AJCP / Original Article
Using the Hemoglobin Content of Reticulocytes (RET-He)
to Evaluate Anemia in Patients With Cancer
Ellinor I. B. Peerschke, PhD,1,2 Melissa S. Pessin, MD, PhD,1 and Peter Maslak, MD1,2
From the 1Memorial Sloan-Kettering Cancer Center, New York, NY, and 2Weill Cornell Medical College, New York, NY.
Key Words: RET-He; Anemia; Iron deficiency; Cancer; Hematology
Am J Clin Pathol October 2014;142:506-512
DOI: 10.1309/AJCPCVZ5B0BOYJGN
ABSTRACT
Objectives: Evaluation of anemia, particularly iron
deficiency, in patients with cancer is difficult. This study
examined using the hemoglobin content of reticulocytes
(RET-He) to rule out iron deficiency, as defined by serum iron
studies (transferrin saturation <20%, serum iron <40 µg/
dL, and ferritin <100 ng/mL), in an unselected cancer patient
population.
Methods: Patients were entered into the study based on the
existence of concurrent laboratory test requests for CBC and
serum iron studies.
Results: Using a threshold of 32 pg/cell, RET-He ruled out
iron deficiency with a negative predictive value (NPV) of
98.5% and 100%, respectively, in the study population (n =
209) and in a subpopulation of patients with low reticulocyte
counts (n = 19). In comparison, the NPV of traditional CBC
parameters (hemoglobin, <11 g/dL; mean corpuscular
volume, <80 fL) was only 88.5%.
Conclusions: These results support the use of RET-He in the
evaluation of iron deficiency in a cancer care setting.
506 Am J Clin Pathol 2014;142:506-512
506 DOI: 10.1309/AJCPCVZ5B0BOYJGN
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Advanced reticulocyte indices, such as the cellular
hemoglobin content of reticulocytes, designated CHr and
RET-He, are reportable parameters on Siemens ADVIA 2120
(Siemens, Tarrytown, NY) and Sysmex XE and XN series
automated hematology analyzers (Sysmex, Lincolnshire,
IL),1 respectively, without additional blood requirements or
technical intervention. Good agreement between CHr and
RET-He measurements has been reported.2-4 These indices
correlate with iron-deficient erythropoiesis and are useful
markers of iron deficiency in infants and children,5 adult blood
donors,6 geriatric patients,7,8 pregnant women,9 and patients
with chronic kidney disease undergoing hemodialysis.3,4,8,10,11
In addition, the hemoglobin content of reticulocytes is a useful
tool for monitoring patients’ response to iron replacement
therapy12,13 and detecting iron-restricted erythropoiesis in
patients receiving erythropoietin therapy.14-18 However, little
is known about the utility of RET-He in the evaluation of
complex anemias in malignancy.
Although iron deficiency is the leading cause of anemia
worldwide, the anemia of chronic disease is more common
among patients with malignancies. In contrast to iron
deficiency anemia caused by inadequate iron stores, the
anemia of chronic disease is associated with decreased iron
availability despite abundant stores.19-21 The hemoglobin
content of reticulocytes may assist in differentiating between
these forms of anemia22,23 and shows a lower degree of within-
patient variability than ferritin or other serum indicators of
iron status.24
In the current outpatient cancer care setting, assessing
iron deficiency requires additional biochemical tests, results
of which are not available in real time. Iron deficiency
anemia may be inferred from limited information provided
© American Society for Clinical Pathology

by automated CBC and reticulocyte analysis, including
hemoglobin (Hb), hematocrit, and RBC indices. However,
measures of mature erythrocyte indices (mean corpuscular
volume [MCV], mean corpuscular hemoglobin, and RBC
distribution width) cannot detect early iron-deficient
erythropoiesis due to the slow turnover of erythrocytes (~120
days) in circulation.25
In contrast, the hemoglobin content of reticulocytes
reflects the recent functional availability of iron12,26-29 for
erythropoiesis, and results are available in real time as part
of automated reticulocyte analysis. The measurement is
not affected by physiologic interferences, except in cases
of thalassemia30 and macrocytosis/megaloblastosis.31 The
present study, therefore, investigated RET-He, reported by
the Sysmex XE 2100, as a tool to rapidly rule out iron
deficiency, defined by results of serum iron studies in an
outpatient oncology setting. Iron deficiency is associated
with significant morbidity in oncology patients and is readily
treated. Thus, the ability to assess iron deficiency, as part
of automated CBC/reticulocyte analysis, may significantly
enhance patient management, particularly in an outpatient
setting. Since the etiology of anemia in patients with cancer
is complex, involving cytopenias secondary to chemotherapy,
bone marrow failure, chronic disease/inflammation, and/or
iron deficiency, the goal of the present study was to establish
an RET-He cutoff that could rapidly rule out iron deficiency.
The data suggest that RET-He, at a threshold of 32 pg/cell,
may be an important discriminator to rule out iron deficiency
anemia in patients with cancer.
Materials and Methods
Patients and Blood Samples
Remnant peripheral blood samples (n = 255), collected
in EDTA anticoagulant, were retrieved from the Hematology
Laboratory between May 25, 2012, and November 7, 2012,
based on the existence of concurrent laboratory test requests
for CBC analysis and ferritin, as well as sample availability
within 6 hours of blood collection. In 209 cases, orders for
ferritin were accompanied by orders for serum iron and
transferrin saturation. RET-He was quantified using the
Sysmex XE 2100. Chart review was undertaken to obtain
laboratory test results and clinical diagnosis. The study
population consisted of 114 males (aged 17 months to 94
years) and 95 females (aged 11-88 years). Patients were
entered into the study without regard for diagnosis or treatment
and consisted of individuals with solid tumors or hematologic
malignancies who were undergoing and/or recovering from
diverse chemotherapy and/or radiation treatment regimens.
This study was approved by the institutional review board of
© American Society for Clinical Pathology
AJCP / Original Article
Memorial Sloan Kettering Cancer Center for the protection of
human subjects.
Although bone marrow examination is the conventional
gold standard for assessing iron stores, it is not performed
routinely at our center for the sole purpose of identifying iron
deficiency because of its invasive nature. Thus, in the present
study, iron deficiency anemia was defined biochemically
using serum iron (<40 µg/dL), transferrin saturation (<20%),
and Hb (<11 g/dL), with or without ferritin (<100 ng/
mL), as described previously,2,20,21 consistent with current
clinical practice recommendations. Transferrin saturation is
a particularly useful biochemical marker of iron deficiency,
since it reflects the interrelationship between bone marrow
iron stores, iron absorption, and the availability of iron in
circulating blood.32 In contrast, ferritin is less useful in
oncology patients due to its elevation in patients with diverse
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solid tumors and its association with more progressive disease
and shorter survival when elevated.33 Indeed, World Health
Organization guidelines indicate that reliable assessment
of iron status is not possible using ferritin in the presence
of inflammation and infection.34 Thus, the present study
evaluated RET-He performance characteristics against
biochemical markers of iron deficiency, including serum iron
and transferrin saturation with and without ferritin.
Statistical Analysis
The ability of RET-He to identify iron deficiency in
a diverse patient population with cancer was evaluated by
calculating sensitivity, specificity, and positive and negative
predictive values35-37 against routinely ordered serum iron
studies as the diagnostic standard. Differences between
selected data sets were evaluated by performing a t test for
independent samples using Excel Windows 2010 (Microsoft,
Redmond, WA). Statistical differences between data sets were
defined by a P value less than .05. Results were not stratified
by type of malignancy or evidence of marrow infiltration
because of limited sample size.
Results
❚Table 1❚ summarizes major characteristics of the study
population. Patients consisted of approximately equal numbers
of males and females. The mean age of the female population
was slightly younger than that of the male cohort (P = .006),
and as expected, the mean Hb was slightly lower in females
than in males (P = .013). However, a similar proportion of
males and females were anemic based on thresholds of Hb
of 12 g/dL and 11 g/dL, respectively. No differences in MCV,
serum iron, transferrin saturation, or ferritin were noted.
The distribution of RET-He values (pg) in the study
population is illustrated in ❚Figure 1❚. No differences were
Am J Clin Pathol 2014;142:506-512 507
507 DOI: 10.1309/AJCPCVZ5B0BOYJGN 507
Peerschke et al / RET-He and Anemia

❚Table 1❚ 35
Study Population Characteristicsa
30
Characteristic Male Female 25

No. of Patients
No. of patients 114 95 20
Age, y 61.6 ± 20.4b 53.9 ± 16.2b
Median age, y 64 58 15
Hb, g/dL 11.8 ± 2.7b 10.9 ± 2.4b 10
Anemia, No. (%)
Hb <11 g/dL 43 (37.7) 50 (52.6) 5
Hb <12 g/dL 62 (54.3)
MCV, fL 89.8 ± 8.1 90.8 ± 9.4 0
RET-He, pg/cell 33.2 ± 4.3 33.1 ± 4.5

2
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40

>41
1
<2

4
Serum iron, µg/dL 92.9 ± 59.0 83.6 ± 63.9
Transferrin saturation, % 31.9 ± 21.6 29.1 ± 21.0 RET-He (pg)
Ferritin, ng/mL 392 ± 791 404 ± 650
❚Figure 1❚ Distribution of the hemoglobin content of
Hb, hemoglobin; MCV, mean corpuscular volume; RET-He, hemoglobin content of reticulocytes (RET-He) in the study population. The solid line
reticulocytes.
a Values are presented as mean ± SD unless otherwise indicated. indicates the reference range for RET-He: 28 to 36 pg.
b Statistically significant difference (P < .05).

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observed between males and females (P = .831) (Table 1).
Values ranged from 19.7 to 47.6 pg. Although reference
intervals vary among published series, RET-He values
ranging from 28 to 36 pg are generally considered normal.28,31
The distribution of RET-He values for the current patient
population was shifted to the right because of an increase
in the number of patients with macrocytic RBCs (95%
confidence interval for MCV, 73-105 fL). Macrocytosis has
been correlated with an increased RET-He.31
RET-He is a measure of the amount of Hb in reticulocytes
and as such has been identified as a convenient indicator of
iron-deficient and iron-restricted erythropoiesis. In the current
study, statistically significant differences (P < .05) in Hb,
MCV, serum iron, and transferrin saturation were noted when
comparing patients with RET-He levels above and below
the published normal population reference range of 28 pg
❚Table 2❚. Consistent with observations that ferritin is of limited
value as an indicator of iron status in patients with cancer,33
serum ferritin did not correlate significantly with RET-He or
Hb (P = .20) and even showed an inverse relationship with
RET-He, suggesting inflammation and chronic disease and/or
the presence of tumor-derived ferritin.27,33
In the current study of 209 patients, 93 were anemic
with Hb levels less than 11 g/dL, the threshold identified
by Steinmetz et al38 for consideration of iron deficiency in
oncology patients. Iron deficiency anemia was identified in
23 of these patients, based on biochemical studies, including
serum iron and transferrin saturation. In contrast, only nine
patients were identified with iron deficiency anemia based
on the combination of decreased serum iron, transferrin
saturation, and ferritin. Iron deficiency without consideration
for anemia was identified in 34 patients based on decreased
serum iron and transferrin saturation alone and in 18 patients
based on the combination of decreased serum iron, transferrin
saturation, and ferritin.
❚Figure 2❚ illustrates the relationship between the
sensitivity and specificity of RET-He for identifying iron
deficiency, defined using biochemical standards, including
serum iron less than 40 µg/dL and transferrin saturation
less than 20%. An RET-He threshold of 31 to 32 pg/cell
was identified, representing the 34th percentile of RET-He
values in the study population. The same RET-He threshold
was identified when the definition of iron deficiency was
expanded to include low ferritin (<100 ng/mL) and/or low Hb
less than 11 g/dL.
❚Table 3❚ summarizes the ability of RET-He at
thresholds of 31 and 32 pg to rule out iron deficiency in
the study patient population. Uniformly good negative

❚Table 2❚
RET-He Correlationsa

     RET-He <28 pg       RET-He ≥28 pg

Characteristic Mean (SD) Median Mean (SD) Median P Value

Hb, g/dL 9.1 (2.3) 8.9 11.7 (2.6) 11.9 .0000009

MCV, fL 82.3 (8.2) 80.5 90.1 (7.4) 89.5 .0000003
Ferritin, ng/mL 567 (752) 288 370 (724) 96.5 .201
Serum iron, µg/dL 55.5 (50.3) 36 94.0 (518) 78 .001
Transferrin saturation, % 26 (24.5) 13 31.4 (20.8) 25 .210

Hb, hemoglobin; MCV, mean corpuscular volume; RET-He, hemoglobin content of reticulocytes.
a RET-He threshold of 28 pg represents the lower limit of the reference range.

508 Am J Clin Pathol 2014;142:506-512 © American Society for Clinical Pathology

508 DOI: 10.1309/AJCPCVZ5B0BOYJGN
 AJCP / Original Article

Sensitivity 11 g/dL for women or less than 12 g/dL for men. However,
Specificity
the positive predictive value of an abnormal RET-He result
1.2
was low because of the high selected cutoff and the high
1.0 rate of inflammation and chronic disease, leading to iron-
restricted hematopoiesis in the study population.
Sensitivity, Specificity

0.8 In a subanalysis of patients with iron deficiency, defined

by serum iron less than 40 µg/dL and transferrin saturation less
0.6 than 20%, iron deficiency was missed in three of 23 patients
using an RET-He cutoff of less than 32 pg. Interestingly, the
0.4
three observed false-negative cases would not have been
0.2 characterized as iron deficient using in-house reference ranges
for serum iron (34 µg/dL). When serum ferritin (<100 ng/mL)
0.0 was included in the definition of iron deficiency, RET-He
20 25 30 35 40
missed two of nine patients with biochemical iron deficiency.
RET-He (pg)
These two false-negative cases did not meet criteria for
❚Figure 2❚ Comparison of the sensitivity and specificity of anemia (Hb of 15.7 and 11.7 g/dL), and ferritin was normal

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the hemoglobin content of reticulocytes (RET-He) at different
thresholds for the identification of iron deficiency defined
by the combination of serum iron less than 40 µg/dL and
transferrin saturation less than 20% in a diverse oncology
patient population. The circled area represents the clinically
significant cutoff.
predictive values were noted. No difference in RET-He
performance was found when applied to the evaluation of
male and female patients. Indeed, RET-He performance
was better (negative predictive value of 98.5%) than that
of traditional CBC parameters (negative predictive value of
88.5%), consisting of MCV less than 80 fL, and Hb less than
by in-house reference ranges (<22 ng/mL for males and <6
ng/mL for females). Similarly, when monitoring patients (n
= 15) receiving oral iron supplementation, RET-He (<32 pg)
identified two of two patients with residual iron deficiency.
However, three of 13 patients, who were deemed iron replete
based on biochemical studies, were classified as iron deficient
by the high RET-He cutoff. Finally, RET-He (<32 pg) ruled
out iron deficiency (serum iron <40 µg/mL and transferrin
saturation <20%, with or without ferritin <100 ng/mL) with
a negative predictive value of 100% in patients (n = 19) with
low reticulocyte counts (<20 × 109/L). Given the small sample
size, additional prospective studies are required to confirm
RET-He performance in these settings.

❚Table 3❚
Performance of RET-He or the Combination of Hb and MCV in the Evaluation of Iron Deficiency/Iron Deficiency Anemia

MCV Negative Predictive Positive Predictive

Condition RET-He and Hb Sensitivity Specificity Value, % Value, %

Iron deficiency
Transferrin saturation <31 0.758 0.830 94.81 45.45
<20% and serum iron
<40 µg/dL <32 0.789 0.750 94.96 37.14

Transferrin saturation <31 0.778 0.782 98.6 15.2
<20% and serum
iron <40 µg/dL and ferritin <32 0.777 0.709 98.5 11.8
<100 ng/mL

Iron deficiency anemia
Hb <11 g/dL with transferrin <31 1.00 0.697 100.00 5.1
saturation <20%, serum iron
<40 µg/dL, and ferritin <32 1.00 0.697 100.00 5.1
<100 ng/mL

Transferrin saturation <20% Hb <11 g/dL (F) 0.24 0.90 88.5 27.3
and serum iron <40 µg/dL and or <12 g/dL (M) and
ferritin <100 ng/mL MCV <80 fL

Hb <11 g/dL and 0.40 0.84 91.0 26.7
MCV <80 fL

Hb, hemoglobin; MCV, mean corpuscular volume; RET-He, hemoglobin content of reticulocytes.

© American Society for Clinical Pathology Am J Clin Pathol 2014;142:506-512 509

509 DOI: 10.1309/AJCPCVZ5B0BOYJGN 509
Peerschke et al / RET-He and Anemia
Discussion
Anemia is a major cause of morbidity in patients
with cancer. There are multiple causative factors, including
absolute iron deficiency due to blood loss and/or nutritional
deficiencies, anemia of chronic disease, and myelosuppressive
effects of chemotherapy, as well as metastatic infiltration of
the bone marrow. The identification of iron deficiency in
patients with cancer is particularly important in patients
being considered for therapy with erythropoiesis-stimulating
agents.38-40 Approximately 30% to 50% of patients with
cancer with chemotherapy-related anemia have been reported
to experience poor to absent responses to erythropoiesis-
stimulating agents,41,42 and iron therapy has been shown to
improve the response in some of these patients. In addition,
iron therapy has been associated with a reduction in the RBC
transfusion requirements in women with gynecologic cancers
treated with chemoradiotherapy.43,44 Since the identification
of iron deficiency has important therapeutic implications in
oncology patients, current recommendations are to investigate
the cause of anemia if the Hb falls below 11 g/dL.38,39,45
The present study supports the use of RET-He to
rapidly rule out iron deficiency, defined by traditional serum
biochemical markers, in a complex patient population with
cancer, during routine automated CBC and reticulocyte
analysis. To evaluate RET-He in the broadest possible oncology
population, we did not stratify patients by malignancy,
staging, treatment, or transfusion therapy. In this unselected
patient population with cancer, a high negative predictive
value for ruling out iron deficiency was achieved using an
RET-He cutoff of 31 to 32 pg, when iron deficiency was
defined by serum iron and transferrin saturation, with or
without ferritin. In this setting, RET-He outperformed the
combination of Hb and MCV. The MCV (mean RBC volume)
is of limited utility in oncology patients, because is it observed
in the later stages of iron deficiency46-49 and is influenced by
reticulocytosis,20 liver disease, myelodysplastic syndrome,
aplasia, myelofibrosis, and impaired DNA synthesis and cell
division due to chemotherapy.50
Despite the excellent negative predictive value of RET-
He for ruling out iron deficiency in the present study, the
corresponding specificity was low. This was expected based
on the high selected RET-He threshold relative to established
reference ranges28,31 and the high incidence of anemia of
chronic disease in the oncology population. All false-positive
cases identified by an RET-He threshold of 32 pg (n = 50)
were examined by chart review. Indeed, in 40 cases, the anemia
was characterized as anemia of chronic disease in the patient
record. Since anemia of chronic disease is characterized by iron-
restricted hematopoiesis, RET-He levels are expected to be low
and to overlap with those found in iron deficiency.23
In the remaining 10 cases with falsely positive RET-
He values and normal biochemical iron studies, one case
510 Am J Clin Pathol 2014;142:506-512
510 DOI: 10.1309/AJCPCVZ5B0BOYJGN
of a hemoglobinopathy combined with b-thalassemia trait
(heterozygous Hb Mississippi/b-thalassemia) was identified
with expected low RET-He,30 two patients were found to be
iron deficient by biochemical tests on a follow-up visit, and
seven patients had borderline serum iron (<45 µg/dL) and
transferrin saturation (<25%). Further studies are required to
determine how well RET-He distinguishes iron-deficient or
iron-restricted hematopoiesis from bone marrow suppression.
Data from a small subanalysis of patients with low reticulocyte
counts in the present study appear promising.
The hemoglobin content of erythrocytes, measured either
as ADVIA 2120 CHr or Sysmex RET-He, has been reported
to be a reliable indicator of iron-deficient erythropoiesis in
diverse patient populations, including pediatrics, geriatrics,
pregnancy, and particularly chronic kidney disease.5-11 Its
performance characteristics in evaluating anemia in patients
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with cancer, however, are not well understood. This study
provides insight into the utility of measuring RET-He in an
oncology setting, in which patients are routinely monitored
for cytopenias, typically in an outpatient setting. Biochemical
iron studies are often ordered in conjunction with a CBC
and other laboratory tests as part of automated order sets
designed to expedite the interpretation of anemia in patients
at risk. Thus, the ability to obtain information regarding
the availability of iron for erythropoiesis, as part of routine
peripheral blood CBC and reticulocyte analysis, performed
in an outpatient setting before the patient is evaluated by a
specialist, would significantly benefit patient management.
To focus on patients at risk for iron deficiency, we
evaluated RET-He only in patients with orders for CBC and
serum iron studies. This method may have introduced an
unintended bias into the analysis, since only those patients
who had further laboratory studies ordered were included in
the current study. This limitation precludes understanding the
role of RET-He in a broader patient population with cancer.
Other limitations of the study include the small number
of patients who actually had iron deficiency anemia, in
the context of a significant population with iron-restricted
erythropoiesis secondary to inflammation and chronic disease.
In addition, the diagnosis of iron deficiency was inferred from
biochemical studies and medical record review, in the absence
of a gold standard for iron deficiency such as bone marrow
iron studies. Standard biochemical tests of iron metabolism,
such as serum iron, ferritin, and transferrin, are affected by the
acute phase response. During the acute phase, serum iron and
ferritin are increased, whereas transferrin is decreased.19-21
Therefore biochemical markers of iron status are less than
ideal standards for diagnosing iron deficiency. In current
clinical practice, however, bone marrow evaluation to assess
iron status is not performed in most patients with cancer
who have anemia, and iron deficiency is diagnosed based on
results of serum iron studies.
© American Society for Clinical Pathology

In summary, the data support the use of RET-He
to rapidly rule out iron-deficient erythropoiesis, reduce
unnecessary iron studies, and provide rapid diagnostic
information when the automated CBC and reticulocyte
counts are reported. Based on data from the present study,
using RET-He to rule out iron deficiency would reduce the
number of required iron studies by 66% (from 209 to 70)
at our center. If a combination of RET-He (<32 pg) and
anemia (Hb <11 g/dL) were used to rule out iron deficiency
anemia, the number of required iron studies would decrease
by approximately 80% (from 209 to 43). Since RET-
He and its CHr equivalent are routinely available with
automated reticulocyte analysis on Sysmex XE/XN series
and Siemens ADVIA hematology analyzers, respectively,
this parameter can be integrated readily into the diagnostic
algorithm of anemia evaluations. RET-He and CHr cutoff
values reported for the identification of iron deficiency
vary widely in the literature, from 26 to 33 pg, depending
on the study patient population.3-11,51 Thus, customizing
the RET-He threshold to rule out iron deficiency in specific
patient populations is recommended. Of note, the RET-He
threshold of 32 pg/cell, selected in the present study to rule
out iron deficiency, was applicable also to an independent
study cohort described by Canals et al23 to correctly rule out
iron deficiency anemia and iron-restricted hematopoiesis.
Address reprint requests to Dr Peerschke: Dept of Laboratory
Medicine, Memorial Sloan-Kettering Cancer Center, 1275 York
Ave, S317, New York, NY 10065; peersche@mskcc.org.
  This work was supported in part by funds from Sysmex America
(Lincolnshire, IL).
  Acknowledgment: We thank Nenita Francisco for expert
technical assistance.
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