We assessed haemolysis in out-patients with end-stage renal disease recruited from the dialysis unit at the Kansai Medical University Hospital from May to November 2019. Patients aged over 20 years who had been established on 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, mechanical heart valves. The study protocol was approved by the ethics committee of Kansai Medical University (No.2018233) and was registered in the University Hospital Medical Information Network (UMIN) clinical trial registry (URL: https://www.umin.ac.jp/ctr/, Unique Identifier: UMIN000036418). All patients gave written informed consent and the investigation conforms to the principles outlined in the Declaration of Helsinki.
Body weight was obtained pre- and post-dialysis in haemodialysis patients. In peritoneal dialysis patients, body weight was measured after discarding dialysate from the peritoneal cavity. After enrolment, blood samples were drawn from all patients to examine erythrocyte creatine, haemolytic markers (reticulocyte count, haptoglobin and lactate dehydrogenase) and other laboratory parameters (haemoglobin, haematocrit, albumin, transferrin saturation and ferritin). Patients with haemodialysis were obtained blood sample immediately before haemodialysis received. A weekly dose of erythropoiesis-stimulating agents (ESA) 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, based on previous reports [9–11]. 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 (g/dL)) . Post-haemodialysis weight was extracted as a body weight in patients receiving haemodialysis.
Haemodialysis and peritoneal dialysis
Haemodialysis was performed via native arterio-venous fistulas utilizing a dual plastic needle with a 16-gauge cannula size. Haemodialysis patients were uniformly administered with a dialysate (D-dry, Nikkiso Co., Ltd, Tokyo, Japan) and an anticoagulant with heparin sodium. Dialysate temperature of extracorporeal circulation was strictly maintained at 36 to 38 °C. Nocturnal intermittent peritoneal dialysis (Baxter Healthcare, Tokyo, Japan) was performed in all patients with peritoneal dialysis. Evaluation and treatment of anaemia, including ESA and iron therapy, were prescribed according to the KDIGO Clinical Practice Guideline 2012 . Utilized ESA therapy employed darbepoetin alfa in haemodialysis patients and epoetin beta pegol in peritoneal dialysis. Quantity of blood flow (mL/min) and intradialytic ultrafiltration rate (mL/h/kg) were collected to assess haemodialysis conditions, which were calculated by an average of 3 consecutive haemodialysis sessions.
Creatine in human packed erythrocytes indicate the mean age of an RBC population . Erythrocyte creatine was assayed enzymatically in accordance with previous reports . Briefly, blood was collected in ethylenediamine tetra-acetic acid-containing tubes and centrifuged to remove the plasma and the buffy coat. After lysis and deproteinization of packed erythrocytes, the supernatant was obtained by centrifugation and filtration. Creatine concentration in the supernatant was measured using the enzymatic assay method. Mean RBC age (days) was obtained by -22.84 × loge(erythrocyte creatine) + 65.83 . Erythrocyte creatine levels represent average or cumulative erythropoiesis up to the present. Therefore, erythrocyte creatine levels are indicative of a chronic rather than an acute haemolytic condition. RBC age in 305 normal subjects was extracted as a healthy control from our previous report [6, 7].
Continuous variables are presented as medians and interquartile ranges, and categorical variables are presented as numbers and percentages. Differences between the two groups were analysed using the Wilcoxon rank-sum tests for continuous variables and the chi-square tests for categorical variables. The relationship between the clinical covariates and erythrocyte creatine was explored through Spearman correlation analysis. A p-value < 0.05 was considered significant. The JMP 14.2.0 software (SAS Institute Inc., Cary, NC, USA) was used for all statistical analyses.