|Year : 2014 | Volume
| Issue : 3 | Page : 91-95
Spectrum of thalassemias and hemoglobinopathies in West Bengal: A study of 90,210 cases by cation exchange high-performance liquid chromatography method over a period of 8 years
Santosh Kumar Mondal1, Senjuti Dasgupta2, Saikat Mondal2, Nikhilesh Das3
1 Department of Pathology, BS Medical College, Bankura, India
2 Department of Pathology, Medical College, Kolkata, West Bengal, India
3 Serum Analysis Center, Shyambazar, Kolkata, West Bengal, India
|Date of Web Publication||30-Sep-2014|
Santosh Kumar Mondal
Teenkanya Complex, Flat 1B, Block B, 204 R N Guha Road, Dumdum, Kolkata - 700 028, West Bengal
Source of Support: None, Conflict of Interest: None
Background: Thalassemias and hemoglobinopathies are highly prevalent in India. Identification of these disorders is important for epidemiologic purposes and for prevention of thalassemia major and clinically severe hemoglobinopathies. Objectives: The aim of this study was to determine the prevalence of thalassemias and hemoglobinopathies in patients of a tertiary care hospital of West Bengal. Materials and Methods: A prospective study was undertaken in which 90,210 cases were included over a period of 8 years. Clinical history and family history were obtained from each patient. The venous blood samples were analyzed for complete blood count, liver function tests, serum iron, ferritin, cobalamin and folate levels. High-performance liquid chromatography (HPLC) was performed on the samples with Biorad Variant using beta thalassemia short program. Confirmatory tests were done whenever required. Results: Normal hemoglobin (Hb) pattern was observed in 79,897 (88.57%) cases and abnormalities were detected in 10,313 (11.43%) patients. β (beta) thalassemia trait was the most common abnormality found in 3870 (4.29%) patients. HbE trait was found in 2418 (2.68%) cases, and then Eβ thalassemia in 1406 (1.56%) patients and β thalassemia major/intermedia in 1135 (1.26%) cases. Other variants detected included sickle cell trait, HbE disease, sickle cell disease, sickle β thalassemia, HbD-Punjab trait, double heterozygous state of HbS and HbE, double heterozygous state of HbS and HbD, Hb Lepore, HbJ-Meerut and HbH. Conclusion: Premarital and antenatal screenings are important measures to prevent birth of children with severe Hb disorders. HPLC is a rapid and reliable technique for identification of various Hb fractions.
Keywords: Hemoglobinopathy, high performance liquid chromatography, prevalence, thalassemia
|How to cite this article:|
Mondal SK, Dasgupta S, Mondal S, Das N. Spectrum of thalassemias and hemoglobinopathies in West Bengal: A study of 90,210 cases by cation exchange high-performance liquid chromatography method over a period of 8 years. J Appl Hematol 2014;5:91-5
|How to cite this URL:|
Mondal SK, Dasgupta S, Mondal S, Das N. Spectrum of thalassemias and hemoglobinopathies in West Bengal: A study of 90,210 cases by cation exchange high-performance liquid chromatography method over a period of 8 years. J Appl Hematol [serial online] 2014 [cited 2018 Jan 16];5:91-5. Available from: http://www.jahjournal.org/text.asp?2014/5/3/91/141993
| Introduction|| |
Thalassemias constitute the commonest single gene disorder known in humans. They are characterized by reduced synthesis of one or more globin chains of the hemoglobin (Hb) molecule. On the other hand, hemoglobinopathies occur due to production of normal amounts of mutant globin chains. The prevalence of thalassemias and hemoglobinopathies varies with geographic locations. It has been estimated that in India, 0.37/1000 fetuses have a Hb disorder. 
The disorders of Hb frequently encountered in India include beta thalassemia, HbE - beta thalassemia, HbE, HbD and sickle cell anemia. The prevalence of beta thalassemia mutations is as high as 17% in some Indian populations. The prevalence of HbD in our country is estimated to be approximately 1.1%.  Worldwide, the Hb disorders are responsible for 3.4% mortality in children below 5 years of age. 
High performance liquid chromatography (HPLC) is a simple and rapid method of detection of different Hb variants. Clinical history and findings of thorough hematologic evaluation, including complete blood count, reticulocyte count and red blood cell morphology are necessary to reach an accurate diagnosis. In some cases, family studies are also required to detect a particular Hb variant. 
The aim of the present study was to determine the common Hb disorders in patients of a tertiary care hospital of West Bengal. The knowledge of the common Hb variants encountered in a particular area is important for the formulation of specific diagnostic, preventive and therapeutic strategies.
| Materials and methods|| |
This study was conducted in the department of Pathology of a tertiary care teaching medical institution of West Bengal over a period of 8 years from June 2006 to May 2014. Patients who opted for premarital or antenatal Hb analysis were included in the study. Transfusion dependent children and adults were also included. Others included were cases of microcytic hypochromic anemia when a coexistent hemoglobinopathy was suspected on the basis of red cell indices. However, patients with a history of blood transfusion within the last 1 month were excluded.
A signed consent form was obtained from all the patients included in the study. A detailed clinical history and family history were obtained from each patient. History of blood transfusion, if present, was noted.
Blood samples were collected in ethylene diamine tetrachloride acetate vials and analyzed with Sysmex automated cell counter for complete blood counts. Clotted blood samples were also collected for performance of liver function tests and determination of serum iron, ferritin, cobalamin and folate levels. For each patient, a peripheral blood smear (PBS) was prepared and stained with Leishman stain.
High performance liquid chromatography was performed with each blood sample on Biorad Variant using beta thalassemia short program (Biorad laboratories, California, USA). HPLC is based on exchange of charged groups on an ion exchange material for charged groups on Hb molecule. Hbs are identified on the basis of retention time that is defined as the time in minutes from sample injection to the maximum point of the elution peak. Quantification of the Hbs is done by determining the area under the corresponding peak in the elution profile. Retention times are used to define the manufacturer assigned windows of chromatogram. 
Confirmatory tests were done whenever required. These included acid and alkaline electrophoresis, sickling test, solubility test and brilliant cresyl blue test for HbH inclusions.
| Results|| |
During the period of 8 years, a total of 90,210 patients were included in the study. Among them, 65,492 (75.6%) patients were females and 24,718 (27.4%) were males. The ratio of females to males was 2.65:1. Female patients were found to outnumber males because routine antenatal check-ups included HPLC analysis of blood of pregnant women. The age of the patients ranged between 7 months and 69 years. The mean age was found to be 27.3 years.
Normal Hb pattern was found in 79,897 (88.57%) cases [Figure 1]. Disorders of Hb were noted in 10,313 (11.43%) patients. The most common Hb abnormality detected was β (beta) thalassemia trait, present in 3870 (4.29%) patients. HbE trait was found in 2418 (2.68%) cases, followed by Eβ thalassemia in 1406 (1.56%) patients. The distribution of different Hb patterns in the study population has been shown in [Table 1]. For each of these groups, the hematologic parameters and the percentage of various Hbs detected in HPLC have been shown in [Table 2].
|Table 2: Hematologic parameters of the patients stratified according to the hemoglobin patterns|
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Interpretation of results of HPLC was done on the basis of retention time, percentage of Hb and peak characteristics.
Twelve (0.01%) cases of HbH were detected, which were suspected on the basis of a significant peak that appeared in each case during the 1 st min of elution. Confirmatory tests were done, which included Hb electrophoresis and test for HbH inclusion bodies.
In this study, no Hb variants were detected in the p1 window (retention time - 0.63-0.85 min). High Hb level in the F window (retention time - 0.98-1.2 min) was detected in cases of β thalassemia major (1.26%), Eβ thalassemia (1.56%), sickle-β thalassemia (0.21%) and sickle cell disease (0.38%).
In 213 (0.24%) patients, significant peak was obtained in the p2 window (retention time - 1.24-1.4 min). Since haemoglobin A1c (HbA1c) is known to elute in this window, these patients were later tested for blood sugar levels. In 192 (90.1%) cases, the patients were found to be diabetics. However, the other 21 (9.9%) cases were lost to follow-up.
Significant peak in the p3 window (retention time - 1.4-1.9 min) was found in 19 (0.02%) cases. Hb electrophoresis at alkaline pH in these cases showed a fast moving band anodal to HbA. These cases were diagnosed as HbJ-Meerut.
A 0 window has a retention time between 1.9 and 3.1 min. Apart from HbA, no other Hb variant was found to elute in this window. HbA 2, HbE and Hb Lepore were found to elute in A 2 window (retention time - 3.3-3.9 min). HbE trait was detected in 2.68% cases, Eβ thalassemia in 1.56% cases and HbE disease in 0.39% patients. Hb Lepore was noted in 4 (0.004%) patients [Figure 1].
HbD-Punjab was found to elute in the D window (retention time - 3.9-4.3 min). In this study, 141 (0.16%) cases of HbD-Punjab trait were reported. A statistically significant difference (P < 0.0001) was found between the mean values of HbA 2 in normal samples (2.7 ± 0.4) and that in HbD-Punjab trait (1.8 ± 0.5).
In the S window (retention time - 4.3-4.7 min), HbS was found to elute. Sickle cell trait was found in 415 (0.46%) cases and sickle cell disease was noted in 343 (0.38%) patients. Sickling test and solubility test were done to confirm the cases of sickle cell disease. The mean value of HbA 2 in sickle cell trait (3.2 ± 0.3) was found to be significantly higher (P < 0.0001) compared to that in normal samples (2.7 ± 0.4). No Hb variant was detected in the C window (retention time - 4.9-5.3 min).
| Discussion|| |
The wide prevalence of thalassemias and hemoglobinopathies has been attributed to migration of people from one region to another and marriages between different communities.  With increasing awareness, detection of these disorders in countries like India, Iran, Turkey and Cyprus mostly occurs during premarital screening. In western European countries detection usually occurs through preconceptional and neonatal screening programs. 
In the present study, the prevalence of Hb disorders in a cross-sectional population of West Bengal was found to be 11.43%. A study conducted in the southern part of West Bengal however reported a higher prevalence (25%) of thalassemias and hemoglobinopathies.  In the north Indian population, incidence of hemoglobinopathies was found to be 12.5%.  The prevalence rate of Hb disorders was reported to be 7% in Bhopal. 
The most common Hb abnormality detected in this study was that of β thalassemia trait (4.29%). Colah et al. reported that nearly 1.5% of the world's population is carriers of β thalassemia.  In rural Bengal, the prevalence of β thalassemia trait has been reported to be as high as 10.38%.  The overall gene frequency of β thalassemia trait reported in northern, and Western India was 4.05%.  In central India, the prevalence of β thalassemia trait has been estimated to be 9.59%.  These data reveal that in most parts of India, β thalassemia trait is the commonest Hb disorder. Interestingly, in Orissa, sickle cell trait was the most common abnormality found.  In the present study, sickle cell trait was found in 0.46% cases.
In this study, HbE trait was found in 2.68% cases and Eβ thalassemia in 1.56% patients. A study conducted in the rural areas of West Bengal reported the prevalence of HbE trait to be 3.86% and that of Eβ thalassemia, 1.25%.  Due to the high prevalence of Hb disorders in various regions of West Bengal premarital screening must be routinely done for prevention of high-risk marriages. 
Other variants detected in the present study included sickle cell trait, HbE disease, sickle cell disease, sickle β thalassemia, HbD-Punjab trait, double heterozygous state of HbS and HbE, double heterozygous state of HbS and HbD, Hb Lepore, HbJ-Meerut and HbH. All the variants identified eluted in known manufacturer assigned windows. Rao et al. reported 3 cases of HbQ-India, which eluted in an unknown window (retention time - 4.7-4.9 min. 
Some rare Hb variants have been reported to elute in the manufacturer assigned windows. Hb hope is found to elute in the p2 window, but its quantity has been found to be much greater than that expected in case of HbA1c. Apart from HbJ-Meerut, 8 variants including HbJ-Oxford and Hb Camden have elution peaks in the p3 window. Hb Koln, an unstable Hb, has been reported to have a characteristic peak in the A 0 window. HbD-Iran, commonly encountered in North-Western India, elutes in the A 2 window, whereas HbQ-Thailand elutes in the S window.  HbD-Agri, an abnormal Hb, was reported from India and it has an elution peak in the S window. This Hb is distinguished from HbS on the basis of negative sickling and solubility tests and molecular tests like polymerase chain reaction (PCR). 
High performance liquid chromatography has been established as a sensitive, specific and accurate technique for the identification and quantification of different Hb fractions. But it has always been emphasized that interpretation of chromatograms must be done only after taking into consideration the clinical history, family history, complete blood count and findings of PBS. Additional tests to confirm the diagnosis must be undertaken whenever necessary.  In this study a similar step-wise approach was followed for each case.
A review of the literature revealed that the present study, in which analysis of 90,210 blood samples was undertaken, is the largest series being reported from India, so far. To the best of our knowledge, this is also one of the largest series being reported in the world till date. Previously Joutovsky et al. had analyzed 60,293 blood samples in a similar study. 
During the interpretation of chromatograms, nutritional anemia must always be taken into account. A low level of HbA 2 may be induced by iron deficiency thus masking β thalassemia trait. Similarly, cobalamin or folate deficiency may raise HbA 2 level, leading to a false diagnosis of thalassemia trait.  However, in a previous study, no significant difference was found in HbA 2 level in patients of β thalassemia trait with and without concomitant iron deficiency anemia. 
High performance liquid chromatography is limited by its inability to detect α thalassemias and normal HbA 2 β thalassemia. Hb variants that elute with same retention time also cannot be separately identified by HPLC. 
Ideally, HPLC must be used as a screening tool, followed by molecular studies, like PCR, amplification refractory mutation system and other similar tests to determine specific mutations responsible for the Hb disorder. In cases of hemoglobinopathies, beta thalassemia mutations, when present, significantly modify the phenotype that is why molecular studies have been considered gold standard for the diagnosis of hemoglobinopathies. ,
The importance of screening programs for Hb disorders in countries with high prevalence cannot be overemphasized. In India, where β thalassemia trait is so rampant, premarital and antenatal screening should be mandatory to prevent birth of offsprings with β thalassemia major. Moreover, knowledge of common Hb patterns in a particular region helps to formulate appropriate preventive and therapeutic strategies. HPLC is a rapid and reproducible technique for determination of different Hb variants. However, the chromatograms must be interpreted only in the light of other relevant investigations.
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[Table 1], [Table 2]
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