Reply to “Normal range for cytokines should be reported”

Dear Editor, We thank you for the opportunity to further discuss our recent article regarding the effects of hydroxyethyl starch 130/0.42 (HES) vs Ringer’s acetate (RA) on cytokine levels in a subgroup of 226 patients with severe sepsis and septic shock from the Scandinavian Starch for Severe Sepsis/Septic Shock (6S) trial. We also thank Professor R. G. Hahn for his interesting and relevant comments which are discussed below. The aim of our study was to elucidate the pathophysiology behind the findings of the 6S trial. Our hypothesis was that the inflammatory response in septic patients receiving HES would differ from the response in those receiving RA. We measured this response as differential changes in tumor necrosis factor alpha (TNFa), interleukin (IL)-6, and IL-10, rather than absolute cytokine values. It is well known that cytokine levels in patients with sepsis are highly variable and inconsistently elevated. Many relatively small cohort studies have reported correlations between different plasma cytokine concentrations at baseline and outcome, whereas others have not. This reflects that numerous factors affect the results of cytokine measurements in septic patients including genetic differences, chronic and acute morbidities, the underlying focus of infection, timing of the sample, handling of the sample, and method of analysis, all of which reduces the utility of cytokines as biomarkers in sepsis. For example, septic patients often exhibit large cytokine fluctuations at the onset of sepsis; but in some patients cytokine levels are not elevated even at admission, and if elevated, cytokines may decline or even normalize within days. The 6S trial included adult patients in need of fluid resuscitations who fulfilled the criteria for severe sepsis within the previous 24 hours, but as with most clinical trials, the time of onset of sepsis remains unclear. The blood we sampled in our subgroup of patients from the 6S trial only represents a snapshot of the patients’ cytokine status at randomization (before exposure to the intervention) and at the end of day 2 and does not represent the full time course of cytokine levels in patients with severe sepsis and septic shock; thus, initial rises in the cytokine concentrations may have gone undetected. However, this does not invalidate the hypothesis of changes in inflammation with the use of HES vs RA and we, therefore, focused on changes from baseline following exposure to the intervention. As Professor R. G. Hahn correctly pointed out, we used the commercially available Human Proinflammatory Panel 1, V Plex from Meso Scale Diagnostics, Rockville, MD, USA for the cytokine measurements in our study. However, the reported normal values are medians and not means. Cytokine measurements in sepsis are rarely normally distributed and typically, a significant number of patients presents with concentrations that are close to the lower detection limits, with a smaller number of patients with markedly elevated concentrations. Mean concentrations are, therefore, disproportionally influenced by outliers and concentrations should probably be reported as medians. To illustrate, the mean cytokine concentrations of TNF-a, IL-6, and IL-10 at randomization in the HES vs RA group were 21.9 vs 41.9 pg/mL, 308.7 vs 399.2 pg/mL and 78.2 vs 146.8 pg/mL respectively—markedly higher than the reported baseline medians. The manual supplied by Meso Scale Diagnostics state that 20 normal individuals had a median [range] of TNF-a, IL-6, and IL-10 in citrate plasma of 0.48 [0.19-3.17], 0.21 [0.12-0.47], and 0.21 [0.092. 21], respectively. So according to the normal values supplied by the manufacturer, patients in the both the HES and RA groups had elevated median cytokine values at randomization and IL-6 most markedly so. This is in concordance with other studies. However, as professor R. G. Hahn correctly states, the interpretation depends on the normal reference material used; others have reported substantially higher median normal values using a magnetic bead-based multiplex assay (Bio Plex, BIO-RAD Laboratories, Milano, Italy). Multiple high-sensitivity multiplex assays are commercially available, but there are substantial differences between their ability to detect and measure plasma cytokine concentrations as well as variability in measurements between laboratories using the same multiplex assay and even between lots. This may contribute to the relatively large differences between “normal” cytokine values reported by manufactures and researches. When evaluating absolute levels of cytokines in different disease conditions, we fully agree with R. G. Hahn that reporting productspecific or even study-specific normal reference material would ease interpretation and eliminate some of the factors inducing variability between studies.