We evaluated 83 patients (57 men and 26 women, mean age of 74 years) aged ≥20 years who had been receiving maintenance haemodialysis 3 times a week for at least 6 months in the Dialysis Unit of Takarazuka Hospital. The exclusion criteria were as follows: bleeding event within 3 months (n=1), infection requiring parenteral antibiotics (n=1) and mechanical heart valves (n=2). None of the patients had concurrent malignancy, haemolytic disease or blood transfusion within 3 months.
Study protocol is shown in Fig. 1. rHuEpo treatment was fixed 3 months prior to the enrollment and was maintained throughout the study period. Baseline blood examination including the erythrocyte creatine and other laboratory examination (red blood cell count, haemoglobin, haematocrit, reticulocyte, haptoglobin, transferrin saturation, ferritin, intact parathyroid hormone, serum calcium, serum phosphorus, serum albumin and C-reactive protein) were performed. Intravenous iron treatment with 40mg ferric saccharate 3 times a week was administered at the end of each haemodialysis session and followed by 100mg of oral iron supplements in patients with absolute iron deficiency, defined as transferrin saturation <20% and serum ferritin <100 ng/ml [15,16]. Patients were divided into 2 groups; patients with haemoglobin <10g/dL and those with haemoglobin >10g/dL according to the Guidelines of Japanese Society for Dialysis therapy  and clinical characteristics, haemodialysis conditions, treatments and laboratory indices were compared between the 2 groups. Haemoglobin was measured 3 months later and percent change in haemoglobin; (3 months minus baseline)/ baseline x 100% was calculated and expressed as percentage. The study protocol was approved by the Takarazuka Hospital ethical committee on human research. All the patients provided written informed consent, and the investigation conformed to the principles outlined in the Declaration of Helsinki.
All patients were dialysed for 4.0-8.0 hours, using a single-use dialyser with a 1.3-2.1 m2 effective surface area of cellulose, poly-sulfone, polyethersulfone or polymethylmethacrylate membrane, and blood flow of 200 ml/min with dialysate flow of 500 ml/min. Haemodialysis was performed via native arteriovenous fistulas with a dual plastic needle and 16-gauge cannula. The patients in the haemodialysis group uniformly received a dialysate (D-dry, Nikkiso Co., Ltd., Tokyo, Japan) and an anticoagulant with heparin sodium. Bolus of heparin sodium 1,000 units was intravenously administrated at the start of haemodialysis followed by continuous administration of 500 to 750 units/hour. The dialysate temperature of extracorporeal circulation was strictly maintained at 36–38 °C. rHuEpo and iron therapy were prescribed in accordance with the Kidney Disease: Improving Global Outcomes Clinical Practice Guideline 2012 . Erythropoietin therapy with epoetin beta pegol was administrated intravenously at the end of haemodialysis. Haemodialysis time (hours/week), intradialytic ultrafiltration rates (ml/hour/kg) were measured and Kt/V, as an index of urea clearance was calculated. These indices were calculated as the average of the values from 3 consecutive haemodialysis sessions. One of the following dialysis membranes was used: cellulose FB, Nipro Corporation, Osaka, Japan), poly-sulfone, (PN, Nikkiso Co., Ltd., Tokyo, Japan), polyethersulfone (PES, Nipro Corporation, Osaka, Japan), or polymethylmethacrylate (NF-H, Toray Medical Co., Ltd., Tokyo, Japan).
Blood samples were drawn immediately before the haemodialysis. Haematologic examinations and reticulocyte counts were carried out with a Sysmex XN 1000 (Sysmex, Kobe, Japan). Haptoglobin was measured by a TIA method with JCA-BM 6010 (JEOL, Tokyo, Japan). Serum iron was measured by a Nitroso-PSAP method with AU 5840 (Beckman Coulter; Tokyo, Japan), unsaturated iron binding capacity (UIBC) was by a Nitroso-PSAP method with BM-6050 (JEOL, Tokyo, Japan) and ferritin by radioimmunoassay with AU-5840 (Beckman Coulter, Tokyo, Japan) and transferrin saturation was calculated as [Serum iron/ (Serum iron + UIBC)] x100 (%). Intact parathyroid hormone was measured by the ECLIA method with Cobas 8000 (Roche Diagnostics, Tokyo, Japan). The other biochemical laboratory measurements were performed by TBA-120 FR automated biochemical analyzer (Canon, Osaka, Japan).
Erythrocyte creatine was assayed enzymatically in accordance with previous report . Measured date are expressed as micromole per gram of haemoglobin (µmol/g Hb). Briefly, blood samples were collected in ethylenediaminetetraacetic acid-containing tubes and centrifuged to remove the plasma and buffy coat. After lysis and deproteinisation of packed erythrocytes, the supernatant was obtained by centrifugation and filtration. The creatine concentration in the supernatant was measured using the enzymatic assay method. Erythrocyte creatine represents the average or cumulative erythropoiesis up to the present [18,19].
Results are expressed as mean ± standard deviation. Statistical analyses between the 2 groups were performed by one-way layout analysis of variance or chi-square analysis followed by Scheffe type multiple comparison method. Change in the haemoglobin from baseline to 3 months after was estimated by 2-way repeated measures ANOVA. Multivariate logistic regression analysis was performed to evaluate the important variables related to change in haemoglobin at 3 months. A probability value of <0.05 was considered significant. Parameters were compared with the use of commercially available statistical software (STATVIEW, Abacus Concepts, Berkeley, CA).