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Artificial Organs 32(12):962–998, Wiley Periodicals, Inc. © 2008, Copyright the Authors Journal compilation © 2008, International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc. Thoughts and Progress Score Model for the Evaluation of Dialysis should include platelets, leukocytes, and red cells, as Membrane Hemocompatibility well as complement proteins, coagulation factors, and a variety of other plasma proteins. *Ansgar Erlenkötter, *Pia Endres, *Bernd Nederlof, Currently available membranes not only differ †Carsten Hornig, and †Jörg Vienken with respect to their physicochemical properties, *Fresenius Medical Care, St. Wendel; †Fresenius such as performance, thickness, pore size, porosity, Medical Care, Bad Homburg, Germany and adsorptive capacities, they also show different grades of hemocompatibility. Reasons for this are Abstract: The interaction of blood with artificial surfaces is not only differences in polymer composition, but of particular interest during hemodialysis treatments with also conditions of manufacturing and of sterilization extracorporeal blood circuits. Components of the extracor- techniques (1). poreal blood circuit are known to have only a moderate, The large surface area of a dialysis membrane sometimes even an unfavorable hemocompatibility, and and the chronic exposure of blood to a foreign thus may provoke adverse biochemical or clinical sequelae. This article describes a newly established hemocompatibil- surface area during hemodialysis treatment has been ity assessment score. This score is based on on a standard- the motivation for extensive research on how to ized series of in vitro tests and is applied to commercially improve the hemocompatibility pattern of dialysis available hemodialysis membranes. It relates to a variety membranes. Standard investigations are still based on of membrane polymers, such as regenerated cellulose, in vitro test systems only. diethylaminoethyl-modified cellulose, polyethersulfone/ polyarylate blends and polysulfone. In order to compare This article describes an in vitro assessment of the different polymers used in the manufacturing of dialysis hemocompatibility pattern of five different polymers membranes, a set of the following hemocompatibility used for commercially available dialysis membranes. parameters was assessed and assembled to an overall Different polymer classes, such as regenerated score: generation of complement factor 5a, thrombin- cellulose, diethylaminoethyl cellulose (DEAE), antithrombin III-complex, release of platelet factor 4, gen- eration and release of elastase from polymorphonuclear polyethersulfone/polyarylate (PEPA) blends and granulocytes, and platelet count. With respect to these polysulfone, as well as their different modes of ster- parameters, the results reveal major differences between ilization, have been assessed and are compared. Test the selected dialysis membranes. This new score model results of the individual polymers have led to a new proves to be an efficient tool to derive objective results, and score model that has been developed for this inves- it may, thus, be used in the future to facilitate the selection of membrane polymers with an appropriate hemo- tigation and allows for both to provide an objective compatibility pattern for dialysis therapy. Key Words: assessment tool for the comparison of different poly- Dialysis membrane—Dialyzer—Hemocompatibility— mers and to guide manufacturers for future mem- Score model. brane development. Biocompatibility has become the number one factor for the characterization of dialyzers, and in MATERIALS AND METHODS particular of the incorporated dialysis membrane. Consequently, the target of membrane development Blood collection in the past was to improve its bio(hemo)compatibility. 450 mL of whole blood was collected from Markers to characterize membranes under investiga- healthy volunteers. A specially designed blood tion must reflect both the cellular and the plasmatic bag system with a 17 G (1.5 mm) dialysis fistula components of the blood–membrane interface. They needle (Fresenius Medical Care, Bad Homburg, Germany) was applied for blood collection. Whole blood was collected in these bags, which had been doi:10.1111/j.1525-1594.2008.00657.x prefilled with 750 IU heparin (Ratiopharm, Ulm, Received April 2007; revised October 2007. Germany) in a 50-mL saline solution, resulting in Address correspondence and reprint requests to Dr. Ansgar a final heparin concentration of 1.5 IU/mL. This Erlenkötter, Biocompatibility Laboratory, Fresenius Medical Care, Frankfurter Straße 6-8, D—66606 St. Wendel, Germany. E-mail: heparin concentration was used in order to simulate [email protected] clinical dialysis as close as possible and reflects an 962 THOUGHTS AND PROGRESS 963 Reservoir Pven FIG. 1. Experimental setup of the recircu- Dialyzer lation model including a sample reservoir, two pressure measure chambers, a pump segment, and a polyvinylchloride tubing (blood flow rate: QB = 200 mL/min, filling Sampling volume: VB = 150 mL, heparinization: point 1.5 IU/mL). Two of these setups were used Part in parallel in order to allow for membrane comparisons under identical conditions. Blood pump appropriate anticoagulation for in vitro testing of lants (Sarstedt Monovetten, Sarstedt, Nümbrecht, dialyzers. This heparin concentration further yielded Germany). Additional samples were taken directly the most pronounced differences in hemocompatibil- from the blood bags immediately before the filling ity pattern between different polymers. All hemo- procedure. These were used as prevalues (0 min). compatibility experiments started within 30 min after blood collection. Membrane polymers For the assessment of dialysis membrane hemo- Recirculation model compatibility, five different, commercially available Figure 1 shows the in vitro recirculation model membranes made from polysulfone and cellulose used in our study. The test system consists of a dia- were chosen: g-sterilized polysulfone (Toraysulfone, lyzer and a tubing system specifically developed for BS-1.3 dialyzer, 1.3 m2, Toray, Tokyo, Japan), our test system using standard bloodline components g-sterilized polyethersulfone/polyarylate (PEPA = (Fresenius Medical Care). In each experiment, two polyester–polymer alloy, FLX-12GWS dialyzer, test systems (test vs. reference dialyzer) were evalu- 1.2 m2, Nikkiso, Tokyo, Japan), steam-sterilized ated in parallel in a standard incubator (Memmert, polysulfone (Helixone, FX60 dialyzer, 1.4 m2 Schwabach, Germany). The temperature was set at [Fresenius Medical Care]), regenerated cellulose 37°C in all experiments. (Cuprophan, GFS 12 dialyzer, 1.3 m2, Gambro AB, The systems were prerinsed with saline solution Hechingen, Germany), and cellulose etherified with (0.9% NaCl) for approximately 30 min. In order to DEAE groups (Hemophan, GFS Plus 12 dialyzer, prevent any ultrafiltration, the dialyzate compart- 1.3 m2, Gambro AB). Membranes were assessed in ment was filled with saline solution and clamped. their original housing. Thus, it was not possible to After the prerinsing step, the test systems were care- achieve absolutely identical, but only similar mem- fully filled with blood. Blood volume in the circuit brane surface areas. Differences are still significant as was adjusted to 150 mL in order to keep the blood the dialyzer with the largest surface area yielded volume for all test series constant. Each donor pro- better results. In each test, the dialysis membranes vided blood for one double experiment. were tested in comparison with the same reference Blood flow rate in the recirculation setup was estab- membrane, that is, polysulfone (Helixone [Fresenius lished at 200 mL/min. Blood samples were taken at Medical Care]), because this material is one of the several time points (15, 30, 60, 120, and 180 min) from most widely used membrane polymers in the world the two circuits into syringes prefilled with anticoagu- today. Artif Organs, Vol. 32, No. 12, 2008 964 THOUGHTS AND PROGRESS FIG. 2. (a) and (c) course of C5a generation (a, n = 2) and platelet loss (c, n = 3) during a single experiment (dialyzer A: left graph/closed circles, steam-sterilized polysulfone membrane (Helixone); dialyzer B: right graph/open circles, g-sterilized polysulfone membrane (Toraysulfone). The gray area under the curve illustrates the integral generation of either C5a or platelet loss during the time course of the experiment. The double-headed arrow represents the calculated mean C5a generation per hour or the mean platelet loss; (b) correlation of C5a score versus normalized mean C5a generation per hour: three pairs of variables were predefined: point A represents the mean C5a generation with no activation of the complement system (0¦0), point B represents the mean C5a generation of the reference dialyzer (100¦20), and point C represents the maximum level of C5a generation by one of the test dialyzers (14 440¦100); (d) correlation of PLT score versus mean PLT loss: three pairs of variates were predefined: point A represents no mean PLT loss (0%¦0), point B represents the mean PLT loss of the reference dialyzer (IPLT,reference¦20), and point C represents the total loss of platelets (100%¦100). The dotted line represents the correlation for a reference value of 5%; the dashed line represents correlation for a reference value of 75%. Determination of hemocompatibility markers Data analysis Hemocompatibility was assessed by measuring The assessment of hemocompatibility data should complement factor 5a (C5a), thrombin-antithrombin be transparent and traceable. Therefore, we have III-complex (TAT), platelet count (PLT), platelet developed a four-step procedure to analyze the raw factor 4 (PF4), and release of elastase from polymor- data. phonuclear granulocytes (PMN-elastase). These 1st step: Calculation of the mean C5a genera- parameters have turned out to reflect a typical selec- tion per hour, the mean TAT generation per tion of well-established hemocompatibility markers. hour, the mean platelet loss, the mean PF4 release Platelet count and hematocrit (HCT) were measured per hour, and the mean PMN-elastase release in triplicate by using an automatic hematology ana- per hour. lyzer (K 4500, Sysmex, Norderstedt, Germany). C5a A typical course is shown in Fig. 2a for C5a- (EIA-3327, DRG Instruments, Marburg, Germany), generation and in Fig. 2c for platelet loss during a TAT (Enzygnost TAT micro, Dade Behring, Marburg, single experiment. The left graph shows the results of Germany), PF4 (1875353 Asserachrom, Roche Diag- steam-sterilized polysulfone membrane (Helixone) nostics, Mannheim, Germany), and PMN-elastase (dialyzer A), while the right graph represents data for (K6840, Immundiagnostik AG, Bensheim, Germany) a g-sterilized polysulfone membrane (Toraysulfone) plasma concentrations were determined in duplicate (dialyzer B). As a measure of the total C5a genera- by applying commercially available enzyme-linked tion and platelet loss, the area under the curve immunosorbent assay kits. (AUC) was calculated. A mean C5a generation rate Artif Organs, Vol. 32, No. 12, 2008 THOUGHTS AND PROGRESS 965 per hour was achieved by multiplying with 120/TI2. Normalized values are similarly calculated for This leads to the index IC5a (Eq. 1) TAT, PF4, and PMN-elastase. A normalized value (IC5a = mean C5a generation per hour, cC5a,pre = C5a was not calculated for the PLT results. concentration in blood bag, cC5a,HCT–cor. = HCT cor- 3rd step: Calculation of the hemocompatibility rected C5a concentration, t = time, TI = total incuba- scores for C5a, TAT, PLT-loss, PF4, and PMN- tion time): elastase. Hemocompatibility scores were introduced in 120 ⎛ 2 t ⎞ order to compare the previously calculated values 2 ⎜ ∫ C 5 a , HCT − cor . IC 5 a = c (t )dt − cC 5 a, pre ∗TI ⎟ (1) among the individual experiments (C5a score, TAT TI ⎝ t1 ⎠ score, PLT score, PF4 score, and PMN-elastase score). Figure 2b shows the correlation of a C5a score versus This index represents the mean increase of the C5a normalized C5a generation per hour. This correlation level during 1 h of recirculation. In Fig. 2a, it is rep- is calculated by predefining three pairs of variables. resented by the double-headed arrow. The param- A normalized mean C5a increase per hour of zero eters TAT, PF4, and PMN-elastase were calculated in results in a C5a-score of zero and reflects no activation an analogous manner. of the complement system (see point A in Fig. 2b). A Figure 2c represents the platelet loss in % versus score of 20 is purposely defined as a 100% normalized time. Platelet loss is only quantified for the initial mean C5a increase per hour as it is predefined for the phase of the in vitro recirculation (0–60 min). In a reference dialyzer (see point B).The dialyzer with the later stage, platelet count increases following a highest degree of activation defines the last pair of detachment of platelets from the surface. These pos- variables and is named “Reference 2.” In the example sibly “dead” cells have lost their overall functionality of Fig. 2b, the normalized C5a increase per hour of and thus cannot be used for the scoring analysis. Reference 2 was 14 440% (see point C). Equation 4 The mean platelet loss is calculated by dividing shows the mathematical solution of this correlation the integral (gray area under the curve) as shown in (nIC5a,reference 2 = normalized mean C5a generation per Eq. 2 by the initial incubation time (IPLT = mean hour of reference 2; IC5a = mean C5a generation per platelet loss [%], t = time, TII = initial incubation hour of the test dialyzer). time). In the figure, it is represented by the double- headed arrow. ⎛ Ig 5 ⎞ ⎜ 1+ + 2 Ig 5 − Ig nIC 5 a , reference 2 ⎟ C 5a score = 4⎝ Ig 4 ⎠ ∗ t2 ⎛ Ig 5 ⎞ 1+ 2 ∫ Δc PLT (t )dt (2) 5 ⎝⎜ Ig 4 + 2 Ig 5 − Ig nI C 5 a , reference 2 ⎠⎟ ∗ (4) I PLT = t1 ⎛ Ig 5 ⎞ + 2 Ig 5 − Ig nIC 5 a ,rreference 2 ⎟ TII ⎜ nIC 5 a ⎝ Ig 4 ⎠ 2nd step: Calculation of the normalized mean C5a This kind of calculation is similarly used for TAT, generation per hour, the normalized mean TAT gen- PF4, and PMN-elastase analyses. eration per hour, the normalized mean PF4 release The calculation of the PLT score is slightly different per hour, and the normalized mean PMN-elastase and illustrated in Fig. 2d. In this calculation, no plate- release per hour, respectively. let loss (IPLT = 0%) results in a PLT score of zero (see The blood donor characteristics have a strong point A). The total loss of platelets is defined as 100 influence on hemocompatibility results. Therefore, (see point C).The reference value is set as a PLT score a normalization step is necessary and is always of 20 once again (see point B1 and B2). Depending on advisable. The previously calculated values are nor- the mean platelet loss of the reference dialyzer, two malized by defining a reference value of 100%. different correlations are shown in Fig. 2d.The dotted For example, the normalized mean C5a generation line represents the correlation for a reference value per hour is calculated as shown in Eq. 3 of 5%, and the dashed line for a reference value of (nIC5a = normalized mean C5a generation per hour, 75%. The correlation of PLT-score and mean PLT IC5a,reference dialyzer = mean C5a generation per hour of loss is described by Eq. 5 (IPLT = mean PLT loss, the reference dialyzer, IC5a,test dialyzer = mean C5a gen- IPLT,REF = mean PLT loss of the reference dialyzer). eration per hour of the test dialyzer): ⎛ Ig 5 ⎞ Ig 5 1− ⎝⎜ Ig 100 − Ig I PLT,reference ⎠ ⎟ − Ig IPLT,reference IC 5 a,test dialyzer PLT score = 100 ∗ IPLT Ig 100− nIC 5 a = ∗ 100 (3) IC 5 ,reference dialyzer (5) Artif Organs, Vol. 32, No. 12, 2008 966 THOUGHTS AND PROGRESS FIG. 3. Dialyzer membrane polymer versus (a) platelet score; (b) TAT score; (c) C5a score; and (d) PMN-elastase score. High scores implicate an unfavorable hemocompatibility and low scores implicate an excellent hemocompatibility. The solid line indicated by the arrow illustrates the predefined value for the standard reference. Data are means ⫾ SD (n = 3). 4th step: Calculation of the total hemocompatibil- RESULTS ity score. In all experiments, the steam-sterilized polysulfone Five hemocompatibility scores could be calcu- membrane (Helixone) in its original housing served lated by the procedure described here. To create a as a standard reference, because this material is cur- total hemocompatibility score (THS) as requested rently the most widely used membrane material. for the selection of polymers for membrane manu- Changes of hemocompatibility pattern are always facturing, weighing factors are defined for each expressed in relation to this reference. Figure 3 shows hemocompatibility category. Following our experi- the results of all hemocompatibility assessments ence, the determination of the weighing factors is comparing different membrane polymers. arbitrarily taken for each hemocompatibility param- eter as 0.25 for the plasmatic immune system (C5a), cell activation (PMN-elastase), and coagulation PLT score (Fig. 3a) activation (TAT). PF4 and platelet count are taken The highest degree of platelet loss in vitro was equally, with their weight summing up to a final observed for the PEPA membrane (PLT score = figure of 0.25. The THS is then calculated by 80 ⫾ 6, in Fig. 3a) leading to a score value of 80. Eq. 6: Further, the g-sterilized polysulfone membrane (Toraysulfone) showed a higher PLT loss (PLT THS = fC 5 a ∗ C 5 a score + fTAT ∗TAT score + fPLT ∗ score = 46 ⫾ 18) in comparison with the reference PLT score + fPF 4 ∗ PF 4 score + (6) material, while a lower PLT loss was recorded only fPMN ∗ PMN score for the cellulosic membranes (Cuprophan, PLT score Artif Organs, Vol. 32, No. 12, 2008 THOUGHTS AND PROGRESS 967 FIG. 4. Calculated THS for steam-steri- lized polysulfone (Helixone), g-sterilized polysulfone (Toraysulfone), PEPA, regen- erated cellulose (Cuprophan), and DEAE cellulose (Hemophan). High scores impli- cate an unfavorable hemocompatibility and low scores implicate an excellent hemo- compatibility. The solid line indicated by the arrow illustrates the predefined value for the standard reference. 16 ⫾ 4 and Hemophan, PLT score = 12 ⫾ 3). The rial (PMN-elastase score: 23 ⫾ 11). A lower level results for the PF4 score were comparable. could be measured for the g-sterilized polysulfone membrane (Toraysulfone, PMN-elastase score: TAT score (Fig. 3b) 14 ⫾ 7). DEAE cellulose membrane (Hemophan) showed the highest TAT score: 100 ⫾ 40. Steam-sterilized Total hemocompatibility score polysulfone membrane (Helixone) showed a TAT From all results of the individual in vitro scoring score of 17 ⫾ 5 as compared with a g-sterilized experiments, a THS could be derived as a summary polysulfone membrane (Toraysulfone) with a TAT (see Fig. 4). score of 28 ⫾ 16. The PEPA membrane exhibited a The THS of the steam-sterilized polysulfone mem- TAT score of 32 ⫾ 25, and the regenerated cellulose brane (Helixone) was found to be 19.6. This closely membrane (Cuprophan) a TAT score of 32 ⫾ 25. resembles the predefined value of 20 for the reference membrane. The THS values further increased in the C5a score (Fig. 3c) following order: g-sterilized polysulfone membrane A lower complement activation score compared (Toraysulfone, THS: 32) < PEPA membrane (THS: with the reference was only found for the PEPA 38) < DEAE cellulose membrane (Hemophan, membrane (C5a score: 5.0 ⫾ 2.5). All other materials THS: 40) and < regenerated cellulose membrane under investigation showed higher levels: g-sterilized (Cuprophan, THS: 63). polysulfone (Toraysulfone, C5a score: 30.1 ⫾ 1.5), DEAE-modified cellulose membrane (Hemophan) DISCUSSION with a C5a score of 41.8 ⫾ 4.1, and the highest acti- In this study, the hemocompatibility pattern of five vation was found for the regenerated cellulose mem- hemodialysis membranes composed of different brane (Cuprophan, C5a score = 100 ⫾ 1). The C5a polymers (regenerated cellulose, DEAE cellulose, score for the steam-sterilized polysulfone (Helixone) PEPA, and polysulfone) was assessed. A standard- in a direct comparison was found to be 19.9 ⫾ 0.1. ized in vitro test system and a newly developed score model was developed for this purpose. Based on the PMN-elastase score (Fig. 3d) results of this in vitro evaluation and by means of PMN-elastase release was found to be highest for the new score model, a total hemocompatibility the regenerated cellulose membrane (Cuprophan, score could be derived for each membrane type and PMN-elastase score: 100 ⫾ 106), followed by the allowed a comparison of its in vitro hemocompatibil- DEAE cellulose membrane (Hemophan, PMN- ity performance. elastase score: 11 ⫾ 5). The PEPA membrane was The selection of a certain polymer type prior to found to be in the same range as the reference mate- the manufacturing of dialysis membrane influences Artif Organs, Vol. 32, No. 12, 2008 968 THOUGHTS AND PROGRESS the hemocompatibility pattern of a membrane. a substitution of hydroxylgroups by DEAE However, further factors, such as potting material, groups, bears positive charges in a physiological sterilization techniques, geometry of the membrane environment. This surface charge gives rise to the capillaries, and the overall manufacturing conditions adsorption of negatively charged heparin to some further influence the hemocompatibility profile. A extent. A lack of freely available heparin due to its straightforward assessment of membranes in a com- adsorption at the membrane surface might explain parable housing should facilitate the selection of the higher TAT values (Fig. 3b). polymers for large-scale production, as revealed in It might be necessary to monitor the effects of our study. As shown here, a membrane–polymer different materials used for the fabrication of dialysis evaluation, even if limited to five parameters but membranes on the level of single parameters. carried out in a recirculation model under standard- However, the complexity of biochemical reactions at ized conditions with respect to blood collection, the blood–membrane interface implies that the heparinization, blood flow rate and blood volume, overall hemocompatibility of a material may not be tubing sets, time of investigation, and temperature appropriately represented by a single parameter came across with results enabling the manufacturer alone (6). This is supported by the observations from to make up his mind for actual or future polymer our study. Here, the individual materials showed a selection. The use of mini-modules have proven to different behavior with respect to each parameter be a practical tool in in vitro or ex vivo experiments, under investigation. Thus, it seems more convenient (2,3) and thus have been used successfully in our to monitor the overall hemocompatibility of a mate- membrane assessments. In addition, studies with rial, taking into account a set of hemocompatibility full-size hemodialyzers in in vitro circuits are com- parameters in order to facilitate the decision in favor parable with the experimental setup of our study or against a certain polymer. Therefore, the devel- (4,5). In this study, we focused on parameters related oped score model, which incorporates and summa- to the activation of the complement system, the acti- rizes the results of the individual experiments, offers vation of blood coagulation, and the activation of an objective total hemocompatibility assessment for cells, such as platelets and leukocytes. each tested polymer. Score models have also been Striking differences for membranes could be employed by other groups before (7). shown for some of the parameters.They can be attrib- uted to both the molecular composition of the CONCLUSIONS membrane bulk as well as the permeability for low- molecular-weight proteins. To avoid effects specific After calculation of the THS value for each for low- or high-flux membranes, we clamped the polymer, it is possible to determine clear-cut differ- ports for dialysis fluids in the respected dialyzer and ences between the polymers used. While regenerated investigated biocompatibility parameters exclusively cellulose had the highest score value and thus the most as a function of the membranes surface properties unfavorable overall hemocompatibility of all poly- and not for their filtration effects. mers tested, other cellulose-based polymers behaved In addition, some activated proteins that are sig- also less well than the three remaining synthetic poly- nificant for blood compatibility might be adsorbed at mers. PEPA had a better overall hemocompatibility the membrane surface. This is valid for the activated than regenerated cellulose or DEAE cellulose. Nev- complement proteins C3a and C5a for some but not ertheless, in the group of synthetic polymers, this all membrane polymers. The evaluated AUC, then, synthetic biomaterial showed the lowest hemo- represents the difference between generation and compatibility. This study also revealed differences loss through adsorption. There is a notion that mem- between the two polysulfone types investigated: The brane biocompatibility is related to free and not g-sterilized polysulfone had a worse overall hemo- to the amount of adsorbed activated complement compatibility than the steam-sterilized polysulfone. proteins. As an example, polyacrylonitrile (PAN) The intention of this article was to establish an in membranes, such as AN69 (Hospal, Lyon, France), vitro score model and not to offer generalized char- are considered to be highly biocompatible due to acteristics of different polymers. This new score their adsorptive capacity for complement proteins model reflects an overall hemocompatibility score for and despite a considerable complement activation. each individual material. This score model delivers Consequently, the assessment of complement activa- objective results and allows for the comparison of tion in this article was done accordingly. materials that are not tested simultaneously but after Further, Hemophan, the cellulosic membrane in certain time intervals. Further, with the help of such a our investigations that has been modified through score model, the selection of membrane materials Artif Organs, Vol. 32, No. 12, 2008 THOUGHTS AND PROGRESS 969 with an appropriate hemocompatibility pattern is possible and can be applied even for other applica- tions of membranes or biomaterials in medical device technology. Acknowledgments: The authors gratefully ack- nowledge the excellent technical support of Ms. I. Werth, M. Reis and H. Hoffmann. REFERENCES 1. Masaki T, Gilson J, Leypoldt JK, Cheung AK. Effect of perme- ability on indices of haemodialysis membrane biocompatibility. Nephrol Dial Transplant 1999;14:1176–81. 2. Schmidt B, Mujais SK. Evaluation of dialysis membrane-blood compatibility: experimental methods. In: Bonomini V, Berland Y, eds. Dialysis membranes: structure and predictions. Contrib Nephrol 1995;113:32–44. 3. Mulvihill J, Cazenave JP, Mazzucotelli JP, et al. Minimodule dialyser for quantitative ex vivo evaluation of membrane hemo- compatibility in humans: comparison of acrylonitrile copoly- mer, cuprophan and polysulphone hollow fibres. Biomaterials 1992;13:527–36. 4. Yang MC, Lin CC. In vitro characterization of the occurrence of hemolysis during extracorporeal blood circulation using a mini haemodialyser. ASAIO J 2000;46:293–7. 5. Ward RA, McLeish KR. Haemodialysis with cellulose mem- branes primes the neutrophil oxidative burst. Artif Organs 1995;19:801–7. 6. Boure T, Vanholder R. Which dialyser membrane to choose? Nephrol Dial Transplant 2004;19:293–6. 7. Remuzzi A, Boccardo P, Benigni A. In vitro platelet adhesion to dialysis membranes. Nephrol Dial Transplant 1991;6 (Suppl. 2):36–9. Artif Organs, Vol. 32, No. 12, 2008 Didactical article Reçu le : 3 avril 2012 Accepté le : Dialyzers biocompatibility and efficiency 20 janvier 2013 determinants of sterilization method choice§ Mise au point sur la stérilisation des dialyseurs : un des critères de choix à prendre en compte en 2012 B. Allard (Pharmacien gérant)a,*, R. Begri (Pharmacien adjoint)a, J. Potier (Médecin praticien hospitalier)b, S. Coupel (Médecin adjoint centre formation)c Available online at a ECHO, pharmacie à usage intérieur, 11, rue Félix-Éboué, BP55, 44401 Rezé, France b Service d’hémodialyse, centre hospitalier du Cotentin, 46, rue du Val-de-Sair, ScienceDirect 50102 Cherbourg, France c www.sciencedirect.com ECHO, site du Confluent, 6, rue de la Gare, BP10095, 44402 Rezé cedex, France Summary Résumé Introduction. When selecting a dialyzer, the criteria of biocompa- Introduction. Les critères de biocompatibilité, entrant dans le tibility entering the choice of the dialyzer are based on the nature of choix du dialyseur, reposent avant tout sur la nature du polymère the polymer constituting the membrane, but rarely on the dialyzer de la membrane, et rarement sur le mode de stérilisation du sterilization methods. dialyseur. Methods. Thus, it seemed necessary to analyze the advantages and the Méthodes. Il est donc apparu nécessaire de définir les modes de disadvantages of the modes of sterilization and to analyze their impact stérilisation et analyser leurs impacts sur la biocompatibilité et les on the performances and the biocompatibility of dialyzers to integrate performances des dialyseurs, de façon à intégrer ce critère dans this parameter to their investment choices for the kidney machine. l’arbre décisionnel de choix du dialyseur. Results. Radiation sterilization induces structural modifications of Résultats. La stérilisation par irradiation s’accompagne de modifi- the dialyzers components. Beta radiation causes fewer modifications cations structurelles des constituants des dialyseurs, l’irradiation than gamma radiation; these structural modifications can release bêta-entraı̂nant moins de modifications que l’irradiation gamma, ces cytotoxic components. Steam sterilization is a reference in terms of modifications structurelles pouvant amener la libération de composés security for thermostable materials with an added benefit for steam cytotoxiques. Les modes de stérilisation par chaleur humide, auto- process, which allows the removal of waste generated during the clave ou vapeur fluente, restent une référence en terme de sécurité dialyzer’s manufacturing. pour les matériaux non thermosensibles, avec un avantage supplé- Discussion. For the same membrane, performances of the dialyzers mentaire pour la vapeur fluente, permettant un effet de chasse des change according to the methods of sterilization, inducing different résidus de fabrication. performances and different uses. Discussion. Pour une même membrane, les performances des dia- Conclusion. To conclude, beyond the choice of the polymer, the lyseurs sont modifiées en fonction du mode de stérilisation, entraı̂- method of sterilization should consistently be taken into account in nant des performances et possibilités d’utilisation différentes. therapeutic choices and purchase of dialyzers. Considering its very Conclusion. En conclusion, au-delà du choix du polymère, le mode high biocompatibility and considering the acquired experience, de stérilisation devrait être systématiquement pris en compte dans § The editorial board of Le Pharmacien Hospitalier et Clinicien considers each published article for funding English translation and online publication of the most relevant article per issue. A research grant was awarded to Allard et al. Pharm Hosp Clin 2013;48 [In press]. DOI of original article: http://dx.doi.org/10.1016/j.phclin.2013.01.011 * Corresponding author. e-mail : [email protected] (B. Allard). 2211-1042/$ - see front matter ß 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.phclin.2013.10.071 Le Pharmacien Hospitalier et Clinicien 2013;48:e15-e21 e15 B. Allard et al. Le Pharmacien Hospitalier et Clinicien 2013;48:e15-e21 moist heat sterilization is to be favored in the choice of dialyzers of les processus de choix thérapeutique et donc rentrer en compte dans equivalent performances. Furthermore, non-heat sensitive products le processus d’achat. have to be developed first and foremost with this mode of sterilization. ß 2013 Elsevier Masson SAS. Tous droits réservés. ß 2013 Elsevier Masson SAS. All rights reserved. Mots clés : Dialyseur, Stérilisation, Biocompatibilité, Performance Keywords: Dialyzer, Sterilization, Biocompatibility, Dialyzer performance Introduction Dialyzers in their definitive packaging are exposed several hours for the gamma radiation, or few minutes only under Dialyzers (so called kidney machine) are sterile medical devi- the electron-beam for the beta brilliance. For short-term ces that are made up of various polymers or cellulose acetate and surge requirements beta sterilization allows high assembled between them in a synthetic polymer hull. productivity. Good biocompatibility and performances for the clearance of Nevertheless, beta radiation allows a more precise radiation. uraemic toxin are the matters of vital concern. Key performance During the gamma radiation sterilization, dialyzers are dry results considered are permeability, surface and uraemic toxin (without an agent of filling) or filled with an aqueous solution cleared according to the technique of dialysis used. Unlike the in the blood and dialysate compartments. The name ‘‘gamma first three criteria, biocompatibility does not set the stage for wet’’ (wet) or ‘‘gamma dry’’ (dry) results from the state of the manufacturers and prescribers performance reporting. dialyzer at the time of the sterilization up to its end use. So, the criteria of biocompatibility entering the choice of the In the sterilization gamma, the agent by filing allows the dialyzer are based on the nature of the polymer constituting distribution of the residues of manufacturing and products the membrane, but rarely on the mode of sterilization of the generated during the sterilization in the aqueous solution. dialyzer. The sterilization technology has evolved significantly Nonetheless, these dialyzers are much heavier and sensitive over the past decades and has an influence on the biocom- to the temperature. patibility of dialyzers as well as on their performances. The table in supplementary data, Appendix 1 summarizes the Impact of the radiation on the dialyzer constituting materials various ranges of Dialyzers, classified according to the manu- facturers of membrane, and specifies the various methods of Depending on the dose, radiation induces structural modifi- sterilization as well as the places of manufacturing and the cations, splits and reticulations leading to a molecular rear- type CE mark attached. rangement that can affect in a more or less important way Thus, it seemed necessary to analyse the advantages and the polymers to be sterilized [1,2]. The absorbed energy is expres- disadvantages of the modes of sterilization and to analyse sed in Gray unit. their impact on the performances and the biocompatibility of Exposure to the beta radiation may induce fewer structural Dialyzers to integrate this parameter to their investment modifications due to a shorter duration and a more precise choices for the kidney machine. radiation dose. However, only scarce data are available [3]. The structural changes observed after gamma radiation, can arise even for microdoses. In the case of the polysulfone, these Various methods of sterilization appear for a 1.66 KGy dose while the Pharmacopoeia sterilizing dose required is 25 KGy [4]. According to the sterilization There are numerous methods of sterilization as specified in method, these modifications may impact biocompatibility the plan below (fig. 1), but today, ethylene oxide sterilization because of the new substances in the unknown toxicity gene- gave way to radiation and heat sterilization (fig. 1). rated and/or because of the alteration of performances of the dialyzer compared to the non-sterilized initial product [5]. Radiation sterilization Furthermore, the structural modifications continue even after the end of the radiation. Principle and implementation These changes would be due to the presence of free radicals Radiation sterilization bombard the medical device with very [1] formed within the polymer during the radiation process high energy ionizing particles altering microorganisms DNA/ that causes accelerated oxidation after the conclusion of the RNA and producing very reactive free radicals. sterilization. The presence of these free radicals can be mini- The powerful ionizing radiation sources used for dialyzers are mized by radiation without oxygen. mainly generated by the gamma radiation of cobalt 60 or by (tables Ia and Ib), according to the works of Uhlenbusch- electron-beam for the beta radiation equipment. Körwer and Nyoman Ruspiasih, shows the main polymer e16 Dialyzers determinants of sterilization method choice Table Ia Effects of the gamma radiation on polymers. Effets de l’irradiation gamma sur les polymères. Monomer/Polymer Gamma irradiation effect Acrylic monomer (PAN membranes) Increased permeability of high molecular weight molecules Cellulose Splitting of the molecular structure Polymethylmethacrylate (PMMA) Splitting of the molecular structure, decreased permeability of high molecular weight molecules (106 to 105 at 35 kGy) Polysulfone (PSU) Simultaneous chemical bridging and splitting reactions above 25 kGY Polyethylene (EVALW and hull) Chemical bridging: hydrocarbon compounds producing aldehydes and organic acid Polycarbonate (hull) Browning Polyvinylchloride (PVC Coque) Splitting of the molecular structure, browning, hydrochloric acid release, possible infiltration of partially decomposed additives that is responsible for an unpleasant smell and accelarated aging process Polyurethane (PUR) Release of mutagenic compounds, especially from aromatic polyurethane From Uhlenbusch-Körver et al. [2] and Nyoman Ruspiasih et al. [4]. modifications known for dialyzers after gamma radiation Moist heat sterilization processes [2,4]. On empotage resins, mainly constituted by aromatic polyure- Principle and implementation thane, gamma radiation induces toxic molecule formation This sterilization mode is based on the heat denaturation of such as the carcinogenic and mutagenic MDA (4.4-methyl membranes and proteins of the infectious agents. For dialy- alcohol dianiline) molecule [6–9]. Conversely, polyurethanes zers, two moist heat sterilization strategies are implemented: of aliphatic origin that do not release MDA may be used. autoclave or steam flow. However, other carcinogenic compounds are formed in the Autoclave: The vapor acts simply as coolant agent. The mem- rat, although the outcome is less pronounced than the aro- brane of dialysis is not in direct contact with the sterilizing matic polyurethane [7–12]. The cost of this raw material vapor. Sterilization consists of submitting the medical device inhibits also its function. to the heat action for a definite time (20–90 min), in a high Table Ib Effects of the gamma radiation on polymers. Effets de l’irradiation gamma sur les polymères. Preferential alteration of polymer induced by gamma irradiation Chemical bridging [TD$INLE] Polyamide Polyethylene Silicone elastomer Splitting reaction [TD$INLE] Polymethylmethacrylate Cellulose Polyvinyl chlorure From Uhlenbusch-Körver et al. [2]. e17 B. Allard et al. Le Pharmacien Hospitalier et Clinicien 2013;48:e15-e21 Table II Possible sterilization methods according to polymer membrane type. Modes de stérilisation possibles selon la nature de la membrane. Types of polymer membrane Gamma Etylene oxide Moist heat irradiation sterilization sterilization Cellulosic polymer Cellulose, cellulose derivatives + + + (cellulose acetate excepted) Cellulose acetate + + Polysulfone and polysulfone derivatives Polysulfone + + + Polyarylethersulfone (ArvlaneW) + + + PES (DiapesW) + + + PolyamideSW/PolyamixW + + + PEPA + + + Other synthetic polymers PAN + + PMMA + EVALW + + Polycarbonate + + Polyamide + + From Uhlenbusch-Körwer et al. [2]. temperature (superior to 121 8C) in a given time and in a host-cell response conditioned by the genetic heritage and pressure superior to the atmospheric pressure (1 in 2 Bars); the environmental conditions of the cell. there is a relation between the pressure and the temperature in the middle of saturated vapor (Regnault’s table), as well as Comparison of cell toxicity according to sterilization a conversely proportional correlation meanwhile and tempe- mode rature [6,13,14]. In their biocompatibility and toxic risks analysis of the steri- The term ‘‘vapor’’ used by certain manufacturers should be lization processes for dialysis devices, Guedri et al. [15] compa- replaced by ‘‘moist heat’’. red the fragmentation of the DNA of lymphocytes taken at the Steam flow: dialyzers are individually sterilized by the passage beginning, the middle and at the end of the dialysis session of a continuous flow of vapor in all the compartments of the (by electrophoresis and spectrometry). The dosage in spec- dialyzer (blood and dialysate) in a temperature of 121 8C during trometry of malondialdehyde stemming from samples leav- 15 minutes. Compared with the sterilization by the heat, the ing the fibres allowed estimating the production of free vapor fluente present the advantage to eliminate by the radicals. The results showed an increased fragmentation of continuous flow of vapor inside and outside of the fibres of the DNA and the production of free radicals after gamma possible residues of manufacturing as well as the pyrogenic sterilization. The method considered the most biocompatible and the present endotoxins after the sterilization. being the moist heat sterilization. During a cytotoxicity study considering the proliferative capa- Impact of moist heat on dialyzer constituents city of the V79/B2 cells of Chinese hamster, Bommer et al. [16] This mode of sterilization by heat is not applicable for heat in contact with eluted samples of dialyzers cuprophane ste- sensitive polymers, such as polymethylmetacrylate, poly- rilized by moist heat or by increasing doses of gamma rays. acrylonytrile and cellulose triacetate. On the other hand, this The samples eluted from dialyzers sterilized by gamma radia- effective and well-mastered process, is the gold standard in tion reduced the efficiency of the examination of cell culture terms of safety, for non-heat sensitive materials [1,2,5] by 30% compared with the control. Similar effects on the and should be recommended for dialyzers sterilization formation of colonies were observed. The cytotoxicity, mini- (table II). mal after moist heat sterilization, increases with the gamma radiation and depending the dose received. According to the Influence of sterilization mode on authors, this phenomenon would be due to the presence of cytotoxic compounds after gamma sterilization (table III). dialyzers cytotoxicity Effects of manufacturing and sterilization residues A number of studies tried to estimate the in vitro impact of on mitochondrial oxidative phosphorylation the manufacturing/sterilization process on the cell toxicity. These works present the advantage to have used a ‘‘standar- The presence of manufacturing and sterilization residues dized’’ biological material, thus reducing the variability of the in dialyzer is not often analysed with regard to the e18 [(Figure_1)TD$IG] Dialyzers determinants of sterilization method choice Chemical Ethylene oxide α Ray (Noyau d’Hélium) (Helium nucleus) β Ray Irradiation (Very high energy electron beam) (ionizing radiation) Dry (without filler) γ Ray (High-energy photons) Wet (with filler) Dry heat Heat Autoclave (Moist heat outside of the medical device) Moist heat Steam (Steam inside the medical device) Figure 1. Dialysers sterilization methods. Méthodes de stérilisation des dialyseurs. biocompatibility in hemodialysis. Brunet et al. [17,18] estimated The results showed differences in the capacity of mitochon- the effect of manufacturing residues of the dialyzer extracted dria to synthesize ATP and respiratory control (capacity to from the rinsing liquid in an in vitro model using isolated rat activate the phosphorylation and in the presence of ADP). The liver mitochondria. most noxious effect was observed with the dialyzer with The study of the oxidative phosphorylation was realized on cellulosic membrane. Among both membranes it polysulfone, the third rinsing liter of a cellulosic dialyzer and two dialyzers the FX obtained a better score than the APS. According to the polysulfone, one sterilized by fluente steam (FX) and the other author’s hypothesis, the flushing effect of vapor fluente one by gamma radiation (APS) [17]. sterilization on the manufacturing residues may explain this difference. Table III Comparative study of cytotoxicity according to the dialysers Environmental studies on the hemocompatibility sterilization method. Étude comparative de cytotoxicité selon le mode de stérilisation In a study of Erlenkotter et al. [19], two polysulfone mem- des dialyseurs. branes, one sterilized by gamma radiation, and the other by 1h 4h 6h the moist heat, presented different scores of hemocompati- Ethylene Oxide sterilization bility. This study is based on the analysis of a score of Vapor hemocompatibility regarding the factor C5a of the comple- 27 kGy + + + ment, complex thrombin–antithrombin III, the release of the 56 kGy ++ ++ ++ platelet factor 4, the synthesis and the release of elastase from polynuclears and platelet count. The polysulfone mem- Bommer et al. [16]. Eluates effects after circulation of culture medium (500 mL during 1 h, 4 h and 6 h) in brane sterilized by gamma radiation presented a score of dialysers sterilized by different methods [ethylene oxide (EtO) and steam gamma irradiation (56 kGy and 27 kGy)] on the proliferative capacity of Chinese hamster cells hemocompatibility inferior to that of the moist heat sterilized V79/B2. : absence of cytotoxicity; +: moderate toxicity; ++: greater toxicity. polysulfone membrane. Effets des éluats obtenus après circulation d’un milieu de culture (500 mL pendant 1 h, 4 h et 6 h) dans des dialyseurs stérilisés selon différentes méthodes [oxyde d’éthylène Because of the more recent and less widespread use, there are (EtO), vapeur et irradiation gamma (27 kGy et 56 kGy)] sur la capacité proliférative des few data concerning the hemocompatibility of dialyzers ste- cellules V79/B2 de hamster chinois. : absence de cytotoxicité ; + : toxicité modérée ; ++ : toxicité plus importante. rilized by radiation beta. e19 B. Allard et al. Le Pharmacien Hospitalier et Clinicien 2013;48:e15-e21 Table IV Effect of the sterilization method on the performance of the Purema membrane. Impact du mode de stérilisation sur les performances de la membrane Purema. Name Manufacturer Polymer Membrane Sterilization Beta2-Microglobulin clearance Albumin losses (g/4 h) mode Le Roy Potier Le Roy Potier et al. [23] (%) et al. [24] (%) et al. [23] et al. [24] XENIUM 210 Membrana Polyethersulfone PUREMA Gamma 80.0 80.8 0.59 <3g (polyether irradiation sulfone) PHYLTER 22S Moist heat 82.4 82.4 6.14 5–10 g sterilization In 2011, Kiaii et al. reported [20] a thrombocytopenia risk sterilization mode, the Purema membrane exhibited different associated with the use of dialyzers sterilized by beta radiation albumin permeability. from a 22 months retrospective cohort study of 1832 patients. The sterilization mode evidenced an effect on the structure The relative risk of thrombocytopenia – defined by a rate of and the permeability of the membrane to solute (and thus to platelets lower than 100 103/mL and a reduction in more a toxin). There are also more marked differences depending than 15% by the platelet number at the end of dialysis – was on the dialysis methods used (HD or HDF pre-dilution, post- significantly more elevated with beta sterilized dialyzers (OR: dilution, and according to the convective volume), in particu- 3.57; CI 95%: 2.54–5.04, P < 0.001). However, this publication lar transmembrane pressures (PTM). The impact of the ste- was very controversial [21,22], since mechanisms bringing to a rilization mode of the structure of the membrane makes some thrombocytopenia were many, multifactorial and with difficult laboratories to propose dialyzers more adapted to the dialysis to analyse through retrospective data. The possible role of the methods (table IV). sterilization by beta radiation beta in thrombocytopenia is still The moist heat sterilization Phylther (Phylther SD) was elimi- to be confirmed by more thorough studies. nated on a gamma sterilization mode (Phylther G) to defeat the two important permeability of Phylther SD in HDF. Influence of sterilization mode on Conversely, the heat sterilization version must be exclusively reserved for a use in conventional HD. performances of dialyzers Differences of performances for same membrane, Performances stability over time for gamma according to sterilization mode sterilized dialyzers Studies comparing the efficiency of the membranes of dialysis It is probable that the dialysis membranes sterilized by gamma for online hemodiafiltration (HDF) reported differences for the radiation are altered over time [1]. Indeed, the Cotentin Hospital same polyethersulfone membrane according to its steriliza- in Cherbourg studied the losses of albumin in HDF of a poly- tion mode by moist heat or gamma. The two criteria compa- sulfone derived membrane as a function of time since steriliza- red were: the b2-microglobulin (PM: 11,800 Da) reduction tion of the dialyzer. This dialyzer exhibited first normal loss of coefficient and the loss of albumin (PM: 66,000 Da). albumin (< 3 g by dialysis session). However, 4 months later, The study of Le Roy et al., realized in HDF compared eleven exactly the same dialyzer taken from the same batch and membranes of high permeability with a surface superior to secondary packaging (box) stored according to the manufac- 2 m2 with regard to the clearance of the small molecules, of turer recommendations, exhibited losses of albumin exceeding the b2-microglobulin and the losses of albumin during 5 g by dialysis session. The tests were made on several dialyzers 4 hours dialysis sessions [23]. of the same batch with the same result. Each of the dialyzers was tested on three patients during three As far as we conceive that the polymerization process of the successive sessions by the patient. Among them, two dialyzers membrane is stable in the medium-term, this fortuitous resulting from the same manufacturer, but from two different observation questions performance stability over the time distributors, presented the same membrane PuremaW with this of the radiation sterilized products. We are conducting a difference that one was moist heat sterilized (Phylther S) and prospective cohort study on this topic. the other one of the gamma radiation (Xenium). The losses of albumin hemodiafiltration with the heat steri- lized membrane was 6.14 g/4 h while that of the gamma Conclusion sterilized membrane was only 0.59 g/4 h. These results confirmed the survey carried out in Cherbourg In the decision tree for dialyzers choice, criteria that should be on the same membranes from ten trials [24]. Depending the taken in account are: e20 Dialyzers determinants of sterilization method choice performances regarding therapeutic objectives for clear- [7] Shintani H, Nakamura A. Formation of 4,40 -methylenedianiline in polyurethane potting materials by either gamma-ray or ance of uraemic toxin; autoclave sterilization. J Biomed Mater Res 1991;25(10):1275–86. practical implementation, established according to the [8] Shintani H. Formation and elution of toxic compounds from techniques used, to the equipment and to the patients sterilized medical products: methylenedianiline formation in population; polyurethane. J Biomater Appl 1995;10(1):23–58. biocompatibility, more difficult to circumscribe for a [9] Mujais SK, Ing T, Kjellstrand C. Acute complications of hemo- heterogeneous population of polypathologic patients. dialysis and their prevention and treatment. In: Jacobs C, Kjellstrand C, Koch KM, Winchester JF, editors. Replacement Nonetheless, the sterilization mode may have a major impact of renal function by dialysis. 4th ed., Dordrecht: Kluwer Aca- on materials used in the manufacturing of dialyzers. With demic Publishers; 1996. p. 688–725. regard to the cytotoxicity of some sterilization modes and [10] Piento RJ, Shah MJ, Lebherz WB, Andrews AW. Correlation of because of the exposure, three times a week in standard bacterial mutagenicity and hamster cell transformation with tumorigenicity induced by 2,4-toluenediamine. Cancer Lett dialysis to six times a week for the daily dialysis, this criterion 1977;3:45–52. must be taken into account in the decision tree of choice of [11] Shanmugam K, Subrahmanyam S, Tarakad SV, et al. 2,4- the dialyzer. Toluene diamines – their carcinogenicity, biodegradation, ana- Considering its very high biocompatibility and considering the lytic techniques and an approach towards development of acquired experience, moist heat sterilization is to be favoured biosensors. Anal Sci 2001;17(12):1369–74. in the choice of dialyzers of equivalent performances. Fur- [12] Gogolewski S. Selected topics in biomedical polyurethanes. Colloid Polym Sci 1989;267:757–85. thermore, non-heat sensitive products have to be developed [13] Hoenich NA, Woffindin C, Ronco C. In: Jacobs C, Kjellstrand CM, first and foremost with this mode of sterilization. Koch CM, Winchester JF, editors. Haemodialysers and asso- ciated devices in replacement of renal function by dialysis. 4th ed., Dordrecht: Kluwer Academis Publishers; 1996. p. 188–230. Disclosure of interest [14] Takesawa S, Ohmi S, Konno Y, et al. Varying methods of sterilisation, and their effects on the structure and perme- The authors declare that they have no conflicts of interest ability of dialysis membranes. Nephrol Dial Transplant concerning this article. 1987;1(4):254–7. [15] Guedri Y, et al. Biocompatibilité et risques toxiques des pro- cessus de stérilisation des dispositifs de dialyse. Nephrol Ther Appendix A. Supplementary data 2008;4:523. [16] Bommer J, Miltenburger HG, Hoerner V, Kilian S. Cytotoxicity of Supplementary data associated with this article can be found, g-ray sterilised dialysis devices. J Am Soc Nephrol 1994;5:408. in the online version, at doi:10.1016/j.phclin.2013.10.071. [17] Brunet P, Simon N, Roubicek C, Papa K, Berland Y, Bruguerolle B. Les résidus de fabrication des hémodialyseurs diminuent la phosphorylation oxydative des mitochondries isolées du rat. References Nephrol Ther 2010;6:576–80. [18] Tabouy LJ, Chauvet-Monges AMT, Brunet PJ, et al. In vitro [1] Dawids S, Handlos VN. 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Electron-beam sterilized hemodialyzers and nomic Publ Inc.; 1983. p. 975–99. thrombocytopenia. JAMA 2012;307(7):665–6. [4] Nyoman Rupiasih N, Vidyasagar PB. Comparative study of effect [22] De Prada L, Lee J, Gillespie A, Benjamin J. Thrombocytopenia of low and medium dose rate of g irradiation on microporous associated with one type of polysulfone hemodialysis mem- polysulfone membrane using spectroscopic and imaging tech- brane: a report of 5 cases. Am J Kidney Dis 2013;61(1):131–3. niques. Polymer Degradation Stability 2008;93(7):1300–7. [23] Le Roy F, Hanoy M, Claeyssens S, Bertrand D, Freguin C, Godin [5] Guillot-Maachi I, Martin S, Pourieux A, Philip V, Saux MC. M. Beta2-microglobulin removal and albumin losses in post- Biocompatibilité des membranes de dialyse. J Pharm Clin dilution hemodiafiltration: membrane effect. Clin Kidney J 1998;17(4):195–200. 2009;2(suppl 2):402. [6] Shintani H, Nakamura A. Analysis of a carcinogen, 4,40 -methy- [24] Potier J, Besselièvre T, Queffeulou G. Quelle membrane en lenedianiline, from thermosetting polyurethane during ster- hémodiafiltration on line (HDF OL) et pour quelle modalité ilization. J Anal Toxicol 1989;13(6):354–7. postD ou preD ? Nephrol Ther 2008;4:517. e21 Riigihangete vaidlustuskomisjon Teie: [email protected] Meie: 01.02.2024 nr 2-6/14-6 Vastus OÜ MP AlpinTech täiendavale seisukohale vaidlustuses riigihankele „Dialüüsi tarvikute ostmine“, viitenumber 272185 Hankija: AS Lääne-Tallinna Keskhaigla registrikood: 10822269 aadress: Paldiski mnt 68, 10617 Tallinn Hankija esindaja: jurist Kaisa Suvi telefon: 6507 399 e-post: [email protected] Vaidlustaja: OÜ MP AlpinTech registrikood: 16209624 Vaidlustaja esindaja: advokaat Ringo Heidmets Adokaadibüroo GLIMSTEDT Estonia OÜ registrikood: 14233883 e-post: [email protected] telefon: 5663 2589 1. 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Kui vaidlustaja esindas vaidlustuses sisuliselt ühispakkuja huve ning vaidlustaja tahtlus esitada ühispakkumine ühispakkujaga tekkis alles peale hankija poolt 23.01.2024 esitatud taotlust vaidlustuse läbi vaatamata jätmiseks RHS § 192 lg 2 p 6 alusel, tuleb hankija hinnangul vaidlustus jätta läbi vaatamata. Vaidlustaja on oma täiendavas seisukohas väitnud, et vaidlustaja avastas vaidlustatud riigihanke ning registreerus selle juurde 17.01.2024. Ei ole eluliselt usutav, et vaidlustaja avastas riigihanke 17.01.2024 kl 11:04, esitas vaidlustuse 17.01.2024 kl 21:36 ning vahepealse 10,5 tunni jooksul jõudis sõlmida koostöökokkuleppe ühispakkujaga. Vaidlustaja menetluskulude nimekirjast lähtuvalt on enne vaidlustuse esitamist jõutud veel 7 tunni ning 42 minuti ulatuses osutada ka õigusabi vaidlustajale. Seega, kui vaidlustaja väidab, et ta tõepoolest alles 17.01.2024 riigihanke avastas, on vaidlustaja vähem kui 3 tunniga jõudnud turult leida dialüüsitarvikute osas koostööpartneri, sõlminud selles osas koostöökokkuleppe, süvenenud koos koostööpartneriga (s.o ühispakkujaga) riigihanke alusdokumentidesse, leidnud alusdokumentidest neid ühiselt häirivad puudujäägid ning kommunikeerinud omapooolsed mured vaidlustaja esindajale. Hankija juhib tähelepanu, et vaidlustaja planeeritav koostööpartner, ühispakkuja, on hanke juurde registreerunud juba 08.12.2023, s.o. 39 päeva enne vaidlustuse esitamist ning 47 päeva enne 25.01.2024 ehk pakkumuste esitamise tähtaega. Hankija tuletab meelde, et riigihanke juurde registreerunud ettevõtjate nimekiri on nähtav ainult hankija meeskonna liikmetele ning sealt vaidlustaja ega ühispakkuja potentsiaalset koostööpartnerit otsida ei saa. Hankijale ei tundu selline situatsioon eluliselt usutav. Hankijale tundub eluliselt usutav, et taoliste suuremahuliste koostööprojektide (esialgselt vaidlustatud osade eeldatav maksumus 1 610 400 EUR, kogu riigihanke eeldatav maksumus 4 105 000,00 EUR), sõlmimiseks kulub kindlasti enam kui 2 tundi ja 50 minutit (aeg, mis jääb üle lahutades 10 tunnist ja 32 minutist väidetav õigusabi osutamine 7 tunni ja 42 minuti ulatuses). Juba ainuüksi koostöölepingu koostamine, rääkimata tõenäolisest pooltevahelistest läbirääkimistest lepingutingimuste osas, võtab kauem aega kui 2 tundi ja 50 minutit. AS Lääne-Tallinna Keskhaigla, Paldiski mnt 68, 10617 Tallinn, Eesti Vabariik Telefon: 650 7393, Faks 650 7265, e-post [email protected] Registrikood: 10822269, Arvelduskonto: EE081010220022881019 SEB Pank Hankija juhib tähelepanu, et vaidlustaja ainukene juhatuse liige Marko Pikani on vaidlustajat esindava Adokaadibüroo GLIMSTEDT Estonia OÜ osanik ning vaidlustaja lepingulise esindaja, vandeadvokaadi abi Ringo Heidmets’a, tööandja. Vaidlustaja on esitanud menetluskulude nimekirja, mille kohaselt on lepingulise esindamise ajakulu kuni vaidlustuse esitamiseni 7 tundi ja 42 minutit. Samas pole menetluskulude nimekirja järgi võimalik kontrollida kas lepingulise esindamise eest tekkinud õigusabi kulud on tekkinud ühispakkuja või vaidlustaja poolsest lepingulise esindaja poole pöördumisest, mis kuupäevadel on tekkinud õigusabikulud, kas lepingulise esindamise eest on esitatud arved ning kellele. Seetõttu on hankijal jätkuvalt kahtlus kas vaidlustajal oli vaidlustuse esitamise ajal reaalne võimalus riigihankes osaleda. Alternatiivselt esitab hankija oma seisukoha täiendavale seisukohale, kui vaidlustust ei peaks läbi vaatamata jätma. 3. RIIGIHANKE OSA 1 Vaidlustaja on oma täiendavas seisukohas nõustunud hankijaga, et suures doosis kiirgus võib dialüsaatorite materjalidele ja patsientidele negatiivselt mõjuda. Vaidlustaja on samuti oma täiendavas seisukohas märkinud, et „kiirgusega steriliseerimise mõju on sügavam, steriliseeritud tulemuse saavutamine oluliselt kindlam [...] erinevalt auruga steriliseerimisest.“ Samas pole vaidlustaja, erinevalt hankijast, lisanud oma seisukoha tõendamiseks ühtegi tõendit. Hankija on oma eelnevale 23.01.2024 vastusele lisanud teadusartikli hankija veendumuse kohta, et auruga steriliseerimine on oluline biosobivuse seisukohast. Hankija lisab käesolevale vastusele lisaks veel kaks teadusartiklit auruga steriliseerimise kohta, Lisa 11 ja Lisa 22. Hankijal on õigus otsustada, millist kaupa ta hankida soovib ning tehnilise kirjelduse koostamisel lai kaalutlusõigus. Vaidlustajal ei ole subjektiivset õigust nõuda, et hangitaks teistsugust kaupa, mis oleks vaidlustaja hinnangul parem või otstarbekam. Vaidlustaja on hankija vabadust hankeeseme määramise osas ka oma vaidlustuses ise möönanud. Hankija on oma eelnevale 23.01.2024 vastusele lisanud kahe juhtiva (ning konkureeriva) dialüsaatorite tootja brošüürid, 1 Erlenkötter, A.; Endres, P.; Nederlof, B.; Hornig, C.; Vienken, J. Score model for the evaluation of dialysis membrane hemocompatibility. Artif. Organs 2008, 32, 962–969. Väljavõte lk 968: „: The γ- sterilized polysulfone had a worse overall hemocompatibility than the steam-sterilized polysulfone.“ 2 Allard B, Begri R., Potier J., Couple S. Dialyzers biocompatibility and efficiency. Determinants of sterilization method choice. Pharm Hospet Clin. 2013;48(4):e15-e21. Väljavõte lk e15: „Radiation sterilization induces structural modifications of the dialyzers components. Beta radiation causes fewer modifications than gamma radiation; these structural modifications can release cytotoxic components. Steam sterilization is a reference in terms of security for thermostable materials with an added benefit for steam process, which allows the removal of waste generated during the dialyzer’s manufacturing.“ Väljavõte lk e20: „It is probable that the dialysis membranes sterilized by gamma radiation are altered over time [1]. Indeed, the Cotentin Hospital in Cherbourg studied the losses of albumin in HDF of a polysulfone derived membrane as a function of time since sterilization of the dialyzer. This dialyzer exhibited first normal loss of albumin (< 3 g by dialysis session). However, 4 months later, exactly the same dialyzer taken from the same batch and secondary packaging (box) stored according to the manufacturer recommendations, exhibited losses of albumin exceeding 5 g by dialysis session. The tests were made on several dialyzers of the same batch with the same result.“ AS Lääne-Tallinna Keskhaigla, Paldiski mnt 68, 10617 Tallinn, Eesti Vabariik Telefon: 650 7393, Faks 650 7265, e-post [email protected] Registrikood: 10822269, Arvelduskonto: EE081010220022881019 SEB Pank kes kasutavad auruga steriliseerimist. Seetõttu ei ole tõene vaidlustaja väide, et hankija on riigihanke koostamisel aluseks võtnud vaid ühe konkreetse tootja tooted ning seeläbi põhjendamatult piiranud konkurentsi. Hankijal on usaldus, et auruga steriliseerimise nõue vastab hankija vajadustele, hankija on põhjendanud sellise nõude vajadust ning näidanud oma 23.01.2024 vastuses, et riigihanke koostamisel pole aluseks võetud vaid ühe konkreetse tootja tooteid. Kui mitmel pakkujal on võimalik tehnilisele kirjeldusele vastavat toodet pakkuda, on riigihange avatud konkurentsile. Eeltoodust lähtuvalt on hankija seisukohal, et vaidlustaja vaidlustus riigihanke osas 1 tuleb jätta rahuldamata. 4. MENETLUSKULUD Hankija on seisukohal, et vaidlustaja menetluskulud tuleb jätta vaidlustaja kanda. Riigihanke tingimused pidid ühispakkujale teada olema juba enne 17.01.2024, sest ühispakkuja on riigihanke juurde registreerunud 08.12.2023, s.o. 39 päeva enne vaidlustuse esitamist ning 47 päeva enne 25.01.2024 ehk pakkumuste esitamise tähtaega. Vaidlustaja on oma täiendavas seisukohas väitnud, et ühispakkumuses tugineb ta ühispakkuja valdkonnaspetsiifilisele kogemusele ja võimekusele ning vaidlustaja osaks jääb lepingu täitmise administratiivne pool. Tuginedes vaidlustaja väitele, on just ühispakkujal erialaspetsiifilised teadmised mõistmaks riigihanke tehnilist kirjeldust ning vähemalt 39 päeva aega pöördumaks hankija poole läbi riigihangete registri ettepanekuga riigihanke alusdokumentide muutmiseks. Ühispakkuja seda pole aga teinud. Hankija hinnangul on 39 päeva enam kui piisav aeg hankijale ettepaneku esitamiseks ning vastuse ära ootamiseks. Kui ühispakkuja oleks pöördunud hankija poole omapoolse ettepanekuga, poleks riigihanke osade 6 ja 35 osas menetlustuskulusid üldse tekkinud. Hankija juhtis eelnevalt käesolevas vastuses tähelepanu, et vaidlustaja ainukene juhatuse liige Marko Pikani on vaidlustajat esindava Adokaadibüroo GLIMSTEDT Estonia OÜ osanik ning vaidlustaja lepingulise esindaja, vandeadvokaadi abi Ringo Heidmets’a, tööandja. VAKO poolt välja mõistetavad õigusabikulud on otsene tuluallikas vaidlustaja juhatuse liikmele, kes omab oma Adokaadibüroo GLIMSTEDT Estonia OÜ osaluse kaudu kontrolli ja motivatsiooni VAKO-le esitatud õigusabikulude suuruse osas. Eelnevast tulenevalt palub hankija vaidlustuskulud jätta vaidlustaja kandja. VAKO vastupidise otsuse korral palub hankija VAKO-l hinnata õigusabikulude suuruse ja mahu põhjendatust arvestades eelpool toodud asjaolusid. Riigihanke osa 1 osas on hankija seisukohal, et vaidlustus tuleb jätta rahuldamata ning menetluskulud jätta vaidlustaja kanda. AS Lääne-Tallinna Keskhaigla, Paldiski mnt 68, 10617 Tallinn, Eesti Vabariik Telefon: 650 7393, Faks 650 7265, e-post [email protected] Registrikood: 10822269, Arvelduskonto: EE081010220022881019 SEB Pank Eeltoodud asjaolusid arvesse võttes palub hankija: 1. jätta vaidlustaja vaidlustus läbi vaatamata RHS § 192 lg 3 p 6 alusel; 2. jätta vaidlustuse esitamisega seotud kulud vaidlustaja kanda; või alternatiivselt: 1. jätta vaidlustaja vaidlustus rahuldamata riigihanke osa 1 osas; 2. jätta vaidlustaja vaidlustus riigihanke osade 6 ja 35 osas läbi vaatamata RHS § 192 lg 3 p 5 alusel ning lõpetada vaidlustusmenetlus RHS § 197 lg 1 p 8 alusel; 3. jätta vaidlustuse esitamisega seotud kulud vaidlustaja kanda; Lugupidamisega (allkirjastatud digitaalselt) Kaisa Suvi Volitatud esindaja Lisatud: Lisa 1 Erlenkötter, A.; Endres, P.; Nederlof, B.; Hornig, C.; Vienken, J. Score model for the evaluation of dialysis membrane hemocompatibility. Lisa 2 Allard B, Begri R, Potier J, Couple S. Dialyzers biocompatibility and efficiency. Determinants of sterilization method choice. AS Lääne-Tallinna Keskhaigla, Paldiski mnt 68, 10617 Tallinn, Eesti Vabariik Telefon: 650 7393, Faks 650 7265, e-post [email protected] Registrikood: 10822269, Arvelduskonto: EE081010220022881019 SEB Pank

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