Journal Reading Hematologi Putaran 2
Nama : Desak Sembah Laksmi Dewi
ORIGINAL ARTICLE
Erythrocyte Indices as Differential Diagnostic Biomarkers
of Iron Deficiency Anemia and Thalassemia
Yeter Düzenli Kar, MD,* Zeynep C. Özdemir, MD,* Büşra Emir, MSc,†
and Özcan Bör, MD*
Introduction: The most common causes of microcytic anemia are
iron deficiency anemia (IDA) and thalassemia trait (TT). This study
investigated the reliability of erythrocyte indices and formulas as
screening tests in the differential diagnosis of IDA and TT before
performing detailed tests for definitive diagnosis.
Materials and Methods: In total, 50 children with β-TT, 31 with
α-TT, 50 with IDA were included. For the 8 erythrocyte indices and
formulas (red blood cells [RBC], red blood cell distribution width
[RDW], red blood cell distribution width index [RDWI], Mentzer
index [MI], Shine and Lal index [S&L], England and Fraser [E&F],
Green and King index [G&K], Srivastava index) the sensitivity,
specificity, positive and negative predictive values (PPVs and NPVs,
respectively) were calculated according to the cutoff values in the
literature and recalculated revised cutoff values.
Results: According to the cutoff values in the literature for the
differential diagnosis of IDA and TT, the ranking of sensitivity,
specificity, PPVs, and NPVs from the highest to the lowest was
RDWI, RBC, E&F, G&K, MI, Srivastava, RDW, S&L. The sen-
sitivity, specificity, PPVs, and NPVs of all the indices according to
the revised cutoff values were higher than those according to the
cutoff values in the literature.
Conclusions: According to both the cutoff values in the literature and
revised cutoff values, the most reliable indices were RBC and RDWI.
Key Words: erythrocyte indices, iron deficiency, microcytic anemia,
thalassemia trait
(J Pediatr Hematol Oncol 2019;00:000–000)
A nemia is characterized by hemoglobin (Hb), hematocrit
(Hct), and erythrocyte counts that fall 2 SD values
below the level accepted as normal for given age and sex.1
Currently, it continues to be an important health problem,
especially in developing countries.2 According to the World
Health Organization (WHO), it is reported that 30% of
children in the 0 to 4 years age group and 48% of children in the
5 to 14 years age group are anemic in developing countries.3 In
Turkey, the most common types of microcytic anemia are iron
deficiency anemia (IDA) and thalassemia trait (TT), and it has
been estimated that there are ~1,300,000 β-thalassemia carriers
(β-TT) in Turkey.4 In 2 screening studies performed in the
Received for publication April 15, 2019; accepted August 5, 2019.
From the *Department of Pediatrics, Division of Pediatric Hematology/
Oncology, Faculty of Medicine, Eskişehir Osmangazi University,
Eskişehir; and †Department of Biostatistics, Faculty of Medicine,
Izmir Katip Celebi, Izmir, Turkey.
The authors declare no conflict of interest.
Reprints: Yeter Düzenli Kar, MD, Department of Pediatrics, Division of
Pediatric Hematology/Oncology, Faculty of Medicine, Eskişehir
Osmangazi University, 26480 Eskişehir, Turkey (e-mail:
yeterduzenli@yahoo.com).
Copyright © 2019 Wolters Kluwer Health, Inc. All rights reserved.
J Pediatr Hematol Oncol  Volume 00, Number 00, ’’ 2019
Copyright r 2019 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.
Pembimbing : Dr. dr. Sianny Herawati, Sp.PK(K)
Presentasi : Jumat, 16 Juni 2023
Adana province and its surroundings in southeastern Turkey,
the newborn α-TT ratio was reported as 2.9% and 4.1%.5
Iron deficiency in children may lead to a decrease in
cognitive, behavioral, and motor functions.6 Therefore, it is
important to be able to distinguish IDA and TT and to treat
IDA accordingly.7 In contrast, unnecessary iron replace-
ment therapy in thalassemia carriers without iron deficiency
may lead to the risk of tissue and organ damage because of
iron accumulation.8
In studies conducted on children with hypochromic
microcytic anemia, Ferrara et al9 reported that none of the
erythrocyte indices and formulas was sufficient for the dif-
ferential diagnosis of iron deficiency and β-TT. Shen et al10
found that the Green and King (G&K), Ricerca, and England
and Fraser (E&F) indices were the most reliable indices for the
differential diagnosis of IDA and β-TT in Chinese children. In
contrast, Demir et al11 reported that red blood cell (RBC) and
red blood cell distribution width index (RDWI) were the most
reliable indices, whereas Veahapoğlu et al12 reported that
Mentzer index (MI) was the most reliable index, and Narchi
et al13 reported that G&K index and red blood cell distribution
width (RDW) were the most reliable indices for the differential
diagnosis of IDA and α-TT.
Standard tests for the differential diagnosis of micro-
cytic anemia include complete blood count and iron and
HbA2 level measurements. If these tests fail to provide a
diagnosis, genetic mutation analysis is then required for the
diagnosis of α-TT.1 Especially in primary health care pro-
viders, it is possible to screen patients better using eryth-
rocyte indices and formulas, which are easily available from
blood counts. It should be kept in mind that these indices
and formulas are not definitive diagnostic methods and that
they cannot replace globin gene analysis.
The aim of this study was to determine the efficacy of
erythrocyte indices and formulas reported in the literature in
distinguishing IDA and β-TT and IDA and α-TT in children
with the hypochromic microcytic anemia. To this end, the
reliability of erythrocyte indices and formulas in differential
diagnosis was investigated according to both the cutoff
values determined previously in the literature and the
redetermined cutoff (revised cutoff) values calculated in
this study.
MATERIALS AND METHODS
Subjects
A total of 151 children who had been admitted to the
Pediatric Hematology/Oncology Outpatient Clinic because
of hypochromic microcytic anemia between January 2016
and October 2017 and who had been diagnosed with IDA
and TT were included in the study. In this cross-sectional
analytical study, the children were divided into groups
|1 www.jpho-online.com
 Düzenli Kar et al
according to their diagnosis: IDA group consisted of 50
children, the β-TT group consisted of 50 children, and the α-TT
group consisted of 31 children. File records of the patients were
retrospectively examined. Ferritin concentration <12 µg/L and
transferrin saturation <16% were considered as indicators of iron
deficiency.3 Patients in whom IDA was ruled out and in whom
the HbA2 value obtained through Hb electrophoresis was
> 3.5% were considered to have β-TT. α-globulin gene analysis
was performed in patients with hypochromic microcytic anemia
and who were excluded from IDA or β-TT diagnosis. Patients
with mutations detected by gene analysis were considered to have
α-TT.1 HbA2 screening was performed in all patients after the
completion of the iron deficiency treatment in patients diagnosed
with IDA and treated with iron supplements. Molecular methods
were performed in a selected number of patients when the iron
studies and Hb electrophoresis were normal to rule out silent β-
TT or α-TT. Overall, 10 patients treated with iron, 3 patients who
received blood transfusion because of microcytic anemia in the
last 3 months, and 7 patients with concomitant IDA and TT
were excluded from the study. Approval for the study was
obtained from the Local Ethics Committee (ethics committee
decision number: 80558721/G-14, ethics committee meeting date:
January 15, 2018).
Blood Sample Analysis
The complete blood count testing was measured using
the Beckman Coulter LH750 automated hematology ana-
lyzer (Kraemer Blvd, Brea, CA). Blood count Hb, Hct, and
red cell indices, mean cell volume (MCV), mean cell
hemoglobin (MCH), mean cell hemoglobin concentration
(MCHC), and RDW values were recorded. Erythrocyte
indices were calculated using the following formulas.
Red Cell Index and Formulas
The discrimination erythrocyte indices and formulas
were calculated using the RBC indices as defined following:
(i) RBC count11;
(ii) RDW11;
(iii) MI: MCV/RBC14;
(iv) Shine and Lal index (S&L): (MCV)2×MCH×0.0115;
(v) E&F: MCV−RBC− (5×Hb) −k (k is calculated to be
5.19 in our counter as described in the original
report)11,16;
(vi) Srivastava index (S): MCH/RBC17;
(vii) G&K: MCV2×RDW/(100×Hb)18;
(viii) RDWI: MCV×RDW/RBC.19
Blood samples for Hb electrophoresis were analyzed
with a capillary electrophoresis system, MinicapFlexPierc-
ing (Sebia, Lisses, France). Ferritin concentration was
determined by chemiluminescent immunoassay method
(Cobas E-602; Roche Diagnostics, Germany). Transferrin
saturation was calculated as serum iron ×100/total iron-
binding capacity. α-globin gene mutations were analyzed
using multiplex-polymerase chain reaction and reverse-
hybridization assay according to the instructions provided
by the manufacturer (α-globin StripAssay; ViennaLab
Diagnostics, Vienna, Austria), covering the following 21
mutations: 2 single-gene deletions (−α3.7; −α4.2), 5 double-
gene deletions (−MED 1; −SEA; −THAI; −FIL; −(α)20.5),
ααα anti-3.7 gene triplication, 2 point mutations in the α 1
gene (cd 14 G > A; Hb Adana), and 11 point mutations in the
α 2 gene (initiation cd T > C; cd 19 −G; IVS1 −5nt; cd 59
G > A; Hb Quong Sze; Hb Constant Spring; Hb Icaria; Hb
Pakse; Hb Koya Dora; αPolyA1; αPolyA2).
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J Pediatr Hematol Oncol  Volume 00, Number 00, ’’ 2019
Statistical Analysis
Statistical analyses were performed using the Statistical
Package for the Social Sciences (SPSS Inc., Chicago, IL).
Descriptive statistics were used mean ± SD for normally
distributed variables, and the median and interquartile dis-
tribution range were used for other variables. Categorical data
were compared using the χ2 test. The Kolmogorov-Smirnov
test was used to verify the normality of distributions of con-
tinuous variables. Continuous variables conforming to a nor-
mal distribution (Hct, Hb, and MCV) were compared using
the independent t test. For non-normal distribution variables
(age, RBC, MCH, MCHC, RDW, transferrin saturation, and
serum iron and ferritin concentrations), nonparametric Mann-
Whitney U test was used. Sensitivity, specificity, positive and
negative predictive values (PPVs and NPVs, respectively), and
positive and negative likelihood ratios (LR+ and LR−) were
calculated for erythrocyte indices and formulas according to
both the cutoff values in the literature and the revised cutoff
values. Receiver operating characteristic curves analysis was
performed to determine the discriminative value of the eryth-
rocyte indices. A P-value of <0.05 was considered statistically
significant.
RESULTS
Table 1 summarizes the clinical features and laboratory
parameters for the IDA, β-TT, and α-TT groups. There
were no differences among the 3 groups in terms of age and
sex (P > 0.05 for all). Hb, Hct, RBC, MCH, MCHC,
transferrin saturation, and serum iron and ferritin concen-
trations were significantly lower in the IDA group than in
the β-TT (P = 0.004 for MCH, P < 0.001 for others) and
α-TT groups (P < 0.001 for all) groups. There was no dif-
ference between the β-TT and IDA groups in terms of MCV
values (P > 0.05). MCV values were significantly lower in
the IDA group than in the α-TT group (P < 0.001). RDW
values were significantly higher in the IDA group than in the
β-TT and α-TT groups (P < 0.001 for both).
The mean HbA2 level in the β-TT group was
5.06 ± 0.6% (min-max: 3.6% to 6.40%). In the α-TT group,
11 patients had single-gene deletion (−α3.7 deletion), 2 had
nondeletional mutation (αPolyA2), and 18 had double-gene
deletion (14 had −(α)20.5 deletion, 2 had heterozygote
−MED 1 deletion, 2 had heterozygote −FIL deletion).
The sensitivity, specificity, PPVs, NPVs, and LR+ and
LR− values of the erythrocyte indices, and formulas used in
distinguishing IDA and TT according to the cutoff values
reported in the literature are summarized in Table 2.
Accordingly, the indices with the highest sensitivity and
specificity for distinguishing IDA and β-TT, and IDA and α-
TT were identified as RDWI and RBC. The differential
power of the S&L index and RDW were significantly low.
The revised cutoff values of the erythrocyte indices and
formulas used to differentiate IDA and TT were determined
with a 95% confidence interval range. Sensitivity, specificity,
PPVs, NPVs, and LR+ and LR− values according to the
revised cutoff values are summarized in Table 3. In differ-
entiating IDA from β-TT, the ranking of the erythrocyte
indices and formulas from the highest to lowest according to
their area under curve (AUC) values was RBC, RDWI,
E&F, G&K, MI, Srivastava index, and RDW. The sensi-
tivity, specificity, PPVs, NPVs, and LR+ ratios of RBC and
RDWI were higher than those of other indices.
In differentiating IDA from α-TT, the ranking of the
erythrocyte indices and formulas from the highest to the
 J Pediatr Hematol Oncol  Volume 00, Number 00, ’’ 2019 Erythrocyte Indices in Microcytic Anemia

TABLE 1. Characteristics and Laboratory Parameters of Iron Deficiency Anemia (IDA), β-Thalassemia Traits (β-TT), and α-Thalassemia
Traits (α-TT)
Mean ± SD
Median (Q1-Q3) P

Characteristics IDA (n = 50) β-TT (n = 50) α-TT (n = 31) IDA/β-TT IDA/α-TT

Age (y) 8.23 ± 5.95 8.48 ± 5.08 7.94 ± 4.03 0.518* 0.942*
8.5 (1.88-14) 7.5 (4-14) 7.5 (4-12)
Sex (male/female) 20 (40%)/30 (60%) 26 (52%)/24 (48%) 18 (58.07%)/13 (41.93%) 0.316‡ 0.176‡
Hb (g/dL) 8.4 ± 1.63 11.05 ± 1.11 11.76 ± 0.95 < 0.001* < 0.001†
8.75 (7.18-9.5) 10.85 (10.1-11.73) 11.6 (11-12.3)
Hct (%) 26.45 ± 4.74 3436 ± 3.19 35.99 ± 2.74 < 0.001† < 0.001*
27.85 (22.9-30.48) 33.9 (31.6-36.35) 35.3 (34.2-37.1)
RBC (10 /L)6
4.43 ± 0.55 5.74 ± 0.48 5.61 ± 0.46 < 0.001* < 0.001*
4.5 (4.15-4.74) 5.78 (5.4-6.12) 5.59 (5.28-5.86)
MCV (fL) 59.29 ± 5.03 59.97 ± 3.9 64.9 ± 5.95 0.452† < 0.001*
59.2 (55.15-62.85) 59.85 (57-62) 63.2 (60.6-67)
MCH (pg) 18.36 ± 2.67 19.33 ± 1.44 21.21 ± 2.39 0.004* < 0.001*
18.05 (16.45-19.68) 19.1 (18.38-20.13) 20.5 (19.4-22.2)
MCHC (%) 30.16 ± 4.28 32.16 ± 0.66 32.62 ± 1.16 < 0.001* < 0.001*
30.55 (29.58-31.73) 32.3 (31.78-32.6) 32.5 (31.8-33.3)
RDW (%) 20 ± 2.39 17.18 ± 2.04 16.94 ± 4.69 < 0.001* < 0.001*
20 (18.18-21.75) 16.8 (15.78-18.43) 15.8 (14.4-17.2)
Serum iron (mg/dL) 29.12 ± 40.89 95.94 ± 28.27 86.97 ± 29.22 < 0.001* < 0.001*
19 (17-26) 99 (66.75-117.5) 78 (66-98)
Transferrin saturation (%) 5.94 ± 5.04 27.89 ± 9.55 28.76 ± 10.99 < 0.001* < 0.001*
4.68 (3.8-5.5) 28.4 (20-34.02) 25.3 (21-38)
Ferritin (ng/mL) 6.85 ± 4.22 43.29 ± 31.03 41.62 ± 25.15 < 0.001* < 0.001*
5.68 (3.78-9.43) 33.35 (24.75-52.03) 36 (22-48.7)
*Mann-Whitney U test.
†Independent Sample t test.
‡Continuity Correction χ2 test.
Hb indicates hemoglobin; Hct, hematocrit; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; MCV, mean
corpuscular volume; RBC, red blood cells; RDW, red blood cell distribution.

TABLE 2. Sensitivity, Specificity, Predictive Value of Each Erythrocyte Index, and Formula According to Cutoff Values in the Literature in
the IDA, β-TT, and α-TT Groups
Indices Sensitivity Specificity
(Cutoff) Groups (95% CI) (95% CI) PPV (95% CI) NPV (95% CI) LR+ (95% CI) LR− (95% CI)
RDWI IDA/β-TT 92 (85-95.9) 92 (85-95.9) 92 (85-95.9) 92 (85-95.9) 11.500 (5.668-23.335) 0.087 (0.043-0.176)
(220)
IDA/α-TT 87.1 (76.2-93.3) 92 (85.2-95.9) 87.1 (76.2-93.3) 92 (85.2-95.9) 10.887 (5.165-22.616) 0.140 (0.069-0.279)
RBC (5) IDA/β-TT 94 (86.9-97.7) 88 (80.9-91.7) 88.7 (82-92.1) 93.6 (86.1-97.5) 7.833 (4.551-11.725) 0068 (0.025-0.162)
IDA/α-TT 88 (81.1-91.7) 90.3 (79.3-96.2) 93.6 (86.3-97.5) 82.4 (72.3-87.7) 9.093 (3.914-24.257) 0.133 (0.087-0.238)
E&F (0) IDA/β-TT 86 (78-91.2) 88 (80-93.2) 87.8 (79.6-93.1) 86.3 (78.5-91.4) 7.167 (3.907-13.438) 0.159 (0.094-0.275)
IDA/α-TT 80.6 (68.6-88.6) 88 (80.6-93) 80.6 (68.6-88.6) 88 (80.6-93) 6.720 (3.531-12.583) 0.220 (0.122-0.389)
G&K (72) IDA/β-TT 94 (86.3-97.8) 76 (68.3-79.8) 79.7 (73.2-82.9) 92.7 (83.3-97.3) 3.917 (2.727-4.845) 0.079 (0.027-0.200)
IDA/α-TT 90.3 (78.5-96.4) 76 (68.6-79.8) 70 (60.8-74.7) 92.7 (83.7-97.3) 3.763 (2.502-4.774) 0.127 (0.045-0.314)
MI (13) IDA/β-TT 98 (91.2-99.6) 54 (47.2-55.6) 68.1 (63.3-69.2) 96.4 (84.2-99.4) 2.130 (1.725-2.246) 0.037 (0.006-0.187)
IDA/α-TT 77.4 (64-87.7) 54 (45.7-60.3) 51.1 (42.2-57.8) 79.4 (67.1-88.7) 1.683 (1.177-2.210) 0.418 (0.205-0.790)
Srivastava IDA/β-TT 98 (91.5-99.6) 32 (25.5-33.6) 59 (55.1-60) 94.1 (75-98.9) 1.441 (1.228-1.502) 0.063 (0.011-0.334)
(4.4)
IDA/α-TT 80.6 (68.2-90.1) 32 (24.3-37.9) 42.4 (35.8-47.3) 72.7 (55.2-86.1) 1.186 (0.900-1.450) 0.605 (0.261-1.311)
RDW (14) IDA/β-TT 0 (0-0) 100 (100-100) — 50 (50-50) — 1.000 (1.000-1.000)
IDA/α-TT 16.1 (9.4-16.1) 100 (95.8-100) 100 (58.2-100) 65.8 (63-65.8) — 0.839 (0.839-0.946)
S&L (1530) IDA/β-TT 100 (100-100) 0 (0-0) 50 (50-50) — 1 (1-1) —
IDA/α−TT 96.8 (96.8-99.3) 0 (0-1.6) 37.5 (37.5-38.5) 0 (0-79.2) 0.968 (0.968-1.009) —
IDA indicates iron deficiency anemia; α-TT, α-thalassemia traits; β-TT, β-thalassemia traits; 95% CI, 95% confidence interval; E&F, England and Fraser
Index; G&K, Green and King Index; LR+, likelihood ratio positive (sensitivity)/(1 specificity); LR−, likelihood ratio negative (1 sensitivity)/(specificity);
MI, Mentzer Index; NPV, negative predictive value (true negative/[true negative+false negative]); PPV, positive predictive value (true positive/[true positive+false
positive]); RBC, red blood cells; RDW, red blood cell distribution width; RDWI, red blood cell distribution width index; S&L, Shine and Lal
Index; Srivastava, Srivastava Index; Sensitivity, true positive/(true positive+false negative); Specificity, true negative/(true negative+false positive).

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 Düzenli Kar et al J Pediatr Hematol Oncol  Volume 00, Number 00, ’’ 2019
TABLE 3. Receiver Operating Characteristic Curve Analysis Results and Sensitivity, Specificity, Predictive Value of Each Erythrocyte Index and Formula According to Revised Cutoff Values in

[true positive+false positive]); RBC, red blood cells; RDW, red blood cell distribution width; RDWI, red blood cell distribution width index; S&L, Shine and Lal Index; Srivastava, Srivastava index; Sensitivity, true positive/(true positive+false
α-TT indicates α-thalassemia traits; β-TT, β-thalassemia traits; 95% CI, 95% confidence interval; AUC, area under curve; E&F, England and Fraser Index; G&K, Green and King Index; IDA, iron deficiency anemia; LR+, likelihood
ratio positive (sensitivity)/(1 specificity); LR−, likelihood ratio negative (1 sensitivity)/(specificity); MI, Mentzer Index; NPV, negative predictive value (true negative/[true negative+false negative]); PPV, positive predictive value (true positive/
0.0095
< 0.0001
< 0.0001
< 0.0001
< 0.0001
< 0.0001
< 0.0001
< 0.0001
< 0.0001
< 0.0001
< 0.0001
< 0.0001

< 0.0001
< 0.0001

< 0.0001
lowest according to their AUC values was RBC, E&F, RDWI,

0.112
P
G&K, RDW, S&L, and MI. The sensitivity, specificity, PPVs,
NPVs, and LR+ ratios of RBC and RDWI were higher than
those of E&F (Table 3).
(0.006-0.3)
LR− (95% CI)

The α-TT patients were divided into 2 groups as double-

(0.03-0.2)
(0.05-0.3)
(0.01-0.2)
(0.02-0.3)
(0.06-0.3)
(0.06-0.4)

(0.08-0.4)

(0.03-0.5)
(0-0.071)

(0.1-0.4)
(0.2-0.6)
(0.2-0.5)
(0.3-0.8)
(0.1-0.4)

(0.1-0.7)
gene deletion (group 1; n = 18) and silent carriers (single-gene
deletion or nondeletional mutation) (group 2; n = 18). In both
groups, the 95% confidence interval and new cutoff values of
0.087

0.049
0.04

0.13

0.08
0.14
0.14
0.22
0.39
0.33
0.52
0.21
0.19
0.32
0.12
0

erythrocyte index and formulas, which were used for the differ-
entiation of TT from IDA, were determined. In differentiating
IDA from α-TT with double-gene deletion, the ranking of the
Sensitivity (95% CI) Specificity (95% CI) PPV (95% CI) NPV (95% CI) LR+ (95% CI)
(3.3-11.8)
(3.2-11.5)
(4.5-29.5)
(5.6-85.2)

(3.4-15.6)
(3.8-20.2)
(3.9-25.9)
(3.0-21.4)

(3.0-12.1)
(2.9-9.7)
(2.9-9.5)

(2.1-6.6)
(1.5-5.5)
(1.8-4.1)

(1.1-1.8)
(1.5-2.9)
erythrocyte indices and formulas from the highest to the lowest
according to their AUC values was RBC, RDWI, E&F, G&K,
MI. The sensitivity, specificity, PPVs, NPVs, and LR+ ratios
11.5
21.77
6.25
6.05

5.33
5.20
7.33
8.71
10
8.06
3.70
2.90
2.69
5.99
1.42
2.13 of RBC and RDWI were higher than all other indexes. In
differentiating IDA from silent carrier α-TT, the ranking of
the erythrocyte indices and formulas from the highest to
(90.4-99.6)
(83.1-94.2)
(86.4-95.0)
(84.7-97.5)
(87.4-98.6)

(84.7-96.0)
(75.1-86.2)
(73.0-83.2)
(65.6-80.7)
(66.5-80.5)
(72.1-90.6)
(81.8-94.6)
(57.7-87.4)
(81.4-98.1)
(93.4-100)

(80.2-93)

the lowest according to their AUC values were; RBC,

S&L, E&F, G&K, RDWI. The sensitivity and specificity of
RBC and the ratios of PPV, NPV, and LR+ were higher
97.7
90.2
92.3
92.3
95.3
88
91.8
82.1
79.3
74.1
74.1
82.9
89.6
75
93.3
100

than all other indexes. According to the revised cutoff

values, the values of the sensitivity, specificity, PPV, NPV,
LR+, and LR− were summarized in Table 4.
(81.4-86.2)
(70.7-81.1)
(84.5-96.1)
(82.4-97.8)
(77.3-87.3)
(67.4-80.0)

(73.5-90.8)

(66.6-92.5)

(47.9-75.8)
(65.4-78.2)
(67.5-86.1)
(52.4-61.8)
(81.4-98.1)
(80.2-93)

(82-96.1)

(68.3-86)

DISCUSSION
The erythrocyte indices and the various formulas
86.2
78.9
91.8
93.1
83.9
76.3
88
84.4
90.9
82.6
78.3
63
72.9
78.8
57.9
93.3

derived from them have been proposed as simple and

inexpensive methods that can be initially used to differ-
entiate β-TT and IDA.20–22 However, an index with a sen-
(70.9-92.8)
(70.9-92.8)
(80.8-97.8)
(86.3-99.5)
(68.6-91.4)
(68.6-91.4)
(75.7-95.5)
(78.2-96.7)
(80.8-97.8)
(80.8-97.8)

(53.3-80.5)
(73.3-94.2)
(24.7-52.8)
(41.3-70.0)

sitivity and specificity of 100% has not been defined to date.

(66.3-90)
(66.3-90)

Studies performed in children with this regard are limited,

and their results are contradictory. In this study, we eval-
uated the reliability of 8 erythrocyte indices in distinguishing
84
84
92
96
82
82
88
90
92
92
80
80
68
86
38
56

IDA and TT according to previously reported cutoff values

in the literature and revised cutoff values. It was found that
7 indices other than S&L index had high predictive values in
distinguishing β-TT and IDA and that RBC and RDWI
(83.3-99.9)
(82.8-94.1)
(70.2-96.4)
(86.3-99.5)
(78.6-99.2)
(75.7-95.5)
(70.2-96.4)

(45.4-80.8)
(59.7-85.4)
(39.1-75.5)
(73.3-94.2)
(66.3-94.5)
(75.7-95.5)
(78.6-99.2)
(92.9-100)

(66.3-90)

were more reliable than were other indices. In addition,

RBC and RDWI were also shown to be the most reliable
indices in distinguishing α-TT and IDA.
RDW is a measure of the degree of variation in RBC
100
96.77

87.10

93.55

87.10

64.52

58.06

83.87

93.55
90

96

88

80

74

86

88

sizes and is widely used in practice to distinguish IDA and

TT. The increase in RDW in TT is explained by eryth-
rocytosis, the presence of target cells, and the high retic-
(0.926-0.997)
(0.897-0.993)
(0.902-0.989)
(0.818-0.958)
(0.887-0.984)
(0.851-0.975)
(0.877-0.979)
(0.804-0.951)
(0.854-0.968)
(0.670-0.862)
(0.759-0.909)
(0.556-0.770)
(0.733-0.892)
(0.753-0.919)
(0.490-0.690)
(0.685-0.872)

ulocyte count.23 RDWI is a value obtained by multiplying

AUC (95% CI)

MI with RDW, and the results of our study showed that

negative); Specificity, true negative/(true negative+false positive).

RDWI was a more reliable index than was RDW or MI

alone (Table 3). RDW is widely used in practice to dis-
tinguish IDA and TT. In our study, RDW was found to
0.978
0.964
0.961
0.904
0.950
0.930
0.942
0.893
0.924
0.776
0.845
0.669
0.822
0.850
0.592
0.790

have low sensitivity and 100% specificity for the differ-

entiation of both traits according to cutoff values in the
IDA/α-TT

IDA/α-TT

IDA/α-TT

IDA/α-TT

IDA/α-TT

IDA/α-TT

IDA/α-TT

IDA/α-TT
IDA/β-TT

IDA/β-TT

IDA/β-TT

IDA/β-TT

IDA/β-TT

IDA/β-TT

IDA/β-TT

IDA/β-TT

literature (Table 2). Sensitivity values were much higher for

Groups

the revised cutoff values; however, the index was ranked

the IDA, β-TT, and α-TT Groups

seventh in differential power for β-TT and fifth for α-TT

(Table 3), indicating that RDW is not very useful in dis-
tinguishing TT and IDA.
Indices (Revised Cutoff)

The results of studies concerning the use of RBC for

the differential diagnosis of IDA from β-TT tend to be
contradictory. Demir et al11 found that RBC and RDWI
Srivastava (3.61)
RDWI (214.02)
RDWI (204.12)

Srivastava (3.6)

were the most reliable indices in the differential diagnosis of

S&L (571.82)
S&L (652.33)
RDW (17.3)
G&K (65.3)
G&K (63.2)
RBC (4.82)
RBC (4.82)

IDA and β-TT, whereas Rahim and Keikhaei24 found that

E&F (1.27)
E&F (2.44)

MI (11.39)
MI (11.43)

RDW (19)

S&L and RBC were the most reliable indices in <10-year-

old children and that RDWI and RBC were the most reli-
able indices in patients older than 10 years. AlFadhli et al23

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 J Pediatr Hematol Oncol  Volume 00, Number 00, ’’ 2019 Erythrocyte Indices in Microcytic Anemia
TABLE 4. Receiver Operating Characteristic Curve Analysis Results and Sensitivity, Specificity, Predictive Value of Each Erythrocyte Index, and Formula According to Revised Cutoff Values in

α-TT indicates α-thalassemia traits; β-TT, β-thalassemia traits; 95% CI, 95% confidence interval; AUC, area under curve; E&F, England and Fraser Index; G&K, Green and King Index; group 1, two-allele deletions with α-TT; group 2,
silent carrier (single-allele deletion/poly A mutation group) α-TT group; IDA, iron deficiency anemia; LR+, likelihood ratio positive (sensitivity)/(1 specificity); LR−, likelihood ratio negative (1 sensitivity)/(specificity); MI, Mentzer Index;
NPV, negative predictive value (true negative/[true negative+false negative]); PPV, positive predictive value (true positive/[true positive+false positive]); RBC, red blood cells; RDW, red blood cell distribution width; RDWI, red blood cell
0.0323
0.7038
< 0.0001
< 0.0001
< 0.0001
0.0107
< 0.0001
< 0.0001
< 0.0001
0.0071
< 0.0001

< 0.0001
0.5521
< 0.0001

< 0.0001
< 0.0001
showed that the E&F index was the most reliable index in
distinguishing α-TT and β-TT from IDA, whereas RBC was
P

the second most reliable index. They also showed that the
(0.004-0.9) reliability of RDWI was much lower than that of the 2
(0.007-0.5)

(0.008-0.5)
LR− (95% CI)

(0.01-0.7)

(0.04-1.3)

(0.05-0.8)

(0.07-0.9)
(0.04-0.7)

(0.07-0.7)

(0.03-0.6)
(0.06-1.1)

0.33 (0.08-1.3)
indices and that S&L was insufficient in the differential

(0.2-1.9)

(0.3-1.5)
diagnosis. In contrast to these studies, Vehapoğlu et al12
(—)

—
reported that MI was the most reliable index, erythrocyte
counts were high in 29.4% of children with IDA, and RBC
0.24

0.19

0.26
0.18

0.21

0.13
0.25
0.092
0.057

0.059

0.062

0.67

0.63
0.056

alone was therefore not reliable. Nalbantoğlu et al25

reported that none of the eight indices investigated in our
(41.9-53.2)
Sensitivity (95% CI) Specificity (95% CI) PPV (95% CI) NPV (95% CI) LR+ (95% CI)

study were useful in differentiating IDA and β-TT. Shen

(8.2-10.9)
(5.6-10.5)

(9.4-17.4)
(14.2-26)
(13.8-18)
(3.6-6.1)

(3.2-5.3)

(5.2-7.7)

(1.4-2.6)
(2.4-9.6)
(8.3-13)

(8-16.7)
et al10 reported that the Srivastava index and S&L had low
(4.7-7)

(1.8-6)

distribution width index; S&L, Shine and Lal Index; Srivastava, Srivastava index; Sensitivity, true positive/(true positive+false negative); Specificity, true negative/(true negative+false positive).
—

reliability. In their study on 77 school-aged children in

Thailand, Pornprasert et al20 reported that the reliability of
19.23
15.74

10.42

12.82

11.54
5.77
47.2

4.70
9.44
7.69

4.17

6.35

1.92
4.81

3.30

Sirdah and Srivastava indices was over 90% and that these
indices were the most reliable tests for differentiating IDA
the IDA, Two-Allele Deletions With α-TT (Group 1), and Silent Carrier (Single-Allele Deletion/Poly A Mutation Group) α-TT (Group 2) Groups

and β-TT. In their study on 67 children with α-TT and 18

(87.5-99.9)
(89.4-99.9)
(83.6-98.8)
(88.9-99.9)
(84.2-99.4)
(88.5-99.9)
(82.6-98.7)

(80.7-98.6)

(84.9-99.5)
(83.3-98.8)

(81.1-97.8)
(72.9-93.4)

(73.3-94.2)

(85.2-100)
(89.6-100)

(83-98.7)

children with IDA, Narchi et al13 reported that the best

indices that could be used to differentiate IDA and α-TT
were G&K and RDW. Similar to our study, Jameel et al26
97.7
98
94.1
97.9
95.3
97.8
93.7
93.9

93

95.6
94

92.2
100
98

85.2

86

reported that RDWI and RBC were more useful than was
RDW in the differential diagnosis of TT and IDA. In a
comprehensive study, Urrechaga et al27 reported that they
(36.1-80.9)
(72.7-99.9)
(51.6-97.9)
(62.1-96.8)
(31.5-76.9)

(37.3-88.8)
(54.4-93.9)

(38.7-78.9)

(47.1-86.8)

(26.3-56.8)
(42.8-94.5)
(19.4-87.7)

(19.2-74.9)
(80.5-100)

(54-92.4)

(46.2-95)

applied 25 different erythrocyte indices and formulas and

found that RDWI was the most useful test in the differential
diagnosis of TT and IDA.
60
94.4
83.3
85
55
77.3
66.7
78.9

60

69.6
76.9
40.9
75
55.6

46.2
100

In our study, RDWI and RBC were determined to be

the most reliable indices in the differential diagnosis of IDA
and TT according to cutoff values in the literature.
According to revised cutoff values, RBC and RDWI were
(70.9-92.8)
(89.4-99.9)
(86.3-99.5)
(83.5-98.7)
(68.6-91.4)
(78.2-96.7)
(78.2-96.7)
(80.8-97.8)

(66.3-90.0)

(73.3-94.2)
(83.5-98.7)
(33.7-62.6)
(83.5-98.7)
(80.8-97.8)

(73.3-94.2)
(92.9-100)

determined to be the most reliable indices. The differential

power of S&L was lower than that of the other indices. The
results of our study have reported that RBC had the highest
84
98
96
94
82
90
90

86
94
48
94
100

92

80
92

86

sensitivity and specificity in differentiating IDA and TT

according to revised cutoff values. The high differential
power of RBC can be explained by the fact that the eryth-
rocyte count is within the normal range in IDA and above
(72.7-99.9)

(72.7-99.9)
(54.6-98.1)
(72.7-99.9)

(58.6-96.4)

(58.6-96.4)

(65.3-98.6)

(38.6-90.9)
(72.7-99.9)

(13.9-68.4)

(19.2-74.9)

the normal range in TT (Table 1).

(81.5-100)
(64-99.8)

(46.2-95)

(46.2-95)

(46.2-95)

In our study, the sensitivity and specificity values of

revised cutoff values used in the differential diagnosis
of IDA and β-TT and IDA and α-TT were higher than those
92.3
94.4
76.9
94.4
84.6
94.4
76.9
83.3

83.3

88.9
76.9

69.2
94.4

38.5

46.2

100

of cutoff values in the literature (Table 3), indicating that

revised cutoff values are relatively more useful in the dif-
ferential diagnosis of IDA and TT in our patient pop-
(0.824-0.973)
(0.935-1.000)
(0.658-0.875)
(0.904-0.998)
(0.759-0.940)
(0.902-0.997)
(0.660-0.876)
(0.863-0.986)

(0.727-0.915)

(0.824-0.970)
(0.632-0.856)
(0.603-0.826)
(0.773-0.948)
(0.940-1.000)

(0.411-0.668)

(0.431-0.686)

ulation. The reliability of erythrocyte indices in the differ-

AUC (95% CI)

ential diagnosis of IDA and TT varies among different

societies. For example, Sirdah and Srivastava indices have
been defined as indices with the highest differential power in
the Thai population, whereas Sirdah, G&K, and RDWI
0.920
0.993
0.780
0.974
0.868
0.973
0.782
0.946

0.836

0.917
0.757
0.724
0.880
0.996

0.542

0.562

have been defined as indices with the highest differential

power in the Palestinian population20; S&L, Srivastava, and
2/IDA
1/IDA
2/IDA
1/IDA
2/IDA
1/IDA
2/IDA
1/IDA

1/IDA

1/IDA
2/IDA
1/IDA
2/IDA
1/IDA

2/IDA

2/IDA

MI in the Indian population28; and G&F and E&F in the

Chinese population.10 These differences among populations
Groups

are thought to be caused by the diversity of genetic

Group
Group
Group
Group

Group
Group
Group
Group
Group
Group
Group

Group
Group
Group
Group
Group

mutations.10,20,29 Besides, the sample size, the mean age of

participating patients, and the heterogeneity of the included
population may be one of the reasons for the perception of
Indices (Revised Cutoff)

different indexes as reliable in different populations.29 If the

definitive diagnosis was not carefully done (like selecting the
subjects from a population with TT concomitant to iron
Srivastava (3.61)
Srivastava (4.88)
RDWI (197.26)
RDWI (204.12)

anemia or like selecting the subjects from patients under

S&L (945.08)
G&K (62.17)

RDW (16.5)
G&K (63.2)

RDW (1..3)
RBC (5.36)
RBC (4.82)

E&F (1.97)
E&F (2.44)

iron deficiency treatment), the results may be inconsistent

MI (11.43)
MI (11.39)

S&L (603)

and misleading.
α-TT is a very heterogenous disease at both clinical and
molecular level. The silent carrier form of α-TT (most

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Copyright r 2019 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.
 Düzenli Kar et al
common: −α3.7, −α4.2), which is a result of the single-gene
deletion, is completely asymptomatic or there are only mild
microcytosis and hypochromia with normal HbA2 and HbF
levels. In the α-TT form (α trait), which is a result of the
deletion of 2 genes (most common: (α)20.5, −MED 1, −FIL,
−SEA, −THAI), patients have clinical characteristics sim-
ilar to β-TT with the clinical findings of a mild hypochromic
microcytic anemia (MCV < 78 fL, MCH < 27 pg). The
nondeletion mutations may lead to the clinical character-
istics of both α trait and silent carrier.30,31 We determined
that the most reliable indexes were RBC and RDWI in the
differentiation of α-TT with double-gene deletion from IDA
and RBC is the most reliable index in the differentiation of
silent carrier α-TT from IDA.
The limitations of our study included the low number
of patients and the fact that it presented the experience of a
single center.
In conclusion, erythrocyte indices and formulas can be
used as initial tests for the differential diagnosis of TT and
IDA. The results of our study showed that RBC and RDWI
were the most useful indices in the differential diagnosis of
IDA and TT, whereas RDW and MI were not useful. Cutoff
values vary among populations; therefore, it would be more
beneficial for different communities to use erythrocyte
indices by determining their specific and appropriate cutoff
values.
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