**4.1.1.3 Biocompatibility**

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, Japan).

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

For complement and WBC activation investigation, various membranes were used from different companies, Cuprophane (Nephross, Netherlands), Cellulose acetate (Nissho, Japan), Hemophane (Ningbo-Yatai, China), Polysulfone (PSF, Fresenius, Germany). Polycarbonate (PC) was obtained from BASF Co. Ltd., and the PC membrane was prepared in our Lab. Complement C3 activation was determined in vitro by enzymelinked immunosorbent assays (ELISA) (Zwirner et al., 1995). For comparing the results, activation for Cuprophane membrane was used as control.

### **4.1.2 Results and discussion**

#### **4.1.2.1 Solute transport**

78 Progress in Hemodialysis – From Emergent Biotechnology to Clinical Practice

Adult hybrid goats (about 20 kg) were used in the experiment. All the animals underwent local anesthesia with 1.0% procaine hydrochloride by injection into the neck muscle. The hair on the neck was cleared away carefully. The animal was laid on its back and fixed on

Extracorporeal circuits were primed with 500ml normal saline solution to remove the bubbles in the circuits and in the dialyzer, then primed with 500ml saline solution containing 10000 IU heparin. 150mg urea and 50mg creatinine were injected to the animal blood before the treatment. At the initiation of the treatment goats received a loading dose (3000 IU) of heparin, and followed by continuous infusion (3000 IU/h). The infusion was

Extracorporeal circuits with left–right neck intravenous cannulation were created on the animal using B. Braun blood tubing lines for hemodialysis. The clearance (K) of small molecules (urea, creatinine, phosphate) were established by sampling from the inlet and outlet segments of the extracorporeal circuit 1 h after the initiation of the treatment, and was calculated using the formula described in section 3.2. The fluid removal ratio during these

Removal of 2-microglobulin was established from the changes in plasma 2-microglobulin levels 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

Electrolyte levels were determined before and after hemodialysis. K+,Na+ and Cl- were determined using electrolyte analyzer (NOVA CRT-4, US), and Ca2+ was determined using

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,

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

For complement and WBC activation investigation, various membranes were used from different companies, Cuprophane (Nephross, Netherlands), Cellulose acetate (Nissho, Japan), Hemophane (Ningbo-Yatai, China), Polysulfone (PSF, Fresenius, Germany). Polycarbonate (PC) was obtained from BASF Co. Ltd., and the PC membrane was prepared in our Lab. Complement C3 activation was determined in vitro by enzymelinked immunosorbent assays (ELISA) (Zwirner et al., 1995). For comparing the results, activation

an Auto Biochemistry Analyzer 7170A (Hitachi Co., Ltd., Tokyo, Japan).

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

for Cuprophane membrane was used as control.

**4.1 Evaluation by animal experiments** 

terminated at 30 min prior to the end of the dialysis.

measurements was maintained at (3 ml/min).

(Cambridge Life Sciences, Cambridge, UK).

**4.1.1.1 Hemodialysis procedure** 

**4.1.1 Experimental** 

the experimental table.

**4.1.1.2 Solute transport** 

**4.1.1.3 Biocompatibility** 

Japan).

Table 1 also summarizes the clearance data and the reduction ratio after the dialysis for small molecules in vivo. Changes in 2-microglobulin during the dialysis for the goats are plotted in Figure 8. The reduction ratio was about 50% after the treatment for 4hrs.

The ultrafiltration coefficient was obtained by the hemodialysis process using the simulated solution with a value of 81ml/h.mmHg, from which we could conclude that the PES membrane was a high-flux hemodialysis membrane.

The PES membrane was able to reduce the plasma burden of 2-microglobulin during the treatment, as shown in Figure 8. The data were analyzed by consideration of actual values and the percentage reductions achieved. The reduction ratio was about 50% after the treatment for 4 hrs; this value is comparable to that for PSF membrane and polyflux (Hoenich & Katopodis, 2002).

As shown in table 1, the reduction ratio for the 2-microglobulin was smaller than that for the urea and creatinine due to the higher molecular weight (p<0.05). The alteration of 2 microglobulin in plasma levels may not simply be a result of trans-membrane transport; the adsorption to the membrane may also play a role in the observed plasma changes (Hoenich & Katopodis, 2002). For the removal of 2-microglobulin, cellulose derived membrane is impermeable to 2- microglobulin due to its dense symmetrical structure which does not permit the easy diffusion or convection of proteins through the membrane, while polyacrylonitrile (PAN), polysulfone and polymethylmethacrylate (PMMA) membrane could be used (Moachona et al., 2002). The PMMA membrane could also adsorb 2 microglobulin. To remove 2-microglobulin more efficiently from plasma, hemodialysis membranes must therefore not simply be considered as filters of low-molecular-weight metabolites but should be equally assessed for their capacity to eliminate potentially deleterious low-molecular-weight plasma proteins. For the PES membrane, 2 microglobulin adsorption is not an important mechanism of removal. The large solute removal by the membrane is mainly caused by the asymmetric structure and the higher ultra-filtration coefficient, which was presumably caused by the larger pore size and the hydrophilicity of the membrane.

Fig. 8. Changes in 2-microglobulin during the dialysis. Data are expressed as the meansSD, n =3 (From reference, Su et al., 2008)

Polyethersulfone Hollow Fiber Membranes for Hemodialysis 81

system results in release of anaphylatoxins into the circulation which have potent physiological effects, thus complement activation has been the most widely used parameter

Hemolysis ratio was determined for the swine blood in vitro and for the goat blood in vivo. Data showed that there was only a slightly hemolysis phenomena (about 1.7%) in vitro, while the hemolysis ratio was zero in vivo (The absorption value for (+) is 0.832, but for the

The red blood cell (RBC) and hemoglobin (RGB) levels were also determined during the dialysis. The RBC level was (2.040.12)1012/L and (1.960.10)1012/L respectively before and after the hemodialysis. And the HGB level was 115.08.0g/L and 110.58.0g/L before

Biochemistry for the blood was analyzed before and after the hemodialysis, and the data were summarized in Table 3. Only the alkaline phosphatase (ALP) level increased. And the others, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), total protein (TP) and plasma albumin (ALB) were slightly decreased. The blood gas was also analyzed as shown in Table 4. The data showed no statistically change during the dialysis. The urine solution was also analyzed before and after the hemodialysis; the pH value, urine protein and urine glucose had no change before and after the hemodialysis. The concentrations of urobilinogen were 3.5mmol/L and 3.7mmol/L before and after the

Red blood cells (RBC) and hemoglobins (HGB) levels decreased slightly after the treatment, and both of the reduction ratios were about 5%. Slightly decreases in alanine aminotransferase (ALT), aspartate aminotransferase (AST), total protein (TP) and plasma albumin (ALB) were also observed. The reduction ratios for all of them ranged 3-10%, which were presumably caused by the dilution of the blood by normal saline solution infused after the hemodialysis process. ALP is produced primarily in the liver and in bone, and the result

> ALT(IU/L) 29.03.0 26.51.5 AST(IU/L) 189.59.5 173.015.5 ALP(IU/L) 284.033.0 266.013.0 TP(g/L) 70.21.4 63.81.8 ALB(g/L) 29.20.4 27.60.4

pH 7.430.0 7.470.02 PCO2(Kpa) 4.50.7 4.30.1 PO2 (Kpa) 9.70.4 9.20.3 HCO3(mmol/L) 24.52.4 25.61.5

pre-dialysis post-dialysis

pre-dialysis post-dialysis

for ALP indicts that the PES membrane has no effect on the liver.

Table 3. Data for biochemistry analysis pre- and post-dialysis

to evaluate hemocompatibility.

and after the hemodialysis, respectively.

hemodialysis, respectively.

\* Data are expressed as the meansSD, n =3

\* Data are expressed as the meansSD, n =3

Table 4. Blood gas values pre- and post-dialysis

sample is 0).

Table 2 shows the electrolyte values in the goat blood before and after the dialysis process. Among the these ions, only the K+ exhibited statistically significant decreases after the dialysis, whereas Na+, Cl- and Ca2+ did not change. As shown in the table, only the K+ exhibited statistically significant decreases during the dialysis (p<0.05), whereas Na+, Cland Ca2+ did not change (p>0.05). The electrolyte balance could be adjusted by the dialysis fluid, and the accurate K+ values are critically important for the management of patients with little or no residual kidney function (Barry 2003; Morgera et al., 2005).


\* Data are expressed as the meansSD, n =3

Table 2. Electrolyte values pre- and post-dialysis

#### **4.1.2.2 Biocompatibility**

Figure 9 summarizes the changes in the goat blood observed during the dialysis in respect of white cells (WBC) and platelets. Both white blood cell and platelet counts have been normalized to pretreatment levels and expressed as a percentage of these values. A small decline in both was noted at the first 30 minutes, which returned to the initial levels after about 2 h. These phenomena have been reported frequently in hemodialysis, hemofiltration, and plasma separation.

Fig. 9. Changes in WBC and platelet during the dialysis in vivo ♦ Platelet; ■ WBC; Data are expressed as the meansSD, n =3 (From reference, Su et al., 2008)

The complement and WBC activation for various membranes were investigated after contacting to blood for 1h. The data showed the correlation between the complement and WBC activation. We also found that the concentration of C3a increased rapidly at the beginning of the contact between the blood and the PES membrane and remained constant after 90 min, which was consistent with the decrease of white blood cells (Zhao et al., 2001). The decrease of WBC is caused by complement activation; the activation of complement

Table 2 shows the electrolyte values in the goat blood before and after the dialysis process. Among the these ions, only the K+ exhibited statistically significant decreases after the dialysis, whereas Na+, Cl- and Ca2+ did not change. As shown in the table, only the K+ exhibited statistically significant decreases during the dialysis (p<0.05), whereas Na+, Cland Ca2+ did not change (p>0.05). The electrolyte balance could be adjusted by the dialysis fluid, and the accurate K+ values are critically important for the management of patients

> K+(mmol/L) 3.710.37 2.980.17 Na+(mmol/L) 144.03.8 142.81.8 Cl—(mmol/L) 105.14.1 101.01.2 Ca2+(mmol/L) 2.120.16 2.020.11

Figure 9 summarizes the changes in the goat blood observed during the dialysis in respect of white cells (WBC) and platelets. Both white blood cell and platelet counts have been normalized to pretreatment levels and expressed as a percentage of these values. A small decline in both was noted at the first 30 minutes, which returned to the initial levels after about 2 h. These phenomena have been reported frequently in hemodialysis, hemofiltration,

Fig. 9. Changes in WBC and platelet during the dialysis in vivo ♦ Platelet; ■ WBC; Data are

0 60 120 180 240 Treatment duration (minutes)

The complement and WBC activation for various membranes were investigated after contacting to blood for 1h. The data showed the correlation between the complement and WBC activation. We also found that the concentration of C3a increased rapidly at the beginning of the contact between the blood and the PES membrane and remained constant after 90 min, which was consistent with the decrease of white blood cells (Zhao et al., 2001). The decrease of WBC is caused by complement activation; the activation of complement

expressed as the meansSD, n =3 (From reference, Su et al., 2008)

Normalized value (%)

pre-dialysis post-dialysis

with little or no residual kidney function (Barry 2003; Morgera et al., 2005).

\* Data are expressed as the meansSD, n =3

**4.1.2.2 Biocompatibility** 

and plasma separation.

Table 2. Electrolyte values pre- and post-dialysis

system results in release of anaphylatoxins into the circulation which have potent physiological effects, thus complement activation has been the most widely used parameter to evaluate hemocompatibility.

Hemolysis ratio was determined for the swine blood in vitro and for the goat blood in vivo. Data showed that there was only a slightly hemolysis phenomena (about 1.7%) in vitro, while the hemolysis ratio was zero in vivo (The absorption value for (+) is 0.832, but for the sample is 0).

The red blood cell (RBC) and hemoglobin (RGB) levels were also determined during the dialysis. The RBC level was (2.040.12)1012/L and (1.960.10)1012/L respectively before and after the hemodialysis. And the HGB level was 115.08.0g/L and 110.58.0g/L before and after the hemodialysis, respectively.

Biochemistry for the blood was analyzed before and after the hemodialysis, and the data were summarized in Table 3. Only the alkaline phosphatase (ALP) level increased. And the others, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), total protein (TP) and plasma albumin (ALB) were slightly decreased. The blood gas was also analyzed as shown in Table 4. The data showed no statistically change during the dialysis. The urine solution was also analyzed before and after the hemodialysis; the pH value, urine protein and urine glucose had no change before and after the hemodialysis. The concentrations of urobilinogen were 3.5mmol/L and 3.7mmol/L before and after the hemodialysis, respectively.

Red blood cells (RBC) and hemoglobins (HGB) levels decreased slightly after the treatment, and both of the reduction ratios were about 5%. Slightly decreases in alanine aminotransferase (ALT), aspartate aminotransferase (AST), total protein (TP) and plasma albumin (ALB) were also observed. The reduction ratios for all of them ranged 3-10%, which were presumably caused by the dilution of the blood by normal saline solution infused after the hemodialysis process. ALP is produced primarily in the liver and in bone, and the result for ALP indicts that the PES membrane has no effect on the liver.


\* Data are expressed as the meansSD, n =3

Table 3. Data for biochemistry analysis pre- and post-dialysis


\* Data are expressed as the meansSD, n =3

Table 4. Blood gas values pre- and post-dialysis

Polyethersulfone Hollow Fiber Membranes for Hemodialysis 83

between samples in one group was calculated by Student-Newman-Keuls (q-test). All the data are shown by mean values and standard deviations (ms), p<0.05 is considered to have

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

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"

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

provoking any adverse symptoms, such as headache or hypotension.

statistical difference.

**4.2.2.1 Solute clearance** 

molecular solutes as 2-microglobulin.

**4.2.2 Results and discussion** 
