Effect of Anticoagulation with Citrate versus Heparin on the Adsorption

Article pubs.acs.org/Biomac

Effect of Anticoagulation with Citrate versus Heparin on the Adsorption of Coagulation Factors to Blood Purification Resins with Different Charge Carla Tripisciano,† André Leistner,‡ Ingrid Linsberger,† Aniela Leistner,‡ Dieter Falkenhagen,† and Viktoria Weber*,† †

Center for Biomedical Technology, Danube University Krems, Dr.-Karl-Dorrek-Straße 30, 3500 Krems, Austria Polymerics GmbH, Landsberger Allee 378, D-12681 Berlin, Germany

‡

S Supporting Information *

ABSTRACT: In liver failure, hydrophobic toxins accumulate in the blood circulation. To support hepatic function, extracorporeal blood purification systems have been developed, in which both cationic and neutral adsorbents are used to remove albumin-bound metabolites from blood. An issue of these systems is the additional removal of coagulation factors containing negatively charged γ-carboxyglutamate (Gla) domains, which, in physiological conditions, are shielded by calcium ions. We hypothesized that complexation of calcium ions by citrate leads to exposure of negative Gla domains, resulting in their binding to the positively charged adsorbents. The data presented here confirm that the binding of coagulation factors containing Gla domains to positively charged polymers is enhanced in the presence of citrate as compared to heparin. This effect increased with increasing charge density of the polymer and has important implications for the clinical application of positively charged polymers.

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synthesized in the liver.7,8 Therefore, the blood compatibility of adsorbents is crucial and their binding of coagulation factors or activation of the coagulation cascade should be minimized. In this context, it is important to mention that a number of factors of the coagulation cascade, as well as the anticoagulant protein C, are of similar molecular mass as albumin. Thus, adsorbents with a pore size optimized for removal of albumin-bound substances will also bind these coagulation factors. There have been reports in the literature showing that fractionated plasma was deprived of factors II, VII, and X, protein C, and protein S upon direct contact with polystyrenedivinylbenzene-based anion exchange resins.9 A common feature of all these factors is that they contain γcarboxyglutamic acid-rich (Gla) domains, which are generated by vitamin K-dependent post-translational carboxylation of the γ-carbon of glutamic acid residues.10 Under physiological conditions, Gla domains are shielded by free calcium ions in blood, with the resulting formation of a structure that mediates the interaction of Gla domains with membrane phospholipids.11−13 In this structure, Ca2+ plays a dual role, maintaining the Gla domain conformation and serving as a bridge between the negatively charged Gla domains and the negatively charged phosphatidylserine residues14 exposed at membrane surfaces

INTRODUCTION Liver failure is a pathological condition characterized by impaired synthetic and metabolic hepatic functions, with a resulting accumulation of uncleared hydrophobic metabolites in the bloodstream.1 Extracorporeal liver support systems have been developed to purify blood from such harmful substances. Whereas small water-soluble toxins, such as ammonium, can be removed via hemodialysis or hemofiltration, the depletion of poorly water-soluble, albumin-bound compounds, such as bilirubin, bile acids, phenolic derivates, and aromatic amino acids, requires combined membrane-adsorption devices,2,3 in which toxins are removed using activated carbons or synthetic organic polymers. Polystyrene divinylbenzene (PS-DVB) copolymers are employed as adsorbents in liver support devices2,4,5 and efficiently bind molecules containing aromatic rings due to hydrophobic interactions. It has been shown that the pore size of neutral polystyrene divinylbenzene adsorbents is crucial to increase their efficiency in removing strongly albumin-bound substances and that the minimal pore size of neutral polymer for efficient bilirubin removal is about 5.5 nm.6 In addition to neutral polymers, anion exchange resins based on PS-DVB have been developed and are clinically used in liver support devices to optimize the adsorption of bilirubin, which contains carboxyl groups. Coagulation is frequently severely compromised in patients with disturbed liver function, because coagulation factors are © 2012 American Chemical Society

Received: October 29, 2011 Revised: December 19, 2011 Published: January 9, 2012 484

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after cell damage or activation.15 The binding of factors on the phospholipid surface in close proximity renders them more active15 and facilitates their interaction, which is necessary for the efficient proceeding of the coagulation reaction, that is, thrombin generation.16,17 We hypothesized that regional anticoagulation with citrate promotes the adsorption of Gla domain-containing factors to anion exchangers due to the complexation of Ca2+ and the exposure of negatively charged Gla domains. Therefore, the aim of this study was to assess the influence of anticoagulation (heparin vs citrate) on the adsorption of vitamin K-dependent coagulation factors. Our results confirm that these factors bind preferentially to cationic adsorbents in the presence of citrate due to a local depletion of Ca2+ and exposure of anionic Gla domains. In contrast, neutral polymers showed a lower binding of these factors and minimal variations in binding in citrate versus heparin anticoagulated plasma.

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accessible imidazole groups. This suspension was then titrated with 0.1 N NaOH using a DL25 automated titrator (Mettler-Toledo, Switzerland) resulting in 2 inflection points in the titration curve, the first indicating the amount of excess HCl and the second (pKs 5.9) indicating the quantity of protonated imidazole groups. The number of protonated imidazole groups (total ion-exchange capacity) per gram of the DVB-VI adsorbent was calculated as c × V/m (with c = molar concentration of the titrant, V = volume of consumed titrant between first and second inflection point, m = weight of dry adsorbent polymer) and determined at 0.1 mval/g. Because the combination of protonated imidazole and imidazole forms a weak acid/base pair and thus a buffer system, the pH value of the surrounding media determines the number of actual surface charges on the polymer. Therefore, under the conditions of the adsorption experiments at pH 7.4, only 10% of the imidazole groups will be protonated, leading to a charge density of 0.01 mval/g. For CP adsorbent, the value of charge density was provided by the manufacturer.22 The accessibility of the pores of each adsorbent was characterized by inverse size exclusion chromatography (iSEC) using the retention times of glucose (180 Da) and dextran standards with molecular masses between 6 and 2000 kDa23 at a flow rate of 0.5 mL/min in 15% (v/v) isopropanol/water. A Waters HPLC System (Milford, U.S.A.) with a Bischoff 8110 refractive index detector was used to determine the retention volume (VR) of each dextran standard and glucose. Distribution coefficients (Kd) were calculated as Kd = (VR − V0)/(VT − V0), where VR is the retention volume, VT the total mobile phase volume, and V0 the interparticle void volume. For each adsorbent, the retention time of glucose was used to calculate VT, and the retention time of Dextran 2000 to calculate V0. A Kd of zero means that a substance is completely excluded from the pores, while a Kd of 1 means that the pores are completely accessible to a given substance. Adsorbent Pretreatment. Prior to use, adsorbents were hydrophilized as described.6 Prior to each batch experiment, adsorbents were washed three times with 0.9% saline solution and stored at 4 °C in 0.9% saline solution until further use. Assessment of Adsorption Characteristics. The adsorption of coagulation factors II, VII, and X, as well as of protein C, was assessed in batch experiments using an adsorbent-to-plasma ratio of 1:9 (v/v). At defined time points (0, 30, 60 min) samples were drawn and centrifuged at 4600g for 4 min. Supernatants were collected, aliquoted and stored at −80 °C until further characterization. Plasma without adsorbents served as a negative control. All experiments were conducted in triplicates. Quantification of Factors II, VII, X, and Protein C. Factors II, VII, and X were quantified by enzyme-linked immunosorbent assay (ELISA; Coachrom Diagnostica, Vienna, Austria), according to the instructions of the manufacturer. A calibrator (Biophen, Hyphen BioMed, Neuville-sur-Oise, France) specific for each coagulation factor was used to prepare the standard curve, whereas a human normal plasma control (Biophen, Hyphen BioMed, Neuville-sur-Oise, France) was used to ensure the validity of the test. Protein C was determined by ELISA (Technoclone, Vienna, Austria). Quantification of Heparin and Citrate. Heparin was quantified with the Coamatic Heparin test (Coachrom, Vienna, Austria) according to the instructions of the manufacturer. The test is based on the addition of a chromogenic substrate and excess of Factor Xa (responsible for hydrolysis of this substrate) to the sample. The inhibition of Factor Xa by the antithrombin−heparin complex competes with the hydrolysis of the substrate by Factor Xa. The concentration of heparin is inversely proportional to the absorbance due to the hydrolysis of the substrate. Citrate was quantified with an automated analyzer (Hitachi 902), using reagent sets from Roche (Penzburg, Germany). Adsorption of Bilirubin and Cholic Acid. Citrated plasma was spiked with bilirubin (300 μM), cholic acid (100 μM), tryptophan (100 μM), and phenol (2 mM). Batch experiments were conducted using an adsorbent-to-plasma ratio of 1:9 (v/v) for bilirubin adsorption, while a ratio of 1:99 (v/v) was used to measure cholic acid removal. After 0, 15, and 60 min of incubation, samples were

MATERIALS AND METHODS

Plasma and Chemicals. Unfractionated heparin was purchased from Baxter (Vienna, Austria). Citrated and heparinised plasma were obtained by drawing fresh blood from healthy volunteer donors into tubes containing trisodium citrate or heparin to a calculated concentration of 11 mM or 5 IU/mL, respectively, in whole blood (Vacuette, Greiner Bio-One, Vienna, Austria). Blood was immediately centrifuged at 3600 g for 10 min at room temperature (RT) to separate plasma from cellular components. Bovine serum albumin, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), o-phenylenediamine, bilirubin, tryptophan, phenol, and cholic acid were purchased from Sigma Aldrich (Vienna, Austria). Adsorbents. Adsorbents were provided by Polymerics GmbH, Berlin, Germany. Two divinylbenzene copolymers were used, namely, a neutral divinylbenzene-styrene (DVB-ST) resin and a divinylbenzene-vinylimidazole (DVB-VI) copolymer. They were produced as previously described.18 In addition, commercial polystyrene divinylbenzene-based resins were tested in this study, namely, Prometh 01 and 02 (Fresenius Medical Care, Bad Homburg, Germany). Prometh 01 (further designated as NP, from Neutral Polymer) is a styrene divinylbenzene resin whereas Prometh 02 (further designated as CP, from Charged Polymer) is a polystyrene divinylbenzene copolymer substituted with trimethylamine groups.4 Characterization of Adsorbents. Particle morphology was analyzed by scanning electron microscopy (SEM) using a Zeiss Supra 50 VP scanning electron microscope (Carl Zeiss NTS) with an acceleration voltage of 5 kV. Dry NP and CP particles were cut with a razor blade, whereas DVB particles were ground with the help of a mortar and pestle. Both series of particles were mounted on a sample holder and coated with palladium (4 nm thickness). A CILAS 1064 laser granulometer (Cilas, France) was used to measure the average particle diameter in isopropanol. To determine the specific surface and the pore size distribution, nitrogen adsorption and desorption isotherms were recorded at liquid nitrogen temperature (−196 °C) at relative pressures p/p0 between 0.001 and 1.0 using an ASAP 2010 porosimetry system (Micrometrics Instrument Corp., Norcross, U.S.A.). The specific surface area (SBET) was calculated using the Brunauer, Emett, Teller (BET) equation.19 The total micropore volume (pore size < 2 nm) was calculated with the Horvath−Kawazoe (H−K) method,20 whereas the mesopore volume (2−50 nm) was obtained via the Barrett, Joyner, Halenda method (BJH).21 Average pore size d was calculated as d = 4V/SBET (with V = maximum adsorbed nitrogen volume). The charge density of the characterized adsorbents was evaluated via titration: 200 mg of DVB-VI adsorbent polymer were washed with methanol and then suspended in water. Subsequently, 2 mL of 0.1 N HCl were added to the polymer suspension in order to protonate all 485

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drawn and centrifuged (4600g, 4 min), and supernatants were collected and stored at −20 °C until further analysis. Plasma without adsorbents served as a negative control. Experiments were conducted in triplicates. Bilirubin and cholic acid were quantified using a Hitachi 902 analyzer (Roche Diagnostic, Penzberg, Germany) and reagent sets from Roche for bilirubin and from Trinity Biotech (Wicklow, Ireland) for cholic acid. Statistical Analysis. Data were evaluated with SPSS Statistic software, version 18.0 (SPSS Inc., Chicago, IL, U.S.A.), using the nonparametric Mann−Whitney U test. Data are expressed as means ± standard deviation. Differences were accepted as significant at p ≤ 0.05.

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RESULTS AND DISCUSSION Physicochemical Characteristics of the Adsorbents. Two series of divinylbenzene-based adsorbents were used in this study. The first series comprised a neutral and a cationic divinylbenzene resin with an average particle size of 74 μm (DVB-ST and DVB-VI, Figure 1a,b) and specific surface areas of 641 and 670 m2/g, respectively. These adsorbents contain mainly mesopores with an average pore size of 8−9 nm, which have been shown previously to be accessible to albumin-bound molecules.6 The second series of adsorbents were polystyrene divinylbenzene resins which are already in clinical application, namely Prometh 01, a neutral polystyrene divinylbenzene resin (NP), and Prometh 02, a trimethylamine-substituted styrene divinylbenzene resin (CP), which functions as a strong anion exchange resin (Figure 1a,c). The physicochemical characteristics for all the tested adsorbents are summarized in Table 1. Scanning electron micrographs (SEM) are presented in Figure 2 for both series of adsorbents. The outer surface of all the tested adsorbents appears to be denser and smoother than the inner sponge-like structure. Nevertheless, a more compact skin on the surface of NP and CP adsorbents is visible, which may impair the pore accessibility of this series of adsorbents. In addition to the assessment of pore size distribution by nitrogen adsorption (as summarized in Table 1), the accessibility of pores was evaluated by inverse size exclusion chromatography (iSEC). The distribution coefficients (Kd) values for glucose and dextrans from the iSEC measurements were plotted against the logarithm of the molecular mass for each adsorbent (Figure S1). Kd approached zero (0.09) already at molecular mass of 6 kDa for CP and was 0.2 for NP, indicating a poor accessibility of the pores of this series of adsorbents even to small adsorbates. In contrast, the Kd value of the DVB-ST and DVB-VI adsorbents approached zero only at molecular mass above 100 kDa, suggesting that molecules under 100 kDa are able to access the pores of these adsorbents. More than 80% of DVB-ST and 60% of DVB-VI pores were accessible to dextran 6 kDa, and nearly 20% of the pores could be accessed by albumin molecules. Adsorption of Toxins Related to Liver Failure. The adsorption efficiency for cholic acid and unconjugated bilirubin for both series of adsorbents was assessed in vitro under static conditions. The results are summarized in Table 2. The neutral resin DVB-ST showed a slightly higher adsorption for cholic acid than the weak anion-exchange resin DVB-VI under the experimental conditions applied, while bilirubin was bound more efficiently by the DVB-VI resin. The same was true when comparing the adsorptive efficiency of NP and CP, although the difference in toxin removal between NP and CP was much bigger, presumably due to the higher charge density of CP. For both metabolites, the amounts removed by DVB-ST and DVB-VI were significantly higher than for NP and CP (p =

Figure 1. Structure of the adsorbents used: (a) polystyrene divinylbenzene adsorbents, DVB-ST and NP; (b) divinylbenzene vinylimidazole adsorbent, DVB-VI; (c) polystyrene divinylbenzene copolymer substituted with trimethylamine groups, CP. R = N+(CH3)3.

Table 1. Physicochemical Characteristics of the Adsorbents Used in This Study adsorbent

charge density (mval/g)

avg particle diameter (μm)

particle density (g/mL)

SBET (m2/g)

DVB-ST DVB-VI NP CP

neutral 0.01 neutral 3.5

75 73 350 350

0.27 0.25 0.31 0.31

640 670 1200 1200

0.05), reflecting the effect of particle size and indicating the importance of the outer surface area for adsorption. In addition to this difference in particle size, differences in porosity may also account for the variation in adsorption characteristics. The presence of a dense skin on the surface of the NP and CP polymers apparently decreases the accessibility to the internal 486

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Table 3. Residual Percentages of Protein C and Factors II, VII, and X in Citrated Plasma Referred to Heparinized Plasma (100%) After 60 min of Incubation with DVB-ST, DVB-VI, NP, and CP, n = 3 NP

Figure 2. Scanning electron micrographs of representatives of the two series of adsorbents used: (a) DVB-ST and (b) CP.

CP

93 ± 8 105 ± 9 95 ± 6 94 ± 15

factor II factor VII factor X protein C

58 74 59 43

± ± ± ±

DVB-ST 7 3 8 13

79 75 79 56

± ± ± ±

5 2 4 9

DVB-VI 60 60 63 30

± ± ± ±

4 4 3 7

study possess comparable values of specific surface area and mean pore size. As shown by iSEC, their pores are accessible to target substances in the molecular mass range of albumin, making the particles efficient adsorbents for albumin-bound substances and smaller molecules. The presence of vinylimidazole moieties influences the adsorptive characteristics as illustrated by the enhanced binding of bilirubin to DVB-VI. However, this goes hand in hand with stronger adsorption of Gla domain-containing factors, especially in citrated plasma. This effect was even more pronounced for CP, which exhibits a higher charge density than DVB-VI (3.5 vs 0.01 mval/g). The binding of Gla domain-containing factors to NP and CP resins is to be attributed to hydrophobic and ionic interactions, respectively. In both cases, the removal of molecules occurs mainly at the outer surface, which is coated by a dense skin, limiting the accessibility of the pores. Adsorption of Citrate and Heparin. Heparin and citrate were quantified during both sets of experiments (Table S3). Citrate and heparin were not substantially removed by DVBVI and there was no adsorption for the neutral beads (DVBST). Thus, while neither DVB-ST nor DVB-VI adsorbed significant amounts of citrate and heparin, there was significant binding of both citrate and heparin by the CP (p = 0.05 with respect to the control values). After 1 h of incubation, citrate was reduced from 23.5 to 14.6 mM and heparin concentration dropped from 8.3 to 0.7 IU/mL. No such reduction was observed for the neutral polymer NP. Overall, an enhanced adsorption of Gla domain-containing factors to the anion exchanger was observed in the presence of citrate. Adsorbents with weak cationic modifications lead to a better bilirubin removal and a removal of Gla domaincontaining factors to a lesser extent than strong ion exchange resins.

pore structure.22 These findings suggest that the balance between porosity and charge density is crucial to improve the performance with respect to binding of albumin-bound toxins. Adsorption of Coagulation Factors. The removal of the coagulation factors II, VII, and X and of the anticoagulant protein C was tested both in citrated and heparinised plasma. Table S1 (see Supporting Information) summarizes the residual percentages of these molecules after 60 min of incubation in the presence of citrate and heparin. Table 3 further reduces these data to the ratio of residual percentages of the coagulation factors in citrated versus heparinised plasma. Factors II, VII, X, and protein C, are serine proteases containing γ-carboxyglutamic (Gla) domains, responsible for high-affinity binding of calcium ions, which induce and stabilize the folding of Gla domains into tightly packed structures with concomitant exposure of hydrophobic residues that enhance lipid binding.12−14,24 Complexation of calcium by citrate induces a disordered dynamic state of the Gla domains. In fact, negative Gla residues, not being neutralized by positive charges of ionized calcium, point away from each other due to charge repulsions.24 As a result, coagulation factors containing Gla domains with exposed negative charges may bind to positively charged groups on ion exchange resins. This effect is less noticed when heparinised plasma is employed, since heparin does interact with calcium ions, but not as strongly as citrate.25,26 A table reporting the molecular masses, number of Gla residues, and isoelectric points for each factor tested in this study is presented in the Supporting Information (Table S2).24,27−32 As shown in Table 3, a higher percentage of Gla domaincontaining factors was removed when ion-exchange resins (CP and DVB-VI) were incubated with citrated plasma as compared to heparinized plasma (p = 0.05). For neutral polymers (NP and DVB-ST), the difference between citrate and heparin was clearly smaller and not significant (0.2 < p < 1). The efficiency of an adsorbent depends on its pore structure, that is, the pore size distribution and the specific surface area, as well as on the hydrophobic/ionic interactions with the target molecules. The DVB-ST and DVB-VI polymers used in this

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CONCLUSIONS Our data support previous findings regarding decreased levels of Gla domain-containing coagulation factors when ionexchange resins are used during extracorporeal blood purification. In addition, we demonstrated that the adsorption

Table 2. Amount of Cholic Acid and Bilirubin Bound to the Adsorbents after 15 and 60 min Incubation cholic acid adsorbed (μmol/g adsorbent)

bilirubin adsorbed

(μmol/mL adsorbent)

(μmol/g adsorbent)

(μmol/mL adsorbent)

adsorbent

15 min

60 min

15 min

60 min

15 min

60 min

15 min

60 min

DVB-ST DVB-VI NP CP

19.8 19.9 3.4 0.8

24.2 22.4 7.9 1.0

5.3 5.0 1.0 0.3

6.5 5.6 2.4 0.3

4.4 6.7 1.2 2.0

7.0 7.7 1.9 4.1

1.2 1.7 0.4 0.6

1.9 2.1 0.6 1.3

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(15) Tavoosi, N.; Davis-Harrison, R. L.; Pogorelov, T. V.; Ohkubo, Y. Z.; Arcario, M. J.; Clay, M. C.; Rienstra, C. M.; Tajkhorshid, E.; Morrissey, J. H. J. Biol. Chem. 2011, 286, 23247−23253. (16) Spronk, H. M. H.; Govers-Riemslag, J. W. P.; ten Cate, H. BioAssay 2003, 25, 1220−1228. (17) Huang, M.; Rigby, A. C.; Morelli, X.; Grant, M. A.; Huang, G.; Furie, B.; Seaton, B.; Furie, B. C. Nat. Struct. Biol. 2003, 10, 751−756. (18) Leistner, A.; Leistner, A. U.S. Patent 7,311,845, 2003. (19) Brunauer, S.; Emmett, P. H.; Teller, E. J. Am. Chem. Soc. 1938, 60, 309−19. (20) Horvath, G.; Kawazoe, K. J. Chem. Eng. Jpn. 1983, 16, 470−475. (21) Barrett, E. P.; Joyner, L. G.; Halenda, P. P. J. Am. Chem. Soc. 1951, 73, 373−378. (22) Vienken, J.; Christmann, H. Ther. Apher. Dial. 2006, 10, 125− 131. (23) DePhillips, P.; Lenhoff, A. M. J. Chromatogr., A 2000, 883, 39− 54. (24) Whinna, H. C.; Lesesky, E. B.; Monroe, D. M.; High, K. A.; Larson, P. J.; Church, F. C. J. Thromb. Haemost. 2004, 2, 1127−1134. (25) Higgins, C. Med. Lab. Obs. 2007, 39, 16−20. (26) Sachs, C.; Rabouine, P.; Chaneac, M.; Kindermans, C.; Dechaux, M.; Falch-Christiansen, T. Ann. Clin. Biochem. 1991, 28, 167−73. (27) Fujikawa, K.; Legaz, M. E.; Davie, E. W. Biochemistry 1972, 11, 4882−4891. (28) Kisiel, W.; Davie, E. W. Methods Enzymol. 1981, 80, 320−332. (29) DiScipio, R. G.; Davie, E. W. Biochemistry 1979, 18, 899−904. (30) Bajaj, S. P.; Rapaport, S. I.; Brown, S. F. J. Biol. Chem. 1891, 256, 253−259. (31) Kisiel, W.; Hanahan, D. J. Biochim. Biophys. Acta 1973, 304, 103−113. (32) DiScipio, R. G.; Hermodson, M. A.; Yates, S. G.; Davie, E. W. Biochemistry 1977, 16, 698−706.

of Gla domain-containing coagulation factors depended on the anticoagulant used. Positively charged resins exhibited stronger adsorption of Gla domain-containing factors in citrated than in heparinised plasma. This effect was more pronounced for strongly charged polymers, such as CP. Our results on the adsorption of coagulation factors and of albumin-bound toxins related to liver failure demonstrate that the optimization of the adsorbent characteristics (porosity and charge density) is crucial to achieve minimal adsorption of coagulation factors and optimal toxin removal at the same time.

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ASSOCIATED CONTENT

S Supporting Information *

In Tables S1 and S3, the adsorption of coagulation factors and the reduction in citrate and heparin levels, respectively, are summarized. Table S2 reports the structure, number of Gla domains, and isoelectric points of the tested coagulation factors. Figure S1 shows data from iSEC analysis. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: ++43 2732 893 2632. Fax: ++43 2732 893 4600. Email: [email protected].

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ACKNOWLEDGMENTS The authors thank Ute Fichtinger for precious technical assistance, Tanja Buchacher for support with ELISA measurements, Stephan Harm for support with iSEC, and Tanja Stoifl for the electron micrographs, which were taken at the School of Pharmacy and Biomolecular Sciences, University of Brighton, U.K. This research was financially supported by the European Commission within the “MONACO-EXTRA” project on “MONolithic Adsorbent COlumns for EXTRAcorporeal medical devices and bioseparations” − Marie Curie (FP7 Grant Number PIAP-GA-2008-218242).

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