Matt Harmon

50 Chapter three Methods Study design, patients and definitions From January 2011 through July 2013, consecutive patients presenting to the mixed intensive care units (ICU) of two Dutch tertiary teaching hospitals (Academic Medical Center in Amsterdam and University Medical Center in Utrecht) were included. Medical Ethical Committees of both centers approved an opt-out consent method (IRB no.10-056C). Data and plasma samples were prospectively collected as part of the Molecular Diagnosis and Risk Stratification of Sepsis (MARS) project (ClinicalTrials.gov identifier NCT01905033). 13,14 A group of trained investigators collected clinical data. The plausibility of infection was scored post hoc classified on a 4-point scale ( none , possible , probable or definite ) 15,16 , as described in detail previously. 14 Shock was defined as the use of vasopressors for hypotension in a dose of 0.1 mcg/kg/min during at least 50% of the day. Acute kidney injury (AKI) and acute lung injury (ALI) were scored using pre-set criteria. 17,18 We selected patients with sepsis, diagnosed within 24 hours of admission, defined as a definite or probable infection 14 combined with at least one parameter of inflammatory dysfunction, hemodynamic dysfunction, organ dysfunction or deranged tissue perfusion (derived from the 2001 International Sepsis Definitions Conference 2 ). Patients with immunodeficiency disorders, use of corticosteroids, immunosuppressive or antineoplastic drugs were excluded. To exclude iatrogenic hypothermia, readmitted patients, patients undergoing active cooling and patients transferred from another ICU or operating room (OR) were also excluded. To control for inadvertent temperatures that may have been entered in the database (i.e. a rectal sensor that has been displaced and is exposed to ambient temperature) patients with unreliable temperatures (below 33°C) were not included. Also patients with a missing minimum temperature were not included. Temperature was measured using a rectal, nasal, inguinal or tympanic temperature probe. Core temperatures were used over inguinal or tympanic measurements. The threshold for hypothermia was set at 36°C, based on previously used cutoff levels. 10,19 Daily (at admission and at 6 a.m. thereafter) left-over EDTA anticoagulated plasma (obtained from blood drawn for patient care) was stored within 4 hours at -80°C. Samples were drawn prior to rewarming patients. Plasma biomarker measurements TNF- α , IL-1 β , IL-6, IL-8, IL-10, IL-13, soluble intercellular adhesion molecule (ICAM)-1, fractalkine and soluble E-selectin were measured using FlexSet