Sanne Hoeks

Chapter 6 88 blood, serum or plasma sample storage. Requiring a small volume of blood, (approximately 15 μl), on a stable and portable filter paper, 42 these samples are less vulnerable to biological degradation. However, even with higher analyte stability, the sample extraction process for DBS results in the dilution of low abundant proteins. This dilution factor greatly influences the cytokine panel being measured and results in the loss of delicate disease associated cytokines. 43 Lysed tissue (biopsies) In order to analyze diseased tissue, biopsies must be taken. Due to the fact that not all cytokine mediated pathological responses can be measured systemically, measuring cytokine levels within the diseased tissue can provide a better understanding of the cellular pathological process. In addition to this, cytokine levels that can be measured systemically only provide a reflection of what is occurring at the sight of inflammation whilst biopsies provide a key primary representation of the tissue specific diseases process. As with all samples, the processing of this tissue is important. When analyzing cytokine levels in whole cells, samples need to initially be lysed. We thus performed an experiment to determine if the type of lysis buffer used could alter cytokine levels measured due to increased background levels. For these experiments, peripheral blood human mononuclear cells where used to mimic biopsy tissue due to the fact that tissue samples where not readily available. Cells were stimulated with LPS (4 h, 37 °C). Following stimulation, cells were lysed with various lysis buffers; Roche Complete Lysis M, Cell Signaling Technology (CST) or Bio-Rad Lysis buffers. Data showed that the Roche lysis buffer had the highest noise/ background levels and resulted in the lowest cytokine measurements, Bio-Rad background levels were lower than those of Roche with partially higher cytokine levels while the CST lysis buffer resulted in the least background activity and produced the highest cytokine measurements, resulting in a better signal to noise ratio (Fig. 5). Our data clearly indicates that depending on the lysis buffer used, cytokine levels measured within tissue can be drastically altered, indicating that lower lysis buffer background levels result in higher cytokine measurements. It is thus important to understand the functionality of any lysis buffer used in tissue sample analysis as this may influence the discovery of cytokine networks that cannot be measured systemically but rather have a localized tissue specific role. 24