**4.2.2 Results and discussion**

82 Progress in Hemodialysis – From Emergent Biotechnology to Clinical Practice

The PES hollow fiber hemodialysis membrane could effectively remove water and waste products, not only small molecular weight solute such as urea and creatinine, but also "middle" molecular solute as 2-microglobulin. Slight neutropenia and platelet adhesion were observed at the initial stage of the hemodialysis and no significant differences were found in electrolyte, blood gas and blood biochemistry before and after the treatment. The results also

Three groups of hemodialysis patients with mature functioning arteriovenous fistula participated in this study. Their mean age was 48 ± 12 yr, and they had been receiving dialysis treatments for 35 ± 14 months with an average frequency of 3 times per wk. For each

Standard midweek hemodialysis sessions were analyzed, and bicarbonate dialysate was used. The dialysate contained 140 mmol/L sodium, 2 mmol/L potassium, 108 mmol/L chloride, 1.50 mmol/L calcium, 0.5 mmol/L magnesium and 32 mmol/L bicarbonate. The blood flow was 200 ml/min and the dialysate flow was 500 ml/min. Three kinds of dialyzers (PES, polysulfone (PSF), and polyamide (PA)) were used for the three groups of

The levels of urea, creatinine, phosphate, total proteins, albumin (ALB), alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) were determined using an Auto Biochemistry Analyzer 7170A (Hitachi Co., Ltd., Tokyo,

The removal of 2-microglobulin was established by the changes in plasma level during the treatment at different time intervals (30, 60, 120, 180 and 240mins). Plasma 2-microglobulin levels were determined using a commercially produced ELISA assay (Cambridge Life

Electrolyte levels were determined before and after hemodialysis. The levels of K+, Na+ and Cl- were determined using electrolyte analyzer (NOVA CRT-4, US), and Ca2+ was determined using an Auto Biochemistry Analyzer 7170A (Hitachi Co., Ltd., Tokyo, Japan).

In order to investigate the complement and immunoglobin activation, complement C3, C4 and immunoglobin G, A, M and E were determined by enzyme-linked immunosorbent

Blood cells including red blood cell (RBC) and white blood cell (WBC), and blood components including hemoglobin (HGB) and platelet were determined using a blood cell analyzer (BC-3000peus, Shenzhen Mairui Biomedical Device Co. Ltd., China). Blood gas was

The software of SPSS 13.0 was used for statistical analysis. The deviation between the three groups was calculated by analysis of one-factor variance (ANOVA), and the deviation

suggested that the PES membrane hemodialyzer could be used for clinical application.

patient, Hct was determined at the beginning of the hemodialysis session.

**4.1.3 Summary** 

**4.2 Clinical evaluation 4.2.1 Experimental** 

patients, respectively.

Sciences, Cambridge, UK).

**4.2.1.4 Statistical analysis** 

assays (ELISA)

Japan).

**4.2.1.2 Calculation of solute clearance** 

**4.2.1.3 Evaluation of blood compatibility** 

determined by a blood gas analyzer (CORNing 238, US).

**4.2.1.1 Hemodialysis procedure** 

All the patients participated in the whole study period. The vital signs were stable with no adverse events during the dialysis, and there were no abnormal findings in laboratory security parameters. During the dialysis by PA membrane dialyzer, some clots were found after 175 minutes in the extracorporeal blood circuit of a male patient who was on a repeated bolus fraxiparine anticoagulation regimen (6000 IU in total), but the patient still finished the treatment. This was the only adverse event during the whole study. All of patients who were treated by PES, PA or PSF membrane dialyzers were performed without provoking any adverse symptoms, such as headache or hypotension.

### **4.2.2.1 Solute clearance**

The clearance of small molecular and middle molecular toxins was expressed as the solute reduction ratio (RR) after 4 hours hemodialysis, and could be calculated by: RR (%) = (1- (post-solute concentration/pre-solute concentration))100%. The blood flow was controlled at 200 ml/min and the dialysate flow was 500 ml/min. Figure 10 shows the RRs of urea, creatinine and 2-microglobulin for the three kinds of hollow fiber dialyzers. As shown in the figure, large amount of the toxins were removed after the hemodialysis. The RRs of urea for PES, PA and PSF membranes were 61.2%, 63% and 62.3%, respectively. The RRs of creatinine were 51.3%, 54.5% and 54.7%, respectively. Meanwhile, the RRs of 2 microglobulin were 60.8%, 51.3% and 57.7%, respectively. The RRs of urea and creatinine for the PES membrane were slightly smaller than that for the PA and PSF membranes, but no statistical difference. However, the RRs of 2-microglobulin for the PES membrane were slightly larger than that for the PA and PSF membranes. It proved that the PES, PA and PSF hollow fiber hemodialysis membranes could effectively remove waste products including not only small molecular weight solutes such as urea and creatinine but also "middle" molecular solutes as 2-microglobulin.

To increase the removal of large molecular solutes, the rates of diffusion and convection should be increased, and the membrane pore size and porosity should be increased. Pore size limitations arise from the concern over potential loss of blood proteins such as albumin. Given that dialysis patients are generally malnourished, and the relative risk of death of dialysis patients increases as the serum albumin concentration decreases, it is desirable to minimize the albumin loss to the dialysate. Furthermore, small albumin losses may be clinically insignificant to the patient, but may lead to practical problems in the dialysis clinic, such as the foam formation in the dialysate drains. An ideal dialysis membrane should have a uniform pore size large enough to allow the passage of 2-microglobulin but small enough to retain albumin (66,000 daltons). Unfortunately, methods currently used to produce dialysis membranes resulted in a non-uniform pore size distribution. In the phase inversion membrane production process, polymer is dissolved in a solvent and then exposed to a non-solvent as it is extruded through an annular die. The breadth of the distribution produced by the phase inversion process resulted from the finite rate of molecular diffusion through the viscous polymer solution during the membrane coagulation phase (Qian et al., 2009). While previous membrane improvements have resulted from reducing the viscosity of the polymer solution, it is unlikely that the breadth of the pore size

Polyethersulfone Hollow Fiber Membranes for Hemodialysis 85

white blood cells was caused by complement activation, thus similar results were obtained in the changes of complement factor C3 and complement C4 during the hemodialysis process. When comparing PES, PA and PSF membranes, these change showed no significant difference, which indicated that the blood compatibility might be the same, though different

Fig. 11. Changes in WBC during the dialysis in vivo. Data are expressed as the meansSD, n =3 The concentration of albumin (ALB) and immunoglobin (GLB) slightly increased after 4 h hemodialysis, and no significant difference among the three membranes. Total protein adsorption of the membranes was also determined, and the amounts for PES, PA and PSF

0 60 120 180 240 Treatment duration (minutes)

Membrane (♦ ) PES (□ ) PA (▲) PSF

Total bilirubin (TBIL), direct bilirubin (DBIL), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) levels were measured after 4 h hemodialysis, and compared with the initial levels for the three kinds of membranes, as shown in Figure 12. There are no significant differences in the changes of TBIL, DBIL, ALT, and AST for the PES and PSF membranes, and both the TBIL and DBIL levels increased compared to the initial levels.

In Figure 12, slightly changes in total bilirubin (TBIL), direct bilirubin (DBIL), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were also observed. The change ratios for all of them ranged 3-10%. There are no significant differences in the changes of TBIL, DBIL, ALT, and AST for the PES and PSF membranes, and both the TBIL and DBIL levels increased compared to the initial levels, which were presumably caused by the dilution of the blood by normal saline solution infused or pachemia after the hemodialysis process. However, for the PA membrane, the TBIL, and AST levels decreased obviously. TBIL, DBIL, ALT and AST are produced primarily in the liver; all of them are lipophilic and hydrophobic. The dialyzer permits diffusive clearance of non-protein-bound, water soluble uraemic solutes, such as urea and creatinine. The corollary is that the substances are tightly protein-bound and present in small quantities in the aqueous phase, or are lipophilic and removed by HD in negligible amounts, if at all. The results indicted that the PES and PSF membrane had no effect on the liver, and might have possibly higher

However, for the PA membrane, the TBIL, and AST levels decreased obviously.

membranes were 12.2, 10.2, and 11.9 g/cm2, respectively.

0

20

40

60

Normalized value (%)

80

100

120

hydrophilicity than PA membrane.

membrane materials were used.

distribution can be significantly reduced by further modification of the phase inversion process. Given a fixed breadth of the pore size distribution, the requirement for albumin retention limited not only the maximum pore size but also the mean pore size. As a result, the sieving coefficient of 2-microglobulin is generally 0.6 or less in order to maintain the albumin sieving coefficient at 0.01 or less. The PES membrane may be adequate to this requirement (Kim & Kim, 2005).

Fig. 10. Reduction ratios of small molecules urea and creatinine, as well as middle molecules 2-microglobulin after four hours hemodialysis at a blood flow rate of 200 ml/min and dialysate flow rate of 500 ml/min. Data are expressed as the meansSD, n =3

#### **4.2.2.2 Biocompatibility**

Figure 11 shows the white cell (WBC) changes in the patient bloods during the dialysis for the three kinds of membranes. The blood cell counts have been normalized to pre-treatment levels and expressed as a percentage of these values. A small decline was noted at the first 30 minutes for all the membranes, and returned to the initial levels after about 1 h, and no significant difference was observed among the three membranes. The changes in platelet, complement factor C3, and complement C4 during the hemodialysis process for the three membranes were also investigated, and similar results were obtained as the change in WBC (Data not shown).

Retrospective analyses have shown that hemodialysis with synthetic dialysis membranes is associated with improved patient survival in ESRD (Kim & Kim, 2005). This observation was mainly attributed to membrane biocompatibility. Synthetic membranes are generally regarded as to be highly biocompatible, since they lead to low complement activation and leucopenia, which are the two classical parameters to characterizing biocompatibility in dialysis (Hakim et al., 1996). However, several other systems become altered during blood– membrane interaction. Among them are the coagulation system and imbalances of the oxidative and anti–oxidative system (Krieter et al., 2007; Klingel et al., 2004).

A slightly decrease in outlet leukocyte counts was observed for the three dialyzers, and significant difference was observed among them, as shown in Figure 11. The decrease of

distribution can be significantly reduced by further modification of the phase inversion process. Given a fixed breadth of the pore size distribution, the requirement for albumin retention limited not only the maximum pore size but also the mean pore size. As a result, the sieving coefficient of 2-microglobulin is generally 0.6 or less in order to maintain the albumin sieving coefficient at 0.01 or less. The PES membrane may be adequate to this

> For urea; For creatinine; For 2-microglobulin

Fig. 10. Reduction ratios of small molecules urea and creatinine, as well as middle molecules 2-microglobulin after four hours hemodialysis at a blood flow rate of 200 ml/min and

PES PA PSF

Figure 11 shows the white cell (WBC) changes in the patient bloods during the dialysis for the three kinds of membranes. The blood cell counts have been normalized to pre-treatment levels and expressed as a percentage of these values. A small decline was noted at the first 30 minutes for all the membranes, and returned to the initial levels after about 1 h, and no significant difference was observed among the three membranes. The changes in platelet, complement factor C3, and complement C4 during the hemodialysis process for the three membranes were also investigated, and similar results were obtained as the change in WBC

Retrospective analyses have shown that hemodialysis with synthetic dialysis membranes is associated with improved patient survival in ESRD (Kim & Kim, 2005). This observation was mainly attributed to membrane biocompatibility. Synthetic membranes are generally regarded as to be highly biocompatible, since they lead to low complement activation and leucopenia, which are the two classical parameters to characterizing biocompatibility in dialysis (Hakim et al., 1996). However, several other systems become altered during blood– membrane interaction. Among them are the coagulation system and imbalances of the

A slightly decrease in outlet leukocyte counts was observed for the three dialyzers, and significant difference was observed among them, as shown in Figure 11. The decrease of

dialysate flow rate of 500 ml/min. Data are expressed as the meansSD, n =3

Reduction ratio (%)

oxidative and anti–oxidative system (Krieter et al., 2007; Klingel et al., 2004).

requirement (Kim & Kim, 2005).

**4.2.2.2 Biocompatibility** 

(Data not shown).

white blood cells was caused by complement activation, thus similar results were obtained in the changes of complement factor C3 and complement C4 during the hemodialysis process. When comparing PES, PA and PSF membranes, these change showed no significant difference, which indicated that the blood compatibility might be the same, though different membrane materials were used.

Fig. 11. Changes in WBC during the dialysis in vivo. Data are expressed as the meansSD, n =3

The concentration of albumin (ALB) and immunoglobin (GLB) slightly increased after 4 h hemodialysis, and no significant difference among the three membranes. Total protein adsorption of the membranes was also determined, and the amounts for PES, PA and PSF membranes were 12.2, 10.2, and 11.9 g/cm2, respectively.

Total bilirubin (TBIL), direct bilirubin (DBIL), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) levels were measured after 4 h hemodialysis, and compared with the initial levels for the three kinds of membranes, as shown in Figure 12. There are no significant differences in the changes of TBIL, DBIL, ALT, and AST for the PES and PSF membranes, and both the TBIL and DBIL levels increased compared to the initial levels. However, for the PA membrane, the TBIL, and AST levels decreased obviously.

In Figure 12, slightly changes in total bilirubin (TBIL), direct bilirubin (DBIL), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were also observed. The change ratios for all of them ranged 3-10%. There are no significant differences in the changes of TBIL, DBIL, ALT, and AST for the PES and PSF membranes, and both the TBIL and DBIL levels increased compared to the initial levels, which were presumably caused by the dilution of the blood by normal saline solution infused or pachemia after the hemodialysis process. However, for the PA membrane, the TBIL, and AST levels decreased obviously. TBIL, DBIL, ALT and AST are produced primarily in the liver; all of them are lipophilic and hydrophobic. The dialyzer permits diffusive clearance of non-protein-bound, water soluble uraemic solutes, such as urea and creatinine. The corollary is that the substances are tightly protein-bound and present in small quantities in the aqueous phase, or are lipophilic and removed by HD in negligible amounts, if at all. The results indicted that the PES and PSF membrane had no effect on the liver, and might have possibly higher hydrophilicity than PA membrane.

Polyethersulfone Hollow Fiber Membranes for Hemodialysis 87

maltophilia. No significant generation of interleukin 1 (IL-1), IL-6 or tumor necrosis factor (TNF) was found in the blood compartment for the PES dialyzer and Fresenius PSF series of dialyzers as compared with sterile controls. However, significant induction of IL-1, IL-6, and TNF was observed for the highly permeable polysulfone membrane DIAPES, suggesting that not all of the polysulfone membranes were alike with regard to their pyrogen permeability due to the different modification methods. The PES, PA, and PSF dialyzers offered important safety features with regard to a possible contamination of the dialysis

The PES hollow fiber membrane hemodialyzer was effective and safe in the therapy for uremic patients. The PES hollow fiber hemodialysis membrane could effectively remove water and waste products including not only small molecular weight solutes such as urea and creatinine but also "middle" molecular solute as 2-microglobulin. Slight neutropenia and platelet adhesion were observed at the initial stage of the hemodialysis and no significant difference was found in electrolyte or blood biochemistry before or after the treatment. The data indicated that the performances of PES, PSF and PA hemodialyzers in the clinical setting were comparable and the PES hemodialyzer might be better than the others. The results indicated that PES hollow fiber membrane had a potentially wide

Polyethersulfone (PES) is one of the most important polymeric materials and is widely used in separation fields. PES and PES-based membranes show outstanding oxidative, thermal and hydrolytic stability as well as good mechanical and film-forming properties. Furthermore, PES-based membranes show high permeability for low molecular weight proteins when used as hemodialysis membranes. However, the blood compatibility of the PES membrane is not adequate, and injections of anti-coagulants are needed during its

Thus, all the PES membranes used for hemodialysis are not the pristine PES membranes, and most widely used modification method for hemodialysis PES membranes is blending. Poly (vinyl pyrrolidone) (PVP) is the most widely used for the modification of PES membranes by blending, and PVP also acts used as a hydrophilic additive and a membrane forming agent. Surface-coating and grafting methods can also be used for the modification of PES hollow fiber membranes. All the modifications are based on the premise that the materials used in the modification give inherently more hydrophilicity and adsorb less

Protein adsorption on material surface is a common phenomenon during thrombogenic formation. Thus, the amount of protein adsorbed on the PES membrane is considered to be one of the important factors in evaluating the blood compatibility. The adhesion of platelets to blood-contacting medical devices is a key event in thrombus formation on material surface. The clearances and the reduction ratios of small molecules (urea, creatinine, phosphate) for the PES membrane after the hemodialysis in vitro were larger than those in vivo. Animal experiments and clinical experiments indicated that the PES-based high-flux hemodialysis membrane had good blood compatibility, and could effectively remove

fluid (Wang et al., 1996; Schiffl & Lang, 2010).

**4.2.3 Summary** 

application for hemodialysis.

**5. Conclusions** 

clinical application.

protein than the underlying substrate.

"middle" molecular solute as 2-microglobulin.

Fig. 12. *Total* bilirubin (TBIL), *direct* bilirubin (DBIL), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) level changes after 4 h hemodialysis. Data are expressed as the meansSD, n =3

We speculated that the high-flux dialysis membrane might possibly let some biocompatibility markers enter dialysis solution so that the plasma levels of these markers could provide biased information. The plasma levels of biocompatibility markers may have also been influenced by adsorption to the membrane surface (Benz et al., 2007; Gotz et al., 2008). The adsorption to the membrane was not determined in our study. However, the protein adsorption capacity was investigated, and no difference was observed. On the basis of our results, we concluded that the designed modifications of the new high-flux PES dialyzer resultED in its higher middle molecule clearance efficacy, and had an effect on thrombogenicity as assessed by platelet behavior and fibrinolysis. Although coagulation system judged by one of the evaluated parameters was slightly higher compared with the other dialyzers, it was still within the biocompatible dialyzer range. In terms of complement activation and changes in leukocyte count, the new dialyzer is also comparable with the other biocompatible dialyzers. Besides the thrombogenicity, complement activation, and WBC count changes, other issues must be considered when evaluating bio(in)compatibility (Krieter et al., 2007; Klingel et al., 2004)

One further aspect merits consideration is that the PES membrane dialyzer series exhibits a higher permeability and thus, cytokine-inducing substances, possibly present in the dialysis fluid, might gain access to the blood stream through internal filtration (backfiltration). Therefore, investigations on the pyrogen permeability of PES membranes have been performed to the studies on the inflammatory response of the membrane. In the study, the dialysate compartment was deliberately contaminated with purified lipopolysaccharides (LPS) from Escherichia coli, as well as with LPS derived from Stenotrophomonas (Sten) maltophilia. No significant generation of interleukin 1 (IL-1), IL-6 or tumor necrosis factor (TNF) was found in the blood compartment for the PES dialyzer and Fresenius PSF series of dialyzers as compared with sterile controls. However, significant induction of IL-1, IL-6, and TNF was observed for the highly permeable polysulfone membrane DIAPES, suggesting that not all of the polysulfone membranes were alike with regard to their pyrogen permeability due to the different modification methods. The PES, PA, and PSF dialyzers offered important safety features with regard to a possible contamination of the dialysis fluid (Wang et al., 1996; Schiffl & Lang, 2010).
