Systemic Inflammation Response Syndrome
after
Transcatheter Aortic Valve Implantation
Final report
Rozanne Tieman
Academy of Technology, Environment and Health
Avans University, Breda
Forensic Laboratory Research
Systemic Inflammation Syndrome Response after Transcatheter Aortic
Valve Implantation
Final report
by
Rozanne Tieman
Rozanne Tieman 2032475 Supervisor:Dr.P.G. Noordzij- Department of Anesthesiology, Intensive Care and Pain Medicine B.Meek- Department of Medical Microbiology and Immunology
St. Antonius hospital, Nieuwegein
K. Rodenburg, Academy of Technology, Environment and Health Avans University, Breda
03/2015
Abstract
Transcatheter aortic valve implantation (TAVI) has become the standard of care in high-risk patients with severe aortic valve stenosis who are not eligible for conventional aortic valve replacement. Despite the less invasive nature of TAVI, mortality remains high. Prior studies1,2 reported that TAVI is associated with the development of
systemic inflammatory response syndrome (SIRS). SIRS is a serious condition in which there is an abnormal regulation of various cytokines that directly cause a decline in remote organ function by mediating the production of nitric oxide leading to a condition of cellular inability to use oxygen. This may lead to multiple organ dysfunction syndrome (MODS), multiple organ failure syndrome (MOFS) and ultimately to death. The reason why SIRS develops following TAVI is yet unclear. It is hypothesized that due to periods of low cardiac output during the procedure, ischemia-reperfusion injury and subsequent SIRS might occur. Other studies3
suggest that bacterial translocation during low cardiac output may induce SIRS.
The main study objectives were to determine if inflammatory biomarkers can confirm and predict post-procedural SIRS and to determine if ischemia/organ damage biomarkers and gram- negative bacteria biomarkers are associated with SIRS after TAVI.
Between July 2014 and January 2015, 19 patients were enrolled for this pilot study. Blood samples were obtained pre-, peri- and post- TAVI procedure. Biomarkers that cause immunological activation or indicate ischemia/organ damage were analysed in patients plasma, using a multiplex fluorescent immunoassay (Luminex) and enzyme-linked immunosorbent assay (ELISA). Pro-inflammatory cytokines IL-1β, IL-6, IL-8, TNFα and anti-inflammatory cytokines IL-1RA (receptor antagonist), IL-10 are expected to reach high concentrations soon after the infectious stimulus. Furthermore, resulting acute phase proteins CRP and Pentraxin 3 were tested. Ischemia/ organ damage markers I-FABP (intestine) and KIM-1 (kidney) were tested in addition to LAL, LBP, EndoCab, sTREM-1 and sCD14, which could indicate translocation of gram-negative bacteria.
In the first 48 hours following TAVI, 11 patients (57,9%), developed SIRS. Four of these patients (36.4%) died during hospital intake. Peri-and post -procedural characteristics that could be potential risk factors for the development of SIRS did not show any significant difference between the SIRS and non-SIRS group. Such as use of rapid ventricular pacing (RVP) (9 vs. 7 P=1.000) number of RVP (1(IQR 0-4) vs. 1 (IQR 0-3) P=0,604) and duration of RVP (14(IQR 9-40) vs. 13 seconds (IQR 10-24) P=0.533), pre-dilatation of the native aortic valve (54.5% vs. 71.4%; P=0,637), valve- in-valve implantation (10% vs. 0%; P=1.000), post-dilatation of the valve prosthesis (45.5% vs.16.7%; P=0.333), and intervention time (89±16 v. 78±11 minutes; P=0,245).
SIRS after TAVI is common and associated with increased levels of pro-inflammatory cytokines IL-6 and IL-8 and resulting acute phase protein CRP. The combination of these cytokines can reliably and early predict the risk of developing SIRS and might enabling early treatment and increase long- term survival. Ischemia/organ damage biomarkers I-FABP and KIM-1 did not show a difference between the SIRS and non-SIRS group. Furthermore, bacterial translocation is not associated with the occurrence of SIRS, despite the elevated concentrations of gram-negative bacteria (LPS) in both groups. Our limited data do not support the hypothesis that SIRS after TAVI is caused by ischemia-reperfusion injury and/ or bacterial translocation. However, it should be noted that due to the small population, this study is purely observational, and interpretation is speculative.
Abbreviations
CARS compensatory inflammatory response syndrome
CRP C- reactive protein
EndoCab Endotoxin core antibodies
h hour
I-FABP intestinal fatty acid binding protein
IL-
interleukin-KIM-1 kidney injury molecule -1
LAL limulus amebocyte lysate
LBP lipopolysaccharide binding protein
LPS lipopolysaccharide
PAMPs pathogen associate molecule patterns
RVP rapid ventricular pacing
SIRS systemic inflammatory response syndrome
sCD14 soluble cluster of differentiation 14
TAVI transcatheter aortic valve implementation
Index
Abstract...3
1. Introduction...6
2. Theoretical background...7
2.1 Transcatheter aortic valve implantation (TAVI)...7
2.2 Systemic Inflammatory Response Syndrome (SIRS)...8
2.3 Biomarkers...8
Cytokines...8
Acute phase proteins...9
Ischemia/Organ damage markers...9
Gram-negative bacteria markers...10
2.4 Enzyme linked immunoassay (ELISA)...11
2.5 Multiplex fluorescence immunoassay (Luminex)...11
3. Method...12
3.1 Blood sample collection...13
3.2 Multiplex fluorescence immunoassay (Luminex)...13
3.3 Enzyme linked immunoassay (ELISA)...14
4. Results...15
4.1 Inflammatory biomarkers...18
4.2 Ischemia/ organ damage markers...19
4.3 Gram-negative bacteria markers...20
5. Discussion...21
6. Conclusion...21
7. Timetable...22
1. Introduction
This is a pilot study of a substudy of the multicentre prospective POPular-TAVI trial. This is a randomized controlled trial in seven participating Dutch centers that evaluates the use of different antithrombotic treatment strategies after TAVI. The study population consisted of patients with severe, symptomatic aortic stenosis who underwent TAVI procedure due to qualified inoperability for open heart surgery or estimated high surgical risk. All patients who, did not meet the exclusion criteria, written the informed consent, participated in the POPular –TAVI trial in the St. Antonius Hospital and were automatically included in this pilot study “SIRS after TAVI”. Despite the minimally invasive nature of TAVI, the incidence of acute kidney injury (AKI) and mortality remains high. Previous studies showed that an increased leukocyte count and the development of systemic
inflammatory response syndrome (SIRS) after TAVI were associated with acute kidney injury and mortality.2 The
reason why SIRS develops following TAVI is yet unclear. It is hypothesized that due to periods of low cardiac output during the procedure, ischemia-reperfusion injury and subsequent SIRS might occur. Other studies suggest that bacterial translocation during low cardiac output states may induce SIRS.3
The objective of this study is to elucidate these hypothesis and to determine if inflammatory biomarkers can confirm and predict post-procedural SIRS. Which might enable adapted or early treatment, following increase of long-term survival.
This report is divided in six chapters. In chapter two all relevant information with respect to the research will be described in the theoretical background. The method, equipment and material that where applied for this research will be discussed in chapter three. The obtained results will be viewed in chapter four and discussed in chapter five. Finally, the conclusion and possible recommendations are given in chapter six, followed by the bibliography and appendices
2.
Theoretical background
2.1
Transcatheter aortic valve implantation (TAVI)
Aortic valve stenosis (AS) is a degenerative process and prevalence increases with age. Patients with AS remain asymptomatic for a long period but when symptoms occur prognosis rapidly worsens, with an annual mortality rate of approximately 30%.4 Treatment of AS consists of aortic valve replacement using cardiopulmonary bypass
and sternotomy.5 However, high-risk patients with multiple comorbidities and severe AS are not suitable
candidates for conventional aortic valve replacement. Since 2002, transcatheter aortic valve implantation (TAVI) is an less invasive alternative therapeutic option for these patients with an elevated surgical risk.1 This
treatment displaces and functionally replaces the native valve with a bioprosthetic valve delivered on a catheter through the femoral artery (transfemoral placement) or the left ventricular apex (transapical placement). See figure 1. Regularly, balloon valvuloplasty was performed when balloon-expandable bioprosthetic valves were used (no self-expandable) and for pre- and post-dilatation of the biopsthetic valves. In order to prevent shifting of the balloon valvuloplasty blood pressure, RVP (180 beats per minute), via the cardiac pacing wire, was perfomed.6 By using TAVI, sternotomy and cardiopulmonary bypass are avoided, which may improve outcome
in this fragile patient group. However, The PARTNER Trial (Placement of Aortic Transcatheter Valves) showed that the mortality rate after a TAVI (24,2%) is almost equivalent to a open-heart surgery (26,8%).7
Despite its minimally invasive nature, TAVI, is invariably associated with a number of complications that affect outcome, such as a higher incidence of major strokes, vascular complications or acute kidney injury (AKI). Another study of Nuis et al. showed that AKI was reported in 21% of the 995 patients who undergo TAVI.8 AKI
was a predictor of both short- and long-term mortality, whereas blood transfusion predicted long-term mortality. These findings indicate that the outcome of TAVI may be improved by a more restrictive use of blood transfusions. This may be
explained by the fact that in association with the transfusion of red blood cells a number of other cellular and molecular substances are administrated that either directly or indirectly (inflammation) induce kidney damage.8 Also, temporary
hypoperfusion of the kidney during the procedure might be responsible for a significant cytokine release culminating in a inflammatory (SIRS MODs) reaction.8
Figure 1. Schematic representation of a TAVI procedure with the two most used type of valves. On the left the placement (transfemoral and transapical) of an Edward SAPIEN XT valve and on the right the placement (transfemoral) of a Medtronic CoreValve.9
2.2
Systemic Inflammatory Response Syndrome (SIRS)
The systemic immune response induced by non-infectious agents is called systemic inflammatory response syndrome (SIRS) and infection- induced systemic immune response is called sepsis. Besides fever (< 36.0˚C or >38.0˚C) the term SIRS is based on; heart rate >90 beats/minute, respiratory rate >20 breaths/minute, or PaCO2
<32mmHg, leukocyte count <4 or >12 (109/L). When two or more of these variables are present, SIRS is
defined.10
The host inflammatory response in SIRS and sepsis is similar. The inflammatory mediators directly affect organ function and cause a decline in remote organ function by mediating the production of nitric oxide leading to mitochondrial anergy and cytopathic hypoxia, a condition of cellular inability to use oxygen.11 This may lead to
multiple organ dysfunction syndrome (MODS), which may progress to multiple organ failure syndrome (MOFS) and ultimately to death.12 See figure 2.
Inflammation is fundamentally a protective response to tissue damage. The inflammatory response is typically well controlled by a decrease in the pro-inflammatory mediators and by the release of endogenous antagonists of which the goal is homeostasis.10 However, an
overshoot of pro- and anti-inflammatory mediators may lead to SIRS. If homeostasis is not resorted, a significant systemic reaction occurs. The predominant effects of cytokines become destructive rather than protective. The anti-inflammatory response can eventually become prevalent, leading to the compensatory
anti-inflammatory response syndrome (CARS), characterized by immune anergy, associated with a high risk of severe infections and death.12
Sinning et al. have investigated, for the first time, the
prevalence of SIRS among 152 patients undergoing TAVI and the impact of SIRS on outcome. SIRS developed in 61 of 152 patients (40,1%) during the first 48 hours after TAVI. Already 4 hours after the procedure a significantly elevated leucocyte count was observed with a maximum level at 48 hours, in patients with post-procedural SIRS.2 The development of SIRS was also significantly related to the occurrence of major vascular complications,
major bleeding and AKI, as well as the amount of contrast dye, which was higher in SIRS patients. In addition, SIRS occurred more frequently in patients with the need of repeated ventricular pacing and post dilatation.2
Figure 2. Schematic representation of the pathogenic mechanisms of SIRS and sepsis.12
2.3
Biomarkers
The following biomarkers that were tested during this research will be described in this paragraph.
Cytokines
Pro-inflammatory cytokines IL-1β, IL-6, IL-8 and TNFα induce acute inflammation response and expected to reach high concentration soon after the infectious stimulus. These will be tested in combination with the anti-inflammatory cytokines IL-1RA and IL-10, that control the pro-anti-inflammatory cytokine response. It is well known that IL-4, IL-10 and transforming growth factor ß (TGF-ß) are considered anti-inflammatory cytokines because, when administered to animals with infection or inflammation, they reduce the severity of disease and reduce the production of IL-1α/β and TNFα.13 These anti-inflammatory cytokines can inhibit cell surface expression (LPS
recognition), PRRs and nuclear factor-κB (NF-κB). Anti-inflammatory cytokines can also promote the synthesis of other anti-inflammatory cytokines or promotes degradation of messenger RNA for pro-inflammatory
cytokines.14 Increased levels of IL-1β, IL-6, IL-8, TNFα, IL-1RA and IL-10 have been previously associated with
SIRS.15-19
Acute phase proteins
Pro-inflammatory cytokines trigger hepatocytes, these cells built up 70-85% of the liver’s mass, to produce acute phase proteins. At least some of these soluble proteins, particularly C-reactive protein (CRP), bind to pathogen associate molecule patterns (PAMPs). Once bound to the surface of a microbe, these soluble molecules stimulate phagocytosis by innate leukocytes and can activate the classical complement pathway.4
Between 4 and 6 hours after stimulation, a considerable increase in CRP levels is observed. These levels double every 8 hours, then peak between 36 and 50 hours after an infection or acute inflammation has occured.20
The CRP is well charactarized as short Pentraxins. Pentraxins are a superfamily of acute phase reactants Pentraxin 3 (PTX3), also known as Tumor necrosis factor-Stimulated Gene 14, is the prototypic long Pentraxin. The protein is locally produced and released by a variety of cell types including macrophages, neutrophils, smooth muscle cells and alveolar epithelium. PTX3 is induced in response to either inflammatory cytokines IL-1 β and TNFα or PAMPs. Just like CRP, PTX3 has a dual role in the regulation of the innate immune response. PTX3 induces classical complement activation and participates in the clearance of apoptic cells. PTX3 is elevated in critically ill patients, with a gradient from systematic inflammatory resonse syndrome to septic shock. 21
Ischemia/Organ damage markers
Fatty acid-binding proteins (FABPs) are a class of cytoplasmic proteins that bind long chain fatty acids. FABPs are small intracellular proteins (~13-14 kDa) with a high degree of tissue specificity. They are abundantly present in various cell types and play an important role in the intracellular utilization of fatty acids, transport and
metabolism. Due to its small size, FABP leaks rapidly out of ischemically damaged necrotic cells leading to a rise in serum levels. Ischemically damaged tissues are characterized histologically by absence (or low presence) of FABP facilitating recognition of such areas. Intestinal FABP (I-FABP) is specifically localized in the epithelium cells of the small bowel. The I-FABP protein is derived from the human FABP2 gene. Normally, I-FABP is undetectable in serum. Many observations indicate that I-FABP is a useful biochemical marker for intestinal cell damage both in vivo and in vitro. Furthermore, SIRS is, according to Kentaro S. et al., related to an increasing concentration of I-FABP in patients plasma.22-24
Kidney Injury Molecule-1 (KIM-1) is a type 1 transmembrane protein, with an immunoglovulin and mucin domain whose expression is markedly up-regulated in the proximal tubule in the post-ischemic rat kidney.
According to Han K. et all. A soluble form of human KIM-1 can be detected in the urine of patients with acute tubular necrosis and may serve as a useful biomarker for renal proximal tubule injury facilitating the early diagnosis of the disease and serving as a diagnostic discriminator.25
Gram-negative bacteria markers
Lipopolysaccharide (LPS) Binding Protein (LBP) is a type 1 acute phase protein that is
constitutively produced by the liver and rapidly upregulated during the acute phase response. LBP plays a central role in the response to LPS. The protein catalyzes the monomerization of LPS and its transfer to soluble cluster of differentiation 14 (sCD14). This way LBP has both a role in the activation pathway of LPS: activation of monocytes by LPS leading to release of inflammatory mediators and in the neutralization of LPS i.e. the uptake of LPS by lipoprotein and subsequent clearing .In plasma of healthy individuals LBP is present at levels of approximately 10 μg/ml, which increase approximately 10-fold during acute phase responses.26
Limulus amebocyte lysate (LAL) is an aqueous extract of blood cells (amoebocytes) from the horseshoe crab. LAL reacts with bacterial endotoxin (LPS) by triggering the turbidity and gel-forming reaction enzymatically. This reaction is the basis of the LAL test, which is the used for the detection and quantification of bacterial endotoxins.27
Several studies show a consistent drop in postoperative levels of circulating anti-endotoxin core antibodies (EndoCab) compared to the preoperative value. This drop has been interpreted as consumption of antibodies to endotoxin by systemic release of endotoxin. A hypothesis is that if the patients pre-operative endotoxin-core level is low, even moderately low, patients may not be able to cope with the efflux of endotoxin, which may have mild to severe clinical consequences. The assay is of interest for various experimental conditions ranging from in vitro LPS neutralization by plasma components to in vivo studies on kinetics of antibodies to endotoxin in health and diseases. 28,29
CD14 expressed mainly on the surface of monocytes/macrophages, and polymorphonuclear cells (PMN), the cells responsible for scavenging of LPS and bacteria. In addition to the function as receptor for LPS, the soluble form of CD14 (sCD14), which lacks the GPI anchor, is also involved in LPS-induced cell activation. Two forms of sCD14 have been described. An approximately 48 kDa form, derived from monocytes membrane CD14, and a 56 kDa form, speculated to be directly released in plasma or supernatant after processing. sCD14 is an intermediate in the transfer of LPS to lipoproteins, resulting into neutralization of LPS. On the contrary, sCD14 facilitates LPS activation of CD14-membrane negative cells like endo- and epithelium. Furthermore, high concentrations of sCD14 were shown to block LPS-induced activation of monocytes. sCD14 thus both enhances and reduces cellular responses to LPS. sCD14 was demonstrated to be present in plasma in levels ranging from 2 - 4 µg/ml and to be enhanced in infectious diseases.30
Soluble human Triggering Receptor Expressed on Myeloid Cells 1 (sTREM-1) is a protein expressed on
granulocytes and peripheral monocytes. It has been described that the molecule can be further upregulated by stimulation of the cells by bacteria and their products. Triggering of the receptor will result in the release of pro-inflammatory cytokines and chemokines and the upregulation of surface activation markers and in that respect is thought to be amplifying inflammatory responses to bacterial infections and potentiate septic shock. It has been observed that the soluble form of TREM-1 is substantially different in serum of patients with sepsis. Measurement of levels of soluble TREM-1 might be valuable to determine progression of the inflammatory status of the patient.31
2.4 Enzyme linked immunoassay (ELISA)
The enzyme-linked immunosorbent assay (ELISA) is a specific and highly sensitive method for quantification of cytokines and other analytes in solution. It is one of the most widely used techniques in clinical and research laboratories. The assay involves a specific monoclonal antibody, which is used to coat a microtiter plate. After
addition of the sample, the specific antibody on the plate will capture the protein of interest (e.g. a cytokine). A second monoclonal antibody, which is used for detection, binds a different epitope on the protein. The
detection antibody is labeled with biotin, which allows subsequent binding of a streptavidin-conjugated enzyme. Any unbound reagents are removed by washing. Streptavidin-peroxidase conjugate will react with the substrate, tetramethylbenzidine (TMB). The enzyme reaction is stopped by the addition of citric acid. After addition of a substrate, a color reaction will develop that is directly proportional to the amount of protein bound. The absorbance will be measured at a specific wavelength with a spectrophotometer . The
concentration of protein in the sample is determined by comparison with a standard curve of known protein concentrations.32 For a schematic review of this principle see figure 3.
.
2.5 Multiplex fluorescence immunoassay (Luminex)
The principle of Luminex multiplex immunoassay is to a large extent similar to the principle of ELISA. Luminex uses proprietary techniques to internally color-code microspheres with two fluorescent dyes. Through precise concentrations of two dyes, 100 distinctly colored ead sets can be created, each of which is coated with a specific capture antibody or cytokine. After an analyte from a test sample is captured by the bead, a biotinylated detection antibody is introduced. The reaction mixture is then incubated with Streptavidin PE conjugate, the reporter molecule, to complete the reaction on the surface of each microsphere. The microspheres are allowed to pass rapidly through a laser, which excites the internal dyes marking the miscosphere set. A second laser excites PE, the fluorescent dye on the reporter molecule .Finally, high-speed digital-signal processors identify each individual microsphere and quantify the result of its bioassay, based on fluorescent reporter signals.34 For a schematic review of this principle see figure 4.
Figure 4. Principle of a multiplex fluorescence immunoassay (Luminex)34
3. Method
After heart team agrees on indication and treatment proposal, patients has provided written informed consent and do not meet any of the following criteria;
1) Need for long-term oral anticoagulation;
2)Drug-eluting stent implantation within 3 months prior to TAVI procedure; 3) Bare-metal stent implantation within 1 month prior to TAVI procedure;
4) Concurrent inclusion or participation in another trial (depending on the character of the study), they are included in the POPular TAVI trial. Patients that are included in the POPular TAVI trial, are also included in this pilot substudy.
Before surgery all patients were screened on pre-existing comorbidities and pre-operative medication. These baseline characteristics are shown in table 1 (chapter 4). Peri-procedural characteristics might be related with the occurrence of SIRS and were documented as well (see table 2 chapter 4).
3.1 Blood sample collection
On the day of surgery (every Thursday max. 3 TAVI patients) blood samples were collected after induction of general anaesthesia, during the procedure, after the TAVI procedure on the index day and daily thereafter for 4 days. These were the specific time intervals for collecting the blood samples: 0,+1,+3,+6,+24,+48,+96h. These blood samples were collected in a labelled 3ml EDTA vile and were centrifuged at 4270 n/min (4˚C) for 10 minutes, immediately after collecting. Thereafter supernatantia (plasma) was divided in four labelled tubes, which then were stored in a freezer at -60˚C.
The following biomarkers were analysed on the plasma samples; pro- inflammatory cytokines: IL-1β, IL-6, IL-8 and TNFα; anti-inflammatory cytokines: IL-1RA and IL-10;
acute phase proteins: CRP and Pentraxin 3;
ischemia/ organ damage markers: I-FABP (intestine) KIM-1 (kidney).
gram-negative bacteria markers: LBP, LAL, EndoCab, sTREM-1 and sCD14 (activated soluble receptors).
3.2 Multiplex fluorescence immunoassay (Luminex)
Pro-inflammatory (IL-1β, IL-6, IL-8 and TNFα) and anti-inflammatory (IL-1RA and IL-10) cytokines were tested with this technique. Only for cytokines IL-6, IL-8 and TNFα a Human Sensitiviy Cytokine kit (Milliplex) was used. The remaining cytokines were analysed in the Wilhelmina Child Hospital, Utrecht.
The following reagents were supplied in this kit: - Standard
- Quality controls 1 and 2 - Serum matrix
- 96-well plate and sealer - Assay buffer
- 10x wash buffer - Detection antibodies - Streptavidin-Phycoerythirin - Mixing bottle
- Anti-Human IL-8 beads - Anti-Human TNFα beads
These reagents were used after the following preparation:
Each antibody-bead vial was vortexed and sonicated for 30 seconds. 48µl from each antibody bead vial was added to the Mixing bottle and brought to a final volume of 3.0 ml with Assay Buffer. These mixed beads were mixed well with the vortex.
Quality control 1 and 2 were reconstitute with 250 µl MilliQ. After the vials were inverted several times and sit for 5-10 minutes, they were transferred in propylene tubes.
30 ml of the 10x wash buffer was diluted in270 ml MilliQ .
The lyophilized Serum Matrix was reconstituted into 5.0 ml MilliQ and was mixed well.
Two Standard vials were reconstituted in 250µl MilliQ (2000 pg/ml). After these were mixed well and sit for 5-10 minutes they were transferred into a labelled polypropylene tube. These were the first standards (S1). Thereafter, seven other polypropylene tubes were labelled (S2, S3, S4, S5, S7 and S8) for each standard. In each of these tubes 200 µl Assay Buffer was added. Thereafter, serial dilutions were done by adding 50µl of S1 into S2, this was mixed well and the procedure was repeated until S8( this was the blanco).
The procedure started by pre-wet the plate by adding 200 µl of wash buffer into each well. After sealing and mixing the plate on a plate shaker for 10 minutes, the wash buffer was removed by inverting the plate above a sink. Residues were removed by tapping it onto absorbent towels several times. Mixed beads were vortexed and 25µl were add to each well. Subsequently, the plate was placed on a magnet for 1 minute and inverted (while on magnet) above the sink. After removing the plate from the magnet, 100 µl wash buffer was added. The plate was sealed and placed on a plate shaker for 30 seconds. Finally, the plate was placed on the magnet again and the followed washing steps were repeated.
50µl of the standards(S1 t/m S7), quality controls 1 and 2, samples and blanco (S8) were added into the appropriate wells. Assay buffer was added (50 µl) to all sample wells and Serum Matrix (50µl) was added to all standard, blanco and quality control wells. The plate was sealed and incubated overnight on a plate shaker at 4°C.
Next morning the plate was washed twice with 100µl wash buffer and subsequently, 50µl detection body was added to each well. The plate was sealed and incubated for 1 hour on a plate shaker at room temperature. Thereafter, streptavidin-phycoerythirin (50µl) was added in each well. The plate was sealed again and incubated for 30 minutes on a plate shaker at room temperature. Wash the plate twice with 200µl wash buffer and 150µl sheath fluid was added to all the wells. Finally, the beads were resuspended on a plate shaker for 5 minutes and was read on the Luminex (LOW RP1 target; 100µl, 50 beads per bead set; gate settings 5000 to 25000)
3.3 Enzyme linked immunoassay (ELISA)
Acute phase protein; Pentraxin3, Ischemia marker; I-FABP and gram-negative marker; LBP, LAL, EndoCab, sTREM-1 and sCD14 were tested with this technique. Hycult biotech HUMAN ELISA kits were used for these biomarkers.
In general, for each of this kit the following reagents were supplied: - Wash buffer
- Dilution buffer - Standard
- Biotinylated detection antibody - Streptavidin-peroxidase
- TMB substrate - Stop solution
- 12 Microtiter strips, pre-coated
Plasma samples, wash buffer, dilution buffer, tracer , streptavidine-peroxidase and standards were pre-diluted with MilliQ or dilutionbuffer before they could be used in the assay. The amount of dilution differed per assay. When these reagents were brought to room temperature and the necessary microwell strips were placed into the supplied frame, 100 µl of standards and samples were transferred into the appropriate wells. The tray was sealed and incubated for 1 hour at room temperature (or 37°C, differs per protocol). Thereafter, wash buffer was used to wash the plates 4 times as follows: The plate was inverted above the sink and tapped on a dry and thick layers of tissues. Wash buffer (200 µl) was added to each well, after 20 seconds the plate was inverted above the sink again and these steps were repeated three times more. Subsequently, 100 µl of diluted tracer was added to each well, using the same pipetting order as the samples and standards were applied. After the tray was sealed and incubated for 1 hour at room temperature, the washing steps were repeated four times again. Thereafter, diluted streptavidin-peroxidase was added to each well using the same pipetting order as the samples and standards were applied. Also, after this step the tray was sealed, incubated for 1 hour at room temperature (or 37°C, differs per protocol) and washed four times again. Subsequently, 100 μl of TMB substrate was added to each well, using the same pipetting order as the samples and standards were applied. After the tray was sealed it was incubated in a dark environment at room temperature. Regularly, the reaction on the plate was controlled and around 30 minutes after incubation, the reaction was stopped with 100 μl of stop solution with the same sequence and timing as the TMB was applied. Finally the plate was read within 30 minutes after addition of stop solution at 450 nm using a plate reader.
4. Results
Pre-existing comorbidities, pre-operative medication use and peri-procedural characteristics might aggravate the post-interventional course of TAVI patients and are expected to be related with the occurrence of SIRS. Therefore the following parameters of the patients shown in table 1 and 2 were documented.
Table 1. Baseline caracteristics according to the occurrence of systemic inflammatory
response syndrome.
Variable SIRS (n = 11) non SIRS (n = 8 ) P-value Age (years) 78.2±7.3 82.8±4.7 0.136 Male [n (%)] 8 (72.7) 6 (75.0) 1.000 BMI [kg/m2 (%)] 26,49 ± 4,3 23,8 ± 4,1 0.189 Comorbidity COPD [n (%)] 3 (27.3) 1 (12.5) 0,603 Diabetes [n (%)] 3 (27.3) 1 (12.5) 0,603 Hypertension [n (%)] 4 (36.4) 6 (75.0) 0,170Coronary artery disease [n (%)] 2 (22.2) 4(50.0) 0.335
Preoperative atrial fibrillation [n(%)] 6 (54.5 ) 2 (25.0) 0,352
Prior myocardial infarction [n (%)] 3 (27.3) 1 (12.5) 0,603
Prior cardiac surgery [n (%)] 5 (45.5) 4 (50) 1,000
Prior TIA or CVA [n (%)] 2 (18.2) 2 (25.0) 1,000
Renal insufficiency[n (%)] 2 (18.2) 2 (25.0) 1,000
Serum creatinin [mg/dL] 92(79-142) 88(76-411) 0.679
eGFR<60 {mL/min/1.73 m2] 4(36.4) 3(37.5) 1.000
Pre-operative medication use
Statin [n (%)] 5 (45.5) 3 (37.5) 1,000 Steroids [n (%)] 5 (45.5) 2 (25.0) 0,633 Beta-blockers [n (%)] 7 (63.6) 4 (50.0) 0,658 Risk scores Logistic euroScore (%) 18 ±10.6 21.8± 13.9 0.511 STS score (%) 6.3± 4.8 7.1± 3.8 0.697
BMI, body mass index; EuroSCORE, logistic European System for Cardiac Operative Risk Evaluation, measures patient risk at the time of cardiovascular surgery. Scores range from 0% to 100%, with higher scores indicating greater risk, EuroSCORE higher than 20% indicates very high surgical riski; STS score, Society of Thoracic Surgeons score, measures patient risk at the time of cardiovascular surgery. Scores ranges
pulmonary disease; DM, diabetes mellitus; PTCA, percutaneous transluminal coronary angioplasty; TIA, Transient Ischemic Attack; CVA,
Cerebral Vascular Accident (CVA); LVEF, left ventricular ejection fraction; eGFR, Estimated Glomerular Filtration Rate.
Nineteen patients were included in this study. The mean age was 80.1 ± 6.6 years, 74% was male and mean BMI was 25.4 ± 4.3. Most common co-morbidities were hypertension (53%), pre-operative atrial fibrillation (42%) and coronary artery disease (35%). Four patients (21%) suffered from pre-operative renal insufficiency, median serum creatinin was 92 mg/dL (76-411). Also, four patients (21%) had COPD and four patients (21%) had diabetes. Nine patients (48%) underwent prior cardiac surgery and four patients (21% ) had a prior diagnosis of myocardial infarction. The majority of the study population used beta-blockers preoperatively (58%). Seven patients (37%) used steroids pre-operatively and 8 patients (42%) used statins. Patients were classified as high surgical risk as reflected by a mean logistic euroSCORE of 19.6 ± 11.9 and a mean STS-score of 6.6 ± 4.3.
Table 2. Peri- procedural characteristics according to the occurrence of systemic
inflammatory response syndrome
Variable SIRS (n = 11 ) non SIRS (n = 8) P- value Access site Trans-femoral [n (%)] 6 (54.5) 6 (75.0) 0.633 Trans-apical [n (%)] 5 (45.5) 2 (25.0) 0.633 Performance of pacing-run [n (%)] 9 (90.0) 7 (87.5) 1.000 Pacing-runs [n] 1 (0 - 4) 1 (0 - 3) 0.604 Duration [s] 14 (9 – 40) 13 (10-24) 0.533 Pre-dilatation [n] 6 (54.5) 5 (71.4) 0.637 Post-dilatation [n] 5 (45.5) 1 (16.7) 0.333 Contrast dye (mL) 105 ± 48 91 ± 52 0.549 Valve-in-valve implantation [n (%)] 1 (10.0) 0 (0) 1.000 Vasopressor requirements [n (%)] 5 (45.5) 2 (25.0) 0.633 Inotrope requirements[n (%)] 2 (18.2) 2 (25.0) 1.000
Intervention time (min) 89 ± 16 78 ± 11 0.245
Twelve patients (63%) underwent trans-femoral TAVI and seven patients (37%) underwent a trans-apical TAVI. Mean intervention time was 84 ± 14 minutes. In sixteen patients (89%) RVP was performed. Median number of pacing runs was 1.5 (1-4) with a median duration of 15 seconds (IQR 9 - 40). Pre-dilatation of the native aortic valve was performed in 11 patients (61%), post-dilatation in 6 patients (36%). Valve-in-valve implantation was performed once (6%). Mean contrast load was 99 ± 48 mL. During the procedure, seven patients (37%) were on noradrenalin and 4 patients (21%) required dopamine or enoximone.
No significant difference were found between SIRS patients and the control group with regard to baseline characteristics, as summarized in table 1. A majority of the non SIRS patients, 6 (75%), underwent a trans-femoral procedure. In patients who developed SIRS, 6 patients (54.5%) underwent a trans-trans-femoral TAVI and 5 patient (45.5%) a trans-apical TAVI. However, this majority is not significant. Other peri -procedural
characteristics that could be potential risk factors for the development of SIRS did not show any significant difference between the SIRS and non-SIRS group as well. Such as use of RVP (9 vs. 7 P=1.000) number of RVP (1(IQR 0-4) vs. 1 (IQR 0-3) P=0,604) and duration of RVP (14(IQR 9-40) vs. 13 seconds (IQR 10-24) P=0.533), pre-dilatation of the native aortic valve (54.5% vs. 71.4%; P=0,637), valve- in-valve implantation (10% vs. 0%; P=1.000), post-dilatation of the valve prosthesis (45.5% vs.16.7%; P=0.333), and intervention time (89±16 v. 78±11 minutes; P=0,245).
Total median length of hospital stay was 6 (1-34) days. 30-day mortality rate was 27% (n=3). In hospital mortality was 36% (n = 4). For these clinical outcomes see table 3.
Table 3. Clinical outcomes for the occurrence of systemic inflammatory response syndrome
Variable SIRS
(n = 11 )
non SIRS (n = 8)
P-value
Length of hospital stay [days] 6 (1-34) 6 (3-15) 0.657
In hospital mortality [n (%)] 4 (36.4) 0 (0) 0.103
30-day mortality [n (%)] 3 (27.3) 0 (0) 0.228
Eleven patients (58%) were diagnosed with SIRS during the first 48 hours following TAVI. Patients who developed SIRS were mostly characterized by a leukocytosis (80%), an increased heart rate (73%) and fever (64%). Figure 5 shows the occurrence of SIRS criteria in non SIRS patients compared to SIRS patients. None of them showed a respiratory rate > 20/min.
Figure 5. Parameters of SIRS in patients after TAVI. Patients are diagnosed with SIRS when two or more of the following criteria are met: temperature < 36.0˚C or >38.0˚C; heart rate >90 beats/minute; respiratory rate >20 breaths/minute, or PaCO2<32mmHg; leukocyte count <4,000 or >12,000 cells/mm3, or > 10% immature neutrophils. Non SIRS patients did not fulfill more than one criterion.
The increase in inflammatory biomarkers after TAVI are summarized in figure 6 and 7. The mean concentrations of the ischemia/ organ damage markers are shown in figure 8 and the mean concentrations of the gram-negative bacteria markers and activated cell receptor markers are shown in figure 9.
4.1 Inflammatory biomarkers
Figure 6. Mean concentrations of pro-inflammatory cytokines; TNFα, IL-6 and IL-8 (pg/ml) and acute phase proteins CRP(mg/l) and Pentraxin3(pg/ml)per time interval.
SIRS was associated with an significant increase in plasma levels of IL-6 at 3h (9.3 pg/ml vs. 48.5 pg/ml; P=0.0651) and 48h (23.14 pg/ml vs. 86.18 pg/ml; P=0.097) after the procedure.
Furthermore, five SIRS patients peaked above a IL-6 concentration of 100 pg/ml at 6h after the procedure, where IL-6 levels of the non SIRS patients did not reached this level. The mean concentration IL-6 of the negative control samples was 5.60 pg/ml. Absolute mean IL-8 levels of SIRS patients and non SIRS patients differ the most at 3h (9.86 pg/ml vs. 24.48 pg/ml; P = 0.257) and 24h (10.08 pg/ml vs. 21.42 pg/ml; P = 0.2012) following TAVI. However, these differences were not significant. The mean concentration IL-8 of the negative control samples was 3.00 pg/ml. Concentrations of TNFα did not show absolute differences between the two groups. The mean concentration TNFα of the negative control samples was 2.70 pg/ml. Pro-inflammatory cytokine IL-1β was left off, because no response was observed and therefore most values were out of range (<OOR).
Acute phase protein CRP raised gradually in the first levels raised gradually in the first 48h (91.88 mg/l vs. 161.2 mg/l; P = 0.1143) and peaked at 72h following TAVI (83.63 mg/l vs. 178.8 mg/l; P = 0.057). CRP levels were associated with the occurrence of SIRS. The mean concentration CRP of a healthy individual is ≤10 mg/l. Acute phase protein Pentraxin3 did not show a significant difference between the two groups. However, absolute levels of Pentraxin3 in non SIRS patients flattens at 6h while the this level in SIRS patients still elevates and peaked at 24 h (5712.85pg/ml vs. 8490.37 pg/ml; P= 0.314. The mean concentration Pentraxin3 of the negative control samples was 1459.8 pg/ml.
Figure 7. Mean concentrations of anti-inflammatory cytokines; IL-1RA and IL-10 (pg/ml) per time interval.
Both anti-inflammatory cytokines IL-1RA and IL-10 did not show a significant difference between the SIRS and non SIRS patients. Mean levels of IL-1Ra showed higher level at baseline in non-SIRS patients compared to SIRS patients (145 pg/ml vs. 412 pg/ml; P = 0.279). Levels peaked at 6h (425 pg/ml vs. 558 pg/ml; P = 0.876) and remained higher in non SIRS patients. Mean levels of IL-10 peaked at 3h (6.91pg/ml vs. 16.98 pg/ml; P= 0.311), whereupon IL-10 levels in SIRS group gradually drops and in non SIRS group gradually raised and peaked at 24h (8.74 pg/ml vs. 13.55 pg/ml; P= 0.449). Due to circumstances no negative control samples (of healthy persons) were included, therefore no reliable comparison can be done.
4.2 Ischemia/ organ damage markers
Figure 8. Mean concentrations(pg/ml) of ischemia marker I-FABP(intestine) and organ damage marker KIM-1 (kidney) per time interval.
High baseline levels of I-FABP were found in both groups (799 pg/ml vs. 803 pg/ml; P = 0.9183). Levels slightly raised 1h after TAVI (1085.2 pg/ml vs. 1141.5 pg/ml; P = 0.8633) and peaked at 3h following TAVI (1032.79 pg/ml vs. 1149.42 pg/ml; P = 0.5089). Levels gradually decreased and almost normalized at 24h following TAVI in both SIRS and non-SIRS patients (429 pg/ml vs. 379 pg/ml; P = 0.9681). In SIRS patients absolute I-FABP levels were slightly higher compared to non-SIRS patients. However, no significant difference were observed between these two groups. The mean concentration I-FABP of the negative control samples was 193.34 pg/ml.
Absolute baseline levels of KIM-1 were higher in SIRS patients compared to non-SIRS patients (188 pg/ml vs. 278 pg/ml; P = 0.234). In case of patients who developed SIRS, absolute KIM-1 levels raised at 48h (241 pg/ml vs. 356 pg/ml; P =0.303) and peaked at 96h (147 pg/ml vs. 376 pg/ml; P = 0.209). Due to circumstances no negative control samples (of healthy persons) were included for KIM-1, therefore no reliable comparison can be done.
4.3 Gram-negative bacteria markers
Figure 9. Mean concentrations gram-negative bacteria (LPS) biomarkers; LAL (EU/ml), LBP (ng/ml)and EndoCab
(GMU/ml)and activated soluble receptor markers sCD14 (LPS)(ng/ml) and sTREM-1(inflammation)(pg/ml) per time interval.
Activated soluble cell receptor sCD14 show a slightly higher baseline value in both groups (1889 ng/ml vs. 1996 ng/ml; P=0.5861) compared to the concentration of the negative control samples (1387.0 ng/ml). From 6h after TAVI, the concentration of sCD14 started to rise gradually in SIRS patients; 24h (2260 ng/ml vs.2645 ng/ml; P=0.2233), 48h (2425 ng/ml vs. 2772 ng/ml; P=0.2625) 96h (2583 ng/ml vs. 2913 ng/ml; P=0.5770). However, no significant difference was observed between the two groups.
In SIRS patients, absolute concentrations of sTREM-1, remains higher than non SIRS patients and show a significant difference at 96h after TAVI (401.2 pg/ml vs. 1115.3 pg/ml; P=0.0406).
Gram-negative bacteria marker LAL showed a significant difference at 1h (0.0836 EU/ml vs. 0.1157 EU/ml; P=0.0495), 3h (0.087 EU/ml vs. 0.133 EU/ml; P=0.0155) and at 6h (0.094 EU/ml vs. 0.145 EU/ml; P=0.0442). Absolute concentrations of LAL remains higher in SIRS patients compared to non SIRS patients. However, the mean concentration LAL and sTREM-1 in negative control samples (of healthy individuals) were higher than the SIRS and non SIRS group( sTREM-1; 1791.08 pg/ml and LAL; 0.35 EU/ml). This data may therefore be disputed, and more negative control samples should be brought in during analyses.
Gram-negative bacteria marker LBP did not show a significant difference between both groups. However, absolute concentrations LBP remains slightly higher in SIRS patients. From 6h after TAVI concentration LBP gradually raised and peaked at 48 h (21529 ng/ml vs. 21786; P=0.9032). The mean concentration I-FABP of the negative control samples was 11429.6 ng/ml.
Absolute baseline levels of EndoCab were higher in SIRS patients compared to non-SIRS patients (93.81 GMU/ml vs. 136.24 GMU/ml; P=0.1543). A significant difference between the two groups was observed at 6h after TAVI (74.91 GMU/ml vs. 135,92 GMU/ml; P=0.0945). The mean concentration EndoCab of the negative control samples was 94.04 GMU/ml
5. Discussion
In total, 11 patients (57,9%), developed SIRS in the first 48 hours after TAVI. Although we never intended to study outcome following SIRS in this pilot study, it is remarkable that the four patients who died were all patients who were diagnosed with SIRS within 48 hours following TAVI. We expect to observe this outcome measure to be significant in larger population.
No significant difference were found between SIRS patients and the control group with regard to baseline characteristics. During TAVI procedures a transient drop in cardiac output may occur several times, predominantly during balloon inflation during pre- or post- dilation. It is previously reported that repeated ventricular pacing runs are independent predictors of SIRS. Possibly due to subsequent tissue hypoperfusion.2
However, in our population, the mean arterial pressure after RVP was globally similar between SIRS and non SIRS patients. This is supported by the fact that ischemia/organ damage markers I-FABP and KIM-1
concentrations are not significantly different between the two groups.
Other peri-procedural characteristics, that could be potential risk factors for the development of SIRS, such as acces route (trans-femoral or trans-apical), were independent. Apparently the activation of a systemic inflammatory response seems to be independent from the invasiveness of the approach. Because the high prevalence, the question of what causes SIRS following TAVI remains. We suggest that not RVP itself induces SIRS but pressure or movement on the calcified native aortic valve activates the immune system. Possibly the release of micro pieces of calc during balloon inflation could provoke the inflammatory response.
Lowering concentrations of I-FABP post-TAVI in both groups is indicative for improved cardiac output The elevated I-FABP concentration during the procedure might explain the elevated gram-negative bacteria compounds found in the blood. Microcirculatory derangements can not be ruled out . However, it seems that it is not the cause of the occurrence of SIRS. To elucidate this, more patients need to be included for this study. Pro-inflammatory cytokine Il-6 and resulting acute phase protein CRP, show a significant difference between the two groups. Pro-inflammatory cytokine IL-8 did not a s significant difference between these two groups, but this might when a larger study population is used. Furthermore, concentrations of IL-1β did not change at all during the study period. This could be explained, togheter with TNFα, by different biochemical characterists of this pro-inflammatory cytokines. IL-1β and TNFα act locally were IL-6 acts as an endocrine cytokine. This means that IL-6 (next to IL-8) has systemic effects, and circulating levels are early detectable. The combination of these cytokines can reliably and early predict the risk of developing SIRS and might be of diagnostic value in the future.
IL-10 was not associated with SIRS. Possibly, these cytokine react later than our follow- up time as a late complication of SIRS is the overshoot of inflammatory cytokines leading to CARS. In case of anti-inflammatory cytokine IL-1Ra levels, non SIRS patients showed higher levels at baseline compared to SIRS patients and remained higher in non SIRS patients. This could confirm that an inadequate response of the anti-inflammatory response plays a considerable role in the development of SIRS.
Limitations
This study has several limitations. Most important, the small size. Therefore our results are hypothesis generating instead of hypothesis testing. Second, due to logistic reasons the analyses of I-FABP, KIM-1, IL10, IL-1RA and IL- 1β could not be performed in three of the 19 patients. Third, some patients were discharged from the ICU within 48 hours and blood gas analysis was not performed at the ward. This may have resulted in underdiagnosis of SIRS.
6. Conclusion
The results of this pilot study show that SIRS after TAVI is common and associated with increased
pro-inflammatory levels of IL-6 and IL-8. When the results of these cytokines are combined and more samples will be included, this analysis may result in early and reliable prediction of development of SIRS, enabling early treatment and increase of long-term survival. I-FABP is a reliable intestine ischemia marker and probably a reliable marker to test the efficiency of the TAVI procedure as well. Ischemia reperfusion –injury and/or bacterial translocation as cause for the occurrence of SIRS is not supported with this data. To find out the cause of de development of SIRS, more research needs to be done, with a larger study population. It should be noted that due to the small population, this study is purely observational, and interpretation is speculative.
Bibliography
1) Schwietz T. et al. (2014) Occurence and prognostic impact of systemic inflammatory response sydrome in
transfemoral and transapical aortic valve implantation with balloon- and self-expandable valves. CLINICAL
RESEARCH, 1-6
2) J.M. Sinning et al.(2012) Systmic inflammatory response syndrome predicts increased mortality in patients
after transcatheter aortic valve implantation. European Heart Journal, 1-10
3) Karhausen J, Qing M, Gibson A, et al. Intestinal mast cells mediate gut injury and systemic inflammation in a
rat model of deep hypothmic circulatory arrest. Crit Care Med [Internet] 2013 [cited 2-14 Mar1]; 41 (9)e 200-10
4) Ronde- TIllmans , M et. al.(2014) Stand van Zaken. 10 jaar transkatheter-aortaklepvervanging een overzicht
van de klinische toepasbaarheid en bevindingen. Ned Tijdschr Geneeskd, 43(A7768), 158.
5) Leon MB, et al.(2010) Transcatheter aortic-valve implantation for aortic stenosis in patients who cannot
undero surgery. N Engl J Med,363(17), 1597-1607.
6) Smith C.R. et al. (2011) Transcatheter versus Surgical Aortic-Valve Replacement
in High-Risk Patients. The New England Journal of Medicine.364 (23), 2187-2198
7) The Partner Trial, Clinical Results (2011) Establishing a New Path Forward for Patients With Severe
Symptomatic Aortic Stenosis. Edwards Transcathether Heart Valve
Program,http://ht.edwards.com/scin/edwards/eu/sitecollectionimages/products/transcathetervalves/partnerr esultsab.pdf
8) Nuis R. et al. (2012) Blood Transfusion and the Risk of Acute Kidney Injury After Transcatheter Aortic Valve
Implantation. Circulation Cardiovascular Interventions, 5, 680-688
9) Dr. T. (2011) Trans catheter aortic valve implantation (TAVI). Cardiac Health,
http://www.cardiachealth.org/trans-catheter-aortic-valve-implantation-tavi
10) Kaplan L. J. (2014) Systemic Inflammatory Response Syndrome. Medscape reference,
http://emedicine.medscape.com/article/168943-overview.
11) Castellheim, A. et al. (2009) Innate Immune Responses to Danger Signals in Systemic Inflammatory
Response Syndrome and Sepsis. Scandinavian Journal of Immunology 69, 479-491
12) Crea F. and Biasucci L. M. (2012) Innate immune inflammatory response to danger: when, how, and why
does a friend become a foe? European Heart Journal, 1-4
13) Torre D. et al. (2000) Anti- inflammatory response of IL-4, IL-10 and TGF-β. Mediators of Inflammation, 9,
193–195
14) Opal S.M. and DePalo V.A (2000) Anti-Inflammatory Cytokines. CHEST, 117, 1162–1172
15) Sablotzki A et al. (2002) The systemic inflammatory response syndrome following cardiac surgery:different
expression of proinflammatory cytokines and procalcitonin in patients with and without multiorgan dysfunctions. Perfusion, 17(2), 103-109.
16) Hirai S.(2003) Systemic inflammatory response syndrome after cardiac surgery under cardiopulmonary
bypass. Ann Thorac Cardiovasc Sur, 9(6), 365-370.
17) Dimopoulou I. (2007) Tumour necrosis factor-alpha (TNFα) and interleukin-10 are crucial mediators in
post-operative systemic inflammatory response and determine the occurrence of complicatins aft major abdominal surgery. Cytokine 2007, 37(1), 55-61.
18) Takenaka K. et al. (2006) Systemic inflammatory response syndrome and surgical stress in thoracic surgery. J
Crit Care, 21(1), 48-53.
19) Miyaoka K. et al. (2005) Clinical evaluation of circulating intleukin-6 and interleukin-10 levels after
surgery-induced inflammation. J Surg Res, 125(2), 144-150.
20) Hong-Xiang Li et al.(2014) Measuring both procalcitonin and C-reactive protein for a diagnosis of sepsis in
critically ill patients. Journal of International Medical Research,1-10.
21) Fazzini, F. et al (2001) PTX3 in small-vessel vasculitides. An independent indicator of disease
activity produced at sites of inflammation. Arthritis Rheum, 44, 2841.
22) Niewold, T et al. (2004) Plasma intestinal fatty acid binding protein (I-FABP) concentrations
increase following intestinal ischemia in pigs. Res Vet Sci, 77, 89.
23) Cronk, D et al. (2006) Intestinal fatty acid binding protein (I-FABP) for the detection of
strangulated mechanical small bowel obstruction. Curr Surg, 63, 322.
24) Kentaro, S et al. (2014) I-FABP is an intestinal injury marker in SIRS following cardiac arrest. Critical Care
Medicine, 42(12), A1425.
25) Han,W et al. (2002) Kidney Injury Molecule-1 (KIM-1): A novel biomarker for human renal proximal tubule
injury. ) Kidney International, 62, 237–244
26) Hycult biotech (2013 ) HK115-02 HUMAN LBP ELISA kit, Product information & manual, 2.
http://www.hycultbiotech.com/neutrophil-proteins-elisa-kit-for-human-lipopolysacharide-binding-protein-lbp-p10935-html
27) Hycult biotech (2013) HK302 Limulus Amebocyte lysate chromogenic endpoint asay, Product information &
manual, 2. http://www.hycultbiotech.com/downloads/dl/file/id/122/hit302.pdf
28) Barclay, G (1995) Endogenous endotoxin-core antibody (EndoCab) as a marker of endotoxin exposure and a
prognostic indicator: a review. Prog Clin Biol Res, 392, 263.
29) Allan, E et al. (1995) Anti-bacteroides lipopolysaccharide IgG levels in healthy adults and sepsis patients.
Immunol Med Microbiol, 11, 115.
30) Hycult biotech (2012) HK320 HUMAN sCD14 ELISA kit, Product information & manual, 2.
http://www.hycultbiotech.com/downloads/dl/file/id/147/hk320.pdf
31) Bouchon A. et al. (2001) TREM-1 amplifies inflammation and is a crucial mediator of septic shock. Nature,
410, 1103–1107.
32) ELISA, The enzyme-linked immunosorbent assay (ELISA) is a sensitive method for quantification of proteins
that is suitable for numerous applications. MABTECH(2014)
https://www.mabtech.com/knowledge-center/assay-principles/elisa-assay-principle
33) ELISA encyclopedia, ELISA Principle Basis and Extension
http://www.elisa-antibody.com/ELISA-Introduction/ELISA-Principle, Sino Biological Inc.
34) Rijkers, G., Houte van, A.J., Claessen, A. (2011) Een blad voor Heleen, 30 jaar Medische Immunologie in het
Appendix
Timetable
Date
(2014-2015) Week What?
Included patients
07-10 / 12-10 1 Introduction/ guided tour/ watch TAVI operation in OK 0
13-10 / 19-10 2 Literature study/ collect samples 2
20-10 / 26-10 3 Literature study 0
27-10 / 02-11 4 Literature study/ concept projectplan 0
03-11 / 09-11 5 Writing theoretical background 0
10-11 / 16-11 6 Writing theoretical background/ collect samples 1
17-11 / 23-11 7
Writing theoretical background/ Practice protocol Luminex/ collect
samples 2
24-11 / 30-11 8 Processing data Luminex protocol/ Writing introduction 0
01-12 / 07-12 9
Practice protocol Luminex/ processing data/ concept report/ collect
samples 1
08-12 / 14-12 10 Writing method/ editing/ collect samples 1
15-12 / 21-12 11 Hand-in projectplan version 1/ collect samples 2
22-12 / 28-12 12 Holiday 0
29-12 / 04-01 13 Holiday 0
05-01 / 11-01 14 Rewrite projectplan 0
12-01 / 18-01 15 Evaluation at school/ tidying SLB/ collect samples 2
19-01 / 25-01 16 Rewrite projectplan/ finishing protocol luminex -> 1
26-01 / 01-02 17 SLB appointment/ Practice protocol Luminex 2
02-02 / 08-02 18 Start Luminex assay and ELISA with collected samples 0
09-02 / 15-02 19 Luminex assay and ELISA with collected samples/ processing data 0 16-02 / 22-02 20 Processing data / statiscal analysis/ writing results 0
23-02 / 01-03 21 Writing results, conclusion, discussion 0
02-03 / 08-03 22 Wrting concept final report 0
09-03 / 15-03 23 Extra week (CAA), start making poster 0
27-03 25 Concept final report 30-03 26 Poster presentation
31-04 26 Deadline final report internship 14-04 28 Final presentation
i ii iii
