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BMJ Paediatr Open
BMJ Paediatr Open
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bmjpo
BMJ Paediatrics Open
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BMJ Publishing Group BMA House, Tavistock Square, London, WC1H 9JR

39209439
10.1136/bmjpo-2024-002736
bmjpo-2024-002736
Original Research
Haematology
1506
Reference values for reticulocyte haemoglobin equivalent in healthy Chinese children under 5 years and its associations with various blood parameters
Tung Keith Tsz Suen 10keith-tung@connect.hku.hk

Chen Chen 10chencici@hku.hk

Chiu Yung Tuen 2elley.chiu@ha.org.hk

http://orcid.org/0000-0002-6188-7587
So Hung Kwan 1hkso@hku.hk

So Chi Chiu 2*scc131@ha.org.hk

http://orcid.org/0000-0002-6797-6898
Ip Patrick 13*patricip@hku.hk

1 Department of Paediatrics and Adolescent Medicine, The University of Hong Kong, Hong Kong, People's Republic of China
2 Department of Pathology, Hong Kong Children's Hospital, Hong Kong, People's Republic of China
3 Department of Paediatrics and Adolescent Medicine, Hong Kong Children's Hospital, Hong Kong, People's Republic of China
none.

DrChi ChiuSo; scc131@ha.org.hk
DrPatrickIp; patricip@hku.hk
KTST and CC contributed equally.

2024
28 8 2024
8 1 e00273605 5 2024
08 8 2024
Copyright © Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.
2024
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Abstract

Background

Reticulocyte haemoglobin equivalent (RET-He) is a useful tool for evaluating recent iron usage irrespective of inflammatory status. This study aims to establish a reference for RET-He among Hong Kong healthy children under the age of 5 years and to investigate the association between RET-He and various blood parameters.

Methods

A total of 946 children aged 2–48 months from July 2019 to December 2022 were recruited in this cross-sectional study. The RET-He and other haematological parameters were measured by the haematology analyser from Sysmex XN-9100/XN-1500. The ferritin test was performed with the electrochemiluminescence immunoassay. Interval 2.5th percentile to 97.5th percentile represented the normal RET-He ranges. Linear multiple regression analysis was performed to examine the relation between RET-He and various blood parameters. Receiver-operating characteristic curve analysis revealed the sensitivity and specificity of RET-He in identifying iron deficiency.

Results

The RET-He in the study population was approximately normally distributed. The age-specific lower limit of RET-He ranges from 25.81 pg (25–36 months) to 27.15 pg (13–24 months). RET-He was found to be lower in the age group 2–6 months (mean=29.47 pg) and 7–12 months (mean=29.41 pg). Changes in RET-He and haemoglobin in relation to age were observed in both sexes (both p<0.001). RET-He was influenced by age, some red blood cell parameters and reticulocyte concentrations (all p<0.05). A cut-off value of RET-He ≤27.8 pg was determined for identifying iron deficiency.

Conclusions

RET-He levels varied with age, with a relatively lower level in infants than in other age groups. The value below the age-specific lower limit of the reference range of RET-He can be used as a limit for preliminary iron-deficiency screening.

Child Health
Infant
Hong Kong Health and Medical Research Fund Vit D-HKU and the Collaborative Research Fund, University Grants Committee C7149-20GF
==== Body
pmcWHAT IS ALREADY KNOWN ON THIS TOPIC

WHAT THIS STUDY ADDS

This study provides the age-specific reference values for RET-He in healthy Chinese children under 5 years for initial iron-deficiency screening.

RET-He is significantly associated with other haematological parameters indicating iron-deficiency status, demonstrating a potential predictive tool as an early indicator of iron deficiency in children solely.

HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY

It is feasible to promote iron-deficiency screening with RET-He in young children in Hong Kong since the availability of advanced hematology analyser equipment and the age-specific reference value of RET-He.

Further research is warranted to evaluate the risk of developing iron-deficiency anaemia when diagnosed with iron deficiency with the established RET-He reference by conducting a prospective study.

Introduction

Iron-deficiency anaemia (IDA) is a leading global health problem affecting approximately 16.42% of children under 5 years globally.1 2 Children in this age group are at a critical stage of physical and intellectual development,3 4 and IDA can negatively impact their mental, physical and social development, leading to poor school performance and work capacity in later years.57 Therefore, early diagnosis and prompt treatment of IDA are critical to prevent the long-term effects of brain iron deficiency (ID).5 8

Although bone marrow biopsy is the gold standard for diagnosing ID, it is invasive, painful and carries a risk of bleeding or infection from the puncture site.8 9 In clinical practice, serum ferritin levels are commonly used to assess iron status.10 However, diagnosing IDA in patients with reticulocyte haemoglobin equivalent (RET-He) in inflammatory conditions can be challenging as ferritin is an acute phase reactant and its levels are elevated in RET-He in inflammatory conditions.11 12 Recent studies have shown that RET-He could be a useful tool in evaluating recent iron usage and the state of erythropoiesis.13 Unlike many iron markers, it is not influenced by inflammation.11 Therefore, it may be more effective than traditional approaches such as bone marrow iron staining.

The diagnostic usefulness of RET-He has been extensively studied in adults, specifically in cases of functional IDA, iron-restricted erythropoiesis in haemodialysis patients undergoing erythropoietin therapy and in evaluating the response to iron treatment.12 14 Similarly, in children, RET-He has been thoroughly investigated in early young healthy populations and was found to be a reliable indicator of ID.15 16 However, there are limited established reference ranges for RET-He in healthy children, especially for Chinese population. One study established reference values of RET-He in healthy children aged 1–11 years of age in Spain but it did not particularly focus on infants, a vulnerable age group of ID.17 Little is known about how RET-He levels vary with age, gender or erythropoietic activity. Therefore, more research is required to better understand the role of RET-He and its interpretation in different populations.

The study aims to establish a reference for RET-He among Hong Kong healthy children under the age of 5 years and to investigate the association between RET-He and various blood parameters,in particular, the effectiveness of RET-He as a diagnostic indicator for identifying ID in children.

Methods

Study population

This cross-sectional study was conducted from July 2019 to December 2022. 1031 children aged 2–48 months were recruited by stratified random sampling in different districts of Hong Kong, including 6 major maternal and child health centres (MCHCs) and 12 kindergartens. To compile a sampling frame of all kindergartens in Hong Kong, a list of kindergartens from the ‘Profile of Kindergarten and Kindergarten-cum-child care Centers Education Bureau’ was used. Infants with any major congenital malformations, being born prematurely, or with low birth weight were excluded from this study.

On obtaining informed consent, parents filled out demographic questionnaires and reported the age and sex of their children. Study participants could not have a known haemoglobinopathy, history of anaemia or received a blood transfusion or iron supplement. Peripheral blood samples were collected with anticoagulant tubes by a well-trained phlebotomist.

Laboratory methods

The haematology analyser from Sysmex XN-9100/XN-1500 was used to measure RET-He and other blood parameters. In brief, the reticulocytes are detected in the RET channel of the analyser. The nucleic acids in the immature erythrocytes are stained by the fluorescent dye and measured by the flow cytometry. The haemoglobin concentration is measured in the HGB channel of the analyser by the sodium lauryl sulfate haemoglobin method. The chemical pathology division carried out the ferritin test using an electrochemiluminescence immunoassay on the Cobas E801 immunoassay analyser. The 18 min test involved two incubations. In the first, a sample was combined with specific antibodies to form a complex. In the second, the complex was bound to a solid phase using microparticles. The reaction mixture was then placed in a measuring cell, where the microparticles were magnetically captured and unbound substances were removed. A voltage was applied, causing chemiluminescent emission, which was measured to determine the results. The results were calculated using a calibration curve generated by a 2-point calibration and a master curve provided by the cobas link. According to WHO guidelines 2011, ID is suggested when serum ferritin <27 pmol/L in children up to 5 years old, in the absence of acute/chronic inflammation.

Statistical analysis

Qualitative variables were represented by percentages, while quantitative variables were represented by mean±SD, including 95% CI and reference interval (RI) of 2.5th–97.5th percentile using a parametric approach. RET-He distribution and percentile values were analysed only for healthy children with haemoglobin concentration ≥110 g/L. The laboratory parameters were compared by sex using Student’s t-test and by age using one-way analysis of variance. Linear multiple regression analysis was conducted with RET-He as the dependent variable and independent variables including sex, age and BMI z-score. The regression models were adjusted for the sex and age of the child and introduced individually. A 5% level of significance was considered statistically significant. The sensitivity and specificity of RET-He as a marker were determined by receiver-operating characteristic (ROC) plots by MedCalc for window, V.22.001, 95% CI was computed for 2.5 and 97.5 percentiles for 2000 bootstrap resamples using the DescTools Package for R V.4.3.2. Other statistical analyses were conducted by SPSS V.26.0.

Results

A total of 946 children (497 boys and 449 girls) were analysed. Participants were stratified into 5 age groups with a roughly similar distribution, with 167 (17.7%) aged 2–6 months, 184 (19.5%) aged 7–12 months, 289 (30.5%) aged 13– 24 months, 134 (14.2%) aged 25–36 months and 172 (18.2%) aged 37– 48 months.

Table 1 displays the RIs and mean values for various blood parameters while table 2 shows RET-He percentiles for different age groups. Figure 1 shows RET-He was normally distributed in the study population. Table 3 displays the references categorised by age, sex and body weight status. Girls had higher serum ferritin (p<0.001) and reticulocyte (p<0.001) compared with boys, while RET-He and haemoglobin values were similar in both sexes. Haemoglobin level was higher in overweight children than in non-overweight children (p=0.018). Haemoglobin concentration was found to be higher among overweight children. Blood parameters varied with age. RET-He was found to be lower in the age group 2–6 months (mean=26.02) and 7–12 months (mean=26.20).

Table 1 Mean values and reference intervals for various blood parameters (N=946)

Variables	Mean±SD	95% CI	Reference interval P2.5–P97.5	
Erythrocytes (×1012 /L)	4.64±0.32	4.62 to 4.66	4.01–5.28	
Haemoglobin (g/L)	123.6±7.3	123.2 to 124.1	111.0–138.0	
MCV (fL)	78.77±3.37	78.55 to 78.98	72.17–85.40	
MCH (pg)	26.72±1.33	26.63 to 26.80	23.80–29.23	
MCHC (g/dL)	33.91±0.87	33.86 to 33.97	32.27–35.60	
HCT (L/L)	0.37±0.03	0.36 to 0.37	0.30–0.41	
RDW (%)	12.48±0.83	12.42 to 12.53	11.30–14.30	
Leukocytes (×109 /L)	8.43±2.34	8.28 to 8.58	4.70–13.73	
Platelet (×109 /L)	343.04±89.73	337.31 to 348.77	181.00–539.28	
MPV (fL)	9.96±0.87	9.91 to 10.02	8.60–11.80	
Mentzer index	17.10±1.71	16.99 to 17.20	14.04–20.74	
Reticulocytes (×109 /L)	61.88±17.18	60.79 to 62.98	34.57–100.43	
RET-He (pg)	30.04±1.54	29.94 to 30.14	26.40–32.90	
Ferritin (pmol/L)	149.88±135.27	141.24 to 158.53	31.00–484.15	
HCThaematocritMCHmean corpuscular haemoglobinMCHCmean corpuscular haemoglobin concentrationMCVmean corpuscular volumeMPVmean platelet volumePpercentileRDWred cell distribution widthRET-Hereticulocyte haemoglobin equivalent

Table 2 Reticulocyte haemoglobin equivalent (RET-He) percentiles according to age groups

	Age group (months)	
RET-He (pg) percentile	2–6 (n=167)	7–12 (n=184)	13–24 (n=289)	25–36 (n=134)	37–48 (n=172)	Total (n=946)	
P2.5	25.92	26.13	27.15	25.81	27.07	26.40	
P5	26.44	26.50	27.70	27.08	27.47	27.24	
P10	27.70	27.45	28.70	28.25	28.50	28.07	
P25	28.70	28.50	29.80	29.30	29.43	29.20	
P50	29.50	29.70	30.70	30.40	30.20	30.20	
P75	30.50	30.60	31.60	30.93	30.98	31.00	
P90	31.42	31.40	32.50	31.50	31.40	31.90	
P95	31.96	32.18	33.10	32.23	31.80	32.40	
P97.5	32.62	32.64	33.45	32.56	32.00	32.90	

Figure 1 Reticulocyte haemoglobin equivalent (RET-He) distribution among all participants.

Table 3 Comparison of mean values and reference intervals for (A) RET-He and ferritin and (B) reticulocytes and haemoglobin, according to sex, body weight status and age

Variables		RET-He (pg)	Ferritin (pmol/L)	
N	Mean±SD	P2.5 (95% CI)	P97.5 (95% CI)	Mean±SD	P2.5 (95% CI)	P97.5 (95% CI)	
(A)								
Sex								
 Male	497	30.01±1.52	26.44 (25.74 to 26.88)	32.76 (32.52 to 33.06)	135.38±98.36	31.00 (22.00 to 33.00)	387.85 (308.36 to 440.70)	
 Female	449	30.08±1.58	26.34 (25.58 to 26.68)	33.18 (33.06 to 33.68)	165.91±165.47	31.00 (22.00 to 33.00)	598.00 (419.00 to 732.35)	
 P value		0.486			<0.001			
Overweight								
 Yes	123	29.87±1.55	26.31 (25.01 to 26.51)	32.88 (32.55 to 33.65)	155.18±111.66	31.25 (13.35 to 33.50)	473.35 (366.70 to 600.90)	
 No	801	30.07±1.55	26.50 (26.00 to 27.00)	32.90 (32.60 to 33.20)	147.97±136.35	31.00 (26.00 to 33.00)	465.00 (417.01 to 529.75)	
 P value		0.186			0.577			
Age (months)								
 2–6	167	29.55±1.53	26.02 (25.43 to 26.49)	32.29 (31.67 to 32.72)	250.35±251.78	31.90 (11.80 to 36.60)	943.50 (798.40 to 1244.90)	
 7–12	184	29.56±1.57	26.20 (25.40 to 26.79)	32.49 (32.13 to 32.87)	114.50±71.76	27.20 (23.40 to 33.30)	311.15 (270.20 to 400.30)	
 13–24	289	30.63±1.55	27.30 (26.88 to 28.10)	33.30 (32.40 to 33.52)	109.34±57.86	31.00 (26.00 to 39.00)	248.80 (191.60 to 282.20)	
 25–36	134	30.02±1.45	26.03 (24.60 to 26.57)	32.47 (32.24 to 33.24)	151.00±84.34	57.63 (43.25 to 66.25)	346.50 (138.00 to 432.98)	
 37–48	172	30.08±1.23	27.23 (26.75 to 27.80)	31.97 (31.08 to 32.25)	158.51±96.49	50.13 (35.25 to 58.68)	349.88 (227.78 to 411.14)	
 P value		<0.001			<0.001			
(B)				
Variables		Reticulocytes (×10 9  /L)	Haemoglobin (g/L)	
N	Mean±SD	P 2.5 (95% CI)	P 97.5 (95% CI)	Mean±SD	P 2.5 (95% CI)	P 97.5 (95% CI)	
Sex							
 Male	497	59.14±16.58	33.12 (30.66 to 36.52)	95.10 (86.40 to 100.20)	123.9±7.4	111.0 (110.0 to 112.0)	138.0 (135.0 to 139.4)	
 Female	449	64.92±17.35	34.98 (30.24 to 36.16)	102.38 (92.16 to 108.82)	123.3±0.73	111.0 (110.0 to 112.0)	137.0 (136.0 to 138.0)	
 P value		<0.001			0.196		
Overweight							
 Yes	123	62.61±15.99	35.22 (32.86 to 41.63)	91.43 (82.56 to 98.30)	125.0±7.9	111.1 (109.1 to 112.1)	139.0 (135.9 to 141.0)	
 No	801	61.61±17.26	34.80 (33.90 to 37.60)	100.10 (94.40 to 105.70)	123.4±7.2	111.0 (111.0 to 112.0)	137.0 (136.0 to 138.0)	
 P value		0.549			0.018		
Age (months)							
 2–6	167	58.50±15.19	34.82 (32.83 to 38.41)	87.82 (75.02 to 92.63)	119.7±6.5	110.0 (109.0 to 110.0)	133.9 (130.9 to 135.9)	
 7–12	184	62.03±16.83	39.22 (36.27 to 43.11)	99.05 (71.90 to 112.80)	121.9±7.0	111.0 (110.4 to 112.0)	135.0 (133.0 to 136.0)	
 13–24	289	61.96±15.75	35.74 (31.68 to 38.26)	95.62 (91.68 to 100.46)	124.0±6.8	112.0 (111.0 to 113.8)	137.0 (134.2 to 138.0)	
 25–36	134	64.17±19.85	27.46 (18.58 to 30.62)	102.62 (75.04 to 113.22)	126.2±7.3	113.3 (110.7 to 116.7)	140.0 (134.1 to 143.1)	
 37–48	172	63.11±19.05	32.25 (27.40 to 33.69)	107.61 (97.76 to 122.94)	126.7±7.0	115.0 (114.5 to 116.0)	139.0 (129.8 to 141.0)	
 P value		0.045			<0.001		

Table 4 shows the variations in blood parameters (RET-He, serum ferritin, reticulocytes and haemoglobin) by sex and age. Both sexes showed similar age-related variations in haemoglobin and RET-He while young children (ages 2–6 months) of both sexes had greater serum ferritin levels. The mean values of the RET-He and haemoglobin in each age group were similar in both sexes. Girls had higher reticulocyte levels than boys in general. A series of linear regression models were conducted to determine which independent variables were associated with RET-He. As shown in table 5, age-adjusted and sex-adjusted models showed that RET-He was influenced by age, red cell counts (RCCs) (haemoglobin, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), MCHC, red cell distribution width) and reticulocytes (all p<0.05). There was no significant association between RET-He and sex, body weight status, HCT, leucocytes and ferritin.

Table 4 Sex-specific mean values and reference intervals for RET-He, ferritin, reticulocytes, and haemoglobin by age group

Variables	N	Mean±SD	P2.5 (95% CI)	P97.5 (95% CI)	P value	
(A)Males (N=497)						
RET-He (pg)					<0.001	
 2–6	84	29.43±1.52	26.41 (25.22 to 27.48)	32.66 (32.43 to 33.73)		
 7–12	96	29.57±1.64	26.28 (25.41 to 26.98)	32.53 (31.96 to 32.95)		
 13–24	156	30.49±1.47	27.30 (26.68 to 28.30)	33.10 (32.20 to 33.64)		
 25–36	74	30.22±1.41	25.95 (23.50 to 26.40)	32.52 (32.14 to 33.50)		
 37–48	87	30.00±1.21	27.22 (26.03 to 27.77)	31.97 (31.94 to 32.26)		
Ferritin (pmol/L)					<0.001	
 2–6	84	187.00±156.01	40.15 (26.10 to 53.30)	500.35 (71.10 to 571.50)		
 7–12	96	107.03±69.06	29.00 (18.02 to 36.25)	312.55 (293.25 to 419.00)		
 13–24	156	108.76±52.68	30.75 (19.13 to 39.50)	238.00 (170.00 to 282.82)		
 25–36	74	139.08±62.54	62.65 (53.30 to 71.30)	308.00 (263.00 to 372.65)		
 37–48	87	161.98±115.79	49.63 (33.63 to 61.00)	441.13 (30.25 to 605.88)		
Reticulocytes (×109/L)					0.023	
 2–6	84	54.52±15.31	33.69 (30.68 to 38.43)	87.93 (67.23 to 102.96)		
 7–12	96	60.08±17.25	37.35 (31.30 to 48.70)	90.35 (29.31 to 103.10)		
 13–24	156	58.88±15.01	35.50 (31.90 to 38.70)	91.25 (82.20 to 99.93)		
 25–36	74	59.31±15.82	26.65 (12.93 to 28.99)	92.83 (84.66 to 104.88)		
 37–48	87	62.89±19.36	32.42 (24.45 to 34.15)	112.89 (108.03 to 134.78)		
Haemoglobin (g/L)					<0.001	
 2–6	84	119.5±6.3	110.0 (108.9 to 110.0)	133.0 (130.2 to 136.2)		
 7–12	96	122.6±7.2	111.0 (109.0 to 111.6)	137.0 (134.5 to 141.0)		
 13–24	156	124.6±7.0	111.0 (109.0 to 112.0)	139.1 (136.3 to 142.3)		
 25–36	74	126.7±7.5	113.8 (110.7 to 117.7)	141.9 (137.8 to 146.8)		
 37–48	87	126.1±6.9	114.2 (112.3 to 114.3)	138.9 (130.2 to 141.7)		
(B) Females (N=449)						
Variables	N	Mean±SD	P 2.5 (95% CI)	P 97.5 (95% CI)	P value	
RET-He (pg)					<0.001	
 2–6	83	29.66±1.54	26.01 (24.49 to 26.49)	32.10 (29.80 to 32.70)		
 7–12	88	29.54±1.49	26.25 (25.11 to 27.08)	32.05 (31.31 to 32.73)		
 13–24	133	30.78±1.63	27.19 (26.15 to 27.88)	33.71 (32.92 to 34.25)		
 25–36	60	29.78±1.49	26.58 (25.16 to 27.75)	32.35 (32.01 to 33.21)		
 37–48	85	30.16±1.24	27.31 (26.57 to 28.27)	31.90 (30.51 to 32.51)		
Ferritin (pmol/L)					<0.001	
 2–6	83	314.46±308.94	31.45 (1.13 to 33.85)	1038.88 (105.28 to 1324.75)		
 7–12	88	122.57±74.12	25.70 (15.40 to 32.70)	300.30 (203.68 to 380.60)		
 13–24	133	110.02±63.60	31.90 (23.80 to 45.80)	258.30 (107.60 to 315.00)		
 25–36	60	165.70±103.91	52.33 (22.80 to 61.65)	470.45 (284.90 to 698.68)		
 37–48	85	154.96±72.12	54.83 (29.83 to 62.65)	340.25 (224.43 to 417.50)		
Reticulocytes (×109/L)					0.086	
 2–6	83	62.52±14.05	35.79 (29.07 to 37.44)	86.10 (71.53 to 89.60)		
 7–12	88	64.17±16.20	39.82 (35.97 to 40.59)	100.77 (76.09 to 116.24)		
 13–24	133	65.58±15.89	40.07 (35.35 to 45.24)	96.18 (84.16 to 101.26)		
 25–36	60	70.16±22.64	32.49 (22.37 to 47.17)	129.25 (126.80 to 162.91)		
 37–48	85	63.33±18.83	32.99 (24.69 to 48.51)	103.79 (81.98 to 120.65)		
Haemoglobin (g/L)					<0.001	
 2–6	83	119.8±6.6	111.0 (110.0 to 112.0)	134.0 (130.9 to 138.0)		
 7–12	88	121.1±6.8	111.0 (109.8 to 112.0)	134.0 (133.2 to 136.0)		
 13–24	133	123.4±6.6	113.0 (112.0 to 115.0)	136.7 (134.4 to 139.3)		
 25–36	60	125.5±7.0	112.4 (107.8 to 114.8)	138.0 (133.0 to 140.0)		
 37–48	85	127.3±7.2	115.1 (112.1 to 119.7)	140.6 (128.0 to 144.4)		
Males (N=497) Females (N=449).

RET-Hereticulocyte haemoglobin equivalent

Table 5 The estimated effect of associated factors on RET-He

Variables	Crude	Adjusted*	
β (95% CI)	R2	P value	β (95% CI)	R2	P value	
Sex							
 Male	−0.03 (−0.09 to 0.04)	0.001	0.486	−0.02 (−0.09 to 0.04)	0.010	0.492	
 Female	1.00		–	1.00		–	
Overweight							
 Yes	−0.04 (−0.11 to 0.02)	0.002	0.186	−0.04 (−0.10 to 0.03)	0.012	0.270	
 No	1.00		–	1.00		–	
Age	0.10 (0.03 to 0.16)	0.009	0.003	0.10 (0.03 to 0.16)	0.010	0.003	
Haemoglobin (g/L)	0.20 (0.14 to 0.27)	0.042	<0.001	0.19 (0.13 to 0.26)	0.043	<0.001	
MCV (fL)	0.53 (0.47 to 0.58)	0.280	<0.001	0.58 (0.52 to 0.63)	0.294	<0.001	
MCH (pg)	0.63 (0.58 to 0.68)	0.400	<0.001	0.65 (0.60 to 0.70)	0.404	<0.001	
MCHC (g/dL)	0.36 (0.30 to 0.42)	0.129	<0.001	0.38 (0.32 to 0.44)	0.152	<0.001	
HCT (L/L)	0.00 (−0.06 to 0.06)	0.000	0.987	−0.03 (−0.10 to 0.04)	0.011	0.361	
RDW (%)	−0.33 (−0.39 to to 0.27)	0.110	<0.001	−0.35 (−0.41 to to 0.29)	0.127	<0.001	
Leukocytes (×109/L)	−0.06 (−0.13 to 0.00)	0.004	0.050	−0.04 (−0.11 to 0.03)	0.011	0.255	
Platelet (×109/L)	−0.11 (−0.17 to to 0.05)	0.012	<0.001	−0.10 (−0.16 to to 0.03)	0.019	0.003	
MPV (fL)	−0.07 (−0.13 to 0.00)	0.004	0.042	−0.06 (−0.12 to 0.01)	0.013	0.080	
Mentzer Index	0.42 (0.36 to 0.47)	0.174	<0.001	0.42 (0.36 to 0.48)	0.182	<0.001	
Reticulocytes (×109/L)	0.18 (0.12 to 0.25)	0.033	<0.001	0.18 (0.11 to 0.24)	0.040	<0.001	
Ferritin (pmol/L)	−0.01 (−0.07 to 0.05)	0.0001	0.751	−0.01 (−0.07 to 0.06)	0.009	0.873	
* Adjusted for sex and age of the child.

HCThaematocritMCHmean corpuscular haemoglobinMCHCmean corpuscular haemoglobin concentrationMCVmean corpuscular volumeMPVmean platelet volumeRDWred cell distribution widthRET-Hereticulocyte haemoglobin equivalent

RET-He was assessed as a diagnostic marker for ID when the ferritin level was below 27 pmol/L, using ROC analysis (figure 2). Children exhibiting potential thalassaemia symptoms, characterised by MCV below the reference interval (RI) and RCC above RI, were not considered part of the normal population. In the iron-deficient group, the area under the curve was found to be 0.842 with p<0.0001. A cut-off value of RET-He ≤27.8 pg was determined, which showed a sensitivity of 70.8% and specificity of 90.9% for identifying ID. Additionally, the RET-He of the children with suspected thalassaemia that was excluded from the ROC analysis was further studied. It was observed that out of 82 suspected thalassaemia cases, 80 had an RET-He value ≤27.8 pg.

Figure 2 Receiver-operating characteristic (ROC) analysis of reticulocyte haemoglobin equivalent (RET-He) in the diagnosis of iron deficiency.

Discussion

This study established reference values for RET-He in healthy children under 5 years old in Hong Kong with the reference interval of P2.5–P97.5. RET-He levels varied with age, with lower levels in infants than other age groups. An age-specific value below the lower limit of the reference range of RET-He can be used as a limit to determine ID. There were no significant gender differences in RET-He levels. RET-He was influenced by factors such as age, haemoglobin levels and reticulocyte levels, among others. These findings contribute to a better understanding of RET-He and its interpretation in the context of iron status in children.

In this study, the mean RET-He level is 30.04 pg in children aged 2–48 months, which is slightly below previously reported data with broad age ranges 30.5 pg (6 months to 5 years),18 30.8 pg (15 days to 19 years),19 30.9 pg (1–11 years).17 As the study has a higher composition of infants, who are more vulnerable to ID than older children,20 the population was further categorised into five subgroups, and RET-He reference values were detailed in each group. Based on the P2.5 level of RET-He, RET-He lower limit reference values were proposed among age groups. These values ranged from 25.92 pg to 27.15 pg in different age groups. Previously reported lower limit reference values for ID in healthy young children range from 24.9 pg to 27.5 pg, including 25.6 and 24.9 pg (4–12 months),21 25.6 pg (1–2 years),17 27.3 pg (3–5 years),17 27.5 pg (9–12 months, 6 months–5 years).16 18 Given the reference variations in different age groups, it is necessary to apply a local age-specific reference in clinical medicine when initially assessing ID status with complete blood count (CBC).

The mean RET-He fluctuated with age with a lower level in infants than children aged over 12 months old. Iron from mothers provides necessary iron for growth at birth but iron storage is depleted around 4–6 months since dietary iron becomes the sole source of iron required for continued growth.22 Breast milk, vegetables, fruits and formula milk tend to be low in iron.22 Infants have increased iron demand to support their rapid growth at this stage, making them more vulnerable to ID.23 No significant gender difference was found in RET-He, aligning with previous studies.17 18 24 Ferritin and reticulocytes were higher in females while haemoglobin did not differ by gender.

Various blood parameters were associated with RET-He. Positive correlations were found between RET-He and parameters reflecting IDA, such as haemoglobin, MCV, MCH, MCHC and reticulocytes. These findings align with a previous study,14 showing that RET-He changes are consistent with erythropoiesis-related parameters. However, no correlation was found between RET-He and ferritin. Ferritin levels showed a U-shaped curve with age, peaking at 2–6 months and gradually increasing from 6 to 48 months. Relationships between RET-He and iron metabolism biomarkers are complex and vary with age, biomarker type and ID severity. Age-specific analysis revealed no correlation between RET-He and ferritin in 12-month-old infants while a positive relationship was observed in 4-month-old infants, and a consistent negative association with transferrin saturation (TSAT) was found at both ages.21 Analysis in American children aged 6 months to 18 years revealed that RET-He significantly correlates with ferritin, TSAT and soluble transferrin receptor, making RET-He a potential marker for monitoring erythropoiesis.25 Another paediatric-population-based study did not demonstrate a linear relationship between RET-He and ferritin, indicating that red cells may reach maximum haemoglobin levels while ferritin continues to rise; RET-He may remain constant until ID is evident.26 The varying results suggest that different iron metabolism biomarkers could be used to describe different ID stages.

RET-He is an early indicator of ID in children as well as a supporting diagnostic tool in diagnosing IDA without an extra iron metabolism test, especially in inflammation status. RET-He reflects iron availability within a 4-day time frame,27 especially in predicting the absence of bone marrow iron stores.28 29 Studies focusing on children have demonstrated the RET-He in identifying ID before the onset of anaemia.30 Rescreening during the second year of life revealed a ninefold greater risk of developing IDA among children aged 9–12 months with an RET-He<27.5 pg and no initial anaemia.16 RET-He separated ID cases from the control group with comparatively high sensitivity and demonstrated improved diagnostic performance in IDA group and ID without anaemia.19 Unlike ferritin increasing in acute phase of diseases,31 RET-He has a higher specificity and a lower coefficient of variation, it is more appropriate to screen ID and IDA accompanied by inflammatory responses.32 It has been reported as a sensitive marker of body iron status to conventional tests for the detection of ID in children accompanied with inflammation.33 Combined with low haemoglobin levels, RET-He is an accurate diagnostic test for ID and IDA in ill infants and children.34 RET-He is also important in iron treatment monitoring, serving as an early response to iron therapy within days after initiating the treatment.35 Nevertheless, the use of RET-He in reflecting the effectiveness of iron supplements in IDA children warrants further study.

In our study, the results indicated that the majority of suspected thalassaemia cases had an RET-He value ≤27.8 pg, which is the cut-off determined from the ROC analysis for screening ID. The finding suggests that CHr level could serve as a diagnostic indicator for both thalassaemia and ID. Low MCV and high RCC in CBC may indicate the presence of a thalassaemia trait, requiring further investigation through a haemoglobin study. RET-He values below the cut-off suggest ID, and measuring ferritin level is advisable for confirmation and further evaluation. Another potential application of RET-He is the differentiation of haematological conditions. Thalassaemia, another microcytic, hypoanaemia, demonstrates a similar change of blood parameters as an ID in CBC.36 Reduced RET-He can also be observed in haemoglobinopathies like α-thalassaemia and β-thalassaemia, which may not be related to ID. Clinical history and genetic testing are crucial for ruling out haemoglobinopathies. Studies show that patients with β-thalassaemia have lower RET-He levels and a smaller percentage of microcytic reticulocytes compared with those with ID.28 37 RET-He testing makes it easier to differentiate between the two conditions compared with traditional assessments, allowing for more accurate and timely identification.28 To distinguish between individuals with thalassaemic traits and early ID, various factors can be considered, such as the micro/hypo ratio, Mentzer index and an algorithm using RET-He, Hb/RET-He ratio and the microratio/hyporatio.38 39

RET-He is also advantageous in clinical practice since it is cost-effective and less invasive. Though the conventional iron biomarker-TSAT is also not influenced by inflammation,33 an extra tube of blood and even a second blood draw are required. The traditional test of ferritin and TSAT parameters costs two times more than the cost of getting CBC but the RET-He can be easily obtained or measured in the same blood tube used in CBC analysis with a specific instrument.25 40 In our study, RET-He is measured by the Sysmex XN-9100/XN-1500 haematology analyser, which is widely used throughout Hong Kong, thus promoting the feasibility of adopting age-specific reference value of RET-He in initial ID screening in young children.

One notable strength of this study is a large representative sample stratified by sampling from MCHCs and kindergartens across the territory of Hong Kong. Several limitations should be considered. First, this study was cross-sectional, and causality could not be established. Second, this study only included healthy children, so the reference ranges established may not be generalisable to children with diseases that may affect iron status. As we did not measure inflammation biomarkers, participants with inflammatory status cannot be excluded. Third, this study only included children from Hong Kong so the reference ranges established may not be generalisable to other populations with different genetic and environmental factors.

Conclusion

This study established reference values for RET-He in healthy children under 5 years old in Hong Kong and explored its relationship with various blood parameters, enhancing our understanding of RET-He in different populations. RET-He can aid in assessing children’s iron usage and erythropoiesis, and early identification and treatment of IDA are essential to preventing long-term developmental damage. Longitudinal studies are necessary to determine how RET-He reference levels can predict future IDA in Hong Kong children. More research on RET-He in various populations and its association with iron status is also required. Understanding how RET-He responds to iron treatment in children should be a focus.

Acknowledgements

The authors would like to thank the support from the Family Health Service of the Department of Health as well as all participants in this study.

Data availability statement

Data are available on reasonable request.

Review Process File
28 08 2024

Funding: This work is supported by the Hong Kong Health and Medical Research Fund (reference No: Vit D-HKU) and the Collaborative Research Fund, University Grants Committee (Reference No: C7149-20GF).

Patient consent for publication: Not applicable.

Ethics approval: This study involves human participants and was approved by the Institutional Review Board of the University of Hong Kong/Hospital Authority Hong Kong West Cluster Research Ethics Committee (UW 13-055, UW 17-491). Participants gave informed consent to participate in the study before taking part.

Provenance and peer review: Not commissioned; externally peer reviewed.

Patient and public involvement: Patients and/or the public were not involved in the design, or conduct, or reporting, or dissemination plans of this research.
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References

1 Gedfie S Getawa S Melku M Prevalence and Associated Factors of Iron Deficiency and Iron Deficiency Anemia Among Under-5 Children: A Systematic Review and Meta-Analysis Glob Pediatr Health 2022 9 2333794X221110860 10.1177/2333794X221110860
2 Safiri S Kolahi A-A Noori M et al Burden of anemia and its underlying causes in 204 countries and territories, 1990–2019: results from the Global Burden of Disease Study 2019 J Hematol Oncol 2021 14 185 10.1186/s13045-021-01202-2 34736513
3 Zhao J Chen S Wan R et al Status of malnutrition and its influencing factors in children under 5 years old in Guangnan District of Yunnan Province in 2009 - 2010 Wei Sheng Yan Jiu 2013 42 67 71 23596710
4 Likhar A Baghel P Patil M Early Childhood Development and Social Determinants Cureus 2022 14 e29500 10.7759/cureus.29500 36312682
5 East P Doom JR Blanco E et al Iron deficiency in infancy and neurocognitive and educational outcomes in young adulthood Dev Psychol 2021 57 962 75 10.1037/dev0001030 34424013
6 Pivina L Semenova Y Doşa MD et al Iron Deficiency, Cognitive Functions, and Neurobehavioral Disorders in Children J Mol Neurosci 2019 68 1 10 10.1007/s12031-019-01276-1 30778834
7 McCann S Perapoch Amadó M Moore SE The Role of Iron in Brain Development: A Systematic Review Nutrients 2020 12 2001 10.3390/nu12072001 32635675
8 Ning S Zeller MP Management of iron deficiency Hematology Am Soc Hematol Educ Program 2019 2019 315 22 10.1182/hematology.2019000034 31808874
9 Hung SC Tarng DC Bone marrow iron in CKD: correlation with functional iron deficiency Am J Kidney Dis 2010 55 617 21 10.1053/j.ajkd.2009.12.027 20338462
10 Stein J Bager P Befrits R et al Anaemia management in patients with inflammatory bowel disease: routine practice across nine European countries Eur J Gastroenterol Hepatol 2013 25 1456 63 10.1097/MEG.0b013e328365ca7f 24100539
11 Mast AE Blinder MA Lu Q et al Clinical utility of the reticulocyte hemoglobin content in the diagnosis of iron deficiency Blood 2002 99 1489 91 10.1182/blood.v99.4.1489 11830506
12 Urrechaga E Borque L Escanero JF Erythrocyte and Reticulocyte Indices on the LH 750 as Potential Markers of Functional Iron Deficiency Anemia 2010 2010 625919 10.1155/2010/625919 21490909
13 Wish JB Assessing iron status: beyond serum ferritin and transferrin saturation Clin J Am Soc Nephrol 2006 1 Suppl 1 S4 8 10.2215/CJN.01490506 17699374
14 Urrechaga Igartua E Hoffmann JJML Izquierdo-Álvarez S et al Reticulocyte hemoglobin content (MCHr) in the detection of iron deficiency J Trace Elem Med Biol 2017 43 29 32 10.1016/j.jtemb.2016.11.001 27836440
15 Kiudeliene R Griniūte R Labanauskas L Prognostic value of reticulocyte hemoglobin content to diagnose iron deficiency in 6-24-month-old children Medicina (Kaunas) 2008 44 673 7 18971604
16 Ullrich C Wu A Armsby C et al Screening healthy infants for iron deficiency using reticulocyte hemoglobin content JAMA 2005 294 924 30 10.1001/jama.294.8.924 16118382
17 López-Ruzafa E Vázquez-López MA Lendinez-Molinos F et al Reference Values of Reticulocyte Hemoglobin Content and Their Relation With Other Indicators of Iron Status in Healthy Children J Pediatr Hematol Oncol 2016 38 e207 12 10.1097/MPH.0000000000000639 27403769
18 Teixeira C Barbot J Freitas MI Reference values for reticulocyte parameters and hypochromic RBC in healthy children Int J Lab Hematol 2015 37 626 30 10.1111/ijlh.12374 25923905
19 Poventud-Fuentes I Chong TH Dowlin M et al Reticulocyte hemoglobin equivalent as a marker to assess iron deficiency: A large pediatric tertiary care hospital study Int J Lab Hematol 2024 46 148 55 10.1111/ijlh.14188 37850393
20 Moscheo C Licciardello M Samperi P et al New Insights into Iron Deficiency Anemia in Children: A Practical Review Metabolites 2022 12 289 10.3390/metabo12040289 35448476
21 Löfving A Domellöf M Hellström-Westas L et al Reference intervals for reticulocyte hemoglobin content in healthy infants Pediatr Res 2018 84 657 61 10.1038/s41390-018-0046-4 30140071
22 Ziegler EE Consumption of cow’s milk as a cause of iron deficiency in infants and toddlers Nutr Rev 2011 69 Suppl 1 S37 42 10.1111/j.1753-4887.2011.00431.x 22043881
23 McMillen SA Dean R Dihardja E et al Benefits and Risks of Early Life Iron Supplementation Nutrients 2022 14 4380 10.3390/nu14204380 36297062
24 Kuehn D Roberts SS Olsen CH et al Reticulocyte hemoglobin content testing for iron deficiency in healthy toddlers Mil Med 2012 177 91 5 10.7205/milmed-d-11-00243 22338987
25 Neef V Schmitt E Bader P et al The Reticulocyte Hemoglobin Equivalent as a Screening Marker for Iron Deficiency and Iron Deficiency Anemia in Children J Clin Med 2021 10 3506 10.3390/jcm10163506 34441801
26 Perchard M Barbaro P Rane M et al Clinical utility of reticulocyte haemoglobin in the assessment of iron deficiency and iron deficiency anaemia in the paediatric population J Paediatr Child Health 2023 59 153 8 10.1111/jpc.16265 36334002
27 Brugnara C Adamson J Auerbach M et al Iron deficiency: what are the future trends in diagnostics and therapeutics? Clin Chem 2013 59 740 5 10.1373/clinchem.2012.182071 23150055
28 Ogawa C Tsuchiya K Maeda K Reticulocyte hemoglobin content Clin Chim Acta 2020 504 138 45 10.1016/j.cca.2020.01.032 32014518
29 Mittman N Sreedhara R Mushnick R et al Reticulocyte hemoglobin content predicts functional iron deficiency in hemodialysis patients receiving rHuEPO Am J Kidney Dis 1997 30 912 22 10.1016/s0272-6386(97)90104-9 9398141
30 Shaker M Jenkins P Ullrich C et al An economic analysis of anemia prevention during infancy J Pediatr 2009 154 44 9 10.1016/j.jpeds.2008.06.038 18760421
31 Kernan KF Carcillo JA Hyperferritinemia and inflammation Int Immunol 2017 29 401 9 10.1093/intimm/dxx031 28541437
32 Kılıç M Özpınar A Serteser M et al The effect of reticulocyte hemoglobin content on the diagnosis of iron deficiency anemia: A meta-analysis study J Med Biochem 2022 41 1 13 10.5937/jomb0-31435 35291499
33 Marković M Majkić-Singh N Ignjatović S et al Reticulocyte haemoglobin content vs. soluble transferrin receptor and ferritin index in iron deficiency anaemia accompanied with inflammation Int J Lab Hematol 2007 29 341 6 10.1111/j.1365-2257.2006.00875.x 17824914
34 Swart PDR Rautenbach K Raubenheimer JE Reticulocyte haemoglobin content as a diagnostic tool for iron deficiency and iron-deficiency anaemia in ill infants and children S Afr J CH 2014 8 23 10.7196/sajch.645
35 Gelaw Y Woldu B Melku M The Role of Reticulocyte Hemoglobin Content for Diagnosis of Iron Deficiency and Iron Deficiency Anemia, and Monitoring of Iron Therapy: a Literature Review Clin Lab 2019 65 10.7754/Clin.Lab.2019.190315
36 Jameel T Baig M Ahmed I et al Differentiation of beta thalassemia trait from iron deficiency anemia by hematological indices Pak J Med Sci 2017 33 665 9 10.12669/pjms.333.12098 28811791
37 Velasco-Rodríguez D Alonso-Domínguez J-M González-Fernández F-A et al Reticulocyte parameters of delta beta thalassaemia trait, beta thalassaemia trait and iron deficiency anaemia J Clin Pathol 2016 69 149 54 10.1136/jclinpath-2015-203034 26265587
38 Mateos ME De-la-Cruz J López-Laso E et al Reticulocyte hemoglobin content for the diagnosis of iron deficiency J Pediatr Hematol Oncol 2008 30 539 42 10.1097/MPH.0b013e31817580ca 18797202
39 Stoffman N Brugnara C Woods ER An algorithm using reticulocyte hemoglobin content (CHr) measurement in screening adolescents for iron deficiency J Adolesc Health 2005 36 529 10.1016/j.jadohealth.2004.09.011
40 Chadha V Mudaliar S Khurana R et al Burden of Iron Deficiency and Correlation of Reticulocyte Hemoglobin Content (CHr) in Iron Deficiency-a Study from Tertiary Care Center in Developing Country Blood 2022 140 11070 1 10.1182/blood-2022-166045
