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Rimmelé et al. Page 2 NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript disease (1–3). In critical care, 5% of patients will undergo renal replacement therapy for acute kidney injury during their stay in the intensive care unit, and besides renal support, some of these extracorporeal therapies (high volume hemofiltration, super high-flux hemofiltration, hemoperfusion, coupled plasma filtration adsorption) are also proposed as a blood purification treatment for septic shock (4–6). Because of blood exposure to foreign materials, the extracorporeal circuit by itself is responsible for inflammation activation (7). Despite recent improvements in circuit biocompatibility, this additional production of locally formed inflammatory mediators is still considered a major adverse effect (8). For research purposes, ex vivo circuits are commonly employed because they allow for evaluation of filters, dialyzers, sorbent cartridges, or other adsorption devices without any exposure to a patient or an animal, and they can be miniaturized, thus permitting rapid screening of materials (9–13). Blood is usually placed in a reservoir and then circulates through a closed loop with multiple passes through the device being studied. Circuit settings are not standardized and experimental conditions vary from one study to another (9–13). These ex vivo extracorporeal circuits are also under the influence of the inflammatory activation due to special environmental conditions such as artificial tubing, temperature, blood movement, and blood–air interface. However, in order to evaluate blood purification devices using ex vivo circuits, it is important to establish conditions that have the least inflammatory activation possible because this activation may interfere with or overshadow the effects of the device itself. One such parameter that is known to have a significant impact on inflammatory cell activation is temperature. However, in ex vivo circuit experiments, it is unclear if work should be conducted at body temperature (37°C) or some other temperature (e.g., room temperature). One could argue that maintaining blood temperature at 37°C with a warmer could be physiological, but if maintaining body temperature is itself proinflammatory, then this effect will confound any attempt to evaluate artificial material. In addition, hypothermia (room temperature) is known to have anti-inflammatory effects by inhibiting leukocyte response following several tissue insults such as ischemic brain or liver injury (14–16). Therefore, the aim of this study was to evaluate the influence of different blood temperature conditions on cytokine production and leukocyte surface markers expression in a miniaturized extracorporeal ex vivo circuit. MATERIALS AND METHODS Study population Ex vivo circuit The study was approved by the University of Pitts-burgh Institutional Review Board. After consent was obtained, 20 healthy volunteers donated blood for the purpose of these ex vivo experiments. Healthy volunteers were defined as being >18 years old and not pregnant, weighing at least 50 kg, having no history of anemia or hemophilia, and having no history of chronic medical illness or acute infection during the previous 2 weeks. Blood was collected from healthy volunteers into standard vacuum-evacuated blood collection tubes containing sodium heparin. Venipuncture was performed in a dedicated room by trained personnel. The experiment consisted of running the blood through three different conditions over a period of 4 h: (i) a miniaturized ex vivo extracorporeal circuit equipped with a blood warmer set to 37°C; (ii) the same circuit without the warmer (blood kept at room temperature ~23°C); and (iii) blood samples placed into a rocking incubator at 37°C (no circuit). These three experimental conditions are represented in Fig. 1. Artif Organs. Author manuscript; available in PMC 2012 June 1.
Rimmelé et al. Page 3 NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript Components of the extracorporeal circuit were polyethylene tubing (Intramedic, Becton Dickinson and Company, Franklin Lakes, NJ, USA), a 15-mL conical bottom tube to serve as the 10-mL blood reservoir (Becton Dickinson), silicone tubing and a mini-pump (Control Company, Friendswood, TX, USA) set up with a blood flow rate of 0.75 mL/min. Unfractionated heparin (heparin sodium for injection, USP, Hospira, Inc., Lake Forest, IL, USA) was added to blood before the beginning of each experiment in order to obtain a heparin concentration of 10 UI/mL. Sampling and cytokine assays Flow cytometry assays Blood samples were pipetted from the circuit blood reservoir or from the tube placed in the incubator at the following time points: baseline, 15 min, 1h, 2 h, and 4 h. The samples were immediately centrifuged at 1500 rpm for 5 min (temperature of centrifugation = 4°C) and plasma was removed and stored in polypropylene microfuge tubes (Fisher Scientific, Pittsburgh, PA, USA) at −80°C until measurement. Granulocyte macrophage colonystimulating factor (GM-CSF), tumor necrosis factor (TNF), and interleukin (IL)-1 β, IL-6, IL-8, and IL-10 concentrations were measured in duplicate by Luminex bead technology using human inflammatory Five-Plex bead kits combined with human IL-10 bead kits (Invitrogen, Camarillo, CA, USA). Plates were analyzed on a Bio-Rad Bio-Plex 200 protein array system with Bio-Plex Manager 4.0 software (Bio-Rad Laboratories, Hercules, CA, USA). Detection limits were as follows: 3.1 pg/mL for GM-CSF; 6.7 pg/mL for IL-1 β; 1.6 pg/mL for IL-6; 3.8 pg/mL for IL-8; 2.3 pg/mL for TNF; and 4.1 pg/mL for IL-10. To assess if the time between blood withdrawal from the healthy volunteers and the start of the experiments (about 20–30 min) had any influence on the cytokine level measurements (blood temperature decreased just after the draw, then increased due to use of warming), we ran an additional experiment in which blood was withdrawn from healthy volunteers and separated in three tubes. One tube was placed immediately in the incubator at 37°C (for the purpose of this subexperiment, draw was performed at the laboratory, in front of the incubator), one tube was placed in the incubator after having been left at room temperature for 1 h, and the remaining tube was left at room temperature for the entire experiment. Samples for cytokine measurements were processed as described above over 4 h. Blood samples were pipetted from the circuit blood reservoir or from the tube placed in the incubator at baseline, 2 h, and 4 h. Red cells were lysed with BD Pharm Lyse lysing solution (Becton Dickinson) and washed in 1% bovine serum albumin in phosphate buffered saline. Fc receptors were blocked with excess IgG. Cells to be stained for surface markers were incubated with appropriate antibodies and fixed in 1% paraformaldehyde before analysis on Beckman Coulter XL-MCL. All antibodies were from Becton Dickinson, except hTACE (R&D Systems, Minneapolis, MN, USA) and annexin V (Millipore, Billerica, MA, USA). NFkB cells were treated with Cycletest Plus Kit (Becton Dickinson) according to manufacturer instruction prior to staining with anti-NFkB antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). All data were analyzed with FCS Express (De Novo Software, Los Angeles, CA, USA). Cells were gated by distinctive sizes for polymorphonuclear neutrophils (PMNs) and monocytes. Human leukocyte antigen (HLA)- DR, CD11b, CD11a, CD62L, tumor necrosis factor alpha converting enzyme (TACE), and NFkB expression was measured by the geometric mean of fluorescence intensity. Apoptosis was measured as percent positive for annexin V staining, with propidium iodide excluding necrotic cells. Artif Organs. Author manuscript; available in PMC 2012 June 1.
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