PAGE 343
2019 July; 25(3): 343-348
p-ISSN 0854-4263 e-ISSN 2477-4685
Available at www.indonesianjournalofclinicalpathology.org
THE HEMOGLOBIN, RDW, AND MEAN CORPUSCULAR VALUES IN
PATIENTS WITH BETA-THALASSEMIA/HEMOGLOBIN E DISEASE
AND BETA-THALASSEMIA TRAIT
Vinisia Setiadji1, Bidasari Lubis2, Adi Koesoema Aman1, Herman Hariman1
1
2
Clinical Pathology Department, Faculty of Medicine, North Sumatera University/Adam Malik Hospital, Medan, Indonesia. E-mail:
Pediatrics Department, Faculty of Medicine, North Sumatera University/Adam Malik Hospital, Medan, Indonesia
ABSTRACT
Beta-thalassemia/hemoglobin E disease is a condition where there is double heterozygosity of beta-thalassemia trait and
hemoglobin E trait. This produces a condition with more severe phenotypic appearance compared to beta thalassemia trait
and hemoglobin E trait. Logically the Mean Corpuscular Values (MCV) of beta-thalassemia/hemoglobin E disease should
also be worsened. The aim of this study was to assess the hemoglobin level, RDW, and MCV between
beta-thalassemia/hemoglobin E disease and beta thalassemia trait. The researchers hereby studied eleven cases from two
families who were detected to have beta-thalassemia/hemoglobin E disease. Family-1 with beta-thalassemia trait had MCV
68 fL and 65 fL, the MCH value was 21 pg and 20 pg, respectively. In Family-2, mother with beta-thalassemia trait, had MCV
60.2 fL and MCH 18. 8 pg. Daughters with beta-thalassemia/hemoglobin E disease from subjects 1 and 2 whose blood were
taken repetitively during visits to the hematology clinic, had mean±SD of MCV 70.8±4.9 fL and Mean Corpuscular Hemoglobin
(MCH) value 22.8±2.3 pg. They were significantly higher than the ones with beta-thalassemia trait (p<0.05). Moreover, there
were found that the MCV from post-transfusion state were significantly higher than the pre-transfusion state (p<0.001). Based
on the study, it could concluded that the MCV from subjects with beta-thalassemia/hemoglobin E disease were persistently
higher than the beta-thalassemia trait. The role of blood transfusion in patients with beta-thalassemia/hemoglobin E disease
seems to play a part in the result of a discrepancy in this matter.
Key words: Beta-thalassemia, hemoglobin E, discrepancy, MCV, RDW
INTRODUCTION
Hemoglobinopathy is the most common genetic
disorder in Southeast Asia. Southeast Asia consists of
ten countries: Indonesia, Malaysia, Singapura, Brunei
Darussalam, Thailand, Myanmar, Kamboja, Laos,
Vietnam, and Filipina. The ethnic origin of the people
living in these countries is very heterogeneous.
Malayo-Polynesian ethnic (Austronesian) live in
Malaysia, Indonesia, Brunei, Philippines, and
countries in the Pacific Ocean. Chinese and Indian
tribes are spread in every country. Globally,
thalassemia is the most common monogenic
disorder, characterized by anemia caused by globin
chain synthesis defects of adult hemoglobin.1,2
Thalassemia is an autosomal recessively inherited
disease caused by the decrease or absent of globin
chain synthesis. Based on the type of globin chain,
thalassemia can be divided into α-, β-, γ-, δ-, δβ-, and
εγδβ- thalassemia. The quantitative defect of globin
chain can be accompanied by qualitative defects
such as HbE, HbC, HbS, on beta-thalassemia and
HbCS (Constant Spring) on alpha thalassemia. As an
autosomal recessively inherited the disease, a person
with heterozygote is state usually asymptomatic and
does not need any therapy. Whereas individual with
homozygote state and some type of heterozygote
are suffered from thalassemia syndrome.2,3
Beta thalassemia carrier/trait is usually
asymptomatic. The peripheral blood morphology
show hypochromic and microcytic as well as an
increase of HbA2 level. The hemoglobin type in
heterozygote beta-thalassemia consist of 92-95%
HbA, >3.8% HbA2, and variable HbF level (0.5 – 4%).4
Hemoglobin E is a variant of hemoglobin caused
by a beta-globin gene mutation that results in
glutamic acid substitution with lysine in codon 26 of
beta-globin gene [codon 26 (GA)]. Hemoglobin E is
the second most common globin abnormality after
sickle cell hemoglobin. HbE is common in Southeast
Asia, as much as 30-40% of all hemoglobin
abnormality.5,6
Hemoglobin E trait is a heterozygote condition
caused by marriage between one person with normal
The Hemoglobin, RDW, and Mean Corpuscular Values - Setiadji, et al.
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Indonesian Journal of Clinical Pathology and Medical Laboratory, 2019 July, 25 (3) : 343 - 348
hemoglobin and the other one with hemoglobin E
variant producing offspring with ββE genotype. The
hemoglobin electrophoresis show HbA, HbE, and
HbA2.5,6
Beta-thalassemia/hemoglobin E disease occurs
when the hemoglobin E trait gene combines with β+
or β0 thalassemia. This condition is commonly found
in Southeast Asia, including Indonesia. Most of the
patient show medium or severe condition with
dependent on blood transfusion. The hemoglobin
electrophoresis detects HbA, HbE, HbF, and HbA2 in
beta+ thalassemia/hemoglobin E and HbE, HbF, and
HbA2 in beta0 thalassemia/hemoglobin E.5,6
In Indonesia, the most common beta-globin gene
mutations are IVS1-nt5 (GàC), IVS1-nt1 (GàT),
codon 15/Cd15 (TGGTriptofanàTAGstop), codon 26 HbE
(GAG Glutamat à AAG Lysin ), and HbMalay/Codon 19
(AAC A s p a r g i n à AGC S e r i n ). Study from Semarang
transfusion unit on a β thalassemic patient with
routine transfusion the most common mutation is
HbE/IVS1-nt5 (55.3%) followed by IVS1-nt5/IVS1-nt5
and HbE/Cd35 each as much as 13.2%.7,8
In Indonesia, according to data released by WHO,
the percentage of pregnant female with beta-globin
gene variant is 3,956% with A/β-thalassemia; 1,872%
with AE, and 0.03% with another variant. Every year
there are 192.842 pregnant females who are
β-thalassemia carrier, 91.063 HbE carrier, and 1.459
carriers of another beta-globin gene variant. If both
parents have the significant beta-globin gene
variant, then each year Indonesia has risks as much as
10.256 birth with homozygous-β-thalassemia, 9.008
birth with β-thalassemia/HbE, and 2.640 birth with a
combination of an undangerous variant. Every year in
Indonesia there are 2.564 conceptions with
homozygous β-thalassemia, 2.252 conceptions with
β-thalassemia/HbE, and 660 conceptions with
undangerous variant.9
The MC values: Mean Corpuscular Volume (MCV),
Mean Corpuscular Hemoglobin (MCH) and Mean
Corpuscular Hemoglobin Concentration (MCHC).
They were first introduced by Wintrobe in 1929 to
define size and hemoglobin concentration of red
blood cells. Those three parameters usually termed
as red cell indices and are very useful in determining
the etiology of anemia. The red cells indices are
calculated based on hemoglobin, hematocrit
(packed cell volume), and red blood cell count. With
the new technology, red cell indices can be
automatically measured.10
The Red Cell Distribution Width (RDW) is the
variation of red blood cells size. The size distribution
of a population of cells is graphically represented by
the red cells histogram (Price-Jones Curves).10
Mean corpuscular volume defines the red blood
cells size in femtoliters (10-15/fL) or in cubic microns
(µm3). The normal values are 87±7 fL. The MCV can
be manually calculated using the formula:10
MCV =
hematocrit
red blood cell count
X 10
The MCH quantifies the amount of hemoglobin in
one red blood cell and, the normal value is 29 ± 2
picograms (pg) per cell. The MCH can be manually
calculated using the formula.10
MCH =
hemoglobin
red blood cell count
X 10
The RDW represents the coefficient of variation of
the red blood cell volume distribution (size). The
normal value is 13±1.5%.10
In everyday life, the majority of clinicians who
work mostly in handling patients with
hemoglobinopathy look first at the result of full
blood count, which are the hemoglobin level, MCV as
well as RDW. When those parameters were found to
be abnormal and tend to side to the finding of A
hemoglobinopathy, they required further
investigation such as hemoglobin electrophoresis,
High-Performance Liquid Chromatography (HPLC),
and when confirmation was made, gene study was
requested. So, the MCV and RDW is the first front
gate for every clinician who has suspicion for
investigating hemoglobinopathy.
This research finding in the hematology clinic,
sometimes the MCV cannot give direct guidance and
sometimes clinicians feel that the MCV are confusing.
The MCV may give a potential benefit as a front gate
for a guide of hemoglobinopathy. So, it was
imperative that a study should be done to clarify if
there was a discrepancy of MCV with a diagnosis of
hemoglobinopathy, as well as the RDW in assisting
the diagnosis.
METHODS
Two patients with beta-thalassemia/hemoglobin
E disease were recruited in the study, and 45 blood
samples were taken during the routine follow up
examinations. For control comparisons, 5 samples
were taken from beta-thalassemia trait patients.
Three from their families and two from other
beta-thalassemia trait cases. They were all cases from
the Department of Pediatrics of the Medical Faculty of
the University of North Sumatera/Adam Malik
Hospital Medan. The beta-thalassemia/hemoglobin E
The Hemoglobin, RDW, and Mean Corpuscular Values - Setiadji, et al.
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Indonesian Journal of Clinical Pathology and Medical Laboratory, 2019 July, 25 (3) : 343 - 348
disease patients received regular blood transfusion
for maintaining the hemoglobin level. Transfusion
was given when the hemoglobin level was below 7
gr/dL or when the patients felt that the hemodynamic
was disturb (such as weakness, shortness of breath,
recurrent fever, and heart palpitation). All betathalassemia trait cases were not given a blood
transfusion.
Six mL of venous blood was taken from the
median vein. Three mL of blood was for the
investigation of full blood count, and the other 3 mL
of blood was for the investigation of genotyping
study.
All beta-thalassemia/hemoglobin E disease and
beta-thalassemia trait patients underwent full blood
count examination every time they visit the clinic. The
full blood count was investigated using Sysmex XN1000. The hemoglobin level was investigated by the
machine based on chemical analysis using lauryl
sulfate, the MCV were automatically calculated by
the machine based on the calculation of hemoglobin,
hematocrit, and red blood cell count. The RDW was
also automatically calculated based on the
distribution width of the red blood cell which size and
shape did not exit coefficient of variation of 15%,
when the coefficient of variation was >15%, the RDW
was considered as abnormally increased. The normal
RDW was regarded as <15%.
The determination of beta-thalassemia trait and
hemoglobin E trait, as well as beta-thalassemia
/hemoglobin E disease, was carried out by means of
capillary electrophoresis from Minicap Sebia where
the machine uses a capillary tube to be filled up by
EDTA blood and that blood in the capillary tube was
run to the mini-chamber of an electrophoretic tank.
The separation was automatically scanned by an
in-machine densitometer and the result was
compared with already made standard of normal
hemoglobin, abnormal hemoglobin, and variant
hemoglobin which was already provided inside the
machine by the company.
The genotyping of the hemoglobin was also
performed by ARMS-PCR and DNA sequencing done
in Eijkman Institute For Molecular Biology in Jakarta.
For this method, the DNA extraction was done by
salting out method. After DNA was extracted, it was
run into the ARMS PCR and DNA sequencing.
Statistical analysis was performed using a t-test
for the sample with normal distribution and
Mann-Whitney U Test for non-normal distribution.
A correlation test was performed with Pearson for
normal distribution and Spearman for non-normal
distribution. This study was approved by the Health
Research Ethical Committee of Universitas Sumatera
Utara (No. 226/TGL/KEPK/ FK USU-RSUP
HAM/2017).
RESULT AND DISCUSSION
From the hemoglobin electrophoresis
investigation, it was found that there were two
families which parents had one beta-thalassemia
trait gene, and the other had hemoglobin E trait
gene.
The father of Family-1 had beta-thalassemia trait
gene and the genotype show IVS1nt5 (GC), while the
mother had hemoglobin E genotype of codon 26
(GA). Together they have five children: one daughter
Table 1. Samples characteristics
Family-1
Father
Mother
Son-1
Son-2
Son-3
Daughter-1
Daughter-2
Family-2
Father
Mother
Daughter
Son
Person-1
Person-2
Age (years)
HbA (%)
50
46
26
24
23
12
9
95.1
73.6
72.6
96.8
94.8
97.1
75.3
32
32
10
8
35
26
70.7
93.5
72.4
97
94.8
95.3
HbA2 (%)
HbE (%)
HbF (%)
4.9
3.5
3.9
3.2
4.9
2.9
3.5
22.9
23.5
15.6
0.3
5.6
3.2
6.2
3.8
3
4.7
4.7
26.1
16.7
-
0.3
7.1
0.5
-
The Hemoglobin, RDW, and Mean Corpuscular Values - Setiadji, et al.
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Indonesian Journal of Clinical Pathology and Medical Laboratory, 2019 July, 25 (3) : 343 - 348
with beta-thalassemia/hemoglobin E disease
[IVS1nt5 (GC)/Codon 26 (GA)], one son with
hemoglobin E trait (codon 26 GA/-), one son with
beta-thalassemia trait (IVS1nt5 (GC)/-), one son and
one daughter that normal.
Family-2: the father had hemoglobin E trait gene
with the genotype show codon 26 (GA). The mother
had a beta-thalassemia trait with the genotype show
codon 41/42 (-TTCT). Together they had two children:
one daughter with beta-thalassemia/hemoglobin E
disease [Codon 41/42 (-TTCT)/Codon 26 (GA)] and
one normal son. The result of the MCV can be seen in
Table 2.
Table 2 shows that the Mean±SD of the MCV of
patients with beta-thalassemia/hemoglobin E
disease is significantly higher compared to
beta-thalassemia trait.
The mean concentration hemoglobin of patients
with beta-thalassemia/hemoglobin E disease was
significantly higher compared to beta-thalassemia
trait.
Figure 1 shows the mean differences between MCV
and MCH in beta-thalassemia/hemoglobin E disease
and beta thalassemia trait. The left Figure shows that
the MCV level of beta-thalassemia/hemoglobin E is
significantly higher than the beta-thalassemia trait
with p-value 0.008 (using an independent t-test). The
right Figure shows that the MCH level of
beta-thalassemia/hemoglobin E is also significantly
higher than the beta-thalassemia trait with p-value
also 0.008 (using Mann-Whitney U Test).
Table 3 shows that the Mean±SD of the hemoglobin
levels of patients with beta-thalassemia/hemoglobin E
disease is significantly lower compared to
beta-thalassemia trait. The RDW of patients with
beta-thalassemia/hemoglobin E disease was
significantly higher compared to beta-thalassemia trait.
Table 2. The MCV of patients with beta-thalassemia/hemoglobin E disease and beta-thalassemia trait
Beta-thalassemia trait
Beta- thalassemia/hemoglobin E disease
MCV
MCV (fL)
MCH (pg)
70.898 ± 4.897
22.831 ± 2.287
80.0
64.640 ± 2.889
19.720 ± 0.968
26.0
p-value =0.008
p-value=0.008
24.0
70.0
22.0
MCV
MCH
75.0
65.0
20.0
50
60.0
18.0
38
19 44
19
38
55.0
16.0
beta trait
HbE/beta
beta trait
HbE/beta
Disease
Disease
Figure 1. The differences between MCV between beta-thalassemia/hemoglobin E disease and
beta-thalassemia trait
Table 3. The hemoglobin levels and RDW of patients with beta-thalassemia/hemoglobin E disease and
beta-thalassemia trait
Beta- thalassemia/hemoglobin E disease
Hemoglobin (gr/dL)
RDW (%)
8.078±2.394
29.340±3.978
Beta-thalassemia trait
12.580±0.896
16.160±0.422
The Hemoglobin, RDW, and Mean Corpuscular Values - Setiadji, et al.
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Indonesian Journal of Clinical Pathology and Medical Laboratory, 2019 July, 25 (3) : 343 - 348
40.0
15.0
35.0
12.5
RDW
30.0
Hb
10.0
25.0
p-value=0.001
7.5
p-value=0.001
20.0
46
5.0
*
15.0
beta trait
HbE/beta
beta trait
HbE/beta
Disease
Disease
Figure 2. The differences in hemoglobin level and RDW between beta-thalassemia/hemoglobin E disease and
beta-thalassemia trait
Table 4. The difference of hemoglobin level, MCV, MCH, and RDW between pre- and post-transfusion state in
beta-thalassemia/hemoglobin E disease
Pre-transfusion
Post-transfusion
p-value
Hemoglobin (gr/dL)
MCV (fL)
MCH (pg)
6.475 ± 0.939
68.757 ± 4.557
21.879 ± 2.305
10.465 ± 1.236
74.835 ± 2.873
24.494 ± 1.159
<0.001
<0.001
<0.001
RDW (%)
31.114 ± 3.125
26.235 ± 3.818
<0.001
Figure 2 show there are a significant difference
of hemoglobin level and RDW between
beta-thalassemia/hemoglobin E and beta-thalassemia
trait with p-value 0.001 and <0.001 respectively (using
Mann-Whitney U Test).
Moreover, divided the beta-thalassemia/hemoglobin
E disease into pre-transfusion and post-transfusion state.
This table show Mean±SD of hemoglobin level, MCV,
MCH, and RDW in pre- and post-transfusion state. The
result shows that there are significant differences
between those four parameters between pre- and
post-transfusion state. Beta-thalassemia/hemoglobin E
disease is a condition where there is a combination of
double heterozygosity of beta-thalassemia trait and
hemoglobin E trait gene. This condition is more severe
than the beta-thalassemia trait alone or hemoglobin E
trait alone. Either in the phenotypic appearances such as
clinical conditions, hemoglobin value, MCV, as well
as RDW. Nonetheless, surprisingly the researchers
found that the MCV of patients with
beta-thalassemia/hemoglobin E disease had better
result compared to beta-thalassemia trait alone. In this
method, it was shown that the MCV and MCH were
significantly higher in beta-thalassemia/hemoglobin E
disease which logically a condition that was more
severe and should produce a worse MCV.
Patients with newly diagnosed
beta-thalassemia/hemoglobin E should not
receive regular transfusion without long period
observation of growth and development, quality
of life, and spleen size. Transfusion should be
administered if the hemoglobin level is <4 gr/dL
and/or if the patient is suspected of having an acute
intermittent infection, showing any problem
suspected to be related to anemia. If a few transfusion
have been administered in the acute situation,
immediate commitment to a regular transfusion
program should not be undertaken. During the
initial assessment period, when the decision to
administer regular transfusion is not decided yet,
it is important to do full blood count examination
every 2-3weeks.11
After 3-6 months of careful observation, a clinical
and laboratory pattern should begin to emerge.
Regular transfusion is not needed if the patients
maintain reasonable appetite, level of energy, quality
of life, develop good growth and development
(height is better in depict growth pattern than
weight), sexual maturation in parallel with bone age,
and spleen size is stable (enlarging rate <3 cm/year).11
The regular transfusion should be considered if
the hemoglobin level falls below 5 gr/dL or if
The Hemoglobin, RDW, and Mean Corpuscular Values - Setiadji, et al.
Indonesian Journal of Clinical Pathology and Medical Laboratory, 2019 July, 25 (3) : 343 - 348
appetite, energy, growth, or developmental
milestone is compromised, and if the spleen
enlarging >3 cm/year.11
In pediatric department of Adam Malik Hospital
beta-thalassemia/hemoglobin E disease received
regular transfusion if the steady-state hemoglobin
level is below 7 gr/dL or if the patient show any
hemodynamic instability sign such as weakness,
shortness of breath, recurrent fever, and heart
palpitation, this is to avoid retardation of growth
(stunting). In this study, those two patients will come
every 3-4 weeks to received blood transfusion or
whenever their parents feel that their children
become more parlor or if their children show
weakness. The pediatrician usually gives packed red
cell and the blood volume given to the patient is
calculated based on the patient's body weight.
This study found that the MC level was
significantly better in beta-thalassemia/hemoglobin
E compared to beta-thalassemia trait. This fact was
confusing because logically, beta-thalassemia/
hemoglobin E should had worse MC level. This might
be due to regular transfusion they received. The data
on our study showed that the MC level were
significantly higher in post-transfusion state
compared to the pre-transfusion state. From this
finding, we can learn that children who come to the
the physician for the first time with low hemoglobin
level, a slight reduction of MC level, and increase
RDW should be asked if they ever received blood
transfusion recently. The iron deficiency anemia can
be excluded. The physician should request full blood
count, peripheral blood morphology, iron profile,
and if needed hemoglobin electrophoresis. This
examination is important in making a diagnosis
before giving transfusion, because patient newly
diagnosed with beta-thalassemia/hemoglobin E
does not need a regular transfusion, instead they
should be observed carefully to determine when
they need a transfusion.
CONCLUSION AND SUGGESTION
This research found a discrepancy of MCV
between beta-thalassemia/hemoglobin E disease
PAGE 348
with beta-thalassemia trait. This finding closely
relates to the administration of blood transfusion.
The researchers recommend that all patients with
beta-thalassemia/hemoglobin E disease who show a
discrepancy of the MC values should be investigated
carefully with RDW as well as information about
possible blood transfusion to avoid a mistake.
ACKNOWLEDGMENT
Thank you very much to The Eijkman Institute For
Molecular Biology for their assistance on performing
DNA analysis study.
REFERENCE
1.
Fucharoen S, Winichagoon S. Hemoglobinopathies in
Southeast Asia. Indian Journal of Medical Research,
2011; 134(4): 498-506.
2. Cao A, Galanello R, Rosatelli C. Genotype-phenotype
in beta-thalassemia. Blood Journal, 1994; 8(1): 1-12.
3. Fucharoen, S. Genotype and Phenotype of
Thalassemia: A discussion. 2005. Annals New York
Academy of Science. Volume 1054. Page 518 – 521
4. Ricchi P, Filosa A, Maggio A, Fucharoen A.
Non-transfusion-dependent thalassemia: A complex
mix of genetic entities yet to be fully discovered. 2015.
Biomed Research International, 2015; 2015: 1-2.
5. Vichinsky E. Hemoglobin E syndrome. American
Society of Hematology, 2007; 2007(1): 79-83.
6. Olivieri N, Pakbaz Z, Vichinsky E. HbE/beta-thalassemia:
A common and clinically diverse disorder. Indian
Journal of Medicine, 2011; 134(4): 522 – 531.
7. Tamam M, Hadisaputro S, Setianingsih A. Hubungan
antara tipe mutasi gen globin-beta dan manifestasi
klinis pada penderita thalassemia. Jurnal Kedokteran
Briwajaya, 2010; 26: 48-52.
8. Wahidayat P, Gatot D, Tjitrasari D. Phenotypic
diversity in Beta-HbE thalassemia patients. Pediatrica
Indonesia,2006; 6 (3-4): 82-86.
9. Modell B, Darlison M. Epidemiological estimates for
haemoglobin disorder: WHO South East Asian region
by country. Modell's haemoglobinopathologist's
almanac. 2007; 1-11.
10. Sarma PR. In: Walker HK, Hall WD, Hurst JW, editors.
Clinical Methods: The history, physical, and laboratory
examinations. Boston, Butterworths, 1990; 152: 721-725.
11. Olivieri N, Muraca G, O'Donnel A. Studies in
hemoglobin E beta-thalassemia. British Journal of
Hematology, 2008; 141(3): 388 – 397.
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