TOWARD OPTIMAL
LABORATORY USE
Reticulocyte Hemoglobin Content
to Diagnose Iron Deficiency in Children
Carlo Brugnara, MD
David Zurakowski, PhD
James DiCanzio, MS
Theonia Boyd, MD
Orah Platt, MD
I
RON DEFICIENCY IS ONE OF THE MOST
common nutritional deficiencies
and is the leading cause of anemia
in children and adult women. Ac-
cording to a recent study, 700 000 chil-
dren aged 1 to 2 years are iron deficient
and 240 000 have iron deficiency ane-
mia.1 Although anemia can be reversed
with iron supplementation, the alter-
ation in cognitive performance ob-
served in children with iron deficiency
may not be fully correctable.2-4 Early rec-
ognition of iron deficiency, even be-
fore the development of anemia, is there-
fore crucial to prevent the systemic
complications of this disease. Such early
diagnosis, by necessity, relies on labo-
ratory testing, a strategy that is expen-
sive and fraught with error.
The diagnosis of simple iron defi-
ciency has been traditionally based on
a panel of biochemical indicators of iron
metabolism, which includes determi-
nation in serum or plasma of iron, trans-
ferrin, transferrin saturation (Tfsat), and
ferritin. The diagnosis of iron defi-
ciency anemia relies on the presence of
anemia with the characteristic morpho-
logic features of iron-deficient eryth-
rocytes (microcytosis, hypochromia)
and elevated erythrocyte zinc proto-
porphyrin (ZPP) in conjunction with
the above mentioned biochemical
markers of iron metabolism. A large
number of articles have been pub-
For editorial comment see p 2247.
©1999 American Medical Association. All rights reserved.
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Context Early identification of iron deficiency in children is essential to prevent the
damaging long-term consequences of this disease. However, it is not clear which in-
dices should be included in a diagnostic panel for iron deficiency and iron deficiency
anemia in children.
Objective To develop an effective approach for the diagnosis of iron deficiency and
iron deficiency anemia in young children.
Design and Setting Retrospective laboratory analysis, carried out over 7 weeks in
1996, using blood samples ordered by pediatricians and sent to a large metropolitan
hospital for analysis.
Patients A total of 210 children (mean [SD] age, 2.9 [2.0] years; 120 were male)
who had a lead screening test (complete blood cell count and plasma lead level) or-
dered by a primary care pediatrician.
Main Outcome Measures Levels of hemoglobin (Hb), iron, transferrin, transfer-
rin saturation (Tfsat), ferritin, and circulating transferrin receptor and reticulocyte Hb
content (CHr) among patients with and without iron deficiency, defined as Tfsat of
less than 20%, and iron deficiency anemia, defined as Tfsat of less than 20% and Hb
level of less than 110 g/L.
Results Of the 210 subjects, 43 (20.5%) were iron deficient; 24 of these had iron
deficiency anemia. Reticulocyte Hb content and Hb levels were the only significant
predictors of iron deficiency (likelihood ratio test [LRT] = 15.96; P,.001 for CHr, and
LRT = 6.59; P = .01 for Hb), and CHr was the only significant multivariate predictor of
iron deficiency anemia (LRT = 30.43; P,.001). Plasma ferritin level had no predictive
value (P = .97). Subjects with CHr of less than 26 pg (optimal cutoff value based on
sensitivity/specificity analysis) had lower Hb level, mean corpuscular volume, mean
corpuscular Hb level, serum iron level, and Tfsat, and increased red blood cell distri-
bution width vs those with CHr of 26 pg or more (P,.001 for all).
Conclusions Reticulocyte Hb content level was the strongest predictor of iron de-
ficiency and iron deficiency anemia in children. It holds promise as an alternative to
biochemical iron studies in diagnosis.
JAMA. 1999;281:2225-2230 www.jama.com
lished on the relative merits and weak- have been added to the diagnostic menu
nesses of these parameters for the di- for iron deficiency. Several studies have
agnosis of iron deficiency in both the shown that serum circulating TfR is
adult and pediatric settings.5-9 useful in the early identification of mild
More recently, measurements of se- iron deficiency, and in the distinction
rum circulating transferrin receptor of anemia of chronic disease from that
(TfR) and reticulocyte cellular indices due to iron deficiency.10-15 We have
Author Affiliations: From the Departments of Pa- Hospital–approved consulting agreement with Bayer
thology (Dr Brugnara), Laboratory Medicine (Drs Brug- Diagnostics (Tarrytown, NY). He has received occa-
nara and Platt), Medicine (Dr Platt), and Biostatistics sional honoraria from Bayer Diagnostics for scientific
(Dr Zurakowski and Mr DiCanzio), The Children’s Hos- presentations and editorial work.
pital, Harvard Medical School, Boston, Mass (Drs Brug- Corresponding Author and Reprints: Carlo Brugnara,
nara, Zurakowski, Platt and Mr DiCanzio), and Bay- MD, Department of Laboratory Medicine, The Chil-
state Medical Center, Springfield, Mass (Dr Boyd). dren’s Hospital, 300 Longwood Ave, Bader 760, Bos-
Financial Disclosure: Dr Brugnara has a Children’s ton, MA 02115 (e-mail: brugnara@A1.tch.harvard.edu).
JAMA, June 16, 1999—Vol 281, No. 23 2225
 IRON DEFICIENCY DIAGNOSIS WITH RETICULOCYTE HEMOGLOBIN CONTENT
demonstrated that reticulocyte hemo-
globin content (CHr) is an early indi-
cator of iron-restricted erythropoiesis
in healthy subjects receiving recombi-
nant human erythropoietin.16,17 There
have been some recent reports on the
use of CHr in the identification of func-
tional iron deficiency and monitoring
of intravenous iron and recombinant
human erythropoietin therapies in di-
alysis patients.18-20
There is no systematic study that
evaluates the performance of these old
and new indices of iron deficiency in
children and it is not clear which ele-
ments should be included in a diagnos-
tic panel for iron deficiency and iron
deficiency anemia in children. We pre-
sent data on the performance of these
indicators in a group of children ran-
domly selected from those followed up
by general pediatric practices that use
our laboratory services.
METHODS
Sample Collection
The study was carried out over 7 weeks
in 1996. On the same day of each week
(Wednesday), a maximum of 35 samples
were selected. Only samples from gen-
eral pediatric outpatient clinics that had
both a complete blood cell count and a
lead level ordered were considered. The
selection was based on the accession
number, which is given at the time the
blood is collected. Starting from the low-
est accession number of the day, the
samples were selected consecutively up
to the maximum number of 35. Since this
selection took place in the evenings, the
samples selected had been collected be-
tween 8 AM and 5 PM. Of the 210 samples
studied, 94 had been collected before 11
AM. The amount of blood collected for
a complete blood cell count is 1.5 mL,
and after analysis, approximately 1 mL
is leftover in the tube. For lead levels, 1.5
mL of blood is collected in heparin, and
after the test is run, there is approxi-
mately 400 µL of plasma leftover.
Researchers were blinded to patient
identity when they analyzed samples.
Therefore, informed consent and insti-
tutional review board approval were not
required. This is consistent with US
2226 JAMA, June 16, 1999—Vol 281, No. 23
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Code of Federal Regulations, Part 46,
Protection of Human Subjects, under
46.101(b), paragraph 4 and with Chil-
dren’s Hospital institutional review
board guidelines.
Analytical Methods
A complete blood cell count (whole
blood collected in EDTA) and plasma
lead determination were routinely or-
dered for all the study subjects. In ap-
proximately 40% of the samples, red
blood cell ZPP was also ordered by the
primary care pediatrician.
The leftover EDTA blood was used
on the same day of collection for re-
ticulocyte analysis. The leftover hepa-
rinized blood was spun down on the
same day of collection. Plasma was col-
lected, aliquoted, and frozen at − 70°C
for biochemical determinations.
Red blood cell and reticulocyte indi-
ces were measured with an automated
flow cytometer (Technicon H*3, Bayer
Diagnostics, Tarrytown, NY).21-23 This
flow cytometry system quantifies the dis-
tribution for cellular indices of erythro-
cytes (mean corpuscular volume [MCV],
mean corpuscular hemoglobin (Hb)
concentration, mean corpuscular Hb
content [MCH], and red blood cell vol-
ume distribution width [RDW]) and
CHr. Reticulocytes were stained using
the dye oxazine 750. Approximately
20 000 red blood cells were counted for
each reticulocyte determination.
The ZPP level was measured in whole
blood with the Protofluor-Z hemato-
fluorometer (Helena Laboratories,
Beaumont, Tex). Results were ex-
pressed as micromoles per mole of
heme. Serum iron and transferrin were
measured using a Hitachi 911 chemis-
try analyzer (Roche Diagnostics, India-
napolis, Ind). Ferritin was measured us-
ing the Bayer Immuno 1 analyzer (Bayer
Diagnostics). Circulating TfR was mea-
sured using the Quantikine human TfR
immunoassay (R&D Systems Inc, Min-
neapolis, Minn).
Statistical Analysis
For all 210 patients, 2 clinical out-
comes were investigated: iron defi-
ciency and iron deficiency anemia. Iron
©1999 American Medical Association. All rights reserved.
deficiency was defined as a Tfsat level
of less than 20% and iron deficiency
anemia as a Tfsat level of less than 20%
and Hb level of less than 110 g/L. The
20% cutoff for Tfsat has been used in
previous studies,7 and has been shown
to have a better diagnostic efficacy than
lower cutoff levels.8 Alternative diag-
nostic criteria were also analyzed based
on levels of Tfsat, ferritin, and ZPP. Sub-
groups were based on these cutoff lev-
els, and mean values of CHr, plasma fer-
ritin, Hb, plasma iron, MCV, MCH, and
RDW were compared with 2-sample t
tests. The Kolmogorov-Smirnov good-
ness-of-fit test24 revealed no signifi-
cant departures from normality for any
of the variables. Logistic regression
analysis25 was performed to determine
the relationship of CHr and ferritin
for each outcome. The likelihood-
ratio x2 test (LRT) was used to assess
the significance of CHr and ferritin.
Strength of the relationship was mea-
sured by the odds ratio and 95% con-
fidence interval. Slope and y-intercept
parameters were used to derive prob-
ability curves.26 In addition, multiple
stepwise logistic regression analysis was
performed to identify the variables
independently predictive of each
outcome.
Receiver operating characteristic
analysis was used to illustrate the di-
agnostic performance of CHr and fer-
ritin with receiver operating character-
istic curves compared by the Wilcoxon
statistic. 27 A CHr cutoff was estab-
lished based on the optimal combina-
tion of sensitivity and specificity. Val-
ues below this cutoff were considered
to be abnormal. To validate the CHr
cutoff, the patient population was di-
vided into healthy and abnormal sub-
groups and plasma iron, Hb, MCV,
MCH, RDW, ferritin, and Tfsat were
compared with 2-sample t tests. Data
analysis was conducted using the SPSS
software package (version 8.0, SPSS Inc,
Chicago, Ill). Areas under receiver
operating characteristic curves were
compared using GraphROC software
(version 2.0, Maxiwatli Oy, Turku,
Finland). All statistical tests were
2 sided.
 IRON DEFICIENCY DIAGNOSIS WITH RETICULOCYTE HEMOGLOBIN CONTENT

RESULTS ability of iron deficiency. According to estimated odds of iron deficiency and
Mean (SD) age for the 210 study sub- this relationship, each unit increase in iron deficiency anemia were reduced by
jects was 2.9 (2.0) years. A total of 90 CHr lowers the risk of iron deficiency 30% and 45%, respectively, with each
samples were collected from females by 30%. Similar results were obtained unit increase in CHr.
(mean [SD] age, 2.7 [2.0] years) and for iron deficiency anemia (data not To minimize the effect of diurnal
120 samples from males (mean [SD] shown), with each unit increase in CHr variation in plasma iron levels, a sepa-
age, 3.1 [2.1] years). lowering the risk of iron deficiency ane- rate statistical analysis was carried out
Using a cutoff value of 20% for mia by 42%. in the 94 samples collected before 11 AM.
Tfsat, 43 subjects (20.5%) were classi- Using cutoff levels from the Na- In this subset, similar findings to those
fied as iron deficient. Twenty-four of tional Health and Nutrition Examina- shown in Table 1 were observed for iron
these subjects were also anemic, based tion Survey for Tfsat and ferritin (Tfsat deficiency (defined as Tfsat ,20%). In
on an Hb cutoff level of 110 g/L. Of the level [1] ,10%, age 1-2 years, [2] addition to the significant predictors of
210 subjects, 41 (19.5%) had anemia ,12%, age 3-5 years, and [3] ,14%, age iron deficiency anemia shown in Table
with Tfsat values greater than 20%. 6-15 years; ferritin level [1] ,10 µg/L, 2, circulating TfR and ferritin become
TABLE 1 compares several hemato- age ,6 years and [2] ,12 µg/L, age $6 significant predictors (P = .04 and
logic and biochemical variables be- years)1 among the 210 subjects in this P = .02, respectively) for this outcome in
tween iron deficient and healthy sub- study population, 18 (8.6%) could be this subset of patients.
jects. The iron deficient group had considered iron deficient. Seven of these Results from the stepwise multiple
significantly lower Hb, MCV, MCH, and 18 were also anemic according to Na- logistic regression analysis revealed that
increased RDW values (P,.001 for all) tional Health and Nutrition Examina- CHr (LRT = 15.96; P,.001) and Hb
compared with the healthy group. In- tion Survey Hb criteria.1 Reticulocyte Hb (LRT = 6.59; P = .01) were the only sig-
terestingly, no significant differences content emerged as a significant predic- nificant multivariate predictors of iron
could be found in plasma ferritin tor of iron deficiency and iron defi- deficiency among the indices listed in
(P = .97). A marked difference was also ciency anemia (P,.001 for both). The Table 1. Given that approximately 60%
noted in the values of CHr, which was
significantly decreased in the iron de- Table 1. Comparison of Hematological and Biochemical Indices in the Diagnosis
ficient group (P,.001). Logistic regres- of Iron Deficiency*
sion indicated that CHr was a signifi- Abnormal Normal P
cant predictor of iron deficiency Index (n = 43) (n = 167) Value
(LRT = 37.28; odds ratio, 0.58 [95% CHr, pg 24.8 (2.5) 27.0 (1.7) ,.001
confidence interval, 0.48-0.71]; Hemoglobin, g/L 106.9 (12.7) 114.8 (7.1) ,.001
P,.001). MCV, fL 74.2 (5.5) 77.7 (3.9) ,.001
Hematologic and biochemical char- MCH, pg 23.9 (2.7) 25.6 (1.8) ,.001
acteristics of the 24 subjects in the iron RDW, % 14.7 (1.6) 13.9 (0.9) ,.001
deficiency anemia subgroup are sum- ZPP, µmol/mol of heme† 56.6 (50.0) 32.5 (19.9) .07
marized in TABLE 2. Significant differ- Transferrin receptor, nmol/L 32.1 (7.4) 29.2 (5.9) ,.01
ences in MCV, MCH, and RDW val- Ferritin, µg/L‡ 34.7 (28.6) 34.5 (21.0) .97
*Transferrin saturation of less than 20% was used as the cutoff point. All data are presented as mean (SD). CHr indi-
ues were detected between the 2 cates reticulocyte hemoglobin content; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; RDW,
subgroups (P,.001 for all). No differ- red blood cell distribution width; and ZPP, zinc protoporphyrin.
†For ZPP, n = 17 for abnormal and n = 64 for normal.
ences in serum ferritin could be dem- ‡For ferritin, n = 38 for abnormal and n = 145 for normal.
onstrated (P = .69), while a marked re-
duction in CHr was noted in the iron Table 2. Comparison of Hematological and Biochemical Indices in the Diagnosis
deficient anemic patients (P,.001). Lo- of Iron Deficiency Anemia*
gistic regression indicated that CHr was Abnormal Normal
a significant predictor of iron defi- Index (n = 24) (n = 186) P Value
ciency anemia among the various in- CHr, pg 24.2 (2.7) 26.8 (1.8) ,.001
dices (LRT = 28.97; odds ratio, 0.57 MCV, fL 72.9 (6.4) 77.5 (3.9) ,.001
[95% confidence interval, 0.46-0.70]; MCH, pg 23.1 (3.0) 25.6 (1.8) ,.001
P,.001). RDW, % 15.0 (1.9) 14.0 (0.9) ,.001
The empirical distributions of CHr ZPP, µmol/mol of heme† 58.1 (51.5) 34.7 (25.1) .19
values among iron deficient and healthy Transferrin receptor, nmol/L 30.7 (7.8) 29.6 (6.1) .42
subjects are depicted in the histogram Ferritin, µg/L‡ 32.7 (31.8) 34.8 (21.3) .69
of FIGURE 1. The superimposed curve *Cutoff values were transferrin saturation of less than 20% and hemoglobin level lower than 110 g/L. All data are pre-
sented as mean (SD). See the asterisk footnote to Table 1 for expansion of the abbreviations.
illustrates the inverse relationship be- †For ZPP, n = 10 for abnormal and n = 71 for normal.
‡For ferritin, n = 22 for abnormal and n = 161 for normal.
tween CHr and the theoretical prob-
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 IRON DEFICIENCY DIAGNOSIS WITH RETICULOCYTE HEMOGLOBIN CONTENT

of the study population was not tested P ,.001). Ferritin, MCV, MCH, RDW, CHr and ferritin in the diagnosis of iron
for ZPP, this index was excluded from and TfR were not significant multivar- deficiency are illustrated in FIGURE 2.
the multivariate analysis. The only sig- iate predictors of either outcome The area under the curve was signifi-
nificant multivariate predictor of iron (P..10 for all). cantly greater for CHr than for ferritin
deficiency anemia among the indices Receiver operating characteristic (P = .004; P = .02 for iron deficiency ane-
listed in Table 2 was CHr (LRT = 30.43; curves comparing the performance of mia, data not shown). A CHr cutoff of
26 pg had a sensitivity and specificity
Figure 1. Empirical Frequency Distributions of Reticulocyte Hemoglobin Content and of 70% and 78%, respectively, in the di-
Theoretical Curve of the Probability of Iron Deficiency by Reticulocyte Hemoglobin Content agnosis of iron deficiency. For iron de-
ficiency anemia, a cutoff of 26 pg had
50 1.0
Iron Deficient 83% sensitivity and 75% specificity. For
40 Normal 0.9 the diagnosis of iron deficiency, CHr
cutoffs of 26.5, 27.0, 27.5, and 28.0 pg
30 0.8 would increase sensitivity to 74%, 81%,
20 0.7
86%, and 91%, respectively, but speci-
ficity would decrease to 63%, 55%, 38%,
10 0.6 and 26%, respectively.
No. of Patients

Probability
TABLE 3 presents the hematologic
0 0.5
and biochemical values for patients with
10 0.4 CHr levels of less than 26 pg or with
CHr levels of 26 pg or more. Differ-
20 0.3 ences were found between the 2 groups
30 0.2
for Hb, MCV, MCH, RDW, Tfsat, and
circulating TfR (P,.001 for all). Dif-
40 0.1 ferences in ZPP were significant
(P,.05) while ferritin showed no dif-
50 0.0
14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 ference (P = .66) between the CHr sub-
CHr, pg groups.
Empirical frequency distributions of reticulocyte hemoglobin content (CHr) for iron deficient and healthy chil-
dren (left axis). Theoretical curve showing the inverse relationship between the probability (right axis) of iron
COMMENT
deficiency and CHr was derived from logistic regression analysis (slope = − 0.5507, y-intercept = 12.99, P,.001). In this study of young children we have
The solid line indicates the theoretical probability curve for iron deficiency.
evaluated 2 relatively new parameters
for the diagnosis of iron-deficient states.
Figure 2. Receiver Operating Characteristic Curves for Reticulocyte Hemoglobin Content
Circulating TfR and CHr have been
1.0 shown to be useful parameters for the
diagnosis of simple iron deficiency or
0.9 functional iron deficiency in patients
0.8
treated with recombinant human eryth-
ropoietin.10-20
0.7 Our data established that CHr is the
True-Positive Fraction

strongest predictor of iron deficiency and

0.6
iron deficiency anemia in children. Fer-
0.5 ritin, a parameter that is traditionally
used in adults to estimate iron stores, had
0.4
little or no diagnostic value in chil-
0.3 dren. We have also shown that TfR and
ZPP were not as informative as CHr in
0.2 Reticulocyte Hemoglobin children. It is also known that serum
Content
0.1 Ferritin
iron, transferrin, and Tfsat have major
limitations based on their biological vari-
0.0 ability.9,28 Thus, a diagnostic approach
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
False-Positive Fraction
based exclusively on hematologic pa-
rameters obtained by the complete blood
Reticulocyte hemoglobin content and ferritin as indicators of iron deficiency. The area under the curve was cell count and the reticulocyte analysis
significantly greater for reticulocyte hemoglobin content (0.78) compared with ferritin (0.57) (P = .004).
is appealing for both its direct assess-
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 IRON DEFICIENCY DIAGNOSIS WITH RETICULOCYTE HEMOGLOBIN CONTENT

ment of iron metabolism and its poten-

tial cost-effectiveness.
There are relatively few conditions
that result in reduced CHr. In addi-
tion to iron deficiency, a and b thal-
assemia result in hypochromia and mi-
crocytosis for both erythrocytes and
reticulocytes. Although several differ-
ent mathematical indices have been pro-
posed for the differential diagnosis of
thalassemia trait and iron deficiency,
none of them is superior to MCV
alone.29 Diagnosis of heterozygous b
thalassemia (b thalassemia trait) can
now be reliably obtained with the de-
termination of the ratio of microcytic
to hypochromic red blood cells ob-
tained from the complete blood cell
count.30 This ratio is greater than 0.9
in b thalassemia trait, which is charac-
terized by significant microcytosis and
mild hypochromia, and is lower than
0.9 in iron deficiency, which is char-
acterized by marked hypochromia and
mild microcytosis. This ratio has a dis-
criminant efficiency of 92.4%, which is
the highest among the various formu-
las described for this kind of analy-
sis.30 The combination of CHr and the
ratio of microcytic to hypochromic red
blood cells will allow distinction, in the
presence of microcytosis, between thal-
assemia and iron deficiency. If thalas-
semia is ruled out by a high ratio, a low
CHr can only be due to iron defi-
ciency. The diagnostic value of CHr in
more complex settings, such as com-
bined iron deficiency and chronic dis-
ease, has not been established.
There is ample evidence to indicate
that changes in red blood cell param-
eters become more apparent late in the
development of iron deficiency.31 Our
previous studies have shown that re-
ticulocyte indices provide a real-time
evaluation of the bone marrow activ-
ity, reflecting the balance between iron
and erythropoiesis of the preceding 48
hours.16,32 Iron deficiency could be de-
tected at an earlier stage, when red
blood cell indicators are still normal but
the iron stores are depleted to the point
of affecting hematopoiesis and induc-
ing production of a certain percentage
of reticulocytes with reduced Hb con-
©1999 American Medical Association. All rights reserved.
Table 3. Comparison of Hematological and Biochemical Indices in the Diagnosis
of Iron Deficiency*
Index
Hemoglobin, g/L
MCV, fL
MCH, pg
RDW, %
Transferrin saturation, %
ZPP, µmol/mol of heme†
Transferrin receptor, nmol/L
Ferritin, µg/L‡
*Reticulocyte hemoglobin content (CHr) of less than 26 pg was used as the cutoff point. All data are presented as
mean (SD). See the asterisk footnote to Table 1 for expansion of abbreviations.
†For ZPP, n = 27 for CHr less than 26 pg and n = 54 for CHr of 26 pg or higher.
‡For ferritin, n = 59 for CHr less than 26 pg and n = 124 for CHr of 26 pg or higher.
tent, resulting in a progressive reduc-
tion of CHr.
There is also experimental evidence
that CHr is an early indicator of re-
sponse to iron therapy in iron defi-
ciency anemia cases. The classic crite-
rion for defining response to iron
therapy is based on observing an in-
crease of at least 10 g/L of Hb after 1
month of therapy. None of the bio-
chemical parameters is helpful in de-
fining response to iron therapy. Stud-
ies of CHr have shown that a response
to oral iron therapy can be identified
after 1 or 2 weeks of oral iron supple-
mentation.16,33 Further studies in chil-
dren are necessary to determine the
value of this parameter in early identi-
fication of responders and nonre-
sponders to iron therapy.
We have not directly compared the
performance of complete blood cell
count and reticulocyte count panel with
the traditional biochemical panel in a
clinical setting. Such a study must now
be performed to validate this alterna-
tive approach. If the value of complete
blood cell count and reticulocyte count
is confirmed by such a study, it could
yield significant reductions in costs. The
cost of the current diagnostic panel for
iron deficiency, which includes a com-
plete blood cell count and evaluation
of iron, transferrin, ferritin, and ZPP,
is $154.33 based on typical fees and
$80.49 based on Medicare nationally
capped fees. 34 A simplified screen,
CHr Value
,26 pg $26 pg P
(n = 67) (n = 143) Value
107.8 (10.7) 115.7 (6.9) ,.001
73.6 (4.8) 78.5 (3.4) ,.001
23.7 (2.3) 26.1 (1.5) ,.001
14.6 (1.4) 13.9 (0.8) ,.001
26.8 (14.7) 35.7 (13.7) ,.001
47.0 (41.7) 32.8 (21.3) ,.05
32.0 (6.6) 28.7 (6.0) ,.001
35.6 (23.6) 34.0 (22.3) .66
which includes complete blood cell
count and reticulocyte counts, would
cost $40.26 based on typical fees and
$20.77 based on Medicare nationally
capped fees.34 Using the published data
on the prevalence of iron deficiency in
children aged 1 to 2 years,1 the use of
the hematologic panel could result in
potential savings of $79.85 million
($41.81 million using nationally capped
fees). Since CHr and microcytic to hy-
pochromic red blood cells ratio are cur-
rently provided by only 1 of the 4 ma-
jor automated hematology analyzers
sold in the United States, these poten-
tial savings will be attainable only when
all manufacturers adopt these 2 param-
eters. Since all of these measurements
can be performed on 1.0 to 1.5 mL of
blood in an EDTA tube, use of this panel
would also result in a significant re-
duction in the amount of blood needed
for the diagnostic workup and the elimi-
nation of the heparin tubes and serum
tubes needed for ZPP and biochemical
determinations. In children, a simple
finger-stick would produce a satisfac-
tory blood sample for this panel.
Our study is limited in the number
of subjects and ages investigated. It is
also difficult to extrapolate from this
data set conclusions that can be readily
applicable to the general pediatric popu-
lation. Future studies should evaluate
this parameter in an unselected popu-
lation of children. The poor diagnos-
tic values of ZPP observed in our study
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 IRON DEFICIENCY DIAGNOSIS WITH RETICULOCYTE HEMOGLOBIN CONTENT
may be due to the limited number of
subjects with ZPP values (80/210). Pre-
vious studies have shown ZPP is help-
ful in identifying children who will re-
spond to oral iron therapy.14
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