Objective: Thalassemia carrier screening in siblings of thalassemia major patients by HbA2 estimation. Methods: This prospective, cross-sectional study was conducted at thalassemia welfare society of JK Lon Hospital, Kota. Siblings of thalassemia major patients registered at our hospital were investigated for chemically bonded ceramics and HbA2 estimation by high performance liquid chromatography (HPLC) method. Results: A total of 121 cases were screened for carrier by HPLC method for HbA2 estimation. Total 59 (48.76%) cases had HbA2 level ≥3.5%, and considered as carrier while 62 (51.24%) cases were non-carrier. Mean HbA2 value in carrier was 5.24% ± 1.14% and in non-carrier was 2.69% ± 0.51%. Conclusion: It was concluded that β thalassemia carriers are more prevalent in siblings of thalassemia major than the normal population

Gulati, R K

Sharma, Dipendra

Sharma, Gopikishan

English

Atharva Scientific Publications (E-Jounals)

Vol 3 | Issue 3 | Jul - Sep 2016 Indian J Child Health 258
Original Article
Thalassemia carrier screening in siblings of thalassemia major patients by HbA2 
estimation
Gopikishan Sharma, Dipendra Sharma, R K Gulati
From Department of Pediatrics, Government Medical College, Kota, Rajasthan, India
Correspondence to: Dr. Gopikishan  Sharma, Assistant Professor, Department of Pediatrics, Goverment Medical College, Kota, 
Rajasthan. E-mail: dr.gopikishansharma6162@gmail.com
Received – 30 May 2016 Initial Review – 27 June 2016 Published Online – 12 August 2016
Thalassemia refers to genetic disorders in globin chain production. 3% of the world’s population carries genes for β thalassemia [1]. It is the most common monogenic 
disorder in India. The prevalence of thalassemia trait varies 
from 1.0-14.9% in various regions of India. Incidence varies 
in different communities, religions, and ethnic group. It is 
estimated that more than 25 million people in India are carriers 
of the β-thalassemia gene and 8000 children are born every year 
with thalassemia major. Only 10–15% of these children receive 
optimal treatment. Community control of hemoglobinopathies 
relies mainly on outreach educational programs and genetic 
counseling with antenatal diagnosis [2].
Accurate and timely detection of various hemoglobin variants 
including β-thalassemia trait can prevent the occurrence of more 
serious disorder like thalassemia major in newborn [3]. There is 
definite need for carrier surveillance in our country. The various 
available options to screen carriers are (1) screening of school 
going children, (2) screening of high-risk community, (3) prenatal 
screening, (4) extended family screening, i.e. screening of relatives, if 
there is a thalassemia major child in family, and (5) routine antenatal 
screening in early pregnancy between 10 and 12 weeks [4]. There 
is a high prevalence of thalassemia in Kota and vicinity. Therefore, 
we performed this study to determine the prevalence of thalassemia 
carrier in siblings of thalassemic children.
MATERIALS AND METHODS
This prospective, cross-sectional study was conducted in the 
Department of Paediatrics, J. K. Lon Mother and Child Hospital, 
Government Medical College, Kota. The study was conducted 
between December 2013 and November 2014 after getting 
approval from the institutional ethics committee. Children aged 
below 18 years who were siblings of thalassemia major patients, 
registered at thalassemia welfare society at J.K. Lon Hospital, 
Kota were included in the study. Children with a history of recent 
blood transfusion in last 3 months and children with any acute 
febrile illness were excluded from the study.
Children were recruited after taking informed consent from 
the parents or legal guardians. For all patients, a careful history 
was taken for any illness or blood transfusion. Family history 
was taken for consanguineous marriage, hemolytic anemia, 
and other significant illnesses. General physical examination 
(including vitals and anthropometry) and systemic examination 
were performed. After taking written consent, 5 ml of venous 
blood was collected and sent for complete blood count and high 
performance liquid chromatography (HPLC).
Complete blood count was performed by automated cell 
counter to get hemoglobin, red cell count, hematocrit, red cell 
distribution width (RDW), and mean corpuscular volume (MCV), 
mean corpuscular hemoglobin (MCH), MCH concentration 
(MCHC), leukocyte, and platelet counts. Cation exchange HPLC 
has been applied to identify hemoglobin variants using Bio Rad 
variant system. It identifies and quantifies hemoglobin by their 
retention time (RT) (the time at which they elute) and computing 
the area under the peak. In “Bio Rad Variant”, hemoglobins are 
separated graphically and quantified spectrophotometrically 
utilizing sophisticated computer software. Before sample 
application, the instrument has to be primed and calibrated 
ABSTRACT
Objective: Thalassemia carrier screening in siblings of thalassemia major patients by HbA2 estimation. Methods: This prospective, 
cross-sectional study was conducted at thalassemia welfare society of JK Lon Hospital, Kota. Siblings of thalassemia major patients 
registered at our hospital were investigated for chemically bonded ceramics and HbA2 estimation by high performance liquid 
chromatography (HPLC) method. Results: A total of 121 cases were screened for carrier by HPLC method for HbA2 estimation. 
Total 59 (48.76%) cases had HbA2 level ≥3.5%, and considered as carrier while 62 (51.24%) cases were non-carrier. Mean HbA2 
value in carrier was 5.24% ± 1.14% and in non-carrier was 2.69% ± 0.51%. Conclusion: It was concluded that β thalassemia 
carriers are more prevalent in siblings of thalassemia major than the normal population.
Key words: HbA2 estimation, Siblings, Thalassemia carrier screening
Vol 3 | Issue 3 | Jul - Sep 2016 Indian J Child Health 259
Sharma et al. Carrier screening in siblings of thalassemia patients
followed by use of control to check reproducibility. The RT is 
stated in relation to that of control for HbA2.
Data obtained was tabulated using version 17 of the Statistical 
Package for Social Sciences (SPSS, published SPSS Inc.). Chi-
square test and Student’s test were used to identify differences 
between groups.
RESULTS
A total of 133 cases were assessed for inclusion in the study; 
out of them, 12 cases were excluded and finally, 121 cases were 
screened for carrier of thalassemia by HbA2 estimation. Out of 
121 cases, 5 cases were <6 months old, 42 aged 6-59 months, 
27 aged 5-10 years, and 47 cases were in 10-18 years age group. 
Out of 121 cases, 72 were male and 49 were female. Total 
59 (48.76%) cases had HbA2 level ≥3.5% (labeled as carrier), 
while 62 (51.24%) cases were non-carrier. Mean HbA2 value 
was significantly higher (P < 0.0001) in carriers (5.24 ± 1.14%) 
than in non-carriers (2.69 ± 0.51%). Mean HbF level was 2.02 ± 
0.41% in carriers and 3.63 ± 0.76% in non-carriers but difference 
was not significant (P = 0.18).
About 2 cases had a history of blood transfusion in the past; 
out of them, one case was carrier. Out of 121 cases, 1 case was 
diagnosed as thalassemia major (HbF 68%). Two cases had HbS 
level 36.2% and 37.8% without any clinical manifestation (sickle 
cell trait) and normal HbA2 level (2.9 and 2.3). HbA level was 
between 85 to 98.6% in rest of the cases. The comparison of 
hematological parameters between carriers and non-carriers is 
given in Table 1.
DISCUSSION
Preventive screening programs to identify carriers are being used 
by many countries where thalassemia is a common disease. Our 
study was done on the concept of cascade screening (extended 
family screening). We included the siblings (1st degree relative) of 
thalassemia major (index case) for screening. In our study, a total 
of 121 cases were screened for carrier of thalassemia by HPLC 
method for HbA2 estimation, and total 59 (48.76%) cases were 
found to be carrier. The WHO reports that 5% of the population is 
carrier of different hemoglobinopathies [5]. According to ICMR 
data, the carrier frequency of hemoglobinopathies is reported 
between 3 and 17% in the different population [6]. The results 
of this study showed a higher prevalence of beta thalassemia 
trait among siblings of thalassemia patients than 5% in general 
population.
The previous studies conducted in Gujarat and West Bengal 
revealed the prevalence of hemoglobinopathies to be 37.5 and 
20.47%, respectively [7,8]. In a study conducted in Maharashtra, 
the prevalence of beta thalassemia trait was 17% [9]. Antenatal 
screening was done in Mumbai and Behrampur (Orissa) shows 
the prevalence of carrier in pregnant mother was 1.99% and 8%, 
respectively [8,10]. Most of the previous studies did screening of 
general population and pregnant women, and only a few studies 
have been done in family, relative and siblings of thalassemia 
cases. As shown in Table 2, the prevalence is higher than the 
studies done by Ahmed et al [11] and Mohanty et al [12] and 
results are similar with Baig et al [13]. The differences in the 
results can be due to the selection of only siblings, geographic 
condition and number of cases and genetic heterogenicity of 
thalassemia gene.
In our study, HbA2 level was between 3.5 and 7.2% in carriers 
and the mean HbA2 was significantly higher (P < 0.001) in carriers 
than in non-carriers. Mean HbA2 in the study done by Mohanty 
et al [12] Mirebehbahani et al [15] and Sujata et al [16] were 
4.99, 5.18 and 4.53 respectively. Mean RDW was lower in carrier. 
RDW measures the coefficient of variation and is higher in iron 
deficiency anemia but not in the thalassemia where a uniform 
microcytic red cell population is seen with a normal RDW.
The mean red blood cell (RBC) count in carrier of 
hemoglobinopathies was increased and considered a useful 
diagnostic adjunct because the thalassemia has microcytic anemia 
with an increase in the RBC number, whereas other causes of 
microcytic anemia including iron deficiency anemia and anemia 
of chronic disease are typically associated with a decrease in the 
RBC number that is proportional to the degree of decrease in Hb 
concentration [8]. MCV is the key indicator for diagnosis and 
screening. Thalassemic individuals have a reduced MCV, and one 
study has suggested that an MCV of <72 is maximally sensitive 
and specific for the presumptive diagnosis of thalassemia [10,17]. 
A low MCH and low MCV are the clues for the diagnosis of 
thalassemia [9].
It is denoted that thalassemia carrier has characteristics 
hematological parameters with mild or no anemia with microcytic 
hypochromic RBCs, increased red cell counts and normal RDW 
Table 1: Comparison of hematological parameters between carrier 
and noncarrier
Mean Carrier Noncarrier t/χ2 P
Hb (g %) 9.89±2.08 9.43±2.42 - 0.21
RDW 14.86±4.00 15.07±5.61 0.22 0.82
HCT 30.79±5.00 29.24±7.13 1.4 0.15
RBC 5.26±1.00 3.75±0.92 8.8 <0.001
MCH 21.35±4.17 25.60±4.37 5.3 <0.001
MCV (fl) 62.08±11 79.7±11.78 8.4 <0.001
MCHC 31.55±3.17 31.65±2.38 0.19 0.84
TLC 8.17±2.56 8.45±4.12 0.48 0.63
Platelet count 261.23±100.11 236.80±145.91 1.1 0.29
RDW: Red cell distribution width, HCT: Hematocrit, RBC: Red blood cell, 
MCH: Mean corpuscular hemoglobin, MCHC: Mean corpuscular hemoglobin 
concentration, TLC: Total leukocyte count, MCV: Mean corpuscular volume
Table 2: Comparison of incidence of carrier in sibling of 
thalassemia major with others
Parameters Present 
study
Ahmed 
et al. [11]
Mohanty 
et al. [12]
S.M. Baig 
et al. [13]
Mishra 
et al. [14]
Year 2014 2012 2008 2008 2013
No. of cases 121 591 25 27 33
No. of carrier 59 183 10 12 25
% 48.76 30.86 40 44.4 73.33
Vol 3 | Issue 3 | Jul - Sep 2016 Indian J Child Health 260
Sharma et al. Carrier screening in siblings of thalassemia patients
and hemoglobinopathies can be suspected on the basis of these 
parameters. We recommend screening to detect carriers in siblings 
of thalassemia major patients, and counseling them to screen the 
forecoming life partener for thalassemia carrier before marriage 
to prevent thalassemia major in their next generation. This study 
was limited by its small sample size and study subjects. A wide-
scale population-based study with a larger sample size is needed 
to assess the exact prevalence of hemoglobinopathies and carriers 
in this part of the country.
CONCLUSION
It is concluded that β thalassemia carriers are more prevalent 
in siblings of thalassemia major than normal population. 
Hemoglobinopathies can be suspected on the basis of 
hematological parameters such as reduced MCV, reduced MCH, 
and elevated RBC count disproportionate to hemoglobin level.
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Funding: None; Conflict of Interest: None Stated.
How to cite this article: Sharma G, Sharma D, Gulati RK. Thalassemia 
carrier screening in siblings of thalassemia major patients by HbA2 estimation. 
Indian J Child Health. 2016; 3(3):258-260.


Thalassemia carrier screening in siblings of thalassemia major patients by HbA2 estimation

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Thalassemia carrier screening in siblings of thalassemia major patients by HbA2 estimation

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