We assessed haemolysis in outpatients with end-stage renal disease who were recruited from the dialysis unit of Kansai Medical University Hospital from May to November 2019. Patients aged ≥20 years who had been receiving maintenance haemodialysis 3 times a week or peritoneal dialysis therapy for at least 6 months were included in this cross-sectional study. The exclusion criteria were as follows: patients undergoing both haemodialysis and peritoneal dialysis, patients with a bleeding event within the last 3 months, blood transfusion within the last 3 months, concurrent malignancy, haemolytic disease, and mechanical heart valves. The study protocol was approved by the ethics committee of Kansai Medical University (No. 2018233) and registered in the University Hospital Medical Information Network (UMIN) clinical trial registry (URL: https://www.umin.ac.jp/ctr/, Unique Identifier: UMIN000036418). All the patients provided written informed consent, and the investigation conformed to the principles outlined in the Declaration of Helsinki.
Haemodialysis and peritoneal dialysis
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 heparin sodium 500 to 1000 units was intravenously administrated at the start of haemodialysis, followed by continuous administration of 500 to 1000 units maintain the pre-haemodialysis activated partial thromboplastin time at 1.5 to 2 times higher than its upper level. The dialysate temperature of extracorporeal circulation was strictly maintained at 36°C–38°C. Nocturnal intermittent peritoneal dialysis (Baxter Healthcare, Tokyo, Japan) was performed in all the patients in the peritoneal dialysis group. Evaluation and treatment of anaemia, including erythropoiesis-stimulating agents (ESAs) and iron therapy, were prescribed in accordance with the Kidney Disease: Improving Global Outcomes Clinical Practise Guideline 2012 . Iron administration therapy with an intravascular supplement (40mg of iron/week) was administrated to the haemodialysis group; and oral supplement (100 mg of iron/day) to the peritoneal dialysis group. ESA therapies with darbepoetin alfa and epoetin beta pegol were administrated to the haemodialysis and peritoneal dialysis groups, respectively. Blood flow (mL/min) and intradialytic ultrafiltration rates (mL/h/kg) were measured to assess the haemodialysis condition, which was calculated as the average of the values from 3 consecutive haemodialysis sessions. One of the following dialysis membranes was used in the haemodialysis group by dialysis unit physicians: cellulose (FB-Uβ, Nipro Corporation, Osaka, Japan), polysulfone, (ABH-PA, Asahi Kasei Corporation, Tokyo, Japan; APS-EA, Asahi Kasei Corporation; NV-X, Toray Medical Co., Ltd., Tokyo, Japan; NVF-H, Toray Medical Co., Ltd.; and VPS-VA, Asahi Kasei Corporation), polyethersulfone (MFX-S, Nipro Corporation; PES-D, Nipro Corporation), polymethylmethacrylate (NF-H, Toray Medical Co., Ltd.), or acrylonitrile-co-methallyl sulphonate (H12-4000, Baxter, Tokyo, Japan).
Body weight was measured before and after dialysis in the haemodialysis group. In the peritoneal dialysis group, body weight was measured after discarding the dialysate from the peritoneal cavity. After enrolment, blood samples were drawn from all the patients to examine the level of erythrocyte creatine, haemolytic markers (reticulocyte, haptoglobin, and lactate dehydrogenase), and other laboratory parameters (haemoglobin, haematocrit, albumin, transferrin saturation, and ferritin). Blood samples were obtained immediately before the patients received haemodialysis. A weekly ESA dose was administered as a darbepoetin alfa equivalent dose. ESA was converted using the following formula: darbepoetin alfa (µg) = epoetin beta pegol (µg) × 0.8 = epoetin (U) × 200, in accordance with previous reports [9-10]. ESA responsiveness was assessed using an erythropoietin resistance index, which was calculated using the following formula: erythropoietin resistance index (U/kg/week/g/dL) = weekly dose of epoetin (U/week)/(body weight [kg] × haemoglobin level [g/dL]) . Post-haemodialysis weight was measured as a body weight in the haemodialysis group.
Creatine is packed in erythrocytes and irreversibly decreases in amount over time. By measuring the erythrocyte creatine level, the mean RBC age can be calculated . Erythrocyte creatine was measured enzymatically in accordance with previous reports . 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. The mean RBC age (days) was −22.84 × loge (erythrocyte creatine) + 65.83 . Erythrocyte creatine levels represent the average or cumulative erythropoiesis up to the present. Therefore, erythrocyte creatine levels are indicative of chronic rather than an acute haemolytic conditions. The RBC ages in 305 healthy subjects in our previous study were extracted as healthy controls [5,6].
Continuous variables are presented as medians and interquartile ranges and categorical variables as numbers and percentages. Differences between the 2 groups were analysed using the Wilcoxon rank-sum test for continuous variables and the chi-squared test for categorical variables. The relationship between the clinical covariates and erythrocyte creatine level was examined using a Spearman correlation analysis. A p value of <0.05 was considered significant. The JMP 14.2.0 software (SAS Institute Inc., Cary, NC, USA) was used for all the statistical analyses.