International Journal of Laboratory Hematology

The Official journal of the International Society for Laboratory Hematology

SHORT REPORT INTERNATIONAL JOURNAL OF LABORATO RY HEMATO LOGY

Co-inherited b-thalassemia trait and HbH disease: clinical

characteristics and interference in diagnosis of thalassemia
by high-performance liquid chromatography
X.-L. YIN*, Z.-K. WU †, X.Y. ZHOU ‡, T.-H. ZHOU*, Y.-L. ZHOU*, L. WANG*, J. HUANG*, X.-H. ZHANG*

*Department of Hematology, 303rd SUMMARY

Hospital of the People’s Liberation
Army, Nanning, Guangxi, China Introduction: To identify the clinical and hematological characteristics
†
Department of Molecular Biology, in a large group of patients with combined HbH disease and b-thalas-
Guang’AnMen Hospital, China
Academy of Traditional Chinese semia trait.
Medicine, Beijing, China Methods: Hemoglobinopathy analysis and full genotyping identified a
‡
Clinical Laboratory Center of cohort of patients with HbH disease, b-thalassemia trait, or combined
Guangxi Zhuang Autonomous
HbH disease and b-thalassemia trait.
Region, Nanning, Guangxi, China
Results: Co-inheritance of b-thalassemia trait and HbH disease signifi-
Correspondence: cantly decreased the mean corpuscular volume (MCV) in 27 patients
Zhang Xin-Hua, Department of when compared to 287 patients with HbH disease alone. The com-
Hematology, 303rd Hospital of the
People’s Liberation Army, Nanning,
bined condition also alleviated anemia in nondeletional HbH disease
Guangxi 530021, China. but not in the deletional cases. Beta-thalassemia trait also signifi-
Tel.: +86 0771 2870523; cantly decreased the expression of HbH, Hb Constant Spring when
Fax: +86 0771 2870523; present, and HbA2, with levels as low as 3.6% on high-performance
E-mail: zxh303xy@hotmail.com
liquid chromatography (HPLC).
doi:10.1111/j.1751-553X.2012.01415.x Conclusion: These cases, although relatively common in the South
Chinese population, may be difficult do diagnose correctly when only
Received 8 September 2011;
examined on HPLC. Therefore, molecular analysis of the a and b glo-
accepted for publication 9 January
2012 bin genes should be done in all cases with hemolytic anemia and
low MCV without clear HbH disease or b-thalassemia parameters.
Keywords
HbH disease, b-thalassemia,
high-performance liquid chromato-
graphy, clinical aspects

assemia syndromes are caused by a-globin chain

INTRODUCTION
deficiency and result in hematological abnormalities
The postnatal hemoglobin molecule is a tetramer con- associated with the degree of deficiency. In hemoglo-
sisting of two a- and two b-globin chains. The a-thal- bin H disease (HbH disease), an a-thalassemia of inter-

 2012 Blackwell Publishing Ltd, Int. Jnl. Lab. Hem. 1

2 X.-L. YIN ET AL. HBH DISEASE ACCOMPANYING b-THALASSEMIA TRAIT
mediate severity, three of the four a-globin genes are
affected. Although the clinical severity of HbH disease
can be quite variable, genotypes involving point
mutations of the a2-globin gene (aTa/–) result in a
more severe phenotype as compared with genotypes
involving gene deletions (-a/–) [1].
As a result of the highly deficient globin chain
synthesis in HbH disease, there are insufficient a globin
chains available for binding to b-globin chains, leading
to the formation of unstable homotetramers (HbH = b4)
, which are the most clear diagnostic parameter and the
cause of pathogenesis. Theoretically, b-thalassemia trait
is considered an ameliorating factor of HbH disease and
several isolated cases have been reported. In these cases,
the disease was masked by the reduction or absence of
HbH [2]. In a previous study, we also found that
co-inherited b-thalassemia heterozygosity slightly, but
not significantly, alleviated anemia in patients with
nondeletional HbH disease [3]. Here, we further sum-
marize the clinical characteristics of patients with HbH
disease in the presence of b-thalassemia heterozygosity
in a larger cohort of patients.
MATERIALS AND METHODS
From October 2009 to March 2011, we performed
hemoglobinopathy analysis and full genotyping on
3256 patients from the province of Guangxi, Southern
China.
Hemoglobin levels and red blood cell indices were
determined using an auto-hematology analyzer (Cell
Dyn 1700, Abbott Park, IL, USA). HbA, HbA2, and
HbF levels were determined using a Bio-Rad Variant
II high-performance liquid chromatography (HPLC)
system (Variant; Bio-Rad, Hercules, CA, USA). The
three most common deletional a-thalassemia genes
(including –SEA, -3.7a, and -4.2a) were detected by
multiplex gap-polymerase chain reaction. The three
common nondeletional a-thalassemia genes [Constant
Spring variant (aCS), Quong Sze variant (aQS), and
Westmead variant (aWS)] and the 17 most common
b-thalassemia genes were detected by reverse dot-
blotting with a genetic diagnostic kit (Yi Sheng Tang
Biological Products, Shenzhen, China) [3]. A group of
314 patients, with three of the four a-globin genes
affected and diagnosed with HbH disease, were exam-
ined for b-globin gene defects. Statistical analyses
were performed using the Statistical Package for Social
Sciences, version 15.0 (SPSS Inc., Chicago, IL, USA).
The data are expressed as median values with ranges
for continuous variables and numbers with percent-
ages for categorical variables. Nonparametric tests
were used to compare continuous variables (the
Mann–Whitney test or the Kruskal–Wallis test) or cat-
egorical variables (Pearson’s chi-squared test) across
subgroups of patients. These tests were followed by a
post hoc Dunn test. P values are two-sided and con-
sidered significant when <0.05.
Medical data were collected from each patient.
Informed consent was obtained from all patients or
parents of pediatric participants.
RESULTS
The cohort
Of the 314 patients with HbH disease, 141 were male
and 173 were female. The average age was 17.0 years,
and 148 cases (47.1%) were younger than 14 years of
age.
The HbH genotypes
The 20 kb deletion that is typical for a-thalassemia
patients in Southeast Asia (–SEA) was the only a defect
found in our cohort. Fifteen patients were diagnosed as
HbH-WS (–SEA/aWSa). One hundred and sixty-six
patients were diagnosed with HbH-CS (–SEA/aCSa), and
eight patients were diagnosed with HbH-QS (–SEA/aQSa),
both of which are classified as nondeletional HbH dis-
ease. One hundred and twenty-five patients were diag-
nosed with deletional HbH disease, including 82 cases of
–SEA/-3.7a and 43 cases of –SEA/-4.2a. Anemia in 15
patients with HbH-WS was mild [3], and therefore these
patients were analyzed separately.
The b-thal defects
Twenty-seven of the 314 HbH cases (8.6%) were het-
erozygous for b-thalassemia. Among these patients, 17
were diagnosed with a b0 mutation (eight cases with
CD41-42(-TCTT), eight cases with CD17(AfiT), and
one case with CD14-15(+G)). The remaining 10
patients carried the b+ mutation [six cases with IVS-2-
654(CfiT), two cases with )28(AfiG), and two cases
bE(CD 26 GfiA)].
 2012 Blackwell Publishing Ltd, Int. Jnl. Lab. Hem.
 X.-L. YIN ET AL. HBH DISEASE ACCOMPANYING b-THALASSEMIA TRAIT 3

Clinical characteristics of HbH/b-thalassemia

0.778

0.095
0.694
0.482
0.772
0.597

M, male; F, female; Hb, Hemoglobin; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin
1.00
heterozygosity

P
b-thalassemia heterozygosity significantly increased

b-thalassemia
the hemoglobin level and delayed the age of diagnosis

26.8 ± 8.5

7.6
2.6

3.7
17

22
Without
in patients with nondeletional HbH disease (Table 1).

(n = 12)

±
±
±
±
±
Conversely, neither the hemoglobin level nor the age

66.5
22.3

23.1
117

289
5/7
of diagnosis differed significantly in patients with dele-
tional HbH disease. For patients with HbH-WS, b-thal-

HbH-Westmead

b-thalassemia
assemia slightly decreased the Hb level, but this was

25.3 ± 4.9

2.5
1.9

1.6
11

26
not significant.

(n = 3)

±
±
±
±
±
With

294
98
68.3
21.1

21.9
1/2
Table 1. Clinical characteristics of patients with different types of HbH disease with or without b-thalassemia trait
Effects of coexisting b-thalassemia on HPLC analysis

0.217

0.725
0.452
0.004
0.105
0.010
0.131
Owing to practical reasons, only 218 patients of 314 were
analyzed on HPLC. All samples were tested within 68 h

P
of being taken. In the three patients with HbH-WS, no

b-thalassemia
HbH fraction was visible either in presence or absence of

19.7 ± 13.8

7.1
3.8

6.3
12

20
(n = 117)
b-thalassemia. Fourteen of the remaining 24 cases of

Without

89 ±
58.9 ±
18.8 ±
281 ±
20.9 ±
HbH disease combined with b-thalassemia were analyzed

50/67
Deletional HbH disease
on HPLC; the HbH fraction was visible in only six cases
(42.9%). This percentage is significantly lower than the
proportion observed among HbH patients without b-thal- b-thalassemia

13.5 ± 11.9
assemia (180/192, 93.8%) (v2 = 38.6, P < 0.001). When

4.1
1.2

1.7
09

15
regrouped according to deletional or nondeletional HbH
(n = 8)

±
±
±
±
±
With

49.4
16.2

17.5
300
92
disease, b-thalassemia still significantly decreased the
amount of free b and the presence of the fraction on 4/4
HPLC (Table 2). HbA2 levels were as low as 3.6% while
0.078

0.798
0.043
0.000
0.021
0.000
0.376
HbE was reduced to 16.6%. In 10 cases with HbH-CS,
P

the presence of HbCS was significantly lower when com-

pared to normal HbH disease without b-thalassemia trait
b-thalassemia

13.7 ± 11.2

(4/10 vs. 103/116, P = 0.001).

7.5
3.2

2.1
17

17
(n = 158)
Without

77 ±
70.5 ±
20.0 ±
198 ±
19.4 ±
73/85

concentration; RDW, red cell distribution width.

Nondeletional HbH disease

DISCUSSION
In our cohort, 8.6% of the patients with HbH disease
With b-thalassemia

were found to be a carrier of b-thalassemia. This figure

is in accordance with the prevalence of the beta-thalas-
21.8 ± 16.8

10.4

semia gene in the Guangxi province [4]. Based on these

2.8

1.9
16

17
(n = 16)

figures, the expected frequency of the combination

±
±
±
±
±

HbH./b-thal is 0.0275% in Guangxi, a figure that is

315
86
59.6
18.0

18.9
8/8

much higher than that in Hong Kong (approximately

0.0007%) [2]. This shows that the combination HbH
Age at diagnosis

disease/b-thalassemia trait is not a rare condition in

Gender (M/F)

MCHC (g/l)

regions with high prevalences of both defects.

MCH (pg)

RDW (%)
MCV (ll)

Although b-thalassemia heterozygosity significantly

(years)

Hb (g/l)

decreased the presence of HbH in both deletional and

nondeletional HbH disease, improved clinical condi-

 2012 Blackwell Publishing Ltd, Int. Jnl. Lab. Hem.

4 X.-L. YIN ET AL. HBH DISEASE ACCOMPANYING b-THALASSEMIA TRAIT
Table 2. Results of high-performance liquid chromatography analysis in patients with different types of HbH disease
with or without ß-thalassemia trait
Deletional HbH disease
With b-thalassemia Without
(n = 4) b-thalassemia (n = 74) P
HbH fraction 1 (25%) 65 (87.7%)
(positive
cases, %)
HbA2 (%) 5.05 ± 1.41 2.05 ± 0.27
HbF (%) 1.15 ± 0.31 0.28 ± 0.43
tions were confined to patients with the more severe
nondeletional HbH disease. As expected, reduction of
the unstable b4 tetramer alone is not enough to allevi-
ate hemolysis in these cases. The improved Hb levels in
patients with nondeletional HbH diseases can also be
ascribed to a lower formation of HbCS tetramers. HbH-
CS was responsible for the overwhelming majority of
nondeletional HbH disease (95.7%). HbCS is caused by
a mutation in the stop codon of the a2-globin gene,
which results in 31 additional amino acids. Oxidized
HbCS is deposited on the membranes of red blood cells
and accelerates the damage to these cells [5, 6].
In HbH-WS, however, concomitant b-heterozygos-
ity tends to aggravate rather than alleviate anemia.
HbH-WS is prevalent in the Guangxi Province. Unlike
other types of nondeletional HbH disease, anemia in
HbH-WS is only mild or even absent [3]. In these
cases, the b-globin defect adds to the severity of the
a-globin gene defects when the HbH-WS genotype
combines with a b-thalassemia trait.
Additionally, we noticed a significant decrease in
the mean corpuscular volume (MCV) levels in cases
of HbH disease in the presence of b-thalassemia trait,
both in deletional and nondeletional HbH disease
patients, in spite of the decreased imbalance of the b/
a ratio and probably due to the lower Hb content
(mean corpuscular hemoglobin) in the red cell.
High-performance liquid chromatography is an
important technology in thalassemia screening. How-
ever, HPLC diagnostics become complex when HbH dis-
ease is combined with b-heterozygosity [7], HbS, or HbC
>Nondeletional HbH disease
With b-thalassemia Without
(n = 10) b-thalassemia (n = 118) P
0.011 5 (50%) 115 (97.5%) 0.011
0.24 4.53 ± 1.05 2.27 ± 0.55 0.000
0.000 3.94 ± 3.74 1.18 ± 1.43 0.045
[8]. The present study showed that the HbH fraction
tends to disappear in the presence of b-thalassemia trait
and that HbA2, still elevated in combinations of b-thal
with (–/aa) or (-a/-a), tends to reach almost normal lev-
els in the presence of HbH disease (–/-a). Moreover,
abnormal fractions, such as HbE, HbS, or HbC, tend to
be much depleted or disappear for preferential binding
of the reduced alpha chains with the normal b, rather
than with the abnormal one. All these have obvious
consequences for the diagnosis when only HPLC is used.
The HbA2 level was only 3.6% in three patients, a
level which is in the ‘gray zone’ and insufficient to
diagnose the b-thalassemia trait in most laboratories.
The cutoff value of HbA2 for the determination of b-
thalassemia without considering the total picture and
the molecular analysis remains controversial [9].
Although 3.5% is the generally accepted cutoff point
in many studies, some authors have used an HbA2 of
more than 4.0% to diagnose b-thalassemia trait with
certainty [10]. Therefore, in areas with a high preva-
lence of both a- and b-thalassemia, it may be more
reasonable to consider a cutoff value of 3.5% when
anemia and low MCV persist in spite of normal iron
and in the absence of alpha thalassemia defects.
ACKNOWLEDGEMENTS
This research was supported by the National Basic
Research Program of China (973 Program, No.
2010CB530406) and the Natural Science Foundation
of Guangxi (No. 2011GXNSFA018196).
 2012 Blackwell Publishing Ltd, Int. Jnl. Lab. Hem.

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