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 1–3 days within the bone marrow and circulate for 1–2 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 con-
tent (CHr or Ret-He) provides an indirect measure of the functional iron available for new red blood cell pro-
duction over the previous 3–4 days. Measurement of reticulocyte hemoglobin content in peripheral blood
samples is useful for diagnosis of iron deficiency in adults (Mast et al., Blood 2002;99:1489–1491) and chil-
dren (Brugnara et al., JAMA 1999;281:2225–2230; Ullrich et al., JAMA 2005;294:924–930; Bakr and Sarette,
Eur J Pediatr 2006;165:442–445). It provides an early measure of the response to iron therapy increasing
within 2–4 days of the initiation of intravenous iron therapy (Brugnara et al., Blood 1994;83:3100–3101).
Sequential measurements of reticulocyte hemoglobin content in patients with iron deficiency anemia
provide a rapid means for assessing the erythropoietic response to iron replacement therapy (Brugnara
et al., Blood 1994;83:3100–3101). It is also an early indicator or iron-restricted erythropoiesis in patients
receiving erythropoietin therapy (Fishbane et al., Kidney Int 1997;52:217–222; Fishbane et al., Kidney Int
2001;60:2406–2411; Mittman et al., Am J Kidney Dis 1997;30:912–922; Tsuchiya et al., Clin Nephrol
2003;59:115–123; Chuang et al., Nephrol Dial Transplant 2003;18:370–377). Thus, reticulocyte hemoglobin
content is a recent addition to an expanding list of biomarkers that can be used to differentiate iron defi-
ciency from other causes of anemia. Am. J. Hematol. 83:307–310, 2008. V C 2007 Wiley-Liss, Inc.

Background also within this range [2,3]. In contrast, serum ferritin had
Diagnostic tests for the evaluation of possible iron defi- an average AUC of 0.95 confirming that it is the best single
ciency anemia include indicators of disrupted heme synthe- test for assessment of body iron stores. However, ferritin is
sis such as zinc protoporphyrin or free erythrocyte proto- an acute phase reactant so its diagnostic utility is limited in
porphyrin, mature erythrocyte indices including MCH, MCV, patients with chronic inflammatory diseases such as rheu-
and RDW, as well as markers of iron stores, uptake, and matoid arthritis or cancer. In studies including anemic
metabolism, which include serum ferritin, serum iron, trans- patients with coexisting inflammatory disease the AUCs for
ferrin saturation, and soluble transferrin receptor. The pub- the diagnosis of iron deficiency by ferritin ranged from 0.57
lished sensitivity and specificity of these markers is highly to 0.89 [1,4,5].
variable. Results vary with patient population and different
‘‘gold standards’’ used to define iron deficiency. The long Technical Aspects and Performance
established gold standard is assessment of stainable mar- CHr is measured during reticulocyte analysis by auto-
row iron; however, due to interobserver variability and the mated hematology analyzers produced by Siemens (Advia
presence of iron stores in patients with anemia of chronic 120 and 2120). It was approved for clinical use on the
inflammation, it is not a perfect gold standard for iron defi- Advia analyzers in the United States by the FDA in 1997.
ciency. Measurement of the erythropoietic response to iron CHr is determined from measurements of light scatter at
therapy is perhaps a better gold standard; yet, it has been two different angles following isovolumetric sphering of oxa-
employed only rarely in published studies. More recently, zine 750-stained reticulocytes. The volume and hemoglobin
multivariate biochemical indices suggestive of decreased concentration of individual reticulocytes are independently
iron stores such as the soluble transferrin receptor (sTfR)- measured from the amount of light scattered at two differ-
ferritin index have been used as a gold standard assay to ent angles [6]. CHr is the product of the cellular volume
define iron deficiency [1]. and the cellular hemoglobin concentration. The hemoglobin
Measures of mature erythrocyte indices obtained on concentration increases and the cell volume decreases as
automated hematology analyzers are not sensitive indica-
tors of early iron deficient erythropoiesis because of the
slow turnover of erythrocytes (120 days) and broad inter- 1
Blood Research Institute, Blood Center of Wisconsin, Milwaukee, WI;
individual variability. Receiver operator characteristics 2
Department of Medicine, Washington University School of Medicine, St.
(ROC) analysis is used to assess the combined sensitivity Louis, MO; 3Department of Pathology and Immunology, Washington Univer-
sity School of Medicine, St. Louis, MO; 4Department of Pediatrics, Washing-
and specificity of diagnostic assays. A test with perfect sen- ton University School of Medicine, St. Louis, MO
sitivity and specificity will have an area under the curve
*Correspondence to: Alan E. Mast, Blood Research Institute, PO Box 2178,
(AUC) of 1.0. The AUC of a test with no diagnostic utility is Milwaukee, WI 53201-2178. E-mail: alan.mast@bcw.edu
0.5. A review of 55 studies assessing the clinical utility of Received for publication 20 July 2007; Revised 6 September 2007; Accepted
mean cellular volume, mean cellular hemoglobin, random 11 September 2007
distribution width, and erythrocyte protoporphyrin displayed Am. J. Hematol. 83:307–310, 2008.
area under the curves (AUCs) between 0.62 and 0.77 Published online 20 November 2007 in Wiley InterScience (www.interscience.
when compared to bone marrow iron content as the gold wiley.com).
standard assay [2]. The AUC for transferrin saturation is DOI: 10.1002/ajh.21090

V
C 2007 Wiley-Liss, Inc.

American Journal of Hematology 307 http://www3.interscience.wiley.com/cgi-bin/jhome/35105

a reticulocyte matures into an erythrocyte; therefore, CHr is transport continues to evolve so will the diagnostic
a more stable parameter than reticulocyte hemoglobin con- approach to anemic patients with suspected iron deficiency.
centration. The Sysmex (XE 2100) hematology analyzer Measurement of either plasma or urine hepcidin holds
measures a similar, but not identical, reticulocyte parameter potential as a useful future test that may help to accurately
called RET-Y that has been available since May 2005 [7]. diagnose iron deficiency in patients with multiple medical
Ret-He is a measure of the forward scatter of stained retic- problems [15].
ulocytes and has a curvilinear relationship with CHr. Ret- CHr has clinical utility in determination of the need for
He values can be mathematically converted into reticulo- iron supplementation in patients with functional iron defi-
cyte hemoglobin concentration values that mirror the CHr ciency. This is particularly important for providing appropri-
values obtained on the Advia analyzers [8–10]. The aver- ate iron therapy to chronic hemodialysis patients receiving
age CHr value for healthy individuals using the Advia ana- erythropoietin, who can have functional iron deficiency and
lyzer has been reported to be 30.8 pg with no difference respond to intravenous iron treatment even with serum fer-
between male and female subjects [3]. ritin over 800 ng/ml [16]. Studies examining the use of CHr
to manage intravenous iron therapy in this group of patients
Applications and Limitations have demonstrated that CHr < 28 pg more accurately pre-
Measures of reticulocyte indices, such as CHr, obtained dicts functional iron deficiency than combined use of ferritin
on automated hematology analyzers have proven to be < 100 ng/ml and transferrin saturation <20%. Additionally,
sensitive indicators of early iron deficient erythropoiesis it has been demonstrated that use of CHr, when compared
because of the 4-day life span of a reticulocyte. Two stud- to use of ferritin and transferrin saturation, significantly
ies have examined the clinical utility of CHr in pediatric reduces intravenous iron exposure in this patient population
patients (age 9 months to 8 years) with comparison to [17–20]. While it is clear that CHr has utility in dosing intra-
other markers. The optimal CHr concentration cutoff value venous iron therapy, it is important to note that no studies
for the diagnosis of iron deficiency differed slightly in these have provided convincing evidence that its use efficiently
two studies at 26.0 and 27.5 pg [11,12]. CHr outperformed limits erythropoietin to the minimum necessary dose [21].
ferritin, MCV, MCH, RDW, and Zn protoporphyrin in detect- Future studies designed to determine if CHr use can effec-
ing iron deficiency defined by decreased hemoglobin and tively limit erythropoietin administration will be particularly
transferrin saturations. In a third study of pediatric patients, relevant in light of recent data indicating potential adverse
a statistically significant discrimination between iron-replete survival in patients with cancer receiving erythropoietin
and iron-deficient children was present using a CHr cutoff therapy [22,23] and increased serious cardiovascular
of 26 pg [13]. In a study of adults presenting for bone mar- events in patients with chronic renal disease treated with
row biopsy, CHr < 28 pg had an optimal sensitivity (74%) erythropoietin [24].
and specificity (73%) for diagnosis of iron deficiency using CHr provides an accurate measure of functional iron
Prussian blue staining of the bone marrow aspirate to available for erythropoiesis over the previous 3–4 days. As
define iron deficiency. In this study, the AUC of CHr such, it is a useful laboratory marker for diagnosis of iron
exceeded that of ferritin, transferrin saturation, and MCV deficiency in adults and children and can be used to iden-
demonstrating that CHr is a useful marker for diagnosis of tify functional iron deficiency in patients receiving erythro-
iron deficiency in adults [3]. poietin therapy. However, because mean cellular volume is
Iron-restricted erythropoiesis can take place despite the used for calculation of CHr, it has diagnostic limitations.
presence of adequate iron. This often occurs in patients CHr is often low in iron replete patients with thalassemia
with an inflammatory disease, such as rheumatoid arthritis and hemoglobinopathies that cause microcytic anemia [3].
and is commonly referred to as the anemia of chronic It can also be elevated in iron-deficient patients with con-
inflammation or the anemia of chronic disease. The meta- founding megalobastic anemia because of the high mean
bolic basis for this so-called functional iron deficiency is cellular volume associated with megaloblastosis [3,25].
thought to be due to inappropriate production of hepcidin, Therefore, it is important that CHr values be interpreted in
an iron regulatory hormone produced by the liver [14]. Hep- the context of the patient’s overall erythrocyte physiology,
cidin is an acute phase reactant that prevents absorption of including knowledge of recent blood transfusions, iron ther-
iron from the gastrointestinal tract, its release from hepatic apy, vitamin B12 or folate deficiency, and the results of he-
stores, and its recycling within the reticuloendothelial sys- moglobin analysis.
tem [15]. In this setting, ferritin and other markers of iron
deficiency may be inappropriately normal so that the diag-
nosis of iron deficiency in patients with underlying inflam- References
1. Thomas C, Thomas L. Biochemical markers and hematologic indices in the
matory disease may be difficult. Thomas and Thomas have diagnosis of functional iron deficiency. Clin Chem 2002;48:1066–1076.
proposed a multivariate classification scheme as an alter- 2. Guyatt GH, Oxman AD, Ali M, et al. Laboratory diagnosis of iron-deficiency
native to a binary outcome (iron deficient or not) applied to anemia: An overview. J Gen Intern Med 1992;7:145–153.
3. Mast AE, Blinder MA, Lu Q, et al. Clinical utility of the reticulocyte hemoglobin
patients under investigation for iron deficiency [1]. These content in the diagnosis of iron deficiency. Blood 2002;99:1489–1491.
authors propose using a ratio of sTfR to the log of serum 4. Markovic M, Majkic-Singh N, Subota V. Usefulness of soluble transferrin re-
ferritin concentration to delineate iron stores and CHr to ceptor and ferritin in iron deficiency and chronic disease. Scand J Clin Lab
assess hemoglobinization of erythrocytes. The use of both Invest 2005;65:571–576.
5. Punnonen K, Irjala K, Rajamaki A. Serum transferrin receptor and its ratio to
sTfR and ferritin limits the influence of inflammatory condi- serum ferritin in the diagnosis of iron deficiency. Blood 1997;89:1052–1057.
tions while CHr marks functional incorporation of iron into 6. Mohandas N, Kim YR, Tycko DH, et al. Accurate and independent measure-
reticulocyte heme. In this manner, patients are classified ment of volume and hemoglobin concentration of individual red cells by laser
into four groups: those with (a) normal erythropoiesis with light scattering. Blood 1986;68:506–513.
7. Buttarello M, Temporin V, Ceravolo R, et al. The new reticulocyte parameter
normal iron stores, (b) normal erythropoeisis with adequate (RET-Y) of the Sysmex XE 2100: Its use in the diagnosis and monitoring of
iron stores, (c) restricted erythropoiesis with adequate iron posttreatment sideropenic anemia. Am J Clin Pathol 2004;121:489–495.
stores, and (d) erythropoeisis restricted by available iron 8. Thomas L, Franck S, Messinger M, et al. Reticulocyte hemoglobin measure-
stores. Such a classification scheme may provide more ment—comparison of two methods in the diagnosis of iron-restricted erythro-
poiesis. Clin Chem Lab Med 2005;43:1193–1202.
practical guidance to managing iron status than individual 9. David O, Grillo A, Ceoloni B, et al. Analysis of red cell parameters on the
biochemical markers of iron status and hemoglobin synthe- Sysmex XE 2100 and ADVIA 120 in iron deficiency and in uraemic chronic
sis. As our understanding of iron absorption and cellular disease. Scand J Clin Lab Invest 2006;66:113–120.

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10. Brugnara C, Schiler B, Moran J. Reticulocyte hemoglobin equivalent (Ret He) 19. Tsuchiya K, Okano H, Teramura M, et al. Content of reticulocyte hemoglobin
and assessment of iron-deficient states. Clin Lab Haematol 2006;28:303– is a reliable tool for determining iron deficiency in dialysis patients. Clin Neph-
308. rol 2003;59:115–123.
11. Brugnara C, Zurakowski D, DiCanzio J, et al. Reticulocyte hemoglobin content 20. Chuang CL, Liu RS, Wei YH, et al. Early prediction of response to intrave-
to diagnose iron deficiency in children. JAMA 1999;281:2225–2230. nous iron supplementation by reticulocyte haemoglobin content and high-
12. Ullrich C, Wu A, Armsby C, et al. Screening healthy infants for iron deficiency fluorescence reticulocyte count in haemodialysis patients. Nephrol Dial Trans-
using reticulocyte hemoglobin content. JAMA 2005;294:924–930. plant 2003;18:370–377.
13. Bakr AF, Sarette G. Measurement of reticulocyte hemoglobin content to diag- 21. Brugnara C. Iron deficiency and erythropoiesis: New diagnostic approaches.
nose iron deficiency in Saudi children. Eur J Pediatr 2006;165:442–445. Clin Chem 2003;49:1573–1578.
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4044. 23. Henke M, Laszig R, Rube C, et al. Erythropoietin to treat head and neck can-
15. Nemeth E, Ganz T. Regulation of iron metabolism by hepcidin. Ann Rev Nutr cer patients with anaemia undergoing radiotherapy: Randomised, double-
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2411. marrow activity and red cell hemoglobinization in iron sufficient beta-thalasse-
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Am J Kidney Dis 1997;30:912–922. matologica 2003;88:631–636.

American Journal of Hematology 309

 The American Journal of Hematology such as rheumatoid arthritis, and is commonly referred to as the anemia of
TEST OF THE MONTH chronic inflammation or the anemia of chronic disease.
What tests are helpful to do with it for a more complete picture?
RETICULOCYTE HEMOGLOBIN CONTENT
When used in combination with ferritin and soluble transferrin receptor a
SUMMARY TABLE complete assessment of the iron stores and functional erythropoiesis of a
patient can be obtained. Performance of additional tests such as serum iron or
What is the test? total iron binding capacity adds little additional information.
Reticulocytes are the youngest erythrocytes released from the bone marrow
What tests provide similar information?
into circulating blood. They mature for 1–3 days within the bone marrow and
No other test provides similar information. Measures of mature erythrocyte
circulate for 1–2 days before becoming mature erythrocytes. Reticulocyte Hb
indices obtained on automated hematology analyzers are not sensitive indica-
content measures the amount of Hb contained in reticulocytes.
tors of early iron deficient erythropoiesis because of the slow turnover of eryth-
How is it measured? rocytes (120 days) and broad interindividual variability.
Reticulocyte hemoglobin content is measured during routine reticulocyte anal-
What tests can be avoided/eliminated?
ysis on automated hematology analyzers from Siemens (CHr) and Sysmex
Studies in pediatric patients have shown that CHr outperformed ferritin, MCV,
(Ret-He). It is the product of the reticulocyte volume and hemoglobin concentra-
MCH, RDW, and Zn protoporphyrin in detecting iron deficiency defined by
tion that are determined by measuring light scatter of individual reticulocytes.
decreased hemoglobin and transferrin saturations. It has been proposed that
What are the normal range values? CHr (either alone or in conjunction with CBC) could be used as a screening
The normal range is 28.9–32.9 pg. Values below 28 pg may indicate the tool for the identification of children with iron deficiency. Serum ferritin, iron, and
presence of iron deficient erythropoiesis. However, low values are also present TIBC could potentially be avoided in assessing iron availability.
in patients with thalassemia and hemoglobinopathy. Values below 27.5 have
How does its use impact treatment?
been shown to have promising sensitivity and specificity to detect iron defi-
Appropriate iron therapy is crucial for patients on chronic hemodialysis
ciency before the development of anemia in healthy 9- to 12-month-old infants.
receiving erythropoietin, who can have functional iron deficiency and respond to
What conditions or types of conditions is it used in? intravenous iron treatment even with serum ferritin over 800 ng/ml. Studies
The reticulocyte hemoglobin content (CHr or Ret-He) provides an indirect examining the use of CHr to manage intravenous iron therapy in this group of
measure of the functional iron available for new red blood cell production over patients have demonstrated that CHr < 28 pg more accurately predicts func-
the previous 3–4 days. In a study of adults presenting for bone marrow biopsy, tional iron deficiency than combined use of ferritin < 100 ng/ml and transferrin
CHr < 28 pg had an optimal sensitivity (74%) and specificity (73%) for diagno- saturation <20%. Use of CHr, when compared to use of ferritin and transferrin
sis of iron deficiency using Prussian blue staining of the bone marrow aspirate saturation, significantly reduces intravenous iron exposure in this patient popu-
to define iron deficiency. In this study, the AUC of CHr exceeded that of ferritin, lation. While it is clear that CHr has utility in dosing intravenous iron therapy, no
transferrin saturation, and MCV demonstrating that CHr is a useful marker for studies have provided convincing evidence that its use efficiently limits erythro-
diagnosis of iron deficiency in adults. poietin to the minimum necessary dose.
In addition to diagnosis of iron deficiency, measurement of reticulocyte hemo-
globin content is useful in determining the need for iron supplementation in
patients receiving erythropoietin, by serving as an early indicator or iron- References
restricted erythropoiesis (with values below the normal range). Measurement of Mast AE, Blinder MA, Lu Q, Flax S, Dietzen DJ. Clinical utility of the reticulocyte hemoglobin content in the
reticulocyte hemoglobin content can significantly impact the treatment of diagnosis of iron deficiency. Blood 2002;99:1489–1491.
Thomas C, Thomas L. Biochemical markers and hematologic indices in the diagnosis of functional iron
patients through rapidly assessing the response to iron therapy in patients with deficiency. Clin Chem 2002;48:1066–1076.
iron deficiency anemia or in patients receiving erythropoietin therapy. Iron- Ullrich C, Wu A, Armsby C, et al. Screening healthy infants for iron deficiency using reticulocyte hemoglobin
restricted erythropoiesis often occurs in patients with an inflammatory disease, content. JAMA 2005;294:924–930.