Reticulocyte parameters for monitoring anemia in patients with chronic kidney disease on peritoneal dialysis

1. Pontifícia Universidade Católica do Paraná, Curitiba, Paraná, Brazil
2. Centro de Hematologia e Hemoterapia do Paraná, Curitiba, Paraná, Brazil

Corresponding author

Júlio C. Merlin
0000-0002-8973-4806
e-mail: merlinjc1960@gmail.com

INTRODUCTION

Anemia in chronic kidney disease (CKD) is multi-factorial, but mainly driven by erythropoietin (EPO) production deficiency and depletion of iron reserves^(1-3). Inflammation is also an important determinant in a sub-group of patients, as it interferes directly with iron mobilization and determines EPO resistance^{(1, 4)}. Inflammation in CKD increases the concentration of inflammatory cytokines, interleukin-6 (IL-6) being the principal cytokine responsible for the activation of hepcidin expression^(5-8). Iron stores can be monitored by ferritin and serum levels by iron transferrin saturation (STI), as well as by hemoblobin⁽⁹⁾. Thus, in inflammatory situation, high levels of hepcidin directly and negatively interfere with the availability of iron in erythropoiesis, and may cause anemia, mainly due to iron functional deficiency. Indeed, relative and absolute reticulocyte count is the principal marker the evaluation of the erythropoietic activity of the bone marrow⁽¹⁰⁾. Although all of these markers are used in clinical practice, they are far from ideal real-time indicators.

Different parameters have been described in the literature, but not much yet evaluated in the CKD population, especially in patients undergoing peritoneal dialysis (PD). Equivalent of hemoglobin concentrations in reticulocytes (RET-He) represents the degree of hemoglobinization that has occurred in the bone marrow which is dependent on iron availability^{(11, 12)}. Thus potentially allows an early evaluation, for clinical diagnosis, monitoring and define intervention, particularly in patients with CKD using erythropoiesis stimulating agents (ESA) and iron supplementation⁽¹³⁾. Additionally, immature reticulocytes fraction (IRF) is an excellent marker of nearly real-time erythropoietic activity since it represents the proportion of younger reticulocytes in the peripheral blood and rises much earlier than the total number of reticulocytes^{(3, 11, 14)}. These parameters may be useful in the monitoring and management of patients with renal anemia, particularly those on PD.

Due to their conceptual characteristics and because they do not suffer direct analytical interference from inflammation, these reticulocytes markers may identify earlier and better the general iron status and erythropoietic activity of the bone marrow in patients with CKD on PD when compared to conventional markers such as ferritin, transferrin saturation index and reticulocytes count.

OBJECTIVE

Analyze the role of the hematological parameters IRF and RET-He in the monitoring and identification of ESA resistance of CKD anemia in peritoneal dialysis patients.

METHODS

This was a prospective, multi-centric study, including patients with stage 5 CKD on continuous ambulatory peritoneal dialysis (CAPD/APD) for at least 30 days, over 18 years of age, with or without therapeutic use of ESA and iron supplements, and who signed the informed consent (project with approval at Comitê de Ética em Pesquisa envolvendo Seres Humanos (CEP)-Universidade Estadual de Londrina (UEL) under number: 357/2011). The venous blood samples for the laboratory tests and for blood counts were obtained in tubes with ethylenediamine tetraacetic acid (EDTA)-K₂ and in dry tubes with no anticoagulant, using separator gel and clot activator for measuring inflammatory biomarkers.

Complete blood count (CBC) were processed on the same day of collection for up to a maximum of 4 hours in a hematological analyzer (Sysmex, model XE-5000) to obtain hemoglobin parameters, RET-He, relative reticulocyte count (RET%), immature reticulocytes fraction (IRF%), and their fractions [low fluorescence reticulocyte fraction (LRF%), medium fluorescence reticulocyte (MFR%) and high fluorescence reticulocyte fraction (HFR%)]. Inflammatory biomarkers were measured using the Luminex^® Multiplexing Instruments (Luminex^® xMAP^® Technology), in a single sample for all of the following markers: interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and monocyte chemoattractant protein-1 (MCP-1).

The demographic and clinical data of the participants were obtained from the medical records, using data from six months before and after the date of sample collection. To evaluate the association between two quantitative variables, the Pearson or Spearman correlation coefficients were estimated. Student’s t-test or the non-parametric Mann-Whitney tests were used for independent samples t for comparison between two groups defined by the erythropoietin resistance index (ERI) that was calculated using the mean weekly dose of the last six months of EPO (micrograms) by their weight (kg) and hemoglobin (Hb) concentration (dose EPO/weight/Hb).

The normality of the variables was evaluated by the Shapiro Wilks test and p-values of 0.05 indicated statistical significance. The data were analyzed using the IBM SPSS Statistics v.20 program.

RESULTS

Population characteristics and clinical and laboratorial data

Thirty-five patients under peritoneal dialysis, at least 30 days previously, accepted to participate and were included in the study. Demographic, clinical and laboratory data are described on Table 1.

Analysis of iron deficiency using ferritin, TSI% and RET-He

Ferritin, TSI%, and RET-He parameters were used to evaluate the availability of iron, following the cutoff criteria proposed by KDIGO (2012)⁽¹⁵⁾ of below 500 μg/l for ferritin, less than or equal to 30% for TSI and less than 29 pg for RET-He. The results are shown in Table 2.

Analysis da erythropoietin resistance

We calculated erythropoietin resistance index (ERI) for 23 individuals and 86.9% (20/23) presented EPO resistance (values greater than 0.02 μg according to KDIGO, 2012⁽¹⁵⁾). However, when the ERI values were classified by quartiles (the cut-off of the 3^rd quartile being 0.102 μg) 30.5% (7/23) were above this cut-off. For those who were in the fourth quartile, there was a significant difference for RET-He (p < 0.05) when compared to other quartiles.

Comparative analysis of erythropoietic activity by RET%, RET#, IRF%, RET-He and ERI

Erythropoietic activity of the bone marrow was evaluated by the reticulocyte parameters (RET%, RET#, IRF%, LFR%, MFR% and HFR% and RET-He), the results of which are shown on Table 3. For each of the reticulocyte variables, we tested the null and alternative hypotheses that the means would be the same or different in the groups, respectively, defined by the fourth quartile (≤ 0.102 or > 0.102). There was a significant difference for the IRF%, MFR% and LFR% (p < 0.05) (Table 4, Figures 1 and 2).

FIGURE 1 – RET# by ERI, 4^th quartile versus other quartiles (0.102)
RET%: count of reticulocytes; ERI: erythropoietin resistance index.

FIGURE 2 – IRF% by ERI, 4^th quartile versus other quartiles (0.102)
IRF%: number of immature reticulocyte; ERI: erythropoietin resistance index.

Analysis of inflammation by biomarkers, reticulocytes parameters and ERI

The values for the inflammatory biomarkers, IL-1β, IL-6, MCP-1 and TNF-α, found in each sample, without exception, were above the maximum reference value reported for each biomarker. IL-6 showed the inverse correlation with reticulocyte parameters (Table 5).

Correlation analysis between hemoglobin and IRF%

Hemoglobin values at the moment of analysis, and after 60 and 180 days were consistently higher in the group of patients with a IRF% lower than 10.5 (Table 6).

DISCUSSION

Anemia is an early and frequent complication in patients with CKD, especially for those on dialysis, and is associated with high morbidity and mortality. However, resistance to ESA treatment associated with chronic inflammation in these patients is an important cause of persistent anemia. In view of limited clinical and laboratory tools currently used for the diagnosis, monitoring and treatment of anemia in CKD, in the present study we analyzed the potential of hematological parameters of reticulocytes, IRF and RET-He, that could improve this process^{(8, 13, 15)}.

In the present study, 32.4% (11/34) of the individuals presented Hb < 11 g/dl, corroborating the findings of Gonçalves & Pecoits-Filho (2013)⁽¹⁶⁾ (38% in a similar population), and Alves et al. (2015)⁽¹³⁾ presenting 34% in patients with CKD on hemodialysis (HD). However, it was lower than that related by Kanbay et al. (2010)⁽¹⁷⁾, who reported about 90% of patients with a glomerular filtration rate of less than 25-30 ml/min with anemia. In view of these findings, the studied population seemed to represent a typical, clinically stable population on peritoneal dialysis, and therefore, adequate for a study of anemia with new diagnostic perspectives.

Iron deficiency is prevalent in 50% of patients with CKD. Ferritin and TSI% are the main available markers of iron stores and systemic iron, however, their values can be overestimated in situations of chronic inflammatory disease such as anemia in CKD and in PD. For this reason, the goal of this study was use reticulocytes parameters IRF and RET-He as markers of iron and erythropoietic activity that are not influenced by inflammation and may better reflect the real status of these two factors. RET-He has been demonstrated to be a marker with good sensitivity and precocity for the assessment of iron deficiency, and the adequacy of iron stores and availability⁽¹⁸⁾. Although there are several studies that suggest reference or cutoff values for RET-He, few refer specifically to patients with CKD and PD. The cut-off value proposed in the present study was suggested by the NKF-KDOQI (29 pg), in agreement with the British Guidelines for Laboratory Diagnosis of Functional Iron Deficiency and with the work of Piva et al. (2014)⁽¹⁹⁾, who demonstrated a higher positive predictive value, when compared with ferritin values below 50 μg/l and STI% < 13%. We observed that only 11.8% (4/34) were below recommended values, which contrasts with the high prevalence of iron deficiency assessed by TSI% or ferritin.

Future studies should establish a reference or cutoff value for monitoring iron availability in based in RET-He. This will potentially add information in clinical practice with no additional cost, since RET-HE are included in the report generated by modern analyzers^{(11, 13)}.

When evaluating the ESA resistance in the complex context of inflammation and iron mobilization, we observed that 86.9% (20/23) of the individuals presented an ERI greater than 0.02 μg, but there was no significant difference when compared to ERI values with those of RET%, RET#, IRF% (LFR%, MFR%, HFR%) and RET-He. However, when ERI was evaluated in quartiles, it seems to be more adequate approach. In this case, there was a significant difference (p < 0.05) when comparing the ERI values of the 4^th quartile and the values for IRF%, MFR% and LFR%, but not for RET% and RET#. This finding is striking, since RET# is the recommended exam for monitoring the management of patients with CKD on dialysis using ESA. As suggested by the International Society of Laboratory Hematology, IRF% and RET# may be useful used together because IRF% means response or acceleration and RET# effectiveness of erythropoiesis activity. Butarello et al. (2016)⁽²⁰⁾ hypothesized that this relationship is variable, independent, and concordant. Elevated IRF% compared to normal or decreased RET# is the standard finding that identifies ineffective erythropoiesis. In the present study we standardized reference values for the parameters RET%, RET#, IRF%, LFR%, MFR% and HFR%, in Sysmex analyzer, model XE 5000, 0.43%-1.36%; 23,000-70,100/μl; 1.6%-10.5%; 89.9%-98.4%; 1.6%-9.5%; 0.0%-1.7%, respectively⁽²¹⁾.

In addition, in the present study, there was a significant difference between IRF% (values ≤ 10.5% and > 10.5%) and values of current Hb, 60 days and 180 days (p < 0.05). We can see that the mean value of Hb was lower for those individuals in whom the IRF% was greater than 10.5%. This fact may suggest two possible and different clinical situations for each group specifically: in the group where IRF% is lower than 10.5% and higher values for hemoglobin, it may indicate a stable and effective erythropoiesis, corroborated by the ERI value, less than 0.102; in the group in which IRF% is higher than 10.5% and with lower hemoglobin values may indicate accelerated but ineffective erythropoietic activity or erythropoietic stress, since in this group ERI values are higher than 0.102, that is, are in the 4^th quartile of resistance to EPO.

Finelly, our study describes IL-6 as the inflammatory marker that demonstrates a correlation with the variables MFR% (p = 0.03) and tendency of association with IRF% and LFR%, both with p values = 0.062, the latter being an inverse correlation. Ganz et al. (2003)⁽²²⁾ positions IL-6 as the main route of activation of hepcidin, directly interfering in the balance of mobility and availability of iron for erythropoiesis. IL-1β and TNF-α suppress the expression of the EPO gene – Canziani et al. (2006)⁽²³⁾.

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