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Brief report

Clinical utility of the reticulocyte hemoglobin content in the diagnosis of

iron deficiency
Alan E. Mast, Morey A. Blinder, Qing Lu, Sherri Flax, and Dennis J. Dietzen

Determination of the reticulocyte hemoglo-
bin content (CHr) provides an early measure
of functional iron deficiency because reticu-
locytes are the earliest erythrocytes re-
leased into blood and circulate for only 1 to
2 days. The CHr in 78 patients undergoing
bone marrow examination was measured to
assess its clinical utility for the diagnosis of
iron deficiency. Twenty-eight patients were
iron deficient, based on the lack of stainable
iron in the aspirate. The diagnostic power of
CHr is limited in patients with high mean
cellular volume (MCV) or red cell disorders
such as thalassemia. However, when pa-
tients with MCV more than 100 fL are ex-
cluded, receiver operator curve analysis of
CHr, ferritin, transferrin saturation, and MCV
demonstrates that CHr has the highest over-
all sensitivity and specificity of these periph-
eral blood tests for predicting the absence
of bone marrow iron stores. (Blood. 2002;
99:1489-1491)
© 2002 by The American Society of Hematology

Introduction
Accurate, early diagnosis of iron deficiency is essential because it may
be the presenting sign of a gastrointestinal malignancy.1 Numerous
peripheral blood tests are performed to diagnose iron deficiency,
including ferritin, transferrin saturation, serum iron, and mean cellular
volume (MCV). Serum ferritin of 12 ␮g/L or less is the most specific
indicator of iron deficiency.2 However, because it is an acute-phase
reactant, ferritin may be normal or increased in iron-deficient patients
with other medical problems. Because reticulocytes are the earliest
erythrocytes released into blood and circulate for only 1 to 2 days, the
reticulocyte hemoglobin content (CHr) provides a measure of iron
available to red cells recently produced by the bone marrow. CHr has
been shown to be an early indicator of iron-restricted erythropoiesis in
patients receiving erythropoietin therapy3-7 and is a strong predictor of
iron deficiency in children.8 However, a study evaluating the ability of
CHr to predict bone marrow iron stores has not been performed.
Study design
Subjects
measurements of light scatter at 2 different angles after isovolumetric
sphering of oxazine 750-stained reticulocytes. From the amount of light
scattered at the 2 different angles, the hemoglobin concentration and
cellular volume of individual reticulocytes are independently deter-
mined.9,10 As a reticulocyte matures into an erythrocyte, the hemoglobin
concentration increases as the cell volume decreases. Thus, CHr (the
product of the hemoglobin concentration and the cell volume) is a more
stable parameter than reticulocyte hemoglobin concentration. Ferritin was
measured in serum by using an Access Immunoanalyzer (Beckman-Coulter,
Brea, CA). The percentage of transferrin saturation was calculated from
serum iron, and the serum total iron binding capacity was measured by
using a Dimension chemistry analyzer (Dade-Behring, Newark, DE). Bone
marrow aspirates were collected in K3EDTA and stained for iron with
Prussian blue. Stainable iron was determined to be absent or present in
aspirates containing marrow spicules by 2 pathologists who were kept
blinded to the peripheral blood test results. Discrepant samples were
referred to a third pathologist for interpretation. Interobserver variability
occurs in histopathologic diagnosis; thus, the bone marrow aspirate is not a
perfect gold standard assay for iron deficiency. The Cohen coefficient ⌲ for
the 2 pathologists initially interpreting the specimens was 0.78, indicating
substantial agreement.11

Seventy-eight patients undergoing bone marrow examination were enrolled

in this study after informed consent. Peripheral blood samples from 34
medical students were obtained anonymously during a pathology course to
establish reference limits. No student was anemic as defined by a hematocrit
less than 36% for women and less than 39% for men.
Analytical methods
Peripheral blood samples were obtained on the day of the bone marrow
examination and were submitted for routine analysis at the Memphis
Veterans Affairs Medical Center clinical laboratories. A complete blood
count and reticulocyte analysis, including CHr, were measured in whole
blood collected in K3EDTA with the use of an Advia 120 hematology
analyzer (Bayer Diagnostics, Tarrytown, NY). The CHr is determined from
Results and discussion
The average CHr in samples from 34 medical students was
30.8 ⫾ 0.90 pg with a range of 28.8 to 32.9 pg. No significant
difference between male and female values was present. To
determine its efficacy in predicting bone marrow iron stores, CHr
was measured in 78 patients undergoing bone marrow biopsy.
Reasons for biopsy included anemia work-up (34%), benign
hematologic disorders such as thrombocytopenia or monoclonal
gammopathy (22%), hematologic malignancy (19%), lymphoma or
multiple myeloma (19%), and other malignancies (6%). As deter-
mined by the absence of stainable iron in the bone marrow aspirate,

From the Research and Pathology Services, Department of Veterans Affairs,
Memphis, TN; the Department of Pathology, University of Tennessee,
Memphis; and the Department of Pathology and Immunology, Washington
University, St Louis, MO.
Submitted April 6, 2001; accepted October 10, 2001.
Supported by the Office of Research and Development, Department of
Veterans Affairs.
Reprints: Alan Mast, Research Service-151, VA Hospital, 1030 Jefferson Ave,
Memphis, TN 38104; e-mail: alan.mast3@med.va.gov.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2002 by The American Society of Hematology

BLOOD, 15 FEBRUARY 2002 䡠 VOLUME 99, NUMBER 4 1489

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1490 MAST et al BLOOD, 15 FEBRUARY 2002 䡠 VOLUME 99, NUMBER 4

Table 1. Test characteristics based on optimal cutoff values determined by ROC analysis
Sensitivity (%) Specificity (%) PPV (%) NPV (%) ROC area*

CHr (no more than 28.0 pg) 60.7 76.0 58.6 77.6 0.642 ⫾ 0.15
CHr† 73.9 73.3 58.6 84.6 0.735 ⫾ 0.14
Ferritin (no more than 50 mg/L) 42.3 93.6 78.6 74.6 0.660 ⫾ 0.14
Ferritin† 52.4 92.9 78.6 79.6 0.690 ⫾ 0.15
Tf sat (no more than 13%) 62.5 73.8 57.7 77.5 0.660 ⫾ 0.15
Tf sat† 65.0 70.3 54.2 78.8 0.637 ⫾ 0.16
MCV (no more than 81 fL) 25.9 94.0 70.0 70.1 0.505 ⫾ 0.15
MCV† 31.8 93.3 70.0 73.7 0.570 ⫾ 0.15

PPV, positive predictive value; NPV, negative predictive value; ROC, receiver operator curve; CHr, reticulocyte hemoglobin content; Tf sat, transferrin saturation; MCV,
mean cellular volume.
*The ⫾ 95% confidence interval was calculated by using the Wilcoxon statistic according to Hanley and McNeil.13,14
†Values determined after excluding patients with MCV of at least 100 fL.

28 (36%) of the 78 patients were classified as iron deficient. The determined by ROC analysis and are presented along with sensitiv-
average CHr in the iron-deficient patients was 28.3 ⫾ 5.2 pg with a ity, specificity, and positive and negative predictive values in Ta-
range of 21.0 to 38.6 pg, whereas in iron-replete patients the ble 1.
average CHr was 30.2 ⫾ 3.4 pg with a range of 22.8 to 43.7 pg. Of The clinical utility of CHr for the diagnosis of the anemia of
the 28 iron-deficient patients, 17 had a CHr of 28.0 pg or less, chronic disease has not been carefully studied. The soluble
giving an overall sensitivity for the assay of 60.7% in this patient transferrin receptor is another peripheral blood test that appears to
population (Table 1). Because the CHr is the product of the cellular predict iron deficiency more accurately than traditionally used
volume and cellular hemoglobin concentration, patients with tests15-17 and is particularly useful in differentiating iron deficiency
combined iron deficiency and megaloblastic anemia may have a anemia from the anemia of chronic disease.18-20 Both CHr and
falsely elevated CHr because of the high MCV associated with
soluble transferrin receptor can be affected by other erythrocyte
megaloblastosis. The MCV among iron-deficient patients ranged
from 61.9 to 120.9 fL, and 7 patients, all with CHr less than 28 pg,
had an MCV less than 81 fL. Of the iron-deficient patients with
CHr more than 28.0 pg, 5 had MCV of 100 fL or more. One patient
had a CHr of 30.5 pg, and the other 4 patients had CHr values
between 36 and 38 pg. An iron-replete patient who developed
pancytopenia and megaloblastic anemia (MCV, 105 fL) secondary
to vitamin B12 deficiency had a CHr of 43.7 pg, 5 pg more than any
other patient in this study, further emphasizing the effects of
megaloblastic changes on CHr. When patients with MCV of 100 fL
or more (5 iron deficient and 5 iron replete) are excluded from
analysis, the average CHr is 26.7 ⫾ 3.9 pg in iron-deficient patients
and 29.6 ⫾ 2.6 pg in iron-replete patients, and the sensitivity for
the assay increases to 73.9%. Of the 6 remaining iron-deficient
patients with CHr more than 28.0 pg, 3 had been clinically diagnosed
with iron deficiency and were receiving oral iron therapy. The CHr is
an early indicator of response to iron therapy, increasing within 2 to
4 days of the initiation of intravenous iron therapy.12 Thus, in
patients receiving iron, the CHr may normalize before bone
marrow iron stores return, resulting in false-negative test values.
The 3 other patients with chronic lymphocytic leukemia, B-cell
lymphoma and diabetes, arthritis and anemia, respectively, had no
obvious reason for normal CHr values. Of the iron-replete patients,
38 of 50 had CHr more than 28.0 pg/cell, giving an overall
specificity of 76.0% for the assay (Table 1). Among the 12 patients
with CHr of 28.0 pg or less, 2 had thalassemia, which is a
recognized cause of low CHr,10 2 had a clinical diagnosis of iron
deficiency, and 3 had hematologic malignancy. The remaining 5
patients had various diseases such as diabetes or arthritis and were
examined for anemia evaluation.
The ability of CHr to predict bone marrow iron stores was
compared with that of ferritin, percentage of transferrin saturation,
and MCV by receiver operator curve (ROC) analysis (Figure 1).
When patients with MCV 100 fL or more are excluded from Figure 1. ROC comparison of peripheral blood tests (ƒ, CHr; , MCV; F,
analysis, the area under the ROC curve is greater for CHr than for transferrin saturation; O, Ferritin) for the detection of absent bone marrow iron
stores. (A) All patients. (B) Patients with MCV more than 100 are excluded from
the other 3 tests, indicating that it has the best overall sensitivity
analysis. Diagnostic cutoff concentrations spanned the full range of sensitivity and
and specificity for iron deficiency in the population of patients specificity for all analytes. Areas under the curves (⫾ 95% confidence interval) are
tested.13,14 The optimal cutoff values for the different assays were presented in Table 1.
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BLOOD, 15 FEBRUARY 2002 䡠 VOLUME 99, NUMBER 4 RETICULOCYTE HEMOGLOBIN CONTENT IN IRON DEFICIENCY 1491

disorders that produce false-positive or false-negative test results

for iron deficiency. Therefore, the correct diagnosis of iron
deficiency requires that these tests are interpreted in the context of
the patient’s overall erythrocyte physiology, including knowledge
of recent transfusions, iron therapy, vitamin B12 or folate defi-
ciency, and the results of hemoglobin electrophoresis.
Acknowledgments
We thank the medical technologists in the Memphis VA Hospital
Clinical Laboratories for assistance in obtaining the bone marrow
aspirates and performance of the peripheral blood tests.

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2002 99: 1489-1491

doi:10.1182/blood.V99.4.1489

Clinical utility of the reticulocyte hemoglobin content in the diagnosis of iron

deficiency
Alan E. Mast, Morey A. Blinder, Qing Lu, Sherri Flax and Dennis J. Dietzen

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