Effect of acetate-free biofiltration on the anaemia of haemodialysis patients: a prospective cross-over study

Carlo Basile, Rosa Giordano, Alessio Montanaro, Pasquale De Maio, Francesco De Padova, Anna Lisa Marangi and Angelo Semeraro

Nephrology and Dialysis Unit, Hospital of Martina Franca, Italy



   Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. The discussion about the pathogenesis of renal anaemia, whether it is primarily due to relative erythropoietin (Epo) deficiency or to uraemic inhibition of erythropoiesis, is still open. Although it has so far not been possible to identify or isolate a substance retained in uraemia with a suppressive action directed specifically against red-cell production, dialysis therapy can improve the effect of both residual endogenous Epo and exogenous rHuEpo. To what extent the mode and/or the dose of dialysis influence Epo efficacy is as yet poorly understood.

Methods. This study was performed as a single-centre trial. The protocol included a run-in period of 4 months followed by a prospective cross-over study including 6 months each of acetate-free biofiltration (AFB) with a high-flux biocompatible membrane and standard bicarbonate dialysis (BD) with a low-flux cellulosic membrane in a random sequence. AFB is a haemodiafiltration technique based on a continuous post-dilution infusion of a sterile isotonic bicarbonate solution. At the start of the run-in period (and for the entire length of the study), rHuEpo administration was withdrawn; patients whose haemoglobin (Hb) levels dropped at a level <8.0 g/dl at one single monthly check, had to be withdrawn from the study. A blood sample was collected every month for the blood gas analysis and for the determination of blood urea nitrogen, serum creatinine, sodium, potassium, calcium, phosphorus, Hb, erythrocyte, reticulocyte, leukocyte and thrombocyte cell counts, mean globular volume and haematocrit. An equilibrated single pool Kt/Vurea>1.2 was mandatory in both treatment modalities. Serum iron, total iron-binding capacity, and ferritin were checked every 3 months.

Results. Twenty-three of 137 haemodialysis patients were considered eligible for the trial on the basis of the entry criteria. Of these, 15 volunteered and only 10 completed the study. No significant differences in the haematological indices, in the biochemical parameters assessing body iron stores, or in i.v. iron dosage was observed when comparing AFB with BD treatments. The equilibrated single pool Kt/Vurea was always >1.2 and in no case was a significant difference observed when comparing AFB with BD treatments. Treatment time was significantly different between the two treatments (262±2 min in BD and 249±1 in AFB, P<0.0001). Neither pre- nor post-dialysis systolic and diastolic blood pressures, pre-dialysis serum bicarbonate and pH, pre-dialysis serum sodium, potassium, calcium, or phosphorus were significantly different when comparing the two treatment modalities. All 10 patients completed the 1-year follow-up without any major side-effects.

Conclusions. Our study did not show any improvement of anaemia when treating a highly selected patient group, in the absence of any Epo therapy, with AFB compared with standard BD. Even though these conclusions cannot be extended in toto to the entire dialysis population, in which there is a large proportion of Epo-treated patients with Hb levels around 11 g/dl, we may nevertheless conclude that when patients are well selected, adequately dialysed, and not iron- and/or vitamin-depleted, the effect of a haemodiafiltration technique with a high-flux biocompatible membrane is less than might be expected from the results of uncontrolled studies.

Keywords: acetate-free biofiltration; anaemia; bicarbonate dialysis; biocompatible membrane; erythropoietin; high-flux membrane



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
The enormous success of treating renal anaemia with recombinant human erythropoietin (rHuEpo) during the last 15 years lets one easily forget that the discussion about the pathogenesis of renal anaemia was dominated for decades by the question of whether it is primarily due to relative Epo deficiency or uraemic inhibition of erythropoiesis [1]. Although it has so far not been possible to identify or isolate a substance retained in uraemia with a suppressive action directed specifically against red-cell production, dialysis therapy can improve the effect of both residual endogenous Epo and exogenous rHuEpo [1]. To what extent the mode and/or the dose of dialysis influence Epo efficacy is as yet poorly understood [210]. We therefore conducted a prospective, randomized cross-over study with the aim of testing whether erythroid suppression due to a still unknown large-molecular-weight uraemic toxin may play a role in the anaemia of haemodialysis patients.



   Subjects and methods
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Study design
This study was performed as a single-centre trial. The aim of this study was to test whether a modified haemodiafiltration technique (acetate-free biofiltration (AFB)) with high-flux biocompatible membrane (AN69, Hospal, Italy) [11] improves anaemia in haemodialysis patients in comparison with standard bicarbonate dialysis (BD) using a cellulose membrane.

The protocol included a run-in period of 4 months followed by a prospective cross-over study including 6 months each of AFB and BD in a random sequence. At the start of the run-in period rHuEpo administration was withdrawn and haemoglobin (Hb) was checked every month; patients whose Hb dropped at a level <8.0 g/dl at one single monthly check were withdrawn from the study; only patients whose Hb remained >=8.0 g/dl at all monthly checks of the run-in period were allowed to participate in the prospective randomized cross-over study.

A strict adherence in assessing and optimizing iron stores to European Best Practice Guidelines for the management of anaemia in patients with chronic renal failure was applied throughout the study [12].

Criteria for admission to the study

(i) Haemodialysis patients aged more than 18 and less than 75 years.
(ii) Routine use of rHuEpo with no change in the 3 months preceding enrolment.
(iii) rHuEpo dosage <120 IU/kg/week and Epo resistance index <10 IU/kg/week/g of Hb [7] in the 3 months preceding the enrolment.
(iv) Assurance from the patients not to use rHuEpo during the study.
(v) Maintenance thrice-weekly BD for at least 6 months previously with a cellulose dialyser.
(vi) Dialysis dosage >=1.2 of equilibrated single-pool Kt/Vurea. (Single-pool Kt/Vurea was estimated according to the algorithm suggested by Casino et al. [13]. Then it was converted to equilibrated single-pool Kt/Vurea utilizing the formula suggested by Daugirdas and Schneditz [14]).
(vii) Negligible residual renal function.
(viii) No change in iron, folic acid, vitamin B12, or ACE inhibitor in the 3 months preceding enrolment.

Criteria for exclusion from the study

(i) Unstable clinical conditions in the 3 months preceding enrolment.
(ii) Treatment with drugs affecting erythropoiesis (steroids, cyclophosphamide, azathioprine, androgen hormones).
(iii) Severe cardiac, hepatic, bone-marrow, connective tissue, or neoplastic diseases.
(iv) Blood transfusions in the 3 months preceding enrolment.

Criteria for continuing to participate in the study after the run-in period
Hb level >=8.0 g/dl (a fall in Hb <8.0 g/dl at two consecutive monthly checks was sufficient for withdrawal from the study) with no need for blood transfusions and/or rHuEpo administration.

Dialysis protocols
AFB is a haemodiafiltration technique based on a continuous post-dilution infusion of a sterile isotonic bicarbonate solution [11]. It was carried out with AN69 dialysers with a surface area of 1.2 m2. Dialysate sodium concentration was about 135 mEq/l; the dialysis bath also contained 1 g/l glucose with calcium and potassium concentrations varying according to clinical needs. The dialysate was completely buffer-free, and acidosis was corrected with a 145-mEq/l sodium bicarbonate solution as substitution fluid. The infusion rate was programmed at 2 l/h, whereas the ultrafiltration rate was equivalent to the sum of the infusion rate and the programmed weight loss rate per hour.

Dialysate sodium concentration of BD treatments was about 139 mEq/l; the dialysate also contained 35 mEq/l bicarbonate, and calcium and potassium concentrations according to the clinical needs. All dialysates contained 1 g/l glucose and 4.0 mEq/l acetate. BD treatments were carried out with cellulose acetate dialysers with a surface area of 1.4 m2 (Baxter, Italy). This choice was dictated by the need of avoiding the risk of back-filtration and subsequent endotoxin transfer to the blood compartment that may have ensued with a high-flux membrane in BD [15]. Thus, we chose cellulose acetate, which has been reported to be more biocompatible than cuprophane, and then closer to the biocompatibility characteristics of AN69 membranes [16].

Five points common to both treatment modalities need to be stressed.

(i) All runs were performed with the same type of monitor (IntegraTM, Hospal, Italy).
(ii) Patients maintained their usual haemodialysis schedule (three times weekly) throughout the study.
(iii) The blood flow rate was about 300 ml/min and the dialysate flow rate about 500 ml/min in both treatments.
(iv) Treatment time was conditioned by equilibrated single-pool Kt/Vurea>=1.2.
(v) The patients were on various outpatient medications (including i.v. iron supplementation), which were not changed throughout the study period.

Measurements
A blood sample was collected every month at the mid-week run for the blood gas analysis (Ciba-Corning 288 Blood Gas System, Italy) and for the determination of blood urea nitrogen, serum creatinine, sodium, potassium, calcium, phosphorus (routine automated methods), Hb, erythrocyte, reticulocyte, leukocyte and thrombocyte cell counts, mean globular volume and haematocrit (Coulter Counter). Single pool Kt/Vurea was estimated monthly according to the algorithm suggested by Casino et al. [13]. It was then converted to equilibrated single-pool Kt/Vurea utilizing the formula suggested by Daugirdas and Schneditz [14].

Serum iron, total iron-binding capacity, and ferritin were checked every 3 months. Pre- and post-dialysis body weight and systolic and diastolic blood pressure were checked and recorded in each run. The duration of HD runs was averaged for all patients, pooling all the data collected in each of the treatment periods.

Statistical analysis
A multifactor analysis was employed for purposes of comparing multiple measurements during each treatment modality and of comparing both treatment modalities. Where the results of the factorial analysis were significant, the difference between means was detected by a least significant difference test [17]. The treatment times were compared using the unpaired Student's t-test. A P value <0.05 was accepted as statistically significant. Data are expressed as means±SD.

Informed consent and ethical surveillance
The patients were informed about the aims of the study, the expected benefits to them and/or to others, the risks and inconveniences involved, and their right to refuse to participate or to withdraw from the study at any time without sanction. Their written consent was obtained. The study was approved by the local ethics committee.



   Results
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Baseline characteristics
Twenty-three out of 137 haemodialysis patients treated in our centre were considered eligible for the trial on the basis of the entry criteria. Of them, 15 volunteered after written consent for the present study. Five patients whose Hb dropped at a level <8.0 g/dl at the monthly checks (four during the run-in period, and the fifth 3 months after starting the study, while in BD control period) were withdrawn from the study. Only 10 patients completed the study. All of them fulfilled the criteria for admission to the study, as shown in Table 1Go.


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Table 1. Baseline characteristics (before the run-in period) of the 10 patients who completed the study

 

Haematological parameters
No significant difference in the haematological indices was observed when comparing AFB with BD treatment (Table 2Go). There was no significant difference in the biochemical parameters assessing body iron stores and in i.v. iron dosage between the two treatment modalities, as shown in Table 3Go.


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Table 2. Follow-up data of haematological indices of the 10 patients who completed the study

 

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Table 3. Follow-up data of parameters assessing body iron stores, i.v. iron dosage, and depurative indices of the 10 patients who completed the study

 

Depurative parameters
The dialysis dose estimated by means of equilibrated single-pool Kt/Vurea was always >1.2 and in no case was a significant difference observed when comparing AFB with BD treatments (Table 3Go). Treatment time was significantly different between the two treatments (262±2 min in BD and 249±1 in AFB, P<0.0001).

Other parameters
Neither pre- nor post-dialysis systolic and diastolic blood pressures, pre-dialysis serum bicarbonate and pH, pre-dialysis serum sodium, potassium, calcium, or phosphorus were significantly different when comparing the two treatment modalities (not shown).

Safety
All 10 patients completed the 1-year follow-up without experiencing any major side-effects.



   Discussion
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Among the factors affecting red cell production in renal failure, iron deficiency, inflammatory mediators, and several others have received considerable attention during recent years, but the role of specific uraemia-associated inhibitors of erythropoiesis has remained unclear [1]. Although direct proof for the existence of substances specifically inhibiting red-cell formation in uraemia is lacking, this does not exclude that a variety of different molecules accumulating in the uraemic state may contribute to an impairment of erythropoiesis [1].

The present study examined the question of whether AFB, a modified haemodiafiltration technique with a large-pore biocompatible membrane, might improve anaemia in comparison with a conventional dialysis technique using a cellulose membrane, with the dialysis dose being kept equal between the two treatment modalities. The main result was that Hb levels were not significantly different between the two treatments.

Our results are similar to those of a recent work by Locatelli et al. [7], who performed a randomized controlled multicentre trial in which they examined the question of whether the use of a high-flux biocompatible membrane (BK-F PMMA, Hoechst, Toray, Japan) might improve anaemia in comparison with a cellulose membrane. Locatelli et al. were careful enough to point out that the negative results of their study (no significantly different Hb level between the two groups) could have been due either to the trial duration (only 3 months) or to the sample size (74 patients, 35 being in the experimental treatment group and 39 in the control group) or to both [7]. These criticisms might be applied to our work: however, as to the first point, our study lasted longer (twice as long) than that by Locatelli et al.; as to the second point, it must be said that our study had a cross-over design, which reduces the need for a large number of patients. However, we cannot exclude the possibility that the enrolment of a larger number of patients into the study would have helped to demonstrate any effects of either biocompatibility or low/high-flux dialysis on erythropoiesis, if such effects exist.

Another possibility put forward by Locatelli et al. for the lack of a difference between the study groups in terms of anaemia correction could be that their patients were not too anaemic in comparison with the general dialysis population [7]. In contrast, our study enrolled patients who were routinely dependent on rHuEpo administration, even though not showing a high Epo resistance index [7]; during the study, the withdrawal of rHuEpo therapy with a consequent relatively low Hb level would have allowed maximization of the beneficial effects of AFB, if any, on renal anaemia without the confounding effect of concomitant rHuEpo administration. The latter statement holds more true when taking into account the fact that Cheung et al. [18] showed that AN69 membrane adsorbed in vitro 30 times more rHuEpo than cuprophane. Even though Opatrny et al. [19] could demonstrate that this phenomenon is not clinically relevant, we designed a study that excluded a priori rHuEpo administration. It was this particular aspect of the study design that limited the enrolment of the patients.

Our results are in contrast with those of Kobayashi et al. [3], Kawano et al. [4], Ifudu et al. [5], and Villaverde et al. [9]. There may be various reasons for this, perhaps the most important being the study design (ours being a randomized cross-over one) and the selection criteria (we selected patients without other known factors affecting uraemic anaemia). Particular care was taken to admit patients with a low Epo resistance index without iron depletion. Finally, the dialysis dose was adequate (equilibrated single pool Kt/Vurea>=1.2). Furthermore, our results are in contrast with those of a very recent paper showing that AFB using an AN69 membrane was able to reduce the dosage of rHuEpo necessary for anaemia control, compared with BD using a low-flux membrane [10]. The reasons for this discrepancy are far from being clear. However, there may be two main explanations: first, that study, even though a randomized control one, had only 10 patients in each arm; second, our study proscribed the use of rHuEpo in order to avoid the confounding effect of the concomitant rHuEpo administration on the results of the study.

Our results are, however, similar to those obtained by Schrander-v.d. Meer et al. [6]. They evaluated, in a year-long study, 11 patients on AFB and nine on BD, all with the AN69 membrane. They did not find differences in anaemia correction and/or rHuEpo consumption. Again there are two possible reasons: the first one, as Eiselt et al. [10] suggest, could be that, when using the AN69 membrane, it makes no difference whether the patient is treated by BD or AFB; the second reason, as we have suggested, could be that neither the mode of dialysis (AFB with a high-flux biocompatible membrane) nor a high-flux biocompatible membrane per se (AN69 in a standard BD) are able to affect erythropoiesis when the dialysis dose is adequate.

Finally, the design of our study did not allow to dissociate between the effects of biocompatibility and high/low flux dialysis on erythropoiesis. Thus, if it had turned out to show any beneficial effects of AFB on anaemia, the specific effects of each of these factors on erythropoiesis would have not been defined.

In conclusion, our study did not show any improvement of anaemia when treating a highly selected patient group, in the absence of any Epo therapy, with AFB compared with standard BD. Even though these conclusions cannot be extended in toto to the entire dialysis population, in which there is a large proportion of Epo–treated patients with Hb levels around 11 g/dl, all the same we can conclude that when patients are well selected, adequately dialysed and not iron- and/or vitamin-depleted, the effect of a haemodiafiltration technique with a high-flux biocompatible membrane is less than might be expected from the results of uncontrolled studies.



   Notes
 
Correspondence and offprint requests to: Dr Carlo Basile, Via Battisti 192, I-74100 Taranto, Italy. Back



   References
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 27. 1.01
Accepted in revised form: 19. 4.01





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