Journal of Global Pharma Technology

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1 Journal of Global Pharma Technology ISSN Available Online at RESEARCH ARTICLE Reticulocyte Hemoglobin Equivalent (RET-HE) As A Predictor and Early Marker for Oral Iron Response in Iron Deficiency Anemia Patients Ketut Suega*, I Made Bakta Hematology-Medical Oncology Division, Faculty of Medicine, Udayana University, Indonesia. *Corresponding Author: Ketut Suega Abstract This study was aimed to determine whether Ret-He has a role for early detection and as a predictor factor for treatment response in iron deficiency anemia patient after oral iron therapy in 4 weeks. Iron deficiency anemia (IDA) patient were diagnosed based on hypochromic microcytic anemia with ferritin level < 20 ng/ml. All patients were taking oral iron pill for 4 weeks. Hemoglobin serum, absolute reticulocyte count (ARC), Ret-He, MCV, MCH, MCHC, and hematocrit were measured. Responders are defined as patients who reach total changes of their hemoglobin level > 2 g/dl in 4 weeks. Data analysis was done by SPSS. There were 26 IDA patients consisted of 15 (57.7%) female and 11 (42.3%) male patients with mean of age was Before treatment, level of Ret-He, absolute reticulocyte count, HB, HCT and MCV were pg (SD 4.43), 0.67 (SD 3.02), 7.94 g/dl (0.74), 26.5 % (SD 2.48), 64.9 fl (SD 0.51) respectively. There was 18 (69.2%) patient considered as responders and 8 (30.8%) patients considered as non-responders. The evaluation showed all markers were increased except for absolute reticulocyte level. On adjusted multiple logistic regression, the only variable that found statistically significant was Ret-He at week 1 with p= 0.031; 95% CI It is suggested that reticulocyte hemoglobin equivalent (Ret-He) proved to be an early and sensitive predictor for oral iron therapy response in patients with iron deficiency anemia. Keywords: Ret-He, Treatment response, Iron deficiency anemia. Introduction Oral iron is considered the best treatment for most of iron deficiency anemia patients especially in the country when financial is still a problem to correct their anemia and to replete iron stored. The potency of their clinical efficiency depends on intestinal bioavailability and intestinal toxicities of its iron formula whether ferric (Fe3*) or ferro (Fe2*). The sequence of response for oral iron therapy can be seen in hour by increasing appetite, after 48 hours there will be bone marrow response with erythroid hyperplasia, reticulocyte peak at 5-7 days later and within 30 days increment of hemoglobin could be seen and iron store depleted in approximately 3-4 months. Ferritin only increased after the iron store is corrected. Several conditions may influence optimal response to iron supplements such as the incomplete course of iron treatment, poor absorption, adverse effect, treatment interrupted and the existence of comorbidities [1]. To monitor and evaluate iron response in patient with anemia, sensitive and immediate parameters are needed in order not to expose least likely beneficial patients from iron toxicities. Several weeks is required before significant hemoglobin increment could be detected. Therefore using hemoglobin as response marker to evaluate successfulness of iron therapy will take 3-4 weeks before iron therapy that being given is considered unsuccessfully [2, 3]. General challenge in monitoring hematologic response hinder by month of mature erythrocyte survival in circulation. Otherwise, hemolytic anemia is presented , JGPT. All Rights Reserved 11

2 erythrocyte survival relatively remain unchanged even in patients with anemia. Around ml of red blood cell will produce daily under normal clinical condition making population of erythrocyte remains constant over time. This situation poses difficulties in using erythrocyte indexes such as MCV, MCHC, and MCH as a marker for immediate iron response. Its take almost 2-3 months elapsing time before changes on MCV clinically detected. In animal experiment, the MCV change was not seen for several weeks after iron deficiency was induced [4, 5]. Bone Marrow erythropoietic activity determines not only by intact stem cells, proper growth factors but also supply of iron molecule in time where it needed the most. Among several of reticulocyte indices such as absolute reticulocyte count (ARC), Immature reticulocyte fraction (IRF) and hemoglobin content of reticulocyte, it is proved that hemoglobin content (CHr = reticulocyte hemoglobin content and Ret-He = equivalent hemoglobin reticulocyte) resemble of iron availabilities in real-time [6, 7]. This is supported by study on intravenous iron with recombinant erythropoietin (r-epo) where administration iron intravenous and r- EPO was not followed by significant increase of ARC and IRF between cases and controls, but hemoglobin content of reticulocyte was found significantly elevated in case when compared to control arm. Another study of r- EPO showed that increasing IRF and percentage of hypochromic cell (%HYPO) in comparable rate suggesting that these indices reflecting bone marrow activities but not instantly iron availability for RBC production. Thus, increment of irf seen on r-epo study simply showed as a marker for erythropoietic response rather than specific information on iron availability real-time [5, 8]. Cellular characteristic of reticulocyte could use as a sensitive response of bone marrow to exclusive iron therapy. Its released to peripheral on common clinical condition to become mature RBC take between 1-2 days except in certain erythropoietic stress such as major bleeding, excessive hemolytic, mature reticulocyte may take longer time 2-3 days. Therefore measurement of hemoglobin content of reticulocyte (CHr and Ret-He) is important to detect immediate iron response in every type of anemia patients due to its short-lived. An immediate iron response marker is better to eliminate unnecessary iron toxicities for those anemia patients where iron therapy is not needed. Parodi et al. study about kinetic response to iron oral in 34 iron deficiency anemia (IDA) children showed that characteristic of reticulocyte cellular content (CHr) prove to be an early response to oral iron therapy. Another study by Miwa et al.(2010) concluded that Ret-He as useful parameter of iron status on patients with regular hemodialysis. Peerschke et al. (2014) measuring Ret-He to evaluate anemia on malignancy and reported that Ret-He is useful tool to evaluate iron deficiency in cancer patients setting [9-11]. This study was aimed to determine whether Ret-He has a rule for early detection and as a predictor factor for treatment response in iron deficiency anemia patient after oral iron therapy in 4 weeks time. Methods Informed consent and study protocol form were approved by hospital institutional review board and study performed principally according to good clinical practice guideline. This study is longitudinal analytic study with repeated measurements. Iron deficiency anemia (IDA) patients were diagnosed based on hypochromic microcytic anemia with ferritin level < 20 ng/ml. Those who fulfill inclusion criteria and did not present with history of prior RBC transfusion, using birth control pills, malignancy, and other severe comorbidities were included in this study. All patient were taking oral iron pill with standard doses for 4 weeks. Start from the beginning and every week afterward hemoglobin serum, absolute reticulocyte count (ARC), Ret-He, MCV, MCH, MCHC, and hematocrit were measured. Ret-He was measured using Sysmex XN series as well as other hematologic parameters. All patients were closely monitored every week for adherence to the oral iron doses, evaluating gastrointestinal toxicities that may be developed and other adverse events while examining all hematologic parameters. Responders is defined for those IDA patients who reach total changes of their hemoglobin level > 2 g/dl in 4 weeks and Non-responders for those who did not. Data were reported as mean and frequency with standard deviation. Distribution of all , JGPT. All Rights Reserved 12

3 data was established by mean of Shapiro- Wilk normality test, mean comparison between responders and non-responders was determined using t independent test. Different within and between group of iron responses measured by repeated measure of generalized linear model, and multivariate logistic model was used to reveal factors that could predict immediate response to oral iron therapy in week four. All tests were done by SPSS for windows and p-value below than 0.05 is accepted. Results There were 26 IDA patients consisted of 15 (57.7%) female and 11 (42.3%) male patients with mean of age patient was years. Patient profile can be seen in Table 1. Table 1: Patients characteristic at diagnosis Variables Case N=26 Mean SD Sex Female (N %) 15 (57.7) Male (N %) 11 (42. 3) Age (Year) Hemoglobin (g/dl) Erythrocyte (10 12 l/l) MCV (fl) MCH (pg) Reticulocyte (10 6 /microl) Ret-He (pg) Serum Iron (micro g/dl) TIBC (micro g/dl) Serum Transferrin (mg/dl) Serum ferritin (micro g/l) HCT (%) All 26 IDA patients completed their four weeks study course taking iron pill for 28 days, and no significant adverse events were noted. By the end of study in which every week all five parameters of iron status were monitored, and results were presented in table 2. There was 18 (69.2%) patient reach total changes of their hemoglobin> 2 mg/dl in 4 weeks considered as responders but 8 (30.8%) patients considered as nonresponders because they did not reach total changes of more than 2 mg/dl in 4 weeks. There was no statistically significant difference in age between responders and non-responders (p= 0.629) as well as no differences on gender also (p= 0.683). Table 2: Comparison of iron parameters from beginning until the end of study Variables Respondent Non-Respondent Value N (%) = 18 N (%)=8 Age (Year) (SD ) (SD ) Sex Male 7 (63.63 %) 4 (36.37%) 0,683 Female 11 (73.33%) 4 (26.67 %) HbW (SD ) (SD ) Hb W (SD ) (SD ) Hb W (SD ) (SD ) Hb W (SD ) (SD ) Hb W (SD ) (SD ) HCT W (SD ) (SD 2,3299) HCT W (SD ) (SD 2,8754) HCT W (SD ) (SD 3,9256) HCT W (SD ) (SD 4,3497) HCT W (SD ) (SD 3,6621) MCV W (SD ) (SD 5,7181) MCV W (SD ) (SD 6,2664) MCV W (SD ) (SD 6,0238) MCV W (SD ) (SD 5,4445) MCV W (SD ) (SD 4,8403) Ret-HeW (SD ) (SD 3,9479) Ret-HeW (SD ) (SD 2,5573) Ret-HeW (SD ) (SD 4,3201) Ret-HeW (SD ) (SD 4,0256) Ret-HeW (SD ) (SD 3,1062) , JGPT. All Rights Reserved 13

4 ReticulocyteW (SD ) (SD 0,1725) Reticulocyte W (SD ) (SD 0,6149) Reticulocyte W (SD ) (SD 0,5231) Reticulocyte W (SD ) (SD 0,2559) Reticulocyte W (SD ) (SD 0,1910) It was also can be seen that every single iron parameter on baseline was not significantly different between responder and nonresponder but our data showed the tendency of more huge differences was detected when more time elapsed toward the end of study at week four. These situations can also be seen in figure 1, where the trend of each iron parameter steadily increased over time base on treatment response. Moreover, this difference was statistically significant (p <0.05). Figure 1: Trend of iron parameters change from baseline until week four Generalize Linear Model Repeated Measure was used to analyze data that weekly measured from baseline until week four. It is usually performed to determine the effect whether within or between subjects as well as interaction effect within subject. Our data on each of iron parameters (Hb, HCT, MCV, Reticulocyte, Ret-He) showed that its sphericity assumption was compromised (p= 0.002) therefore the degree of freedom was adjusted using Greenhouse-Geisser correction because of e < 7.5. Table 3: Performance of several iron parameters as a predictor for treatment response Hemoglobin and treatment response Within subject effect p = Interaction effect within subject p = Between subject effect p = Hematocrit and treatment response Within subject effect p = Interaction effect within subject p = Between subject effect p = MCV and treatment response Within subject effect p = Interaction effect within subject p = Between subject effect p = Reticulocyte and treatment response Within subject effect p = Interaction effect within subject p = Between subject effect p = Ret-He and treatment response Within subject effect p = Interaction effect within subject p = Between subject effect p = , JGPT. All Rights Reserved 14

5 The overall mean of Hb, HCT, MCV, ARC, Ret-He for responder group was significantly higher than for non-responder group (between effect). The increment of Hb, HCT, MCV, ARC, Ret-He each week after oral iron therapy differ significantly than sampling error would predict (within effect). However, interaction of response group with oral iron therapy was significant for Hb and HCT only, meaning that profile of mean of Hb and HCT have different shape (table 3). On multiple logistic regression after adjusting for age, gender and level of hemoglobin at diagnosis, the only variable that found statistically significant was Ret-Heat week 1 with p= 0.031; 95% CI This study result revealed that Ret-He on first week was a significant and important predictor and also an early marker for iron deficiency patients who given oral iron therapy. On comparison of percentage of changes for each of iron parameters as seen on figure 2, showed that even though reticulocyte count increased significantly on week 1 but then followed by very steeply downfall toward the end of the study. Among 5 iron parameters used, this figure depicted that Ret-He stands up as a best predictor when compared to the other parameters. Figure 2: Percentage of changes of Hb, HCT, MCV, Retic, Ret-He from baseline until the end of study Discussion Our study was performed to reveal an early predictor of patient respond when iron pills are given to prevent unnecessary iron toxicities for those anemic patients at least likely benefit from it, since significant clinical response by determining hemoglobin level take approximately 3-4 weeks to be seen. In normal condition, there is an increment of 0.1 mg/dl Hb in a day beginning on day fourth onward [12]. A study by Okam et al. showed that on their meta-analysis of 5 randomized controlled trials Hb was found to be a predictor of longterm and sustained treatment respond after 14 days [3]. During this period, while awaiting Hb level raising to a justifiable clinical switch whether oral iron is needed, definitive early predictor markers may allow clinicians to separate those in need for iron and those who do not in an immediate time. There are no predictor markers that regularly used to predict which iron deficiency anemia patient will and won t respond to oral iron therapy. Oral iron rechallenge test where 60 mg iron is given then 1-2 hours later serum iron is measured. Positive response is considered if serum iron increased. However, this test is not clinically being validated and not commercially available. Other preliminary study revealed that hepcidin level could also be used to differentiate in which patient will have probable response when their hepcidin level is low and is not probable when their hepcidin level is normal or elevated [12, 13]. Reticulocyte hemoglobin content (Ret-He and CHr) could be interpreted after assessing general physiology of the red blood cell (RBC) itself including history of previous transfusion, Vitamin B12 and folic acid , JGPT. All Rights Reserved 15

6 deficiency, iron therapy, thalassemia syndrome and other hemoglobinopathies. Since CHr, not like Ret-He has limited diagnostic value due to their technical measurement that influenced by not only hemoglobin concentration but also mean cell volume, therefore level of CHr will slightly increase once cell volume diminuted as reticulocyte become more mature into RBC. On the other hand, Ret-He, which is also a similar but unidentical reticulocyte content measure by Sysmex 2100, does not depend on cell volume but measure forward scattered of stained reticulocyte and has a linear relationship with CHr [2,10,14]. Iron availability for erythropoietic response was studied by Urrechaga et al. in 90 healthy subjects, 85 patients with IDA and 65 on regular hemodialysis (HD) and 91 patients with IDA. They reported that in IDA patients, Ret-He provide information about individual cell characteristic and represent iron-restricted erythropoiesis. Ret-He provides an accurate tool to identify functional iron deficiency and cellular iron availability. Its level is not influenced by clinical condition such as inflammation and changes in the volume of RBC. Ret-He also important and beneficial since easily measured using standard hematoanalyzer on peripheral blood. There is no requiring additional reagent and can be automatically ordered by previous device simply by installment of software. Ret- He is more markedly early being detected than another conventional iron indicator, knowing that changes of hemoglobin content in precursor cell of RBC occur early and could predict future hemoglobin respond to oral iron and response in Ret-He is acceptably more impressive when compared to changes of hemoglobin content in mature RBC [6, 10]. In our study among Hb, HCT, MCV, ARC and Ret-He that were significantly difference (p <0.05), only Ret-He on first week was found important and statistically significant as an early predictor for treatment respond (p = 0.031; 95%CI ) after adjusting by age, gender, and Hb level. This study result revealed that Ret-He on first week was a significant and important predictor and also an early marker for iron deficiency patients who given oral iron therapy. In a study of Peerschke et al., using Ret-He to evaluate anemia in cancer patients found that Ret-He can rule out iron deficiency anemia apart from other causes of anemia in cancer patients with a negative predictive value (NPV) of 98.5% and 100% respectively, in the study population of 209 patients. This study results outperformed to combination of Hb and MCV to define iron deficiency anemia patient in cancer care setting [11]. In the dialysis population, Ret-He was proved as an excellent diagnostic efficiency to evaluate iron availability to support erythropoietin therapy in chronic kidney patients [15]. In renal anemia, the intense used of r-epo required the amount of iron availability immediately to correct the anemia properly. In a study by Miwa et al. this availability of cellular iron status was measured by Ret-He. They found that Ret-He is a new predictor as well as CHr, is a sensitive and specific marker of iron status in dialysis patients [10]. Study of the effect of iron supplementation on iron deficiency erythropoiesis in patients with pregnancy especially in third trimester also showed that Ret-He proves to be important predictor along with Ret-He/Hb ratio. In this study, Schoorl et al. wanted to establish short-term effect of oral iron on Ret- He and RBC-He in pregnancy patient with suspected iron deficiency erythropoiesis [16]. Another marker for reticulocyte cellular content CHr also found to be a useful indicator for iron deficiency anemia in pediatric population where CHr proved as an early indicator of exclusive oral iron in children with iron deficiency anemia. Multivariate logistic models showed a higher probability of complete responder to absolute reticulocyte count and CHr at 7 days (OR 3.18; 95%CI ) [9]. Beside Ret-He, our study also found other reticulocyte index such as absolute reticulocyte count (ARC) showed a kinetic response assessed within first week from onset of oral iron therapy and our study revealed that Ret-He and ARC even on first week could be used as a sensitive predictor separating those patient will and won t responding to oral iron and eliminating four week delay before switched to other therapies. However, the trend of ARC has the , JGPT. All Rights Reserved 16

7 least consistent with the study period as Ret- He was, due to the reticulocyte maturation indices would shorten when erythropoietic stressed is subside (Figure 2). Indices of reticulocyte and cellular characteristic of reticulocyte could use as a sensitive response of bone marrow to exclusive iron therapy. Reticulocyte will be released to peripheral circulation on normal physiologic condition to become mature RBC in between hours except in certain stress erythropoietic such as major bleeding, excessive hemolytic, maturation of reticulocyte may take time longer between 2-3 days. Therefore, measurement of cellular characteristic of reticulocyte (Ret-He and CHr) important for being used as a marker for early response of oral iron therapy in every type of anemia patients as well as a useful diagnosis of iron deficiency anemia in infant, adults and in children [2,5,17,18]. Ret-He provide an early measure of the response to iron therapy increasing within 2-4 days after the start of iron therapy and repeated measurement of reticulocyte hemoglobin content provide a rapid means to see the erythropoietic response in IDA and kidney failure patients with functional iron deficiency due to short-lived nature of reticulocyte [19,20]. Assessment of iron response status can also be done by determining conventional biochemical marker such as a ferritin, serum iron, TIBC, soluble transferrin receptors, hemoglobin but these biomarkers prone to be compromised by inflammation making them less accurate (misfitted as an indicator). They also fluctuated base on their diurnal phase and difficult to interpret if the measurement is not done within the same period of time. Lots of studies found these traditional markers are highly variable in term of sensitivity and specificity. Thomas and Thomas (2002), reported multivariate biomarker indices such as the index ratio of soluble transferrin receptor (stfr)-ferritin suggestive to define iron deficiency [21]. Despite the limitation of small cohort study, baseline check for thalassemia, ironrefractory IDA and other macrocytic anemia have not done, and this may contribute to minor non-responder patients for oral iron therapy beside non-adherence and incomplete treatment because of gastrointestinal toxicities. Non-adherences due to absorption and toxicities estimate to range from 10% at days 14 to 32% after 2 months [12]. Conclusion Our data suggest that reticulocyte hemoglobin equivalent (Ret-He) prove to be an early and sensitive predictor for oral iron therapy response in patients with iron deficiency anemia and at the same time preventing a month delay in separating those who least likely benefit from iron therapy if hemoglobin is used as a predictor for oral iron therapy. Clinician also could use Ret-He at baseline and during follow-up of iron deficiency erythropoietic activities to define iron supplement response. A Much larger cohort of patients is needed to corroborate our result. References 1. Gupte S, Gupta RK, Gupta R. (2001) Iron deficiency anemia: management and prevention in children. JK science 3: Mast AE, Blinder MA, Dietzen DJ (2008) Reticulocyte hemoglobin content. American Journal of Hematology 83: Okam MM, Koch TA, Tran MH (2015) Iron deficiency anemia treatment response to oral iron therapy: a pooled analysis of five randomized controlled trials. Haematologica (Epub ahead of print). 4. Brugnara C (2000) Reticulocyte Cellular Indices: A New Approach in the Diagnosis of Anemias and Monitoring of Erythropoietic Function. Critical Reviews in Clinical Laboratory Sciences 37(2): Brugnara C, Schiller B, Moran J. (2006) Reticulocyte hemoglobin equivalent (Ret He) and assessment of Iron Deficient states. Clin. Lab. Haem 28: Urrechaga E, Borque L, Escanero JF (2013) Erythrocyte indices in the assessment of erythropoiesis activitity and iron availability. IntJnl. Lab.Hem 35: Piva E, Brugnara C, Spolaore F, Plebani M (2015) Clinical utility of reticulocyte parameters.clinics in Laboratory Medicine 35: , JGPT. All Rights Reserved 17

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