Red Blood Cell Size Factor (Rsf) Potential Marker of Iron Restricted Erythropoiesis in Hemodialysed Patients

Research Article

Chronic Dis Int. 2014;1(2): 5.

Red Blood Cell Size Factor (Rsf) Potential Marker of Iron Restricted Erythropoiesis in Hemodialysed Patients

Eloisa Urrechaga1*, Ramón Saracho2, Luís Borque3 and Jesus F Escanero3

1Hematology Laboratory, Hospital Galdakao – Usansolo Galdakao, Spain

2Department of Nephrology, Hospital Galdakao – Usansolo Galdakao, Spain

3Department of Pharmacology and Physiology, University of Zaragoza, Spain

*Corresponding author: Eloisa Urrechaga, Hematology Laboratory, Hospital Galdakao–Usansolo, 48960 Galdakao, Vizcaya, Spain

Received: December 08, 2014; Accepted: December 15, 2014; Published: December 16, 2014


Background: Reticulocyte hemoglobin content (CHr) is incorporated to National Kidney Foundation (NKF-K/DOQI) guidelines for the monitoring of erythropoiesis-stimulating agents therapy in dialysed patients. The measurement of CHr has been restricted to the analyzers of a single manufacturer, Siemens. Red blood cell size factor (RSf) is a parameter provided by Beckman- Coulter which averages the volume of erythrocytes (MCV) and the volume of reticulocytes (MRV)

RSf= MCV*MRV MathType@MTEF@5@5@+=feaagCart1ev2aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLnhiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr4rNCHbGeaGqiVu0Je9sqqrpepC0xbbL8F4rqqrFfpeea0xe9Lq=Jc9vqaqpepm0xbba9pwe9Q8fs0=yqaqpepae9pg0FirpepeKkFr0xfr=xfr=xb9adbaqaaeGaciGaaiaabeqaamaabaabaaGcbaGaamOuaiaadofacaWGMbGaeyypa0ZaaOaaaeaacaWGnbGaam4qaiaadAfacaGGQaGaamytaiaadkfacaWGwbaaleqaaaaa@3F56@

Aim: we aimed to investigate the reliability of RSf for assessing iron status in hemodialysed patients, in comparison with CHr.

Methods: A prospective study included 90 healthy individuals, 70 Iron Deficiency Anemia (IDA) and 55 dialysed (HD) patients; the hemograms were run in parallel in LH 750 (Beckman-Coulter) and Advia 2120 (Siemens) analyzers.

HD patients were included in a longitudinal study along one month of follow up.

Results: Good correlation between CHr and RSf was observed, r =0.85.

ROC curve analysis for RSf in the diagnosis of iron restricted erythropoiesis, defined by CHr < 29 pg.

AUC 0.952 (95 % CI 0.902-0.981) sensitivity 80.0 % (95 % CI 68.7- 88.6 %), specificity 92.5 % (95 % CI, 83.4- 97.5 %), cut off 92.2 fL. Along the four weeks follow up of the longitudinal study Hb, CHr and RSf remained constant, correlation between CHr and RSf r=0.744.

Conclusion: This study shows a good level of agreement between RSf and CHr. RSf could be a reliable parameter for the study of erythropoiesis in dialysed patients.

Keywords: Anemia; Haemodialysis; Iron monitoring; Red blood cell size factor; Reticulocyte haemoglobin content


Anemia is one of the most characteristic manifestations of Chronic Renal Disease (CKD) and may be a product not only of decreased erythropoietin production, but also of decreased iron availability, both conditions lead to impaired erythropoiesis.

Recombinant human erythropoietin (rHuEpo) has been available for treatment of renal anemia since 1989 [1]. However, rHuEpo therapy results in functional iron deficiency, due to insufficient iron stores for the accelerated erythropoiesis [2].

Iron deficiency is the main cause of suboptimal response to erythropoietin in dialysis patients. Maintenance iron supplementation is required to successfully treat anemia. Long term orally administered iron therapy is limited by noncompliance, gastrointestinal side effects, insufficient absorption and drug interaction; intravenous iron compounds are used to treat dialysis patients who become iron deficient [3].

Monitoring erythropoietin treated patients’ iron status is important to detect iron deficiency and avoid the adverse effects of iron medication [4-6].

Biochemical indicators of iron metabolism (serum iron, transferrin saturation, serum ferritin) although widely used, may be influenced by the acute phase response, which complicates clinical interpretation of the test results. Serum ferritin, an indicator of iron storage but not of iron supply, is an acute phase reactant and its levels are affected by inflammation, serum ferritin levels might not reflect true iron stores.

Transferrin is a negative acute phase reactant, rendering the calculation of transferrin saturation unreliable in this case [7,8]. Transferrin fluctuates due to the diurnal variation of serum iron and is affected by nutritional status, leading to a lack of sensitivity and specificity in assessing iron’s availability [9]. For these reasons, an iron deficient erythropoietic response to rHuEpo may occur despite normal serum ferritin and transferrin values.

Interest has been generated in the use of erythrocyte and reticulocyte new indices available on the modern analyzers based on flow cytometry technology.

Measurement of reticulocyte number and cellular characteristics may provide useful information about marrow erythropoietic activity [10]. The potential use of new reticulocyte parameters in the diagnosis of anemia and in monitoring the bone marrow erythropoietic activity has been assessed [11-13].

The measurement of reticulocyte hemoglobin content (CHr) is an assessment of the incorporation of iron into hemoglobin and thus an estimate of the recent functional availability of iron into the erythron [14,15].

CHr has been used as a diagnostic tool, together with biochemical markers, to distinguish Iron Deficiency Anemia (IDA) from Anemia of the Chronic Diseases (ACD), and is incorporated to National Kidney Foundation, Kidney Disease Outcomes Quality Initiative (NKF-K/DOQI) guidelines for the monitoring of recombinant human erythropoietin rHuEPO therapy [16-18].

The measurement of CHr has been restricted to the analyzers of a single manufacturer, Siemens (Siemens Medical Solutions Diagnostics, Tarrytown NY, USA).

Beckman-Coulter (Beckman Coulter Inc., Miami Fl, and USA) has proposed a new parameter Red blood cell Size Factor, RSf

RSf= MCV*MRV MathType@MTEF@5@5@+=feaagCart1ev2aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLnhiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr4rNCHbGeaGqiVu0Je9sqqrpepC0xbbL8F4rqqrFfpeea0xe9Lq=Jc9vqaqpepm0xbba9pwe9Q8fs0=yqaqpepae9pg0FirpepeKkFr0xfr=xfr=xb9adbaqaaeGaciGaaiaabeqaamaabaabaaGcbaGaamOuaiaadofacaWGMbGaeyypa0ZaaOaaaeaacaWGnbGaam4qaiaadAfacaGGQaGaamytaiaadkfacaWGwbaaleqaaaaa@3F56@

RSf averages the volume of mature red cells (MCV) and the volume of reticulocytes (MRV), both related to erythropoietic activity and hemoglobinization.

When MCV is measured what is measured is the average size or volume of the red blood cells produced in a period of 120 days before the sample is drawn; on the other hand, when MRV is measured what is measured is the size of the recently produced red cells, within a period of less than 2 days: the measurement of reticulocyte number and volume provides real time data regarding certain aspects of erythropoiesis, such as iron availability, that can influence the dimensions of red cells

Both in mature red cells and reticulocytes above 90 % cellular contents are Hb the size of these cells directly correlates with the Hb content of the cells. RSf, as a product function of MCV and MRV reflects indirectly the cellular Hb content of both cell populations; adding the square root averages both volumes and the units in which RSf is expressed are femtoliters.

Examining both precursors and mature red cells provides an opportunity to detect and monitor acute and chronic changes in cell volume related to cellular hemoglobin status and the amount of the iron supply.

RSf reference range and values in different clinical conditions have been reported and its value for the study of bone marrow erythropoietic activity, with the same clinical meaning as CHr, assessed [19].

The aim of this study was to evaluate the overall performance of the new parameter RSf in a cohort of dialysis patients in terms of concordance with CHr as well as the potential clinical utility in monitoring functional iron deficiency in those patients.

Material and Methods

Patients selection

Healthy group: 45 male and 45 female healthy adult subjects, with no clinical symptoms of disease and results within reference ranges in the complete blood count and biochemical iron metabolism markers, were recruited.

Iron deficiency group: IDA patients fulfilled traditional diagnostic criteria for Iron deficiency anemia diagnosis, Serum Iron < 7.5 μmol/L, Transferrin saturation <20%, Ferritin <50 μg/L, and Hemoglobin (Hb) <12.0 g/dL (females) or < 13.0 g/dL (males), were included before iron treatment.

Hemodialysed group (HD): 55 patients (33 male, 22 female, mean age 61±8 years). All of them were being treated with intermittent in center hemodialysis (standard 4 h bicarbonate dialysis three times per week) and were treated with rHuEPO for at least 3 months and were in the maintenance phase of their treatment with stable rHuEPO doses (80-120 units/Kg/) for at least 4 weeks, in order to meet the recommendations of the European Best Practice Guidelines target hemoglobin concentration of 11.0 g/dL [20]. All were given regular iron therapy (intravenous iron gluconate 30 to 60 mg/week), to maintain the iron availability and transferrin saturation 20 %, and were receiving folate (5 mg orally twice a week) and vitamin B12 (1000 μg three times a week) supplementation.

None of the patients had received blood transfusions nor were affected of bleeding episodes in at least the previous month before starting the study.

A prospective study was conducted including the three groups of patients healthy, IDA and HD (baseline data).

HD patients were included in a longitudinal study and followed during a four weeks period. Blood samples were drawn before the hemodialysis session corresponding to the first day of the dialysis week.

Four samples of each patient had been analysed, at the end of the longitudinal study 220 samples have been processed.

The dose of rHuEPO remained unchanged during the whole period under study.

The protocol was approved by the Ethics Committee of Galdakao - Usansolo Hospital.

Laboratory assays

Blood samples were collected in K2EDTA anti-coagulant tubes (VacutainerTM Becton-Dickinson, Rutherford NJ, USA), were run sequentially on both the LH 750 (Beckman Coulter Inc., Miami Fl, USA) and ADVIA 2120 (Siemens Medical Solutions Diagnostics, Tarrytown NY, USA), within 6 h of collection.

Serum Iron, ferritin and transferrin were measured using a Cobas c 711 chemistry analyzer (Roche Diagnostics, Mannheim, Germany) and reactive from Roche Diagnostics, (Mannheim, Germany).

Serum iron was measured using an assay based on a FerroZine Submit your Manuscript | reaction (Iron Gen2). Serum ferritin was measured using an immunoturbidimetric assay (Tina-quant Ferritin Gen3). Transferrin was measured was measured using an immunoturbidimetric assay (Tina-quant TF2).

Statistical analysis

Statistical software package SPSS (SPSS; Chicago, IL 60606, USA) version 19.0 for windows was applied for statistical analysis.

Kolmogorov-Smirnoff test was applied to verify the normal distribution of values of the tests under investigation.

Correlation coefficients were calculated by Pearson method.

Independent samples t test was performed in order to detect statistical deviations between the groups of patients; p values less than 0.05 were considered to be statistically significant.

Receiver Operating Characteristic (ROC) curve analysis was utilized to illustrate the diagnostic performance of RSf in the assessment of erythropoietic function. Iron restricted erythropoiesis was defined by CHr < 29 pg.

For the longitudinal study changes in the variables were examined by means of a linear mixed model.


The biochemical and hematological data of the patients are summarised on Table 1; data of the HD group are the values in the first week of the study (baseline).