Mediators of Inflammation / 2014 / Article / Tab 1

Review Article

Cytokines as Biomarkers in Rheumatoid Arthritis

Table 1

Summary of reported preanalytical precautions to be enforced to measure some of the main cytokines. A large amount of the literature is contradictory, most likely due to different analytical discrepancies in the evaluation of the effect of preanalytical conditions. This table aims to provide a review of the literature available; however, there is no thoroughly enough conducted study allowing us to suggest definitive guidance as to the best condition to process samples universally (i.e., allowing for any cytokines to be tested). Furthermore, the inflammatory nature of RA further complicates such issues.

Serum or plasmaDelays in separation
(whole blood pending processing)
Storage condition (after separation)Sensitivity to freeze-thawing (F/T) cycles

IL-1
(alpha and beta)
(i) Both are used [26]
(ii) Higher heparin plasma concentrations compared to serum [27]
(iii) Higher levels in EDTA plasma than in heparin plasma [28]
(i) Increased levels with delays in processing when kept at RT
(ii) No significant change for up to 4 days of delayed processing in samples from healthy people; however, there is significant decrease in samples from trauma patients [29]
(iii) Significant increase in serum, after delay of 48 h at 4°C, with RA patients, but in plasma there is an increase only if kept at RT [30]
(iv) Prolonged delays before separation result in increased endotoxin-induced cytokine release in contaminated tubes [31, 32]
(i) Storage at 4°C results in an
increase
(ii) Heparin plasma showed time-dependent increases in concentration [31]
No significant change in stability in plasma/serum for up to 6 F/T cycles [26]

IL-2(i) Heparin plasma concentrations are higher than in serum [27]
(ii) Comparable or higher levels in EDTA plasma compared to heparin [28]
No significant change for up to 4 days of delayed processing in samples from healthy people; however, there is a significant decrease when processing samples for trauma patients [29]

IL-4(i) Heparin plasma concentrations are higher than in serum [27]
(ii) Higher levels of IL-4 in EDTA plasma than in heparin [28] and higher concentration in serum than in heparin plasma [33]
(i) No significant change for up to 4 days of delayed processing [29]
(ii) No significant change for serum or EDTA plasma stored before centrifugation at 4°C, RT, and 35°C [30]

IL-5Slightly higher levels in EDTA plasma than in serum [30] (i) No significant change for up to 4 days of delayed processing [29]
(ii) Plasma levels significantly increased if separation delayed by 4 h stored at 4°C. Further increase if stored at RT [30]
(iii) Serum levels increased with delayed processing for 24 h at 4°C or 4 h at RT [30]

IL-6(i) Serum and EDTA plasma samples are comparable while levels in heparin and citrate plasma are lower [34]
(ii) Serum levels are higher than EDTA plasma [30]
(iii) Serum and EDTA, citrate, or heparin plasma gave comparable results [28, 35]
(iv) Heparin plasma levels are higher than those of serum [27] and this anticoagulant is not recommended due to ex vivo Il-6 release prior to assay [31]
(v) Endotoxin contamination (LPS) triggers release in heparin compared to EDTA plasma [32, 36]
(i) Reduced levels when samples are left unseparated for 24 h at 4°C or RT [26] or 4 h at RT [34]
(ii) Significant reduction in stability and recovery with time at RT [26]
(iii) Increased levels with delays in processing when left at RT
(iv) No change in samples stored at 4°C for 24 h before centrifugation [35]
(v) No change when left at 4°C or 20°C for up to 4 days before centrifugation [37]
(vi) No significant change for up to 4 days of delayed processing in samples from healthy people; however, there is a significant decrease when processing samples from trauma patients [29]
(vii) Plasma levels unchanged when stored for up to 3 h at 37°C but afterwards, an increase is observed [31]
(viii) Increased endotoxin-induced cytokine release in contaminated tubes with delays in processing [32]
No change in levels in serum stored at 4°C, −20°C, and −30°C [37] (i) No significant change for up to 6 F/T cycles
(ii) No significanceobserved after 2, 3, and 4 times of repeated F/T cycles [37]
(iii) No significant effect for up to 3 F/T cycles in EDTA plasma and serum but inconsistent stability in heparin plasma [26, 34]

IL-7(i) No significant difference between plasma and serum IL-7 levels [38]
(ii) Serum levels are significantly higher than in plasma [33]
(iii) Heparin plasma concentrations are higher than in serum [27]
(i) 2 to 4 hours of delayed processing decrease IL-7 plasma levels [38]
(ii) With 2 to 4 hours of delayed processing, serum levels are stable [38]
(iii) No significant change for up to 4 days of delayed processing [29]
Stable for up to 3 F/T cycles

IL-8(i) Comparable levels in heparin plasma and in serum [27]
(ii) Higher serum levels than in heparin plasma [33]
(iii) Lower levels in EDTA plasma than in heparin [28]
(iv) LPS induced release in whole blood is up to 100 times higher in heparin versus EDTA plasma [36]
(i) Increased levels with delays in processing if left at RT [29]
(ii) Stable levels if stored at 4°C

IL-9No significant change for up to 4 days of delayed processing [29]

IL-10(i) Higher levels in serum than in plasma [39]
(ii) Lower levels in EDTA plasma than in heparin [28]
(i) Increased levels with delays in processing if left at RT
(ii) The longer the delay, the less stable the levels
(iii) No significant change for up to 4 days of delayed processing in samples from healthy people; however, there was a significant decrease in samples from trauma patients [29]
Storage temperature affects stability: the higher the temperature, the faster the decline [37] No significant decline in levels observed after 2, 3, or 4 times of repeated F/T cycles [37]

IL-12
(p70 & p40)
Heparin and EDTA plasma levels are higher than in serum [27, 28, 30, 33] (i) Levels decrease with delayed processing [29]
(ii) No significant change for up to 4 days of delayed processing [29]
(iii) Increase in serum after 48 h of delayed processing at 4°C and 4 h at RT [30]
(iv) Stable in plasma for over 48 h at 4°C and for up to 48 h at RT [30]

IL-13(i) Heparin plasma levels are higher than those of serum [27]
(ii) Comparable levels in EDTA and heparin plasma [28]
No significant change for up to 4 days of delayed processing [29]

IL-15No significant change for up to 4 days of delayed processing [29]

IL-16Decrease after the 5th F/T cycle [40]

IL-17(i) Lower levels in EDTA plasma than in heparin [28, 33]
(ii) Higher levels in serum than in any plasma (EDTA, citrate, and heparin) [33]
(iii) Higher levels in EDTA plasma than in serum [30]
(i) No significant change for up to 4 days of delayed processing in samples from healthy people; however, there was a significant decrease in samples from trauma patients [29]
(ii) Plasma levels increased if separation delayed by 4 h at 4°C with further increase with time (up to 24 h) [30]

IL-18Similar levels in serum and EDTA plasma [30] No changes in EDTA levels over 48 h at 4°C, and significant increase after 24 h at RT [30]

TNF-alpha(i) Comparable results in serum and EDTA plasma [39]
(ii) Lower levels in sodium citrate plasma
(iii) Higher heparin and EDTA plasma levels than in serum [26, 27, 30]
(iv) LPS induced release of TNF-alpha 20 times higher when in heparin compared to EDTA plasma [36]
(v) Endotoxin induces high release
[32, 41]
Contradictory data:
(i) Reduced levels with delays in processing when kept at 4°C and RT [26, 42]
(ii) Increased levels with delays in processing if left at RT [34, 43]
(iii) No significant change for up to 4 days of delayed processing [29]
(iv) Time-dependent increases in levels with delays at 37°C in heparin plasma [31]
(i) Reduction in samples kept at RT for 20 days
(ii) Relatively stable in samples stored at 4°C [39]
(iii) Stable at −70°Cfor over 9 months [42]
Contradicting data:
(i) Levels increased with successive F/T cycles [26, 34]
(ii) No differences reported in plasma and serum for up to 10 F/T cycles [39]

TGF-beta 1(i) Higher levels in serum than plasma (citrate, EDTA) due to platelet degranulation during the clotting process [30, 4446]
(ii) EDTA plasma is not recommended because of the extreme interindividual variation of PLT activation and concurrent in  vitro GF release [44]
(iii) Sodium citrate can be used but is not as effective or reliable [44]
(iv) CTAD (citrate theophylline dipyridamole adenosine) is recommended as it blocks the in vitro release of growth factors from PLTs
(v) Plasma concentrations should be corrected by simultaneous measurement of markers of platelet degranulation [47]
(i) Increased levels with delay when plasma is left at RT or 37°C [48] due to platelet degranulation and release [45]
(ii) Lower level in serum if left at 4°C than at RT [49]
(iii) Speed of centrifugation affects recovery in plasma (2,500 ×g for 30 min yields lower levels than 1,200 ×g for 10 min) [49]
<5% deviation from baseline value in serum upon successive F/T cycles (for up to 100 F/T cycles) [48]

sCD40-ligand(i) Use of platelet poor/free plasma is recommended as it is [50] higher in serum than in plasma (EDTA, citrate, and heparin) due to clot retraction and sCD40L shedding from the platelet surface [33, 50]
(ii) EDTA anticoagulated plasma samples are not appropriate for sCD40L measurements [51]
(i) Increased levels after 3 h of delay in processing [50]
(ii) Serum levels increase with time in delayed processing [50]
(iii) No significant changes in serum or plasma levels detected after storage at 4°C for up to 48 h
(iv) Significant loss observed in serum and plasma, left at RT [52]
(v) Decreased levels with increasing centrifugation values (200–13 000 g), which gradually deplete plasma of platelets [52]
(i) Loss in serum and plasma kept for over 4 h at RT [40]
(ii) No change while stored at 4°C
(iii) Significant decrease after 24 h at 37°C [40]
(i) Stable for up to 3 F/T cycles [52]
(ii) Increased after 5 or 10 F/T cycles [40]

IFN-gamma(i) Collection in sterile pyrogen free tubes is very essential
(ii) Serum levels are higher than in plasma (EDTA, citrate, and heparin) [33]
(iii) Heparin plasma levels are higher than in serum [27]
(iv) Levels in heparin plasma are higher than in EDTA plasma [33]
(v) Levels in EDTA plasma are higher than in heparin plasma [28]
(i) Significant reduction with time at both 4°C and RT in serum and EDTA tubes [26]
(ii) IFN-gamma decreases if processing is delayed [29]
Stable for up to six F/T cycles [26]