Download TARGETED INTEGRATION OF MOBILE ELEMENTS AS A MODEL

S1.4
IN VITRO MICRODIALYSIS OF CYTOKINES
Kirbs, C., Kloft, C.
Department of Clinical Pharmacy and Biochemistry, Freie Universitaet Berlin
Objectives: Cytokines provide a wide range of potential biomarkers for the monitoring of
inflammation, infection, allergy and drug therapy. However, cytokine concentrations in
plasma in most cases do not represent concentrations in irritated tissues. The objective of
this investigation was to apply microdialysis (µD) as a tool for the determination of
macromolecules in the interstitial fluid (ISF), i.e. at the site of inflammation. To characterize
the performance of µD for cytokines prior to in vivo application in vitro investigations of
relative recovery (RR) and relative delivery were performed.
Methods: Microdialysate (µDialysate) was sampled (n=3) at flow rates (FR) of 0.3, 0.5, 1.0,
2.0 and 5.0 µL/min using 3 linear µD probes (CMA 66, 100 kDa cut-off) fixed in a
standardized in vitro microdialysis system [1]. A mixture of Ringer’s solution and human
albumin solution (ratio 9+1) was used as microperfusate and probe-surrounding medium.
This matrix was spiked with 4 model cytokines, namely interleukin 6, 8 and 10 (IL-6, IL-8, IL10) and tumour necrosis factor alpha (TNF-α). Samples were measured using a BD™
Cytometric Bead Array (CBA) adapted to the µDialysate matrix and the BD FACSArray™
Bioanalyzer device [2].
Results: Overall at FR from 5.0 to 0.3 µL/min RR ranged from 2.62%-26.1% for IL-6, 16.9%89.8% for IL-8, 0.79%-9.57% for IL-10 and 0.75%-29.8% for TNF-α (CV: ≤30.9% interprobe
(3 probes, 3 samples) and ≤25.0% intraprobe (n=3)). Relative delivery results deviated from
RR and occasionally showed negative values. Possible reasons for this observation are fluid
loss by ultrafiltration through the probes membrane and subsequent analyte accumulation in
the decreased µDialysate volume.
Conclusion: RR of all 4 model cytokines could be determined (IL-8 > IL-6 > TNF-α > IL-10)
with high variability. The deviation between RR and relative delivery inhibits the applicability
of retrodialysis which is in any case restricted for application to determine in vivo RR due to
physiological reasons: Administration of cytokines into the ISF would lead to immunological
reactions which may result in tissue irritation and inflammation. As in vitro results are
predictive but cannot be used for in vivo ISF concentration calculations an alternative in vivo
probe calibration method has to be developed.
[1] Simmel et al. Int J Clin Pharmacol Ther. 2010
[2] Kirbs. Diploma thesis. 2011