TEST OF THE MONTH

Reticulocyte hemoglobin content

Alan E. Mast,1* Morey A. Blinder,2,3 and Dennis J. Dietzen4
Under normal conditions, reticulocytes are the youngest erythrocytes released from the bone marrow into circulating blood. They mature for 13 days within the bone marrow and circulate for 12 days before becoming mature erythrocytes. Measurement of cellular hemoglobin concentration has long been reported by automated hematology analyzers as one of the red blood cell indices. The reticulocyte hemoglobin content (CHr or Ret-He) provides an indirect measure of the functional iron available for new red blood cell production over the previous 34 days. Measurement of reticulocyte hemoglobin content in peripheral blood samples is useful for diagnosis of iron deciency in adults (Mast et al., Blood 2002;99:14891491) and children (Brugnara et al., JAMA 1999;281:22252230; Ullrich et al., JAMA 2005;294:924930; Bakr and Sarette, Eur J Pediatr 2006;165:442445). It provides an early measure of the response to iron therapy increasing within 24 days of the initiation of intravenous iron therapy (Brugnara et al., Blood 1994;83:31003101). Sequential measurements of reticulocyte hemoglobin content in patients with iron deciency anemia provide a rapid means for assessing the erythropoietic response to iron replacement therapy (Brugnara et al., Blood 1994;83:31003101). It is also an early indicator or iron-restricted erythropoiesis in patients receiving erythropoietin therapy (Fishbane et al., Kidney Int 1997;52:217222; Fishbane et al., Kidney Int 2001;60:24062411; Mittman et al., Am J Kidney Dis 1997;30:912922; Tsuchiya et al., Clin Nephrol 2003;59:115123; Chuang et al., Nephrol Dial Transplant 2003;18:370377). Thus, reticulocyte hemoglobin content is a recent addition to an expanding list of biomarkers that can be used to differentiate iron deC 2007 Wiley-Liss, Inc. ciency from other causes of anemia. Am. J. Hematol. 83:307310, 2008. V

Background Diagnostic tests for the evaluation of possible iron deciency anemia include indicators of disrupted heme synthesis such as zinc protoporphyrin or free erythrocyte protoporphyrin, mature erythrocyte indices including MCH, MCV, and RDW, as well as markers of iron stores, uptake, and metabolism, which include serum ferritin, serum iron, transferrin saturation, and soluble transferrin receptor. The published sensitivity and specicity of these markers is highly variable. Results vary with patient population and different gold standards used to dene iron deciency. The long established gold standard is assessment of stainable marrow iron; however, due to interobserver variability and the presence of iron stores in patients with anemia of chronic inammation, it is not a perfect gold standard for iron deciency. Measurement of the erythropoietic response to iron therapy is perhaps a better gold standard; yet, it has been employed only rarely in published studies. More recently, multivariate biochemical indices suggestive of decreased iron stores such as the soluble transferrin receptor (sTfR)ferritin index have been used as a gold standard assay to dene iron deciency [1]. Measures of mature erythrocyte indices obtained on automated hematology analyzers are not sensitive indicators of early iron decient erythropoiesis because of the slow turnover of erythrocytes (120 days) and broad interindividual variability. Receiver operator characteristics (ROC) analysis is used to assess the combined sensitivity and specicity of diagnostic assays. A test with perfect sensitivity and specicity will have an area under the curve (AUC) of 1.0. The AUC of a test with no diagnostic utility is 0.5. A review of 55 studies assessing the clinical utility of mean cellular volume, mean cellular hemoglobin, random distribution width, and erythrocyte protoporphyrin displayed area under the curves (AUCs) between 0.62 and 0.77 when compared to bone marrow iron content as the gold standard assay [2]. The AUC for transferrin saturation is
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also within this range [2,3]. In contrast, serum ferritin had an average AUC of 0.95 conrming that it is the best single test for assessment of body iron stores. However, ferritin is an acute phase reactant so its diagnostic utility is limited in patients with chronic inammatory diseases such as rheumatoid arthritis or cancer. In studies including anemic patients with coexisting inammatory disease the AUCs for the diagnosis of iron deciency by ferritin ranged from 0.57 to 0.89 [1,4,5]. Technical Aspects and Performance CHr is measured during reticulocyte analysis by automated hematology analyzers produced by Siemens (Advia 120 and 2120). It was approved for clinical use on the Advia analyzers in the United States by the FDA in 1997. CHr is determined from measurements of light scatter at two different angles following isovolumetric sphering of oxazine 750-stained reticulocytes. The volume and hemoglobin concentration of individual reticulocytes are independently measured from the amount of light scattered at two different angles [6]. CHr is the product of the cellular volume and the cellular hemoglobin concentration. The hemoglobin concentration increases and the cell volume decreases as
Blood Research Institute, Blood Center of Wisconsin, Milwaukee, WI; Department of Medicine, Washington University School of Medicine, St. Louis, MO; 3Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO; 4Department of Pediatrics, Washington University School of Medicine, St. Louis, MO
2 1

*Correspondence to: Alan E. Mast, Blood Research Institute, PO Box 2178, Milwaukee, WI 53201-2178. E-mail: alan.mast@bcw.edu Received for publication 20 July 2007; Revised 6 September 2007; Accepted 11 September 2007 Am. J. Hematol. 83:307310, 2008. Published online 20 November 2007 in Wiley InterScience (www.interscience. wiley.com). DOI: 10.1002/ajh.21090

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a reticulocyte matures into an erythrocyte; therefore, CHr is a more stable parameter than reticulocyte hemoglobin concentration. The Sysmex (XE 2100) hematology analyzer measures a similar, but not identical, reticulocyte parameter called RET-Y that has been available since May 2005 [7]. Ret-He is a measure of the forward scatter of stained reticulocytes and has a curvilinear relationship with CHr. RetHe values can be mathematically converted into reticulocyte hemoglobin concentration values that mirror the CHr values obtained on the Advia analyzers [810]. The average CHr value for healthy individuals using the Advia analyzer has been reported to be 30.8 pg with no difference between male and female subjects [3]. Applications and Limitations Measures of reticulocyte indices, such as CHr, obtained on automated hematology analyzers have proven to be sensitive indicators of early iron decient erythropoiesis because of the 4-day life span of a reticulocyte. Two studies have examined the clinical utility of CHr in pediatric patients (age 9 months to 8 years) with comparison to other markers. The optimal CHr concentration cutoff value for the diagnosis of iron deciency differed slightly in these two studies at 26.0 and 27.5 pg [11,12]. CHr outperformed ferritin, MCV, MCH, RDW, and Zn protoporphyrin in detecting iron deciency dened by decreased hemoglobin and transferrin saturations. In a third study of pediatric patients, a statistically signicant discrimination between iron-replete and iron-decient children was present using a CHr cutoff of 26 pg [13]. In a study of adults presenting for bone marrow biopsy, CHr < 28 pg had an optimal sensitivity (74%) and specicity (73%) for diagnosis of iron deciency using Prussian blue staining of the bone marrow aspirate to dene iron deciency. In this study, the AUC of CHr exceeded that of ferritin, transferrin saturation, and MCV demonstrating that CHr is a useful marker for diagnosis of iron deciency in adults [3]. Iron-restricted erythropoiesis can take place despite the presence of adequate iron. This often occurs in patients with an inammatory disease, such as rheumatoid arthritis and is commonly referred to as the anemia of chronic inammation or the anemia of chronic disease. The metabolic basis for this so-called functional iron deciency is thought to be due to inappropriate production of hepcidin, an iron regulatory hormone produced by the liver [14]. Hepcidin is an acute phase reactant that prevents absorption of iron from the gastrointestinal tract, its release from hepatic stores, and its recycling within the reticuloendothelial system [15]. In this setting, ferritin and other markers of iron deciency may be inappropriately normal so that the diagnosis of iron deciency in patients with underlying inammatory disease may be difcult. Thomas and Thomas have proposed a multivariate classication scheme as an alternative to a binary outcome (iron decient or not) applied to patients under investigation for iron deciency [1]. These authors propose using a ratio of sTfR to the log of serum ferritin concentration to delineate iron stores and CHr to assess hemoglobinization of erythrocytes. The use of both sTfR and ferritin limits the inuence of inammatory conditions while CHr marks functional incorporation of iron into reticulocyte heme. In this manner, patients are classied into four groups: those with (a) normal erythropoiesis with normal iron stores, (b) normal erythropoeisis with adequate iron stores, (c) restricted erythropoiesis with adequate iron stores, and (d) erythropoeisis restricted by available iron stores. Such a classication scheme may provide more practical guidance to managing iron status than individual biochemical markers of iron status and hemoglobin synthesis. As our understanding of iron absorption and cellular

transport continues to evolve so will the diagnostic approach to anemic patients with suspected iron deciency. Measurement of either plasma or urine hepcidin holds potential as a useful future test that may help to accurately diagnose iron deciency in patients with multiple medical problems [15]. CHr has clinical utility in determination of the need for iron supplementation in patients with functional iron deciency. This is particularly important for providing appropriate iron therapy to chronic hemodialysis patients receiving erythropoietin, who can have functional iron deciency and respond to intravenous iron treatment even with serum ferritin over 800 ng/ml [16]. Studies examining the use of CHr to manage intravenous iron therapy in this group of patients have demonstrated that CHr < 28 pg more accurately predicts functional iron deciency than combined use of ferritin < 100 ng/ml and transferrin saturation <20%. Additionally, it has been demonstrated that use of CHr, when compared to use of ferritin and transferrin saturation, signicantly reduces intravenous iron exposure in this patient population [1720]. While it is clear that CHr has utility in dosing intravenous iron therapy, it is important to note that no studies have provided convincing evidence that its use efciently limits erythropoietin to the minimum necessary dose [21]. Future studies designed to determine if CHr use can effectively limit erythropoietin administration will be particularly relevant in light of recent data indicating potential adverse survival in patients with cancer receiving erythropoietin therapy [22,23] and increased serious cardiovascular events in patients with chronic renal disease treated with erythropoietin [24]. CHr provides an accurate measure of functional iron available for erythropoiesis over the previous 34 days. As such, it is a useful laboratory marker for diagnosis of iron deciency in adults and children and can be used to identify functional iron deciency in patients receiving erythropoietin therapy. However, because mean cellular volume is used for calculation of CHr, it has diagnostic limitations. CHr is often low in iron replete patients with thalassemia and hemoglobinopathies that cause microcytic anemia [3]. It can also be elevated in iron-decient patients with confounding megalobastic anemia because of the high mean cellular volume associated with megaloblastosis [3,25]. Therefore, it is important that CHr values be interpreted in the context of the patients overall erythrocyte physiology, including knowledge of recent blood transfusions, iron therapy, vitamin B12 or folate deciency, and the results of hemoglobin analysis. References
1. Thomas C, Thomas L. Biochemical markers and hematologic indices in the diagnosis of functional iron deciency. Clin Chem 2002;48:10661076. 2. Guyatt GH, Oxman AD, Ali M, et al. Laboratory diagnosis of iron-deciency anemia: An overview. J Gen Intern Med 1992;7:145153. 3. Mast AE, Blinder MA, Lu Q, et al. Clinical utility of the reticulocyte hemoglobin content in the diagnosis of iron deciency. Blood 2002;99:14891491. 4. Markovic M, Majkic-Singh N, Subota V. Usefulness of soluble transferrin receptor and ferritin in iron deciency and chronic disease. Scand J Clin Lab Invest 2005;65:571576. 5. Punnonen K, Irjala K, Rajamaki A. Serum transferrin receptor and its ratio to serum ferritin in the diagnosis of iron deciency. Blood 1997;89:10521057. 6. Mohandas N, Kim YR, Tycko DH, et al. Accurate and independent measurement of volume and hemoglobin concentration of individual red cells by laser light scattering. Blood 1986;68:506513. 7. Buttarello M, Temporin V, Ceravolo R, et al. The new reticulocyte parameter (RET-Y) of the Sysmex XE 2100: Its use in the diagnosis and monitoring of posttreatment sideropenic anemia. Am J Clin Pathol 2004;121:489495. 8. Thomas L, Franck S, Messinger M, et al. Reticulocyte hemoglobin measurementcomparison of two methods in the diagnosis of iron-restricted erythropoiesis. Clin Chem Lab Med 2005;43:11931202. 9. David O, Grillo A, Ceoloni B, et al. Analysis of red cell parameters on the Sysmex XE 2100 and ADVIA 120 in iron deciency and in uraemic chronic disease. Scand J Clin Lab Invest 2006;66:113120.

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10. Brugnara C, Schiler B, Moran J. Reticulocyte hemoglobin equivalent (Ret He) and assessment of iron-decient states. Clin Lab Haematol 2006;28:303 308. 11. Brugnara C, Zurakowski D, DiCanzio J, et al. Reticulocyte hemoglobin content to diagnose iron deciency in children. JAMA 1999;281:22252230. 12. Ullrich C, Wu A, Armsby C, et al. Screening healthy infants for iron deciency using reticulocyte hemoglobin content. JAMA 2005;294:924930. 13. Bakr AF, Sarette G. Measurement of reticulocyte hemoglobin content to diagnose iron deciency in Saudi children. Eur J Pediatr 2006;165:442445. 14. Roy CN, Mak HH, Akpan I, et al. Hepcidin antimicrobial peptide transgenic mice exhibit features of the anemia of inammation. Blood 2007;109:4038 4044. 15. Nemeth E, Ganz T. Regulation of iron metabolism by hepcidin. Ann Rev Nutr 2006;26:323342. 16. Kopelman RC, Smith L, Peoples L, et al. Functional iron deciency in hemodialysis patients with high ferritin. Hemodial Int 2007;11:238246. 17. Fishbane S, Shapiro W, Dutka P, et al. A randomized trial of iron deciency testing strategies in hemodialysis patients1. Kidney Int 2001;60:2406 2411. 18. Mittman N, Sreedhara R, Mushnick R, et al. Reticulocyte hemoglobin content predicts functional iron deciency in hemodialysis patients receiving rHuEPO. Am J Kidney Dis 1997;30:912922.

19. Tsuchiya K, Okano H, Teramura M, et al. Content of reticulocyte hemoglobin is a reliable tool for determining iron deciency in dialysis patients. Clin Nephrol 2003;59:115123. 20. Chuang CL, Liu RS, Wei YH, et al. Early prediction of response to intravenous iron supplementation by reticulocyte haemoglobin content and highuorescence reticulocyte count in haemodialysis patients. Nephrol Dial Transplant 2003;18:370377. 21. Brugnara C. Iron deciency and erythropoiesis: New diagnostic approaches. Clin Chem 2003;49:15731578. 22. Leyland-Jones B. Breast cancer trial with erythropoietin terminated unexpectedly. Lancet Oncol 2003;4:459460. 23. Henke M, Laszig R, Rube C, et al. Erythropoietin to treat head and neck cancer patients with anaemia undergoing radiotherapy: Randomised, doubleblind, placebo-controlled trial. Lancet 2003;362:12551260. 24. Singh AK, Szczech L, Tang KL, et al. Correction of anemia with epoetin alfa in chronic kidney disease. N Engl J Med 2006;355:2085 2098. 25. Skarmoutsou C, Papassotiriou I, Traeger-Synodinos J, et al. Erythroid bone marrow activity and red cell hemoglobinization in iron sufcient beta-thalassemia heterozygotes as reected by soluble transferrin receptor and reticulocyte hemoglobin in content. Correlation with genotypes and Hb A(2) levels. Haematologica 2003;88:631636.

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The American Journal of Hematology TEST OF THE MONTH

such as rheumatoid arthritis, and is commonly referred to as the anemia of chronic inammation or the anemia of chronic disease. What tests are helpful to do with it for a more complete picture? When used in combination with ferritin and soluble transferrin receptor a complete assessment of the iron stores and functional erythropoiesis of a patient can be obtained. Performance of additional tests such as serum iron or total iron binding capacity adds little additional information. What tests provide similar information? No other test provides similar information. Measures of mature erythrocyte indices obtained on automated hematology analyzers are not sensitive indicators of early iron decient erythropoiesis because of the slow turnover of erythrocytes (120 days) and broad interindividual variability. What tests can be avoided/eliminated? Studies in pediatric patients have shown that CHr outperformed ferritin, MCV, MCH, RDW, and Zn protoporphyrin in detecting iron deciency dened by decreased hemoglobin and transferrin saturations. It has been proposed that CHr (either alone or in conjunction with CBC) could be used as a screening tool for the identication of children with iron deciency. Serum ferritin, iron, and TIBC could potentially be avoided in assessing iron availability. How does its use impact treatment? Appropriate iron therapy is crucial for patients on chronic hemodialysis receiving erythropoietin, who can have functional iron deciency and respond to intravenous iron treatment even with serum ferritin over 800 ng/ml. Studies examining the use of CHr to manage intravenous iron therapy in this group of patients have demonstrated that CHr < 28 pg more accurately predicts functional iron deciency than combined use of ferritin < 100 ng/ml and transferrin saturation <20%. Use of CHr, when compared to use of ferritin and transferrin saturation, signicantly reduces intravenous iron exposure in this patient population. While it is clear that CHr has utility in dosing intravenous iron therapy, no studies have provided convincing evidence that its use efciently limits erythropoietin to the minimum necessary dose.

RETICULOCYTE HEMOGLOBIN CONTENT SUMMARY TABLE

What is the test? Reticulocytes are the youngest erythrocytes released from the bone marrow into circulating blood. They mature for 13 days within the bone marrow and circulate for 12 days before becoming mature erythrocytes. Reticulocyte Hb content measures the amount of Hb contained in reticulocytes.

How is it measured? Reticulocyte hemoglobin content is measured during routine reticulocyte analysis on automated hematology analyzers from Siemens (CHr) and Sysmex (Ret-He). It is the product of the reticulocyte volume and hemoglobin concentration that are determined by measuring light scatter of individual reticulocytes.

What are the normal range values? The normal range is 28.932.9 pg. Values below 28 pg may indicate the presence of iron decient erythropoiesis. However, low values are also present in patients with thalassemia and hemoglobinopathy. Values below 27.5 have been shown to have promising sensitivity and specicity to detect iron deciency before the development of anemia in healthy 9- to 12-month-old infants.

References
Mast AE, Blinder MA, Lu Q, Flax S, Dietzen DJ. Clinical utility of the reticulocyte hemoglobin content in the diagnosis of iron deciency. Blood 2002;99:14891491. Thomas C, Thomas L. Biochemical markers and hematologic indices in the diagnosis of functional iron deciency. Clin Chem 2002;48:10661076. Ullrich C, Wu A, Armsby C, et al. Screening healthy infants for iron deciency using reticulocyte hemoglobin content. JAMA 2005;294:924930.

What conditions or types of conditions is it used in? The reticulocyte hemoglobin content (CHr or Ret-He) provides an indirect measure of the functional iron available for new red blood cell production over the previous 34 days. In a study of adults presenting for bone marrow biopsy, CHr < 28 pg had an optimal sensitivity (74%) and specicity (73%) for diagnosis of iron deciency using Prussian blue staining of the bone marrow aspirate to dene iron deciency. In this study, the AUC of CHr exceeded that of ferritin, transferrin saturation, and MCV demonstrating that CHr is a useful marker for diagnosis of iron deciency in adults. In addition to diagnosis of iron deciency, measurement of reticulocyte hemoglobin content is useful in determining the need for iron supplementation in patients receiving erythropoietin, by serving as an early indicator or ironrestricted erythropoiesis (with values below the normal range). Measurement of reticulocyte hemoglobin content can signicantly impact the treatment of patients through rapidly assessing the response to iron therapy in patients with iron deciency anemia or in patients receiving erythropoietin therapy. Ironrestricted erythropoiesis often occurs in patients with an inammatory disease,