C. After 4h at RT most analytes significantly increased and levels continued to rise significantly over the 48 h. In order to expand on these data, we chose to analyze the effect of temperature, time and tube type on the level of a set of cytokines during a 48 h period. Our analysis revealed that plasma storage temperature (RT or 4 °C), time and collection tube type have an influence of total cytokine levels. In general, it was seen that at 4 °C there is less fluctuation in cytokine levels with minimal variation between plasma collection tubes while serum exhibited a partially higher total marker level compared to plasma samples stored at 4 °C. The data seems to indicate that there is no difference in anticoagulant tube effect or time at 4 °C even after 48 h (Fig. 3). The partially higher levels seen in serum samples can be explained by higher chemokine levels known to occur in serum samples when compared to plasma. 26 At RT data showed that, over a 48 h period, cytokine levels increase drastically with heparin tubes showing the greatest increase (89 ± 28%) followed by serum (56 ± 16%), EDTA (45 ± 13%) and finally citrate (30 ± 12%). The substantial increase in heparin tubes illustrates that although this sample tube type is ideal in multiple measurements for recovery, it is less than optimal when used to collect samples that are left at RT for longer than 4 h. If donor plasma samples are to be left at RT for up to 48 h citrate tubes would be the best for blood collection, showing the smallest change over time. The general increase in analyte levels after 48 h at RT can be explained by the fact that at RT, soluble receptors may begin to release bound analytes into solution resulting in an increase in the measurable amounts of these compounds. 35 It has been shown that blood samples stored at 35 °C have a lower mean concentration of inflammatory markers than those stored at RT however, these levels are higher than those measured in samples stored at 4 °C. 34 Serum isolated from clotted blood stored at 4 °C shows the lowest increase in inflammatory marker concentration while at 35 °C samples have the greatest increase, exhibiting a mean fold increase of 320 when compared to control serum samples. At RT most inflammatory markers are significantly increased and after 48 h general levels are significantly larger ranging from a 15 to 1700 fold increase. As a whole, serum samples do not show a significant decrease in analyte concentration. In addition to this, it is known that activation of the coagulation cascade occurs in serum tubes and that above RT, large amounts of chemokines are released from the blood cells. 34, 36 Taking all this data into consideration, it is of vital importance that whole blood be kept 4 °C until separation into plasma or serum and that this separation occur within 4 h in order to prevent the absorption, release or degradation of cytokines. 37 If procedure specific tube types cannot be used then, any tube can be used as long as it is the same type within an experiment. Healthy donor samples that are stored at the same temperature and for an equal amount of