Membrane oxygenators (MO) are integral components for extracorporeal circulation (ECC) in open heart surgery, as well as for mechanical assist systems such as extracorporeal membrane oxygenation (ECMO) and interventional lung assist (iLA), which are successfully applied in the management of acute respiratory distress syndrome (ARDS).1–3 In open heart surgery, ECC is applied for a brief period only, whereas ECMO and iLA are long-term treatment modalities which may be employed for several weeks.4,5
Most of the ECC components are not suitable for long-term ECMO treatment. In fact, ECMO requires particularly enhanced biocompatibility such as heparin-coating, low blood flow resistance, and long-term durability. The current development in ECC technology concentrates on high-performance microporous polypropylene (PPL) capillary MO and nonmicroporous poly 4-methyl-1-pentene (PMP) diffusion capillary MO,6 where the molecular structure of PPL and PMP directly affects the physical characteristics of the gas exchange.7–9 The wall of the plasma-tight PMP membrane consists of a highly porous support matrix and a thin tight membrane on the blood side of the matrix, which constitutes a solid barrier between blood and gas. The homogenous tight membrane and the complete separation of blood and gas phases obviously provide a better biocompatibility with less blood trauma. The crossing of microbubbles caused by a lowered pressure on the blood side compared to the gas side, as well as a plasma leakage does not occur due to the tightness of the membrane.9 Recent improvements and device innovations in ECMO technology favor the application of MO with PMP capillaries.10–15 The PMP MO is highly resistant to plasma leakage and has an acceptable function durability, allowing long-term extracorporeal application.13,14 However, despite heparin-coating and continuous systemic low-dose heparin infusion, these MOs can occasionally require early replacement, due to cellular and thrombotic deposits in the blood path with a consecutive increase in blood flow resistance and a consecutive reduction in the gas exchange capacity.16
Patients with severe ARDS, who could not be stabilized with standard treatment protocols, were treated with femoro-jugular venovenous ECMO using an optimized ECMO-Set (CE licensed for use of 14 days in Europe and also approved for Canada while the approval for China still is pending) consisting of a Quadrox PLS (Permanent Life Support Set) MO, and a Rotaflow RF 32 centrifugal blood pump (Maquet Cardiopulmonary AG, Hirrlingen, Germany). All components of the ECMO-Set, including the tubing and cannulas are tip to tip coated with heparin (Bioline, Maquet). The MO has a gas exchange area of 1.8 m2 and is composed of poly-(4-methyl-1-pentene) capillary fabrics (Membrane, Membrana GmbH, Wuppertal, Germany) for the gas exchange. The recommended blood flow rate is 0.5–7.0 L/min. Our ECMO-Set has a very low priming volume of 600 ml. The priming process consisted of 300 ml colloid expander (Gelatinpolysuccinat, Braun, Melsungen, Germany) and 300 ml normal saline without any additional drugs, In all patients, systemic low-dose heparin infusion was administered to maintain a partial thromboplastin time of 50–60 seconds (= 1.5 to 2 fold of normal value). If our ARDS patients required volume substitution at the intensive care unit (ICU), it consisted mainly of crystalloids. At a hemoglobin value of <8.5 g/dl patients received packed red blood cells resulting in a mean substitution of 1 erythrocyte concentrate every 3 to 4 days.
Membrane oxygenators replacement criteria were the appearance of a reduced CO2 elimination and oxygenation transfer capacity, which were considered to be the consequence of thrombotic deposits on the gas exchange capillaries. A further criterion was an increase in blood flow resistance with a pressure decline (dpMO = pMOin − pMOout) across the MO of more than 70 mm Hg at a blood flow of 3 L/min indicating the necessity for replacement of the device (Figure 1).
Preparation of Oxygenators
After removal of the MO from the ECMO circuit, the oxygenators were extensively rinsed with both 0.9% NaCl (500 ml) and 0.1 M phosphate buffer (PB; 600 ml). Subsequently fixative (4% paraformaldehyde in 0.1 M PB and a pH of 7.2) was perfused for 30 minutes at room temperature at 20 ml/min followed by 10%, 20%, and 30% saccharose solution in PB (each 20 ml/min for 30 minutes at 4°C) as a cryoprotectant. The oxygenators were then frozen at −20°C over night and stored at −80°C until further assessment.
The frozen oxygenators were dissected longitudinally in 1 cm slices by a band saw avoiding thawing of the blocks. The frozen specimens were wrapped in aluminum foil and stored at −80°C until further processing and analysis. For analyzing deposits around single capillaries of the MO, pieces of the frozen blocks were embedded in Tissue Tek OCT (Sakura Finetek), cut at 5–10 μm on a Leica CM3050S cryostat (Leica Microsystems) and transferred to Superfrost plus slides (Menzel GmbH, Braunschweig, Germany). For the analysis of the deposits on the capillaries, fabric pieces of a block were thawed in phosphate buffered saline (PBS) and the capillary layers were positioned onto microscope slides.
Nuclei of cells in frozen sections and on the surface of the PMP capillaries were stained using DAPI (4,6-diamidin-2-phenylindol-dihydrochlorid). A drop of VectaShield mounting medium with DAPI (1.5 μg/ml; Vector Laboratories, Petersborough, England) was applied to each sample on the microscope slide and topped with a coverslip. The specimens were analyzed using a Leica DMRBE fluorescence microscope (Bensheim, Germany). Images were digitized using a Spot 2000 camera (Diagnostic Instruments, Stirling Heights, MI) under software control (Metamorph, Universal Imaging Corporation, West Chester, NY). Using the 40x lens of the microscope resulted in a resolution of 0.175 μm per pixel, for overviews, using the 10x lens the resolution was 0.695 μm per pixel.
Scanning Electron Microscopy (SEM)
Thawed sheets of PMP fibers were extensively rinsed with PBS and H2O, and air dried. The specimens were mounted on aluminum stubs with carbon disks and colloidal quick drying silver paint (Balzers SCD 040, PROVAC GmbH, Liechtenstein). Then, all specimens were coated with gold-palladium, by means of a sputter-coater (Balzers SCD 040, Liechtenstein). Surface topography was visualized by scanning electron microscopy (SEM) using a FEI Quanta 400 (Eindhoven, Netherlands).
Over the last 2 years, 31 patients with severe ARDS (mean age, 54 ± 14 years, range, 20–76 years) required an ECMO system in our institution. The cumulative support time was 352 days and averaged out at 11.0 ± 6.5 days (range, 2–33 days). A total of 36 gas exchange modules were used (1.1 oxygenators per patient). Whereas 28 patients were provided with only 1 oxygenator, 3 patients required oxygenator replacement (1 patient with 3 and 2 patients with 2 oxygenators) due to thrombotic deposits despite heparin coating and systemic heparinization. Representative examples of respective MO are shown in Figure 2.
Scanning electron microscopy analysis of the dismantled oxygenator demonstrated deposits on the gas exchange fibers forming fibrous networks with platelets and red blood cells embedded. Several PMP fibers showed large areas with a “pseudomembrane” formation (Figure 3).
For further characterization of these deposits, we surveyed the layer thickness using phase contrast microscopy of PMP capillary cross sections. The layer thickness around the hollow fibers measured 30–45 μm (Figure 4) whereas the primary wall thickness of the PMP capillaries was 75 μm. Additional nuclear staining of tissue sections is shown in Figure 5 and revealed the presence of many cell nuclei in these deposits covering the capillaries. The distribution of the cells on the surface of the PMP capillary fabric is shown in an overview of adjacent fibers (Figure 6). Especially next to the seams connecting neighboring capillaries (arrows in Figure 6) and around the contact zones with the overlying fiber sheet (arrow in Figure 6) cells accumulated. The remaining regions of the fibers that were exposed to higher levels of blood flow showed a variable coverage with single cells and small clusters of cells.
Polymethylpentene MOs are superior in durability as compared to other MOs, and therefore used for long-term ECMO treatment in patients with ARDS. Currently, PMP MOs are approved for a usage of 14 days. While in 90% of ECMO patients, a single oxygenator works well during the entire treatment interval, as has also been seen in our patient cohort, in about 10% of cases the MO fails due to deposits on the gas exchange capillaries, leading to an increase in blood flow resistance and diffusion path. Interestingly, the amount of deposits does not correlate with the time period of usage. Four of our 31 patients were supported with the PMP MO for 12 to 33 days. We could not identify a predictable correlation between patient characteristics and durability of oxygenators. Presumably, an already activated coagulation cascade may lead to premature oxygenator dysfunction. However, all of our patients had severe ARDS and many were in a condition of septic shock with high vasopressor requirements. Some had disseminated intravascular coagulopathy and thrombocytopenia before implantation of the ECMO. So it is hardly possible to measure a potential additional inflammatory stimulus caused by the extracorporeal circuit. Also it could be speculated that the substitution of packed red blood cells may augment an inflammatory response. Koch et al.17 reported recently, that the transfusion of blood products with a longer storage time can increase the complication rate after cardiac surgery. Due to the small number of studied oxygenators, we could not demonstrate a dependency of oxygenator function on the number of substituted blood products. It also has to be taken into consideration that generally patients needing large amounts of blood products are sicker than patients with little demand.
The underlying pathophysiological mechanisms of MO failure consist of two main pathways: 1) Contact of blood with a foreign surface activates the coagulation cascade18 which leads to clot formation and consecutive occlusion of the blood compartment of the MO. Scanning electron microscopic analysis identified specific formation of fibrin strands with integration of thrombotic deposits on the gas exchange surface (Figure 3). The increase in blood flow resistance in a pump-driven system like the ECMO is usually counteracted with an augmentation of the pump speed (round/min), which raises mechanical stress on cellular blood components and induces further cell damage and additional activation of the coagulation cascade.19,20 2) Cellular deposits on the PMP capillaries may increase the diffusion path of the gas exchange fibers. Cross-sections of the overgrown PMP membranes demonstrated a thickness of the layer around the hollow fibers almost reaching the magnitude of the PMP capillary wall thickness (Figures 4 and 5). In such a situation, the gas exchange capacity collapsed without an increase in the blood flow resistance. Nuclear staining of the PMP samples of removed MO demonstrated cellular accumulations especially localized at the seams and the contact points with adjacent layers of capillaries, presumably areas of low blood flow (Figure 6). It appears that areas of high blood flow rate show a lower density of cell deposition, which is well understandable. One limitation of our study was the small number of tested oxygenators. A larger number of examined oxygenators and the inclusion of patient’s characteristics and technical criteria as well may allow a better correlation of the cellular deposits and the time of usage or the identification of factors related to the observed deposits. This is the objective of future studies, which may result in a surface modification to avoid cellular or fibrous deposits.
So far, the type of cells and their proliferation status remain unclear, but several assumptions have been made with regard to cell adherence. LaIuppa and coworkers demonstrated that hematopoietic stem cells adhere well on PMP.21 Adsorption of proteins from the serum onto the surface of the material may promote cellular adherence.21 An additional aspect in protein adsorption was the heparin coating of the PMP surfaces. In this context, Niimi and coworker demonstrated that heparin coating of the oxygenator capillaries can decrease platelet adhesion without affecting adsorption of major adhesive proteins.22 In contrast, the adsorption of fibronectin is increased by heparin coating. This protein was identified as a putative stimulator of adhesion and proliferation of adipose-derived stem cells.23,24 In our opinion, the nuclear cells we observed are macrophages or progenitor cells. We cannot exclude that progenitor cells may adhere and proliferate on the heparin coated PMP surface during extended ECMO application. Future experiments with selected surface antibodies may allow the identification of these cells.
In conclusion, ECMO systems with a heparin-coated PMP MO are appropriate for long-term oxygenation of patients with respiratory failure. However, in some cases, cellular deposits on the gas exchange membrane require replacement of the MO. A detailed study is needed to characterize the adherent cells, to analyze potential patient characteristics leading to earlier occlusion and to further improve the surface properties of PMP to prevent accumulation of these cells.
The authors thank the excellent technical assistance of Leykauf C, Bielenberg K, and Bergman S. Supported by Maquet (Hirrlingen, Germany).
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