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ORIGINAL ARTICLE  
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Prevalence of hemoglobinopathies using high-performance liquid chromatography as diagnostic tool in anemic patients of tertiary care center of Western India


1 Department of Zoology, Om Sterling Global University, Hisar, Haryana, India
2 Department of Immunohematology and Blood Transfusion, Armed Forces Medical College, Pune, Maharashtra, India
3 Department of Pathology, Command Hospital, Pune, Maharashtra, India
4 Department of Zoology, CCS HAU, Hisar, Haryana, India

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Date of Submission22-May-2022
Date of Acceptance03-Jul-2022
Date of Web Publication26-Sep-2022
 

   Abstract 

CONTEXT: Hemoglobinopathies are the most common heterogeneous group of monogenetic disorder in the world and its prevalence varies with geographical regions. India is developing country and many studies show a significant burden of hemoglobinopathies in India.
AIMS: The aim of the present study was to check the prevalence of various hemoglobinopathies in anemic subjects using high-performance liquid chromatography (HPLC) method in Pune region which has multiple ethnic population groups from all parts of India.
SETTINGS AND DESIGN: The present study was conducted at the department of IH and BT on anemic patients referred from different outpatient department and Wards of the hospital and informed consent were taken from all participants.
SUBJECTS AND METHODS: The present study included a total of 2698 individuals' age ranging from 1.5 to 67 years. The HPLC test was performed using Bio-Rad D-10 analyzer once a week.
RESULTS: Out of a total of 2698 cases, we found 543 (20.12%) cases with abnormal hemoglobin fractions and 2155 (79.88%) cases free from hemoglobinopathies. Out of the total hemoglobinopathies detected 250 (46%) were male and 293 (54%) were female. The major abnormality detected was beta-thalassemia trait (BTT) with 425 (15.75%) cases, followed by sickle cell disorders 58 (2.15%), HbE 38 (1.41%), hereditary persistence of fetal hemoglobin 6 (0.22%), HbD Punjab 13 (0.48%), HbD Iran 2 cases and 4 cases of compound heterozygous for HbS beta-thalassemia. Forty (1.48%) cases were detected as borderline with HbA2 level ranges from 3.6% to 3.9%.
CONCLUSIONS: In our study, we found a high prevalence of hemoglobinopathies among anemic subjects. The most common disorder detected was BTT. Most of the hemoglobinopathies found in our study could be accurately quantified by HPLC which is a rapid, sensitive, and reproducible method for the detection of different hemoglobinopathies.

Keywords: Genetic disorder, hemoglobinopathies, high-performance liquid chromatography, sickle cell anemia, thalassemia


How to cite this URL:
Singh V, Biswas AK, Baranwal AK, Asthana B, Dahiya T. Prevalence of hemoglobinopathies using high-performance liquid chromatography as diagnostic tool in anemic patients of tertiary care center of Western India. Asian J Transfus Sci [Epub ahead of print] [cited 2022 Dec 4]. Available from: https://www.ajts.org/preprintarticle.asp?id=356893



   Introduction Top


All the genetic disorders of hemoglobin are termed hemoglobinopathies and are the most common inherited single-gene red cell disorders globally. It can result due to genetic mutations such as insertion, deletion, or substitution in the amino acid sequence in either of the α- or non-α globin genes.[1] About 7% of the world's population carries a clinically significant hemoglobinopathy with more than 270 million carriers globally.[2] It contributes to nearly 3.4% of mortality in children up to 5 years of age worldwide. It is detected in nearly 7% of pregnancies and approximately 1% of couples are at risk for this condition.[3] About 3–5 lakhs newborn with serious hemoglobinopathies are born every year, of which nearly 90% are born in middle- or low-income countries.[4]

India being a developing country and located in the thalassemia belt of the world (Dolai et al., 2012) has multiple geographical, ethnic, religious, and language regions with different beta-thalassemia trait (BTT) frequencies ranging from 0% to 17% (Kumar and Devisri, 2019).[5],[6] In India, the prevalence of significant hemoglobinopathies is 1.2/1000 live births, with an annual birth of 32,400 babies with a hemoglobin disorder.[7] India constitutes 10% of the total thalassemia burden in the world with about 10,000 children being born with thalassemia major each year. HbS is the most common hemoglobin variant found with an average frequency of 4.3%.[3] HbE is the second-most common hemoglobin variant found in the eastern half of the Indian sub-continent and Southeast Asia with a carrier frequency of 50%–60%.[4],[8]

The clinical manifestations of these different types of hemoglobin disorders can vary from asymptomatic to severe transfusion-dependent anemia with other complications, hence, an early identification and precise diagnosis of these disorders is necessary to prevent the incidence of severe hemoglobinopathies and lessen the economic and psychological burden related to a womb-to-tomb disorder.[3] The present study aimed to observe the prevalence of the various hemoglobinopathies in the Pune region, of Western Maharashtra so that an early intervention strategy can be formulated for its identification, diagnosis, and prevention of more severe hemoglobinopathies in future generations. We also analyzed the efficacy of high-performance liquid chromatography method in detecting these various hemoglobin disorders because most of the center in India now use high-performance liquid chromatography (HPLC) analysis for carrier detection for different hemoglobinopathies.[8]


   Subjects and Methods Top


The present study was carried out in Pune region of Maharashtra situated in Western India from October 2020 to March 2022. A total of 2698 study population ranging from 1.5 to 67 years of age were included in this study, Of which 1122 (41.5%) were male and 1576 (58.4%) were female. The patients with abnormal complete blood count (CBC) results such as microcytic hypochromic anemia, hemoglobin <12 g/dl, and a significant family history were included and the patients with transfusion history during the past 3 months were excluded from the study. Consent was taken from all study participants and ethical clearance from our institutional ethical committee.

Procedure

Three milliliters of whole blood samples from all the study participants were collected in ethylenediaminetetraacetic acid (EDTA) (Becton Dickinson) vacutainer. CBC with PBS was performed on the same day for patients who did not have a CBC report and then the samples were refrigerated at 2°C–8°C for HPLC analysis. The HPLC test was performed within a week. All the samples were performed using HPLC in Bio-Rad D-10 analyzer (Bio-Rad Laboratories, California, USA) [Figure 1]. The accuracy of the procedure was confirmed by analyzing two levels of A2 controls and calibrators which run with every batch of patient samples. The instrument runs the samples only after passing the calibrator and control. If the control or calibrators are not valid, then the instrument automatically stops the sample for processing and the controls are required to be rerun. The inaccuracy of HPLC method is shown in [Table 1].
Figure 1: Bio-Rad D-10 analyzer for HPLC analysis. HPLC: high-performance liquid chromatography

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Table 1: Inaccuracy of high-performance liquid chromatography method in Bio-Rad D-10 dual program analyzer

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Reagents

The dual Kit (D-10recorder pack) for HPLC analysis carries a blood primer, two calibrators (1and2), elusion buffer 1 and 2 with different ionic strength, wash reagent which acts as the mobile phase, sample vial used to make the hemolysate, and a cartridge which are negatively charged and acts as the stationary phase.

Principle or methodology of test

The Bio-Rad D-10 is a fully automated analyzer which performs a test from a primary whole blood tube (EDTA), followed by sample dilution. A total test run time of 6 min is required for analysis. The samples are automatically diluted with wash diluents provided in the pack and this act as the mobile phase which is then injected into the stationary phase (i.e., the negatively charged cartridge) where the different hemoglobin fragments interact based on their respective charges. Then, the instrument pushes the elution buffer of increasing ionic strength into the cartridge where the hemoglobin fragments separate based on their ionic interactions with negatively charged the stationary phase. These hemoglobin fractions were separated based on their retention time in the stationary phase. The retention time with assigned windows was provided by the manufacturer. The amount of separated hemoglobin fraction is then measured photometrically at 415 nm wavelength. Then, the instrument generates a chromatogram based on the amount of hemoglobin eluted in each peak with its respective retention time.[3]

A sickling test was performed using freshly prepared sodium meta-bisulfate solution to rule out all cases of sickle cell hemoglobin. One percent sodium meta-bisulfate solution is freshly prepared by mixing 0.2 g of sodium metabisulfite with 10 ml of distal water which acts as a reducing agent. One drop or about 50 ul of this 1% sodium meta-bisulfate solution on a clean glass slide is mixed with approximately 3–5 ul of whole blood and a glass cover slip is placed over it.[9] The cover slip is sealed with Dibutylphthalate Polystyrene Xylene (DPX) or vaseline to prevent the air influx and incubated at room temperature in a wet petri dish. The slides were observed under the microscope at 40× after 4, 8, 12, and 24 h for sickling. Sodium meta-bisulfate solution used in the sickling test reduces the oxygen content very quickly, which results in sickling of RBC as shown in [Figure 2].
Figure 2: Sickling test showing the sickle cells

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   Results Top


All the samples were analyzed using Bio-Rad D-10 HPLC analyzer. Various types of the hemoglobin abnormalities were detected on the basis of their retention time and area percentage by HPLC. The total acceptable area was 1–5 million. Values outside this range should not be reported and samples in case it was above the higher limit should be manually prediluted and rerun. The retention time with windows assigned to various Hb fractions provided by the manufacturer is shown in [Table 2].
Table 2: Various windows and retention time assigned by the manufacturer for Bio-Rad D-10 analyzer

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A total of 2698 cases were studied out of whom, 543 (20.12%) cases had an abnormal hemoglobin fraction while 2155 (79.88%) cases should normal Hb fraction on HPLC. Out of 543 patients with abnormal Hb fraction, 250 (46%) were male and 293 (54%) were female. Among the total hemoglobinopathies noted, βTT was the major abnormality with 425 (15.75%) cases, followed by sickle cell disorders (58 cases) which were further classified into sickle cell disease 22 (0.82%) and sickle cell trait 36 (1.33%). Other abnormal hemoglobins found included HbE homozygous in 18 cases (0.67%), HbE heterozygous in 20 cases (0.74%), HbD Punjab in 13 cases (0.48%), hereditary persistence of fetal hemoglobin (HPFH) in 6 cases (0.22%), and HbD Iran in 2 cases. Four cases of compound heterozygote for HbS-beta-thalassemia and 4 cases of HbS with borderline A2 (3.5%–3.9%) were also found. The HbA2 level ranging from 3.6% to 3.9% were considered as borderline and included 40 (1.48%) cases. The distribution of these various hemoglobinopathies is shown in [Table 3]. Hb fractions with their retention times and mean ± standard deviation in various hemoglobinopathies are shown in [Table 4].
Table 3: Different types of hemoglobin pattern among the study population (n=2698) with age and sex

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Table 4: Haemoglobin fractions on high performance liquid chromatography Bio-Rad D-10 analyzer with retention times and mean±standard deviation percentage in various hemoglobinopathies

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The total βTT detected was 425 cases, out of which 230 (54.11%) cases were from Maharashtra, 53 (12.47) from Uttar Pradesh, 37 (8.70%) from Bihar, 25 (5.66%) from West Bengal, and remaining 79 (18.55%) cases from rest of the states. In the case of sickle cell hemoglobinopathies, 24 (43.10%) cases were from Maharashtra, 6 (10.34%) from Jharkhand, 4 (6.89%) from Assam, 3 (5.17%) cases each from Orissa, Manipur, Andhra Pradesh, and remaining cases from rest of the states. In the case of HbE, majority of cases were from Northeast region of the country, i.e., West Bengal 12 cases (31.6%), Assam 9 cases (23.7%), Bihar 5 cases (13.1%), and Manipur 4 cases (10.5%). The geographical and state-wise distribution of different hemoglobinopathies is shown in [Figure 3] and [Figure 4], respectively.
Figure 3: Regional distribution of various hemoglobinopathies among the study population

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Figure 4: States-wise distribution of various hemoglobinopathies among study population

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The major abnormality detected in our study was βTT which was diagnosed by HbA2 value. The retention time for HbA2 ranges between 3.2 min and 3.9 min and a cut-off value of >3.9% was considered for a diagnosis of βTT. A sickling test was done on all HbS-positive cases for confirmation of sickle cell disorder, to exclude any other hemoglobin variants with a similar retention time that may be eluted along with HbS. The HPFH cases were advised further molecular analysis for confirmation and further typifying them into deletional and nondeletional categories.


   Discussion Top


Hemoglobinopathies are one of the major public health problems in the world including in India, which possesses a large burden on the health and financial resources of the country affected. As hemoglobinopathies should be a geographical variation, it is important to know about the actual prevalence of this lifelong disorder by conducting various regional studies. The early precise and accurate diagnosis of these conditions is also very important because it prevents the occurrence of clinically sense hemoglobinopathies and thus lessens the economic and psychological burden of this lifelong health problem.[3] Therefore, we conducted this study to determine the prevalence of various hemoglobinopathies in a city of Western Maharashtra, located in western India. The study was carried out in Pune region which not only represents the Western Indian population but also has a large migrant population from eastern and northern parts of the country.

Most of the centers in India now use HPLC as a standard method for diagnosing various hemoglobin disorders.[8] This is because, the conventional technique like alkaline and acid electrophoresis for hemoglobin variants, HbA2 quantification by ion-exchange column chromatography, and HbF quantification by alkali denaturation or radial immunodiffusion for thalassemia are time-consuming and labor-intensive and at a times are unable to detect many Hb variants having similar electrophoretic mobility. The conventional methods are further incapable of detecting multiple Hb fractions in a single-step procedure. Thus, the identification of Hb variants by these conventional techniques is often speculated.

Cation exchange HPLC is accepted as the gold standard for screening of various hemoglobinopathies because it accurately quantifies most of the commonly found Hb variants except some rare variants which get co-eluted with other normal and Hb variants having similar retention times. In such cases, further molecular studies are required for their definitive diagnosis. HPLC has the advantage of quantifying HbF and HbA2 along with detecting other variants such as HbS, HbD, and HbE in a single-screening test. It is very sensitive, reproducible, rapid, and easy to perform. The major limitation of HPLC method is that it requires a skilled and qualified person for the interpretation of results and some Hb variants having the same retention time, get co-eluted in one common window.[10]

In our study, we used Bio-Rad D-10 fully automated analyzer for HPLC analysis of various hemoglobinopathies. It detected most of the commonly found hemoglobinopathies very accurately, precisely, and rapidly as compared to the conventional techniques. Thus, the CE-HPLC can be used routinely for screening of various hemoglobinopathies, especially in high throughput laboratories.

In the present study, we found 543 (20.12%) cases out of 2698 cases with various hemoglobin disorders with occurrence in female preponderance as compared to male (54% vs. 46%). This was in conflict from other studies which were conducted in this geographical area. The prevalence of various hemoglobinopathies found in our study was higher than the study conducted by Philip et al. and Gupta et al. in which they reported 15.8% and 14.3% prevalence in anemic patient's in Pune region of Maharashtra, respectively.[3],[11] The study conducted by Pant et al., in Delhi and Mondal and Mandal, in Bengal, in which they reported 6.04% and 12.17% incidents of abnormal hemoglobin, respectively, which was also lower than our study.[10],[12] Balgir reported 65.7% prevalence of hemoglobinopathies in Orissa which was higher than our study.[13]

The major abnormality found in most of the studies was βTT. We found 385 (14.27%) patients with βTT, which was higher than the prevalence of 10.49% reported by Philip et al. 9.53% reported by Gupta et al. 8.03% reported by Biswas and Philip in the state of Maharashtra and 4.60% reported by Mondal and Mandal in rural areas of Bengal.[3],[11],[12],[14] However, in view of higher prevalence of βTT in our study and also in some of the previous studies, there is a need to set a program for premarital and antenatal screening to prevent thalassemia major in the offspring.

In our study, the average HbA2 in βTT was 5.21% which was higher than 4.8% reported by Philip et al. and lower than 5.4% reported by Chandrashekar and Soni, in South Indian population.[3],[15] The borderline HbA2 cases were carefully interpreted and checked for iron or folate/B12 deficiency because iron deficiency may lead to a low A2 level which may mask the βTT whereas B12 deficiency may lead to slightly increased in HbA2 level which may result in false diagnosis of βTT. Therefore, these types of cases should be correlated with serum iron studies.[11]

HbS is the most common hemoglobin variant found in the world. HbS was the second-most common abnormal Hb detected in our study after βTT with occurrence of 2.14% which was higher than the study conducted by Philip et al. (1.35%) and Biswas and Philip (1.23%) in Pune region and Mondal and Mandal (0.98%) in rural areas of Bengal and was lower than a study conducted by Balgir (9.13%).[3],[12],[13],[14] Of the total HbS cases, 36 (1.33%) were detected as HbS heterozygous (AS) as compared to 22 (0.82%) cases of HbS homozygous (SS). The mean abnormal hemoglobin S detected was 61.8 ± 7.14% in case of HbS homozygous and 31.14 ± 5.23% in the case of HbS heterozygous. The mean HbF detected was >16.5% in HbS homozygous and 1.39 ± 1.0% in HbS heterozygous. Compound homozygous for HbS + BTT constitutes of 5 (0.18%) cases. The mean HbS and HbF levels were 68.57 ± 6.59% and 17.7 ± 5.13%, respectively, in the case of compound homozygous cases. HbS is a beta chain variant that occurs due to the substitution of a single polar amino acid residue to nonpolar one (i.e., glutamate to valine) at 6th position of beta-globin chain. The HbS containing RBC change their shape from oval to sickle in low oxygen tension.[16]

HbE was the third most common abnormality after sickle cell disorders with a prevalence of 1.44% in our study. It was lower than 3.36% found by Mondal and Mandal and slightly higher than 1.31% reported by Philip et al. and 1.07% by Philip et al. in Pune region.[3],[10],[11] HbE is an asymptomatic condition except it present in compound heterozygous state with beta-thalassemia which has more moderate to severe symptoms. It is a beta chain variant caused by the substitution of glutamate residue by lysine residue at 26 positions of beta-globin chain. It usually elutes in A2 window when run on HPLC.[17] The mean HbA2 level was 82.5 ± 14.85% in HbE homozygous and 27.9 ± 4.93% in the case of HbE heterozygous.

HbD Punjab is another beta chain Hb variant with relatively low frequency with the highest prevalence of 2%–3% in Punjab, 1% in Gujarat.[3] We found 0.48% prevalence of HbD in our study which was lower than 1.09% in Ludhiana and higher than the prevalence of 0.19% in Bengaluru, 0.20% in Kolkata, 0.21% in Mumbai, and 0.34% in Vadodara (Mohanty et al.) Philip et al. in Pune reported 0.21%, and Mondal & Mandal in Bengal reported 0.09 prevalence which was also lower than the present study.[3],[12],[18] HbD is also a beta chain variant caused due to single amino acid substitution at position 121 of beta chain in which glutamic acid residue is replaced by a glutamine residue. In India, HbD is more frequently observed in Punjab and is known as HbD Punjab.[19] HbD Iran is another beta chain variant caused by single amino acid substitution in which glutamate is replaced by glutamine residue at position 22 of beta-globin chain.[20] We detected 2 cases of HbD Iran in our study. The HbD Iran gets eluted in A2 window whereas HbD Punjab eluted in D window on CE-HPLC. Therefore, HPLC is accurately distinguishes HbD Punjab from HbD Iran.


   Conclusions Top


We conclude that HPLC can accurately quantify most of the hemoglobinopathies or Hb variant found in our study and also it is rapid, sensitive, and reproducible methods for the detection of most of the hemoglobinopathies which are commonly found in India. Therefore, it is used as an ideal alternative to the conventional techniques for routine clinical laboratories with heavy workload for screening of various hemoglobinopathies along with CBC and iron studies. As there is a high prevalence of various hemoglobinopathies among anemic subjects which suggests that there is a need to set a screening program of anemic individual through premarital and antenatal screening by the collaboration of central or state governments, nongovernment organizations, government, and nongovernment hospitals to prevent the more severe form of hemoglobinopathies in the offsprings which can reduce the economic and psychological burden of patients and their families.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

1.
Higgins T, Schnabl K, Savoy M, Rowe P, Flamini M, Bananda S. A novel double heterozygous, HbD Punjab/HbQ India, hemoglobinopathy. Clin Biochem 2012;45:264-6.  Back to cited text no. 1
    
2.
Hoppe CC. Prenatal and newborn screening for hemoglobinopathies. Int J Lab Hematol 2013;35:297-305.  Back to cited text no. 2
    
3.
Philip J, Sarkar RS, Kushwaha N. Microcytic hypochromic anemia: Should high performance liquid chromatography be used routinely for screening anemic and antenatal patients? Indian J Pathol Microbiol 2013;56:109-13.  Back to cited text no. 3
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4.
Weatherall D, Akinyanju O, Fucharoen S, Olivieri N, Musgrove P, Jamison DT, et al. Inherited disorders of hemoglobin. In: Disease Control Priorities in Developing Countries. 2nd ed. Washington (DC): The International Bank for Reconstruction and Development/The World Bank; 2006.  Back to cited text no. 4
    
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Dolai TK, Dutta S, Bhattacharyya M, Ghosh MK. Prevalence of hemoglobinopathies in rural Bengal, India. Hemoglobin 2012;36:57-63.  Back to cited text no. 5
    
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Kumar M, Devisri Y. Detection of hemoglobinopathies in patients of anaemia using high performance liquid chromatography (HPLC) – A hospital based prospective study. Trop J Pathol Microbiol 2019;5:51-7.  Back to cited text no. 6
    
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Mathew A, Sobti PC. The burden of thalassemia in Punjab: A roadmap forward. Pediatr Hematol Oncol J 2017;2:85-7.  Back to cited text no. 7
    
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Colah R, Italian K, Gorakshakar A. Burden of thalassemia in India: The road map for control. Paediatr Hematol Oncol J 2017;2:79-84.  Back to cited text no. 8
    
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Old J, Harteveld CL, Traeger-Synodinos J, Petrou M, Angastiniotis, Galanello R, et al. Prevention of Thalassaemias and Other Haemoglobin Disorders: Laboratory Protocols. 2nd ed., Vol. 2, Ch. 2. Nicosia (Cyprus): Thalassaemia International Federation; 2012.  Back to cited text no. 9
    
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Pant L, Kalita D, Singh S, Kudesia M, Mendiratta S, Mittal M, et al. Detection of abnormal hemoglobin variants by HPLC method: Common problems with suggested solutions. Int Sch Res Notices 2014;2014:257805.  Back to cited text no. 10
    
11.
Gupta PK, Kumar H, Kumar S, Jaiprakash M. Cation exchange high performance liquid chromatography for diagnosis of haemoglobinopathies. Med J Armed Forces India 2009;65:33-7.  Back to cited text no. 11
    
12.
Mondal SK, Mandal S. Prevalence of thalassemia and hemoglobinopathy in eastern India: A 10-year high-performance liquid chromatography study of 119,336 cases. Asian J Transfus Sci 2016;10:105-10.  Back to cited text no. 12
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13.
Balgir RS. Prevalence of hemolytic anemia and hemoglobinopathies among the pregnant women attending a tertiary hospital in Central India. Thalassemia Rep 2015;5:16-20.  Back to cited text no. 13
    
14.
Biswas AK, Philip J. Incidence of hemoglobinopathies and haemoglobin variants using HPLC as a diagnostic tool in 6500 anemic patients in a tertiary care center in Western India. Indian J Appl Res 2016;6:214-8.  Back to cited text no. 14
    
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Chandrashekar V, Soni M. Hemoglobin disorders in South India. ISRN Hematol 2011;2011:748939.  Back to cited text no. 15
    
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Bankar MP. Study of Sickle Cell Hemoglobin. [Doctoral thesis, B. J. Medical College Pune]; 1992. Available from: http://hdl.handle.net/10603/151976. [Last accessed on 2022 Apr 03].  Back to cited text no. 16
    
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Bain BJ, Wild B, Stephens A, Phelan L. Variant Haemoglobins: A Guide to Identification. Hoboken, new Jersey: Wiley-Blackwell; 2011.  Back to cited text no. 17
    
18.
Mohanty D, Colah RB, Gorakshakar AC, Patel RZ, Master DC, Mahanta J, et al. Prevalence of β-thalassemia and other haemoglobinopathies in six cities in India: A multicentre study. J Community Genet 2013;4:33-42.  Back to cited text no. 18
    
19.
Madan N, Sharma S, Sood SK, Colah R, Bhatia LH. Frequency of β-thalassemia trait and other hemoglobinopathies in Northern and Western India. Indian J Hum Genet 2010;16:16-25.  Back to cited text no. 19
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Shanthala Devi AM, Rameshkumar K, Sitalakshmi S. Hb D: A not so rare hemoglobinopathy. Indian J Hematol Blood Transfus 2016;32:294-8.  Back to cited text no. 20
    

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Correspondence Address:
Virender Singh,
Department of Immunohematology and Blood Transfusion, Armed Forces Medical College, Pune - 411 040, Maharashtra
India
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ajts.ajts_62_22



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