Cheng et al. BMC Pediatrics(2024) 24:541
https://doi.org/10.1186/s12887-024-05000-w

BMC Pediatrics

RESEARCH

Open Access

The role of reticulocyte hemoglobin
equivalent on the evaluation of irondeficiency and iron deficiency anemia
in pediatric cyanotic heart disease:a diagnostic study in Indonesia
William Cheng1*, Piprim Basarah Yanuarso1, Pustika Amalia Wahidiyat1, Syarif Rohimi2, Partini Pudjiastuti Trihono1,
Muzal Kadim1 and Dina Muktiarti1
Abstract
Background In light of prolonged hypoxia, children with cyanotic heart disase (CHD) are at a high risk of developing
iron deficiency iron deficiency (ID) and iron deficiency anemia (IDA). Reticulocyte hemoglobin equivalent (Ret-He)
is a novel and dependable indicator for assessing iron status. However, there has been no previous study regarding
cut-off value in pediatric CHD group. The purpose of this study is to assess the role of Ret-He and to establish cut-off
points in the diagnosis of iron deficiency and IDA in pediatric cyanotic heart disease.
Method This study was conducted in two tertiary hospitals in Jakarta, Indonesia. 59 children with CHD, aged 3
months to 18 years, were enrolled consecutively. To determine iron status, hematological parameters (hemoglobin,
hematocrit, mean corpuscular volume, mean corpuscular hemoglobin) and biochemical parameters for ironstatus (serum ferritin, transferrin saturation) were analysed and compared to Ret-He levels. The receiver operating
characteristic (ROC) analysis was performed for the Ret-He cut-off points for ID and IDA. Sensitivity, specificity, positive
and negative predictive value were calculated for each cut-off point.
Result Normal iron status was identified in 27 (45.8%) subjects, ID in 8 (13.5%) subjects, and IDA 24 (40.7%) subjects.
The ID cut-off value for Ret-He is 28.8 pg (sensitivity 75%, specificity 85.2%, PPV 60%, NPV 92%, and AUC 0.828) and
the Ret-He cut-off point for IDA is 28.15 pg (sensitivity 75%, specificity 88.9%, PPV 85.7%, NPV 80%, and AUC 0.824).
Hemoglobin should be used in conjunction with Ret-He. ID might be detected in this cohort with Ret-He 28.8 pg andhemoglobin > 16,5 g/dL. While Ret-He 28.15 pg or Ret-He 28.15–28.8 pg with hemoglobin 16.5 g/dL could be used todiagnose IDA.
Conclusion The reticulocyte hemolgobin equivalent could be utilised as an iron status parameter in pediatric CHD,
with a cut-off value of 28.8 pg for ID and 28.15 pg for IDA.

*Correspondence:
William Cheng
william.chengcw@gmail.com
Full list of author information is available at the end of the article© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0
International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you
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Cheng et al. BMC Pediatrics(2024) 24:541

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Keywords Pediatric, Iron deficiency, Iron deficiency anemia, Cyanotic heart disease, Reticulocyte hemoglobin
equivalent (Ret-He)

Background
Iron deficiency and iron deficiency anemia (IDA) are
the most common micronutrient problem worldwide,
especially in developing countries [1]. Pediatric are thehigh-risk group for iron deficiency due to an imbalance
between the production rate of red blood cells and ironstorage in the body, specifically during the growth spurt[2, 3]. Based on World Health Organization (WHO)2015, South East Asia has a higher risk of IDA prevalence on toddler (53.8%). The IDA prevalence in pediatric population in South East Asia is higher than in Africa,around 66% versus 60% [4]. Its prevalence in Indonesia
was 40.5% among children under 5 years old and 47.2%
among school-aged children [1]. Iron deficiency has
short and long-term effects on psychomotor and cognitive development, and many of them are irreversible [1,4].
The impact of IDA on certain groups such as children
with cyanotic heart disease (CHD) may be greater. In
pediatric CHD, secondary erythropoiesis as a form ofadaptation to chronic hypoxia increases iron requirement
continuously. Additionally, hepcidin suppression due tohypoxia and a higher risk of erythrocyte destruction also
increases the risk of iron loss. The previous study statedthat iron deficiency in the pediatric CHD population was
47.5% [5] and IDA was 52.2% [6]. Therefore, early diagnosis is important to reduce morbidity and mortality ofpediatric cyanotic heart disease [5].
American Academy of Pediatrics (AAP) recommendation of IDA diagnosis with the measurement of (1) ferritin and CRP, or (2) reticulocyte hemoglobin equivalent(Ret-He), as an additional examination for hemoglobin
levels [7]. Since serum ferritin is an acute phase reactant, it is necessary to support the validity of the resultwith other inflammatory markers such as C-reactive
protein (CRP) [7]. While Ret-He represents the synthesis of hemoglobin precursors in the bone marrow and is
not affected by inflammation, infection, malignancy, orchronic disease and therefore it is a reliable parameter ofiron status []. The usage of Ret-He in healthy children andneonates has been widely applied [8, 9]. However, its use
in pediatric CHD is still limited [10]. The objective of thisstudy is to evaluate the application of Ret-He in pediatric
CHD population and to determine cut-off off points in
detection of iron deficiency and iron deficiency anemia.

and IDA on children, aged of 3 months until 18 years old,with CHD, from December 2021 to March 2022, at Cipto
Mangunkusumo Hospital and Harapan Kita Women andChildren’s Hospital, Jakarta. Both hospitals are tertiaryreferral hospitals located in the capital of Indonesia.

MethodVariables

Study design and setting

Iron deficiency was defined by:This is a prospective cross-sectional diagnostic test studyof Ret-He as a diagnostic parameter for iron deficiency

Participants

Individuals who met age criteria and diagnosed with cyanotic heart disease in both hospitals, were approachedand given information of the study. Research team
explained the literature using written information andgave opportunity for potential subjects to ask questions.
Informed written consent was obtainted from the parents
and assent was obtained from children and adolescents.
Cyanotic heart disease was confirmed using echocardiography by pediatric cardiologists, marked by a significant right-to-left shunt and the mean daily oxygen
saturation of all subjects was lower than 90%. The reference growth parameter is 2006 WHO curves for less than
5 years old (Z score) and the 2000 CDC curve for over
5 years old (the percentage of ideal body weight according to the Waterlow criteria), based on gender. According
to these criteria, subjects will be categorized as follows:
normal (with a Z score between − 2 and 2 SD for WHO
or > 90% for Waterlow criteria); mild-moderate malnutrition (with a Z-score between − 3 to -2 SD for WHO or
70–90% for Waterlow criteria); and severe malnutrition(with a Z score below − 3SD for WHO or less than 70%
for Waterlow criteria) [11].
The blood sample was taken consecutively at the Clinical Pathology Laboratory of Cipto Mangunkusumo Hospital and Harapan Kita Women and Children’s Hospital.
Subjects’ absence on test series, received iron supplementation or therapy during the last 3 months, and CRP
levels ≧ 5 mg/L were excluded. This study was approvedby the Medical Ethics Committee of the Universitas
Indonesia.
Study size

Sample size calculation was based on an expected sensitivity of 80%, with a margin of error of 15% and type I
error of 5% [12]. Prevalences of ID and IDA were 47.5%
for ID [5] and 52.2% [6] for IDA. Therefore, we aimed for
59 subjects for acquiring the diagnostic parameters forthis study.

1. Hemoglobin level ≥ 16.5 g/dL.

Cheng et al. BMC Pediatrics(2024) 24:541

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Fig. 1 Study flow chart

2. Serum ferritin < 12 ng/mL for < 5 years old subjectsor < 15 ng/mL for ≥ 5 years old subjects, ortransferrin saturation < 15% [13].
3. Mean corpuscular volume (MCV) < 80 fL or mean
corpuscular hemoglobin (MCH) < 27 pg. [14, 15]
Iron deficiency anemia (IDA) was defined by hemoglobin levels < 16.5 g/dL [15] and met the criteria for irondeficiency.
Data analysis

Data were analysed using IBM Statistical Product andService Solutions (SPSS) version 26 (IBM Corp. Armonk,
NY, USA). Univariate analysis was used for frequencyof data distribution. Normality study used KolmogorovSmirnov test. The normal data distribution was presented
by mean and standard deviation, whereas, abnormal
data distribution by median, minimum, and maximum.
One-way ANOVA was used for normally distributed
numerical data and Kruskal-Wallis non-parametric test
was used for abnormal data distribution. Statistic’s significance used p < 0.05. The ability of Ret-He discrimination in determining iron status was expressed by receiver
operating characteristic (ROC) analysis.

ResultA total of 59 subjects were recruited as study subjects
from Cipto Mangunkusumo Hospital and Harapan Kita
Women and Children’s Hospital (Fig. 1).
The median age was 23 months (ranging from 3 to
209 months), mostly categorized as 0–2 years old group(49.2%). The gender proportion was equal, with 31(52.5%) boys and 28 (47.5%) girls. As many as 8 (13.5%)
subjects had an iron deficiency and 24 (40.7%) had an
IDA. Most of the subjects was categorized as mild-moderate malnutrition (26 [42.6%]) and stunted (42 [71.2%]).
The clinical characteristic of the subjects presented inTable 1 The clinical characteristic of study subject
Characteristic

Age (months)*
Age group, n (%)
0–2 years
2–5 years
5–12 years
12–18 years
Gender, n (%)
Boys
Females
Nutritional status, n (%)
Normal
Mild-moderatemalnutrition
Severe malnutrition
Stature, n (%)
NormalStuntedPrematurity, n (%)
Premature
Aterm

Total
(n = 59)

Normaliron
status(n = 27)
23 (3-209) 32 (4-174)

Iron deficiency(n = 8)95 (68)Iron deficiency
anemia(n = 24)
16 (3-209)

29 (49.2)
17 (28.8)
7 (11.9)
6 (10.2)

13
7
4
3

1
2
3
2

15
8
0
1

31 (52.5)
28 (47.5)

11
16

7
1

13
11

21 (35.6)
26 (42.6)

12
11

1
5

8
10

12 (20.3)

42

6

17 (28.8)
42 (71.2)

8
19

4
4

5
19

6 (10.2)
53 (89.8)

3
24

1
7

2
22

Note*Showed on mean (standard deviation) or median (min-max)

Table 1. The most prevalent type of CHD was the tetralogy of Fallot (TOF) in 20 (33.9%) subjects. Six subjects
underwent palliative operations; however, their oxygen
saturation was < 90%, so they were included in this study.
Table 2 showed CHD distribution among study subjects.
The prevalence of low iron levels was 32 (54.2%) subjects, of which 8 (13.5%) had iron deficiency withoutanemia and 24 (40.7%) had IDA. Table 3 showed the laboratory characteristic of the study subjects. There were 37(62.7%) subjects who had anemia (Hb levels < 16.5 g/dL)
in this study. Iron deficiency was found as the etiology ofCheng et al. BMC Pediatrics(2024) 24:541

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Table 2 Cardiovascular abnormality distribution on study
subjects
Type of congenital heart disease
Type of CHD
Tetralogy of Fallot
Double-outlet right ventricle
Pulmonary atresia with ventricular septal defectTransposition of great Arteries
Tricuspid atresia
Truncus arteriosus
Total anomalous pulmonary venous drainage(TAPVD)
Ebstein anomaly

Total (n, %)20 (33.9)
14 (23.7)
12 (20.3)
5 (8.5)3 (5.1)2 (3.9)2 (3.9)1 (1.7)anemia on 24 (64.9%) subjects with anemia. Of 24 subjects with IDA, 20 subjects had microcytic hypochromic
erythrocyte index and 4 subjects had normocytic normochromic. Besides that, 13 subjects were categorized asanemia with normal iron status, of which 9 subjects withmicrocytic hypochromic and 4 subjects with normocytic
normochromic anemia. Figure 2 presented iron profile ofstudy subjects.
The median Ret-He for all subjects was 28.7 pg (14.4
to 36.5 pg). The mean value of Ret-He in the group withnormal iron status was 31.6 (SD 3.0) pg and decreased
according to the level of iron status, 25.3 (NB 4.7) pg in
the iron deficiency group, and 23.4 (SB 6.4) pg in the IDA
group. Post hoc Mann-Whitney analysis found a statistically significant difference between the mean Ret-He in
the normal iron status in iron deficiency group (p = 0.001)
and normal iron status in the IDA group (p < 0.001). However, the mean difference between the iron deficiency and
IDA group was not statistically significant (p = 0.428).
Receiver operating characteristic curve showed Ret-He
cut-off of < 28.8 pg has an area under the curve (AUC)of 0.828 (95%CI 0.664–0.934) for iron deficiency withoutanemia as shown in Fig. 3. The sensitivity is 75%, 95%CI
24.9–96.8; specificity 85.2%, 95%CI 66.3–95.8); PPV 60%,

Fig. 2 Iron profile of study subjects

95%CI 35.8–80.1; NPV 92%, 95%CI 77.4–97.5; LR + 5.1,
95%CI 1.9–13.6; and LR- 0.3, 95%CI 0.1–0.9. Table 4
depicts the diagnostic test result of Ret-He for iron deficiency without anemia.
ROC curve showed Ret-He cut-off of < 28.15 pg results
to area under the curve 0.824 (95% CI 0.691–0.916) for
iron deficiency anemia as shown in Fig. 4. The sensitivity
is 75%, 95%CI 53.3–90.2; specificity 88.9%, 95%CI 70.8–97.6); PPV 85.7%, 95%CI 66.8–94.7; NPV 80%, 95%CI
66.4–89; PPR 6.75, 95%CI 2.3–20.1; and NPR 0.3, 95%CI
0.1–0.6. Table 5 shows the diagnostic test result of RetHe for IDA.

Discussion
Iron deficiency anemia is a significant risk factor formorbidity and mortality in pediatric CHD because of
its relation to worsening hyperviscosity conditions [15,16]. It increases the risk of cerebrovascular events, cyanotic spell, endothelial dysfunction, anorexia, and physical activity disturbance [5]. Pediatric CHD have a higher

Table 3 Laboratorium characteristic of study subject
Parameter
Hemoglobin (g/dL)
Hematocrit (%)
MCV (fL)
MCH (pg)
MCHC (g/dL)
Ferritin (ng/mL)
Serum iron (µg/dL)
TIBC (µg/dL)
Transferin saturation (%)
RDW(%)
Ret-He (pg)Total(n = 59)
15.5 (9.7–24.7)**
50.3 (11.5)*
77.2 (8.4)*
25 (4.2)*
32.9 (22.7–36.6)**
22.1 (1-553.7)**
52 (13–211)**358,2 (78.2)*
16 (3–58)**
16.7 (12-32.8)**
28.7 (14.4–36.5)**

Normal iron status(n = 27)17.1 (3.8)*
50.8 (11.4)*
81.1 (6.8)*
27.3 (2.6)*
33.7 (1)*48 (15.4-553.7)**
80 (42–211)**296 (159–450)**30 (12.3)*
16.2 (3.4)*
31.6 (3)*Iron deficiency(n = 8)20.2 (2.1)*
65.1 (7)*77.9 (7.7)*
24.3 (3.6)*
31.11 (1.9)*9.7 (3.7)*
40.8 (17.6)*
432.3 (54.5)*
11.1 (7.5)*
20.8 (4.6)*
25.3 (4.7)*

Iron deficiency anemia(n = 24)
13.6 (1.9)*
44.7 (7.9)*
72.5 (8.1)*
22.6 (4.4)*
30.87 (3.5)*
9.2 (1-185.4)**
39.3 (17.7)*
386.7 (67)*10.6 (5.3)*
19.4 (12-32.8)**
23.4 (6.4)*

Note *The value shown as a mean (standard deviation); **The value shown as a median (min-max); +One-Way ANNOVA test; #Kruskal-Wallis test

P value< 0.001#< 0.001+
0.003+
0.001+
0.001#< 0.001#< 0.001#< 0.001+< 0.001#
0.009#< 0.001#

Cheng et al. BMC Pediatrics(2024) 24:541

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Table 5 Diagnostic test result of Ret-He for iron deficiency
anemia (n = 51)
Ret-He(pg)< 28.15≥ 28.15
Total

Fig. 3 Ret-He ROC curve to evaluate iron deficiency

Table 4 Diagnostic test result of Ret-He for iron deficiency
without anemia (n = 35)
Ret-He(pg)< 28.8≥ 28.8
Total

Iron deficiency
Yes
6
2
8

Total
No
4
23
27

Fig. 4 Ret-He ROC curve to evaluate iron deficiency anemia

10
25
35

Iron Deficiency Anemia
Yes
No
18
3
6
24
24
27

Total
24
27
51

risk to develop iron deficiency and IDA due to chronic
hypoxia could stimulate secondary erythrocytosis, suppress hepcidin, and increase erythrocyte destruction [9].
From this study, the proportion of pediatric CHD withiron deficiency without anemia and IDA were 54.2%(13.5% iron deficiency and 40.7% IDA). The prevalenceof iron deficiency without anemia in pediatric CHD
aged 6–48 months in the previous study was 63.6% [17].
Another study stated that the prevalence of iron deficiency was 47.06% in the age group of 0–12 months[16]. The most common type of CHD in this study was
tetralogy of Fallot (TOF), around 32.8%, as also foundin a previous study [14–16]. Our least common CHD in
accordance with the rare incidence rate [18]. The proportion of gender in this study was equal, boys and girls ratioof 1.1:1. This was in accordance with the previous study
stating that there was no gender predisposition to CHD[5, 18].
The majority of study subjects were 0–2 years old age
group (49.2%). A study on the survival of CHD reported
90% of mortality occurred in the first five years of life[19]. Cohort studies in pediatrics and adults reported
that the hazard ratio in CHD was 65.07 (95% CI 53.39–
76.89) [20]. The relationship between age and iron deficiency and IDA could not be drawn in this study due tothe wide range of age distribution. Preschool children
and adolescents are prone to iron deficiency because ofhigher iron demand for growth [2, 21]. Prematurity profile on this study was less useful due to the wide rangeof age. It may serve as a risk factor during the first six
months of life [22].
Children with CHD has a higher risk for malnutrition[5]. Stunting was a significant nutrition problem in thisstudy. This result was in accordance with the previous
study, with a prevalence of 60% [5], and 68% [23]. Chronic
hypoxia was also a contributing factor to disrupting linear growth [24, 25]. Iron deficiency affects the synthesisof insulin-like growth factor-binding protein (IGFBP-1),
which facilitates growth [25]. Children with congenital
heart diseases mostly had mild to moderate malnutrition,
and acute malnutrition significantly happened to CHD(45.83%) and all types of CHD (74.1%) [26]. The calories
needed were increased to 20–50% normal need for age,inability to fulfill leads to acute malnutrition [27, 28]. The
previous study showed a relationship between acute malnutrition with IDA (OR 2.89, 95%CI 1.45–5.76) [29].

Cheng et al. BMC Pediatrics(2024) 24:541

The previous study stated that early increment ofiron storage (compensated secondary erythropoiesis)
was marked by increasing hemoglobin, hematocrit, anderythrocyte levels. This study demonstrated the presence of compensated secondary erythropoiesis, as seenby the higher mean hemoglobin level in the iron deficiency without anemia group (20.2 g/dL) compared to
the normal iron status group (17.1 g/dL). Subsequently,
as the progression occured, uncompensated secondary
erythropoiesis was followed by decreased hemoglobin
and hematocrit levels [30]. In this study, uncompensatedsecondary erythropoiesis seen on the mean hemoglobin
level on IDA was 13.6 ± 1.88 g/dL and mean hematocrit
44.73 ± 7.92.
The utilization of erythrocyte index (MCV, MCH,
MCHC, and RDW) to assess iron deficiency in pediatric
CHD had conflicting results in previous studies. MCV
and RDW are stable parameters on secondary polycythemia due to chronic hypoxia. MCV < 80 fL was used
as a cut-off value for the progression of iron depletion inpediatric CHD [6, 15, 30]. In contrast to another study,
MCV could be influenced by secondary erythropoiesis,
which physiologically increased the production of young
erythrocytes with a greater volume [14]. Therefore, theirusage should be combined (MCV < 80 fL and MCH < 27pg) [17, 30]. This study showed a lower mean value of
MCV and MCH in all iron status groups, in accordancewith a previous case-control study on pediatric CHD [5].
Serum ferritin is limited as a marker of iron deficiency
as it is an acute phase reactant which will be increased
in inflammation, chronic infection, and other diseases[31, 32]. Due to this reason, serum ferritin level mayvary in IDA, necessitating the use of other hematological(hemoglobin, hematocrit, MCV, MCH) and biochemistry (transferrin saturation) parameters [15]. Specifically,
the application with transferrin saturation was suggested.
The distinction between iron depletion and iron deficiency could be made based on the decrease in transferrin saturation, as observed in a previous study [10, 18].
Therefore, this study utilized both markers as indicator
based on the fulfillment of one of the criteria.
The previous study stated that Ret-He is a stableparameter as it is not influenced by inflammation, malignancy, or chronic disease [2, 33]. Its usage on healthy
neonates and pediatric showed a good diagnostic value[8, 9]. The cut-off value of Ret-He could be influenced byseveral factors, such as race and age [34]. Previous studies stated the Ret-He cut-off for iron deficiency withoutanemia differs between age groups, such as 29 pg for 3–4
months (AUC 0.86; sensitivity 85%; specificity 73%) [9],28.55 pg for 6–24 months (AUC 0.819; sensitivity 76.6%;
specificity 78.4%) [13], and 28.5 pg for 6–16 years (sensitivity 43.5%; specificity 91.1%) [2]. The majority of participants in this study were between the ages of 0 and 2

Page 6 of 8

years (49.2%), so the ID cut-off of 28.8 pg without anemia
was within range.
A study of Ret-He in Indonesia to determine iron status
on healthy pediatric aged 6–18 years old had done with acut-off value of 28.9 pg for ID without anemia (sensitivity 78.9%; specificity 56.2%) and 27 pg for IDA (sensitivity
75%; specificity 80%) [34]. The AUC for ID without anemia in this previous study was inferior with a cut-off of28.9 pg (0.801) versus a cut-off of 28.8 pg (0.824) in thisstudy. The current research exhibits greater sensitivity
and specificity for ID without anemia in comparison to
the previous study. Moreover, the AUC for IDA in theprevious study was inferior with a cut-off of 27 pg (0.757)
versus a cut-off of 28.15 pg (0.824), also the sensitivity
and specificity due to the difference in anemia definition
between studies. The hemoglobin levels in previous study
used WHO criteria based on age, whereas in this study
used hemoglobin levels on CHD (Hb < 16.5 g/dL) [15].
Although there are differences in the cut-off value ofhemoglobin levels, this study found that the Ret-He values in the pediatric CHD population with lower hemoglobin levels (Hb < 16.5 g/dL) also had lower Ret-He
values. The mean value of Ret-He in the normal iron status group was 31.6 pg and significantly lower in the IDA
group at 23.4 pg. This mean difference was statisticallyand clinically significant. However, the mean value of
Ret-He in the iron deficiency without anemia was 25.3 pg
compared to the IDA group 23.4 pg, so that it was clinically and statistically not significant (p = 0.428). Therefore, Ret-He could not be used as a single parameter todifferentiate pediatric CHD with iron deficiency withoutanemia and IDA; instead, it should be used in combination with hemoglobin. This result corroborates the priorresearch that Ret-He alone is insufficient to differentiate
between IDA and iron deficiency without anemia [35].
Nevertheless, this outcome differed from another study
that indicated that Ret-He could serve as a single parameter for IDA and iron deficiency without anemia, without
taking into account other iron parameters [36].
Strength and limitation

There were several strengths of this study. To the best
knowledge of the authors this was the first study to investigate the role of Ret-He in the evaluation of iron deficiency and IDA in pediatric CHD between the ages of 3
months and 18 years. Second, this study excluded somepossible confounders in the evaluation of iron status,
such as CRP ≧ 5 mg/dL and more than 3 months old.
There were also several limitations of this study. First,
there was no universally recognized cut-off for hemoglobin levels in children with CHD across the globe. In thisstudy, we applied a cut-off of hemoglobin less than 16.5 g/
dL, which might be perceived relatively high [15]. Consequently, the use of a high cut-off level of hemoglobin

Cheng et al. BMC Pediatrics(2024) 24:541

may lead to overdiagnosis of IDA. Second, there was no
peripheral blood smear for erythrocyte index confirmation, used to reduce the risk of error in automatic blood
sample processing. Finally, as a cross-sectional diagnostic
test, chronic hypoxia was evaluated on recruitment of thestudy subjects (daily oxygen saturation < 90%), and no differentiation of hypoxia severity. It would be beneficial toconduct a larger sample of diagnostic study in order togather additional information regarding the use of RetHe in this particular population.

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Data availability
The datasets used and or anlysed during the current study are available fromthe corresponding author on a validated request.

Declarations
Ethics approval and consent to participate
This study received approval ethics from Health Research Ethics CommitteeFaculty of Medicine Universitas Indonesia and Cipto Mangunkusumo Hospital(HREC-FMUI/CMH) for feasibility studies at both hospitals (number: KET-1190/
UN2.F1/ETIK/PPM.00.02/2021). Informed written consent was obtainted fromthe parents and assent was obtained from children and adolescents before
commencement of the study.

Conclusion
Reticulocyte hemoglobin equivalent is a good diagnostic
parameter to evaluate iron deficiency and IDA in pediatric CHD. The usage of Ret-He should be accompanied byhemoglobin. In pediatric CHD, iron deficiency withoutanemia could be diagnosed with Ret-He < 28.8 pg withhemoglobin ≥ 16,5 g/dL. While IDA could be diagnosed
with Ret-He < 28.15 pg or Ret-He 28.15-28,8 pg withhemoglobin < 16,5 g/dL. Further studies should be considered for determining the Ret-He cut-off in several age
groups and different hypoxia severity.

Consent for publication
Not applicable.

Abbreviations
AAP	American Academy of Pediatrics
AUC	Area under the curve
CDC	Centers for Disease Control and Prevention
CHD	Cyanotic Heart Disease
CRP	C-Reactive Protein
ID	Iron deficiency
IDA	Iron deficiency anemia
MCH	Mean corpuscular hemoglobin
MCHC	Mean corpuscular hemoglobin concentration
MCV	Mean corpuscular volume
NPV	Negative predictive value
PPV	Positive predictive value
OR	Odd ratio
RDW	Red cell distribution width
Ret-He	Reticulocyte hemoglobin equivalent
ROC	Receiver operating characteristic
TOF	Tetralogy of FallotWHO	World Health Organization

References
1. Rungngu SLP, Wahani A, Mantik MFJ. Reticulocyte hemoglobin equivalent for diagnosing iron deficiency anemia in children. Paediatr Indones.
2016;56:90–4.
2. Vázquez-López MA, López-Ruzafa E, Ibáñez-Alcalde M, Martín-González
M, Bonillo-Perales A, Lendínez-Molinos F. The usefulness of reticulocyte
haemoglobin content, serum transferrin receptor and the sTfR-ferritin index
to identify iron deficiency in healthy children aged 1–16 years. Eur J Pediatr.
2019;178:41–9.
3. Domellöf M, Braegger C, Campoy C, Colomb V, Decsi T, Fewtrell M, et al.
Iron requirements of infants and toddlers. J Pediatr Gastroenterol Nutr.
2014;58:119–29.
4. Sundararajan S, Rabe H. Prevention of iron-deficiency anemia in infants andtoddlers. Pediatr Res. 2020;1:1–11.
5. Mir AB, Shah JA, Mushtaq S, Jan M. Prevalence, profile of malnutrition andiron deficiency anemia in children with cyanotic congenital heart defects: acase control observational study in a tertiary care hospital in North India. Int J
Contemp Pediatr. 2019;6:1303–5.
6. Olcay L, Ozer S, Gugey A. Parameters of iron deficiency in children withcyanotic congenital heart disease. Pediatr Cardiol. 1996;17:150–4.
7. Baker RD, Greer FR, Bhatia JJS, Abrams SA, Daniels SR, Schneider MB, et al.
Clinical report - diagnosis and prevention of iron deficiency and irondeficiency anemia in infants and young children (0–3 years of age). Pediatrics.
2010;126:1040–50.
8. Mateos ME, De-La-Cruz J, López-Laso E, Valdés MD, Nogales A. Reticulocyte
hemoglobin content for the diagnosis of iron deficiency. J Pediatr Hematol
Oncol. 2008;30:539–42.
9. Lorenz L, Arand J, Büchner K, Wacker-Gussmann A, Peter A, Poets CF, et al.
Reticulocyte haemoglobin content as a marker of iron deficiency. Arch Dis
Child Fetal Neonatal Ed. 2015;100:198–202.
10. Wicaksana RA, Yanuarso PB, Setyohandryastuti R, Wahidiyat PA. Studi preliminari: serial kasus peran pemeriksaan reticulocyte hemoglobin equivalent
untuk menilai defisiensi besi pada anak dengan penyakit jantung bawaan
sianotik. [thesis]. [Jakarta]. Universitas Indonesia; 2020.
11. Sjarif DR, Nasar SS, Devaera Y, Tanjung CF. Rekomendasi Ikatan Dokter AnakIndonesia: Asuhan Nutrisi Pediatrik (Pediatric Nutrition Care). Jakarta: Badan
Penerbit Ikatan Dokter Anak Indonesia; 2011. p. 5–9.
12. Bujang MA, Adnan TH. Requirements for minimum sample size for sensitivity
and specificity analysis. J Clin Diagn Res. 2016;10:1–6.

Acknowledgements
We thank the participants, staffs at the participating laboratories, and medical
staffs at the Cipto Mangunkusumo Hospital and Harapan Kita Women andChildren’s Hospital who helped process the data and specimen collections.
Author contributions
All authors contributed to the design of this protocol. WC, PBY, and PAW
initiated the study. WC drafted the protocol and was refined by PPT, PBY, and
PAW. PPT supervised the statistical and methodological aspect of the study.
PBY and SR were conducted or supervised the echocardiography for diagnosisof pediatric cyanotic heart disease and ensured the speciment collection.
PPT, PAW, MK, and DM were responsible for verifying the underlying data. WC,
PBY, PAW, PPT, SR, MK, and DM drafted the first version of the manuscript. All
authors approved the final manuscript. The corresponding author declaresthat all listed authors meet authorship criteria and that no others meeting thecriteria have been omitted.
Funding
The authors received no specific funding for this study from any funding
agency in the public, commercial, and governmental sectors.

Competing interests
The authors declare no competing interests.
Author details
1
Department of Child Health, Faculty of Medicine, Universitas Indonesia,
Cipto Mangunkusumo Hospital, Jakarta, Indonesia
2
Harapan Kita Women’s and Children’s Hospital, Jakarta, Indonesia
Received: 8 September 2023 / Accepted: 8 August 2024

Cheng et al. BMC Pediatrics(2024) 24:541

13. Sandy IM, Andriastuti M. Evidence base case report: The role of reticulocyte
hemoglobin content (RET-He) to detect iron deficiency in children. Sari
Pediatri. 2019;20:16–22.
14. Animasahun BA, Itiola J, Adekunle MO. Comparison of erythrocyte indicesand haematological indices as markers of iron status of lagos children withcyanotic congenital heart disease. Ann Blood. 2020;5:1–12.
15. Binh TQ, Thi N, Suong B, Dinh NH, Anh VT, Thanh VT, et al. Iron deficiency
anemia in children with congenital heart disease and the usefulness oferythrocyte indexes as a screening tool. Arch Gen Intern Med. 2018;2:1–6.
16. Mukherjee S, Sharma M, Devgan A, Jatana SK. Iron deficiency anemia inchildren with cyanotic congenital heart disease and effect on cyanotic spells.
Med J Armed Forces India. 2018;74:235–40.
17. Onur CB, Sipahi T, Tavil B, Karademir S, Yoney A. Diagnosing iron deficiency incyanotic heart disease. Indian J Pediatr. 2003;70:29–31.
18. Puri K, Allen HD, Qureshi AM. Congenital heart disease. Pediatr Rev.
2017;38:471–84.
19. Yeh SJ, Chen HC, Lu CW, Wang JK, Huang LM, Huang SC, et al. Prevalence,mortality, and the disease burden of pediatric congenital heart disease in
Taiwan. Pediatr Neonatol. 2013;54:113–8.
20. Mandalenakis Z, Rosengren A, Skoglund K, Lappas G, Eriksson P, Dellborg M.
Survivorship in children and young adults with congenital heart disease in
Sweden. JAMA Intern Med. 2017;177:224–30.
21. Powers JM, Buchanan GR. Disorders of Iron Metabolism: New Diagnostic andTreatment approaches to Iron Deficiency. Hematol Oncol Clin North Am.
2019;33:393–408.
22. Wu AC, Lesperance L, Bernstein H. Screening for Iron Deficiency the early
introduction of whole cow milk. Pediatr Rev. 2011;23:171–8.
23. Okoromah CAN, Ekure EN, Lesi FEA, Okunowo WO, Tijani BO, Okeiyi JC. Prevalence, profile and predictors of malnutrition in children with congenital heart
defects: a case-control observational study. Arch Dis Child. 2011;96:354–60.
24. Steltzer M, Rudd N, Pick B. Nutrition care for newborns with congenital heartdisease. Clin Perinatol. 2005;32:1017–30.
25. Mohammed SH, Larijani B, Esmaillzadeh A. Concurrent anemia and stunting
in young children: prevalence, dietary and non-dietary associated factors.
Nutr J. 2019;18:1–10.
26. Hassan BA, Albanna EA, Morsy SM, Siam AG, Al Shafie MM, Elsaadany HF, et al.
Nutritional status in children with un-operated congenital heart disease: an
Egyptian Center experience. Front Pediatr. 2015;3:1–5.

Page 8 of 8

27. Chinawa AT, Chinawa JM, Duru CO, Chukwu BF, Obumneme-Anyim I.
Assessment of Nutritional Status of Children with congenital heart disease: acomparative study. Front Nutr. 2021;8:1–7.
28. Rathod B. Anthropometric profiles of children with congenital heart disease.
Pediatr Educ Res. 2017;5:23–7.
29. Yang W, Li X, Li Y, Zhang S, Liu L, Wang X, et al. Anemia, malnutrition and theircorrelations with socio-demographic characteristics and feeding practices
among infants aged 0–18 months in rural areas of Shaanxi province in northwestern China: a cross-sectional study. BMC Public Health. 2012;12:1–7.
30. Ossei I, Buabeng KO, Ossei PPS, Nguah SB, Ayibor WG, Anto BP, et al. Irondeficiency anaemia in children with congenital heart diseases at a teaching
hospital in Ghana. Heliyon. 2020;6:1–5.
31. Allali S, Brousse V, Sacri AS, Chalumeau M, de Montalembert M. Anemia inchildren: prevalence, causes, diagnostic work-up, and long-term consequences. Expert Rev Hematol. 2017;10:1023–8.
32. World Health Organization. WHO guideline on use of ferriti concentrations toassess iron status in individuals and population. 2020. p. 20–31.
33. Gelaw Y, Woldu B, Melku M. The role of reticulocyte hemoglobin content for
diagnosis of iron deficiency and iron deficiency anemia, and monitoring ofiron therapy: a litterature review. Clin Lab. 2019;67:1–7.
34. Melita, Andriastuti M, Satari HI. Reticulocyte heaemoglobin content sebagai
parameter skrining alternatif untuk mendeteksi defisiensi besi pada anak[thesis].[Jakarta]. Universitas Indonesia; 2019.
35. Aedh AI, Khalil MSM, Abd-Elkader AS, El-Khawanky MM, Alshehri HM, Hussein
A, et al. Reticulocyte hemoglobin as a screening test for Iron Deficiency
Anemia: a New Cut-Off. Hematol Rep. 2023;15:201–11.
36. Neef V, Schmitt E, Bader P, Zierfuß F, Hintereder G, Steinbicker AU et al. The
reticulocyte hemoglobin equivalent as a screening marker for iron deficiency
and iron deficiency anemia in children. J Clin Med. 2021;10–5.

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