New red blood cell and reticulocyte parameters and reference values for healthy individuals and in chronic kidney disease

Introduction: The importance of local references values has been well described in the literature; this is because the characteristics of the population may influence the laboratory tests. Objective: To establish the reference range for traditional and extended red blood cell parameters and reticulocyte indices in order to investigate its application in patients with chronic kidney disease (CKD). Materials and methods: 249 blood donors (125 males and 124 females) were selected to establish the reference values. The hemodialysis sample consisted of 62 patients with terminal CKD (48 male and 14 female). All analyzes were performed using the Sysmex XE-5000 automated hematology analyzer. Results: Differences between reference values was observed in relation to gender: red blood cells (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular hemoglobin concentration (MCHC), percentage of hyperchromic red blood cells (%HYPER), percentage of microcytosis (%MICRO), percentage of macrocytosis (%MACRO), absolute reticulocyte count (RET), reticulocyte hemoglobin content (RET-He), immature reticulocyte fraction (IFR), low fluorescence reticulocytes (LFR), medium fluorescence reticulocytes (MFR), and high fluorescence reticulocytes (HFR). Individuals with CKD presented RBC, HGB, HCT, MCHC, red cell distribution width expressed as coefficient of variation (RDW-CV), percentage of hypochromic red blood cells (%HYPO), percentage of reticulocytes (RET%), RET (female group), IFR, LFR, MFR, and HFR results compatible with the anemic state, which can be observed in 91.8% of patients. All studied parameters were in the area under the curve (AUC) > 0.4. In male group, %HYPO (AUC: 0.806) and IFR (AUC: 0.762) presented higher AUC values, while female group presented %HYPO (AUC: 0.806), %HYPER (AUC: 0.815), and IFR (AUC: 0.660). Conclusion: The medical advancement, the development of new techniques and hematological parameters have revealed important information about functional integrity of bone marrow, diagnosis of anemia and recombinant human erythropoietin monitoring therapy used in hemodialysis patients.

intRoDuCtion Automated analysis of red blood cells (RBC) is performed using the impedance methodology for obtaining red blood cell count and determination of its volume, and spectrophotometry to measure the concentration of hemoglobin (HGB) (1) .From these measurements, the RBC indices are calculated to obtain the mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) (2) .Currently, additional analyzes are performed, allowing the evaluation of extended RBC parameters, as percentage of hypochromia: percentage of hypochromic red blood cells (%HYPO), percentage of hyperchromic red blood cells (%HYPER), percentage of microcytosis (%MICRO), and percentage of macrocytosis (%MACRO), as well as its precursor cell, the reticulocyte.
The technique used for reticulocyte counts underwent a remarkable evolution, especially with the introduction of 10.5935/1676-2444.20150014automated counting by flow cytometry, which promoted an increase in accuracy and precision, when compared to manual counts (3,4) .Likewise, there was the introduction of reticulocytes indices that analyze the maturity characteristics of reticulocytes by measuring the fluorescence intensity of the cell, classifying reticulocytes for high, medium and low fluorescence, depending on the degree of cell maturation (5,6) .Another parameter inserted, evaluates the reticulocyte hemoglobin content (RET-He), and reflects the amount of iron available for hemoglobin production in bone marrow, thus it is proposed as a marker of iron stores in the erythrocyte (7,8) .
The clinical application of new parameters is clear and have been demonstrated in the differential diagnosis of microcytic anemia (9) , evaluation of thalassemia (9,10) , and differentiation of iron deficiency anemia in patients with chronic diseases (11) , providing previously unattainable information on erythropoiesis (12) .The assessment of response to therapy with erythropoietin and iron supplementation in chronic renal failure patients undergoing hemodialysis is an important target for treatment follow-up and requires new markers.

oBjECtivE
The objective of this study was to establish the reference range for the traditional and extended erythrocyte parameters and reticulocytes indices in a healthy population, in order to investigate its use in patients with chronic kidney disease (CKD).

Study design
The study was conducted at the Clinical Pathology Laboratory of Hematology Department, together with the Nephrology and Blood Bank services at Hospital São Lucas of Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), Brazil.To establish the reference values, we selected 249 blood donors, which were 125 male and 124 female.The hemodialysis sample consisted of 62 CKD terminal patients of both gender (48 male and 14 female), later, these patients were classified for statistical analysis in anemic and non-anemic patients.According to the definition of the World Health Organization (WHO), hemoglobin below 12 g/dl for women and 13 g/dl for men is indicative of anemia (13) .All included individuals aged 18 years or older and signed the Informed Consent Form.

Laboratory variables
Samples were collected in vacuum tubes with ethylenediamine tetraacetic acid (EDTA) anticoagulant and processed within 4 hours after collection.
All hematologic analyzes were performed using the Sysmex XE-5000 automated hematology analyzer (Sysmex, Kobe, Japan), which allows the evaluation of traditional (erythrocyte count, HGB, hematocrit

Data analysis
The reference values calculation was carried out according to the Clinical and Laboratory Standards Institute (CLSI) guidelines C28-using the MedCalc Statistical Software version 13.3 (Ostend, Belgium) (14) .The other statistical analyzes were performed using the Statistical Package for the Social Sciences (SPSS) version 20.0 for Windows (Chicago, EUA).Kolmogorov-Smirnov test was used to verify the Gaussian distribution of traditional and extended red cell and reticulocyte parameters.The parameters with normal distribution were presented as mean and standard deviation; furthermore, in cases where it was indicated to reject normality, the results were reported as median and interquartile range.Student t-test and Mann-Whitney U-test were performed to detect statistical differences between data from parametric and non-parametric groups, respectively.A p < 0.05 was considered significant.When no statistically significant difference between women and men were observed, the values were analyzed together.The receiver operating characteristic (ROC) curve was used to evaluate the diagnostic performance of new parameters.

Ethical aspects
This study was approved by the Scientific Committee of the Hospital São Lucas of PUCRS, and by the Research Ethics Committee of PUCRS, under protocol No. 09/04527.

Reference values
During the study period, the results of 249 healthy patients were included in the data set, represented by 125 male and 124 female.The reference values were calculated according to CLSI guidelines and are presented in Table 1.
In the comparison between genders, the parameters with p < 0.05 values must be interpreted in different ways, corresponding to RBC, HGB, HCT, MCHC, %HYPER, %MICRO, %MACRO, RET, RET-He, IFR, LFR, MFR, and HFR.However, the other parameters (MCV, MCH, RDW-CV, RBC-He, %HYPO, and RET%) should be considered as a single reference value for both men and women.

Comparison between healthy individuals and CKD patients
The results of traditional and extended erythrocyte parameters and reticulocytes indices of healthy individuals were compared with patients on hemodialysis (Table 2).
The previously described parameters were observed (Table 1) as having a different interpretation between men and women.The groups are made up as follows: men only, composed of 125 healthy individuals and 47 with CKD; only women, 124 healthy and 14 with CKD; and both, men and women, represented by 249 healthy individuals and 62 with CKD.

ROC curve
The results of ROC curve analysis are shown in the Figure.The variables that showed distinct reference values between genders were described in two groups, male (n = 172), with 57 individuals classified as anemic, and female (n = 138), with 33 anemic women.In addition, a third group was composed of both sexes (n = 310), with 90 anemic.All the studied parameters showed area under the curve (AUC) > 0.4.The discriminative ability to predict anemia were: for the male group %HYPO (AUC: 0,806), IFR (AUC: 0,762) with their respective fractions LFR (AUC: 0,778), MFR (AUC: 0,779), and HFR (AUC: 0,767); for the female group %HYPO (AUC: 0,806), %HYPER (AUC: 0,815), IFR (AUC: 0,660) with their respective fractions LFR (AUC: 0,660), MFR (AUC: 0,673), and HFR (AUC: 0,529) (Table 3).renal failure is the %HYPO.Bovy et al. (20) study demonstrated a significant increase in the value of %HYPO parameter iron deficient patients, as well as in the present study, in which individuals with CKD had an increased value of this parameter in relation to the healthy individuals group, indicating the presence of hypochromic red cell in hemodialysis patients, confirmed by the of the area under the curve analysis (AUC: 0.806), and was considered a good marker for iron deficiency.
The reticulocytes indices show a direct view of the bone marrow and the supply and use of iron, along with a response to erythropoietin therapy (21) .In our study, RET% and RET (female group) in individuals with CKD showed statistical difference (p < 0.001) compared to healthy subjects, showing that in these patients, the bone marrow is trying to compensate for the HGB and iron decrease, increasing the number of reticulocytes in peripheral blood.However, there was no statistical difference in the RET-He parameter, unlike observed by Brugnara et al. (7) , who reported the use of RET-He as a reliable marker for identifying the presence of iron deficiency with an AUC 0.913.In this study there was an increase of immature reticulocyte fractions (IFR, LFR, MFR, and HFR) in patients with CKD, as observed by João et al. (6) and Choi et al. (22) , who reported that iron deficiency anemia is associated with an increase to approximately twice of IFR and reticulocyte fraction with average levels of fluorescence, and an increase four times of the reticulocytes with high fluorescence levels.The fluorescence intensity is directly related to the RNA intracellular level, and, therefore, with the degree of reticulocytes maturation, suggesting that iron deficiency anemia is associated with an increase in the proportion of IFR, related to increased erythropoietic activity of bone marrow.
The variation of sensitivity and specificity (ROC curve) of new parameters indicated important results for the establishment of cut-off points and their clinical applications.All correlated parameters had an AUC > 0.4, demonstrating that it was able to discriminate patients and healthy individuals.In addition, the study also demonstrated satisfactory performance with an AUC > 0.7 for the reticulocytes indices (%HYPO, %HYPER, IFR, LFR, MFR, and HFR), ensuring reliability of the results obtained and potential clinical application (23) .

ConCLuSion
With the advancement of medicine over the years and the emergence of new hematological techniques that bring more reliability to the results of parameters described in the literature (RBC, HGB, HCT, MCV, MCHC e RDW-CV), as well as for new extended erythrocyte parameters and reticulocyte indices, increasingly mentioned in studies, it has been obtained important
Our study addressed the establishment of local reference values for traditional and extended red cell and reticulocyte parameters as a tool so that clinical laboratories obtain their own results in a standardized way, and we deepen furthermore the subject about the use of new red cell parameters and its application in individuals with CKD, in an attempt to review the use of old exams (HGB and HCT), proposing new markers to assist the monitoring of erythropoietic response in these patients.
[HCT], MCV, MCH, MCHC and red cell distribution width expressed as coefficient of variation [RDW-CV]) and extended (red blood cell hemoglobin content [RBC-He], %HYPO, %HYPER, %MICRO, and %MACRO) erythrocyte parameters, and reticulocytes indices (absolute reticulocyte count [RET] and percentage of reticulocyte [RET%], low fluorescence reticulocytes [LFR], medium fluorescence reticulocytes [MFR], high fluorescence reticulocytes [HFR], immature reticulocyte fraction [IFR], and [RET-He]).In the reticulocyte channel, blood cells are stained with a fluorescent dye, polymethine, which is specific for identification of ribonucleic acid (RNA)/deoxyribonucleic acid (DNA), and then analyzed by flow cytometry.The daily quality control was performed using the three levels of commercial control Sysmex e-Check.The interlaboratory quality was monitored by participating in Laboratory Proficiency Testing program (Proficiência em Ensaios Laboratoriais [PELM]) of the Brazilian Society of Clinical Pathology (Sociedade Brasileira de Patologia Clínica).

taBLE 2 −
Comparison between traditional and extended erythrocyte parameters and reticulocyte indices of healthy individuals and CKD patients Individuals with CKD showed RBC, HGB, HCT, MCHC, RDW-CV, %HYPO, RET%, RET (female group), IFR, LFR, MFR, and HFR results compatible with the anemic state, which can be observed in 91.8% of these patients.