Introduction
One million particles per patient may be infused daily in an intensive care setting, where a potential risk appears against the background of debilitation and impaired host responses [
1,
2]. Mechanisms of particle damage to various organs have been postulated, including effects on microcirculation causing thrombi and granuloma formation [
3‐
5]. Use of in-line filtration almost completely prevents particulate infusion [
6]. Recent studies have evaluated the clinical effect of in-line filtration and showed significant reduction in thrombophlebitis [
7] and a trend towards reduction of complications in neonates such as sepsis, thrombosis, and necrotizing enterocolitis [
8].
To evaluate the effects of microfilters in the pediatric intensive care unit (PICU), we carried out detailed examinations of filters after their use. Electron microscopy (EM) visualized particles adhering to the membrane, and energy dispersion spectroscopy (EDX) revealed their chemical compositions [
9]. Based on these data immune-modulating effects on human umbilical vein endothelial cells (HUVEC) and murine macrophages (RAW 264.7) were analyzed in vitro.
Materials and methods
Prior to this study, the infusion regiment in the PICU was optimized to prevent precipitation and incompatibilities using a computer-based analysis. Solutions and therapeutics were prepared according to the manufacturers’ recommendations. Approval by the ethics committee and parental consent were obtained.
EM and EDX
Twenty-five Pall ELD96LLCE/NEO96E filters with 0.2 μm pore size (Pall, Dreieich, Germany) were analyzed. Twenty filters from nine patients were randomly chosen after 72 h. Five unused filters served as controls (see Table 1, ESM). For detailed information see ESM.
Cell culture
To simulate the in vivo situation, glass particles (2–20 μm) were chosen to mimic the silicon content of particles found on the filters. Contamination with lipopolysaccharide (LPS) was excluded. Inflammatory effects were analyzed on RAW 264.7 (gamma NO-, ATCC CRL-2278) and HUVEC (CC2517). Cytotoxic particle load was determined by release of adenylatkinase (ToxiLight test) and lactate-dehydrogenase (LDH-cytotoxicity test). Experiments were performed at subtoxic loads. Cells were incubated with glass particles at contents from 0 to 500 μg/ml. Release of interleukin 1-beta (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor alpha (TNFα) was measured after 4, 8, and 24 h with ELISA kits for IL-1β (DY201), IL-6 (DY406), IL-8 (DY208), and TNFα (DY410; all kits by R&D Duoset).
Cells were additionally incubated with 1 μg/ml LPS, and HUVEC cells with 1 ng/ml TNFα as well. Results of ELISA are given as the median of repeated determinations. ToxiLight test results reflect the mean of four trials and considered values within three standard deviations of the controls as nontoxic. Statistics were processed by Dunnett’s test [
10].
Discussion
Our results confirmed the existence of particulate contamination in infusions despite a stringent infusion regime in the PICU. Different contents of particles were found, and data suggest that particle exposure is dependent on the complexity of infusions and medications. These findings are in keeping with the results of Mehrkens et al. [
1], who showed that bolus injections mainly increase particulate contamination of the administered fluids.
Considering the detection limit of 5 μm, the detected amount underestimates the particle exposure. Fifty percent of the analyzed membranes showed conglomerates containing uncountable numbers of particles. Smaller particles were retained in the three-dimensional meshwork of the membrane and were disregarded. Most particles were between 5 and 50 μm, which is more than the diameter of lung and tissue capillaries [
11] and may cause obstruction. This supports the findings of Puntis et al. [
4] that particles cause granulomatous arteritis after parenteral nutrition as demonstrated by post-mortem study of 73 infants.
In accordance with other authors, we identified silicon as a major chemical component [
12], which originates mainly from storage in glass ampoules [
13].
In addition to mechanical obstruction, inflammatory response of different organ systems after particle exposure is possible [
3,
14,
15]. To mimic these mechanisms, we exposed macrophages and endothelial cells to particles similar to those found on the filters. Our experiments with endothelial cells showed significant suppression of IL-1β after incubation with particles for 8 and 24 h. Comparable results were assessed for IL-8. This effect was aggravated by LPS. In macrophages, different particle loads suppressed the secretion of TNFα. Similar effects were evident for IL-6, which was significantly suppressed after 8 h incubation. Increased IL-8 was found after incubation with 300–500 μg/ml particles. This was probably due to cytotoxicity as was seen for comparable particle contents in pre-tests.
Previously published data suggest an activation of the immune system by particles [
15]. In contrast, our experiments showed a reduced cytokine release. Immune paralysis is an accepted phenomenon in sepsis or after ischemia or trauma, as confirmed by clinical and experimental studies [
16‐
19]. The importance of the exact regulation of the chemokine release and the balance of pro-inflammatory and anti-inflammatory mediators in this phase of the disease is not exactly clear. Small changes may have an influence and alter the process of overcoming the disease. Foreign particles may interfere with this phase of the disease when the patient is especially vulnerable.
Lehr et al. [
5] demonstrated that stress increases the harmful effects of particles. Their animal model revealed a reduction in capillary density in post-ischemic muscles after particle infusion, in cases without preceding ischemia, no change in density was detectable.
A documented benefit of filtration is the prevention of infusion phlebitis [
7]. Less information exists for the influence of filtration on severely ill patients. One single-center trial on 88 preterm newborns showed a trend toward decreased morbidity in the treatment group [
8]. However, the incidence of sepsis could not be reduced by in-line filtration [
20].
According to the data of a preceding analysis, 70% of our PICU patients were provided with a central-venous line with three lumina for standardized infusion therapy. Despite this standard, our examinations demonstrate that particle exposure continues to be an unpredictable risk for patients receiving infusion therapy.
Review of literature and our data suggest that particles are capable of modulating cytokine secretion and may exert harmful effects beyond mechanical obstruction. Whether particles affect the outcome of PICU patients has to be further evaluated.
Acknowledgments
We thank Dr. Capewell from the Pall Corporation for his support and B. Braun and Pall Corporations for the financial support. This study was funded by Pall Corporation, Dreieich, Germany and B. Braun Corporation, Melsungen, Germany. Dr. Jack and Dr. Sasse received lecture and travel fees form Pall Corporation and B. Braun Corporation. Dr. Boehne received travel fees from Pall Corporation. All authors indicate that their institutions have received research grants from Pall Corporation and B. Braun Corporation.