Sanne Hoeks

Cytokine assays 83 6 SAMPLE STANDARDIZATION Various methodologies are being optimized and designed to allow the precise measurement of cytokine levels within biological fluids however, even in the presence of assay improvement, a key factor needs to be monitored to achieve accurate cytokine measurements, that of sample standardization. The fact cytokines have a short-half life, are released by cells during storage as well as the possibility of degradation during sample handling make it of vital importance that we understand how to treat and store these samples for optimal analysis. 14 As a whole, unless standardization is achieved, data produced by these analytical protocols will remain incomparable and will not represent the true nature of the biological processes at hand. Various types of samples have been used for cytokine analysis. For most patients, these samples can comprise of blood (plasma or serum), 15 urine, 16 saliva, 17 synovial fluid, 15 cerebrospinal fluid, 18 bronchoalveolar fluid, 19 aqueous humour eye fluid, 20 intestinal fluid, 21 exhaled breath condensates, 22 middle ear effusion 23 or lysed biopsies of a diseased organ 24, 25 thus, for each of these sample types, standardization needs to occur. The following section will illustrate which factors influence cytokine levels within common clinical sample types (blood and biopsies), outlining which conditions allow maximum cytokine measurement, stability and representation within a biological process. Sample collection and handling Sample handling and storage are pivotal in biomarker discovery as mishandling of these samples can drastically alter experimental outcomes and produce data that are not reflective of the biological situation. It has been shown for plasma collected in EDTA, sodium heparin (heparin) and sodium citrate (citrate) tubes (see de Jager et al. for methodology 26 ) that, in healthy donors, cytokines are expressed at low levels while chemokines are expressed at higher levels (Fig. 2). In addition, data did not seem to indicate a difference in cytokine levels between anticoagulant effects, as cytokine levels were expressed in a similar range. However, following a consecutive measurement from the same donor (matched data), the general total recovery of spiked cytokines was more stable for heparin and EDTA tube samples, with EDTA tubes exhibiting decreased recovery of IL-15 and IL-18. 26 Citrate tubes showed lower levels of recovery for IL-1β, IL-2 and IL-6 while serum samples had significantly higher levels of CXCL8 and lower IL-6. 26 In addition to this data, it is known that platelet associated chemokines CCL3, CCL5, CXCL4 and TGF- β increase in serum as a result of ex vivo platelet degranulation, this activity could explain the higher levels of CXCL8 recorded in serum samples. 27, 28 Along with platelet degranulation, white blood cells release IL-1β during the clotting process resulting in increased levels of this cytokine in serum samples. 29