|Year : 2016 | Volume
| Issue : 2 | Page : 111-115
Patterns of hemoglobinopathies diagnosed by high-performance liquid chromatography in and around Pune (Western Maharashtra, India): A pilot study
Archana Buch1, Banyameen Iqbal1, Rajiv Bordawekar2, Ambuj Jain1, Pratik Jariwala1, Hetal Rathod3
1 Department of Pathology, Dr. D. Y. Patil Medical College, Hospital and Research Centre, Pune, Maharashtra, India
2 Department of Laboratory Medicine, Millenium Speciality Laboratory, Pune, Maharashtra, India
3 Department of Preventive and Social Medicine, Dr. D. Y. Patil Medical College, Hospital and Research Centre, Pune, Maharashtra, India
|Date of Web Publication||24-May-2016|
Department of Pathology, Dr. D. Y. Patil Medical College, Hospital and Research Center, Pimpri, Pune - 411 018, Maharashtra
Source of Support: None, Conflict of Interest: None
Background: Hemoglobinopathies are one of the major public health problems in the state of Maharashtra, India. Their prevalence shows regional and ethnic variations. Pune, now becoming educational and IT hub cater populations from different regions. Aim: To study the pattern of hemoglobinopathies diagnosed by high-performance liquid chromatography (HPLC) in Western Maharashtra, India. Settings and Design: This pilot study was aimed to find the prevalence of hemoglobinopathies in and around Pune (Western Maharashtra, India) and identify the change in the demographic profile Materials and Methods: A total of 3465 cases received from January 2012 to December 2014 for hemoglobin variant analysis at the referral laboratory were studied for hemoglobinopathies. Samples were received from various private hospitals and laboratories in and around Pune. The samples were run on instrument manufactured by Bio-Rad Laboratories based on the principle of HPLC. Based on retention time and proportion of hemoglobin (Hb) variants different hemoglobinopathies were diagnosed and their prevalence were analyzed. Results and Conclusions: Out of total 3465 patients screened for suspected hemoglobinopathies 175 (5.05%) were found positive for abnormal Hbs with maximum number in the age group 20-30 years. We found that a maximum number of patients was of thalassemia trait with a prevalence of 3.7%, followed by double heterozygous states of Sickle cell with beta thalassemia trait, sickle cell trait, and sickle cell disease in decreasing order. Other rare variants such as Hb E disease, Hb D disease, Hb E trait, double heterozygous Hb SD, and Hb SC were also identified. We found more heterozygous states as compared to homozygous states. Prevalence was on the lower side of the normal range. Conclusion: Continuous awareness programs, mass screening of the population especially childbearing age and school going children will help in early detection of heterozygous states. This further helps in preventing severe hemoglobinopathies and reducing the morbidity and mortality.
Keywords: Hemoglobinopathies, heterozygous states, Pune, thalassemia
|How to cite this article:|
Buch A, Iqbal B, Bordawekar R, Jain A, Jariwala P, Rathod H. Patterns of hemoglobinopathies diagnosed by high-performance liquid chromatography in and around Pune (Western Maharashtra, India): A pilot study. J Med Soc 2016;30:111-5
|How to cite this URL:|
Buch A, Iqbal B, Bordawekar R, Jain A, Jariwala P, Rathod H. Patterns of hemoglobinopathies diagnosed by high-performance liquid chromatography in and around Pune (Western Maharashtra, India): A pilot study. J Med Soc [serial online] 2016 [cited 2021 Jun 21];30:111-5. Available from: https://www.jmedsoc.org/text.asp?2016/30/2/111/182921
| Introduction|| |
Hemoglobinopathies are one of the major public health problems in the state of Maharashtra, India. Hemoglobinopathies can be either quantitative or qualitative.  WHO figures estimate that 5% of the world population is a carrier for hemoglobinopathies  and of these, thalassemia syndromes, particularly beta thalassemia major is serious and a major cause of morbidity. The frequency of β-thalassemia in India ranges from 3.5% to 15%, in general, population.  Every year 10,000 children with thalassemia major are born in India, which constitutes 10% of the total numbers in the world. 
Most of the cases of hemoglobinopathies in India are diagnosed by conventional methods, which include clinical and family history, red cell indices, complete blood counts, Hemoglobin (Hb) F estimation, sickling test, and Hb electrophoresis. These conventional methods have limitations in the identification of Hb variants with same electrophoretic mobility and diagnosing certain compound heterozygous states.  To overcome this problem, cation exchange high-performance liquid chromatography (CE-HPLC) is used to separate and estimate various normal and abnormal Hb fractions.  It offers a definitive tool for early and accurate detection of hemoglobinopathies, thereby aiding in their prevention and management. 
This pilot study was undertaken to find out the incidence of different hemoglobinopathies in and around Pune.
| Materials and methods|| |
This study analyzed 3465 blood samples received from January 2012 to December 2014 at the Millennium Speciality Laboratory, which is a referral laboratory catering samples from various laboratories and hospitals in and around Pune. Patients with anemia, generalized weakness, fever, splenomegaly, and pregnant women with suspected hemoglobinopathy on the basis of clinical and family history were included for study on Hb electrophoresis. Patients with a history of recent blood transfusion and inadequate sample were excluded from the study. Five milliliters of blood was collected in dipotassium ethylenediaminetetraacetic acid vacutainers and was run in Sysmex autoanalyzer for hemogram and red cell indices; the same was used for HPLC. The samples were run on instrument manufactured by Bio-Rad Laboratories. The instrument utilizes the principle of HPLC. An HbA2F calibrator and two levels of control were analyzed at the beginning of each run. Based on retention time and proportion of Hb variants, different hemoglobinopathies were diagnosed and their prevalence was analyzed. Manufacturer assigned windows for Bio-Rad variant HPLC system is shown in [Table 1]. Wherever required, hemolysate preparation was run on agarose gel electrophoresis at alkaline pH of 8.6 from the same sample. The presence of Hb H was confirmed by using brilliant cresyl blue test for Hb H inclusions.
|Table 1: Manufacturer assigned windows for Bio-Rad variant high-performance liquid chromatography system|
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| Results|| |
Laboratory records of total 3465 patients screened for suspected hemoglobinopathies were analyzed. One hundred and seventy-five (5.05%) were found positive for abnormal Hbs. There were 107 (61%) males and 68 (39%) females. The age group ranged from 27 days to 80 years with a maximum number of patients in the age group 20-30 years. Criteria for suspecting hemoglobinopathy in these cases included: Results of screening tests such as various discriminant functions so obtained on hematology cell counters, findings obtained from peripheral smear examination, family history, and relevant clinical signs and symptoms suggestive of hemoglobinopathy. The different hemoglobinopathies found are as shown in [Table 2]. We found that a maximum number of patients were of thalassemia trait with the prevalence of 3.7%. Beta thalassemia trait was diagnosed based on high levels of HbA2 (4-8%). These patients presented with mild anemia, low mean corpuscular volume, and low mean corpuscular hemoglobin [Figure 1]. Fetal Hb was not raised. HbA2 of 3.5-3.9% was considered as borderline. These cases were advised iron status studies and repeat HbA2 levels after iron therapy. Sickle cell trait was considered in patients with Hb A 60-75%, Hb S 25-40% and Hb F <1% [Figure 2]. They had mild anemia, normochromic normocytic with mild anisopoikilocytosis, and few target cells. Sickling test was positive. The patients with Hb S 70-90%, Hb F 10-30% and Hb A 0-10% were diagnosed as sickle cell disease [Figure 3]. We found one case of Hb D disease. This patient had mild hemolytic anemia with splenomegaly. Hb electrophoresis revealed 90-95% Hb D, which moved in the region of Hb S and could be differentiated from Hb S by carrying out sickling test and noting the difference in the retention time.
The other sickling syndromes (double heterozygotes with one gene Hb S and the other gene for some other Hb) diagnosed were Hb S beta thalassemia, Hb SD disease, and Hb SE disease with the highest number of cases of sickle thalassemia trait [Figure 4]. Thalassemia major was diagnosed in only one patient based on Hb F > 90% [Figure 5].
One case each of Hb E trait and disease was identified. Hb E trait had Hb A 70-80%, Hb E 20-30%. Retention time 3.60 min [Figure 6]. Whereas Hb E disease was diagnosed by Hb E > 80% with a retention time of 3.75 min [Figure 7].
Elevated Hb F was found in three cases. These cases were of below 1 year of age, hence repeat Hb F was advised after few months.
| Discussion|| |
India is an ethnically diverse country with marked regional variation. Pune being the educational hub and a place of choice for multinational companies, cater to people of varied ethnic groups of different states. Due to migration, there is constant mixing of people from different regions. We attempted this pilot study to see the difference in the prevalence of various hemoglobinopathies in and around Pune.
Appropriate laboratory tests are required for diagnosis and confirmation of these disorders. The most common investigational tools for diagnosis of hemoglobinopathies and thalassemias are alkaline and acid gel electrophoresis, HbA2 quantification by ion exchange column chromatography, and Hb F quantification by alkali denaturation method. The identification of Hb variants by conventional techniques are often presumptive.  HPLC offers the distinct advantage over classic Hb electrophoresis as it can more accurately identify and quantitate abnormal Hbs.  HPLC has been shown to be rapid, sensitive, specific, and reproducible alternative to conventional Hb electrophoresis.
We found that the maximum number of cases were of Beta thalassemia trait and were diagnosed in the age group of 20-30 years. The high incidence of thalassemia trait calls for the need of antenatal screening and screening of marriageable age groups. This will help in the prevention of thalassemia major in the offsprings. This trend has probably started in the educated and affording population. This could be the reason for detecting less number of cases of thalassemia major in our study group. We think that antenatal screening or screening of higher secondary school children to detect hemoglobinopathies, counseling of the individuals with hemoglobinopathies will definitely help in drastically reducing the incidence of the thalassemia major and sickle cell disease. Similar study done by Patel et al. in Gujarat has reported much higher incidence of thalassemia major (5.63%) and sickle cell disease (4.89%).  In our study, we found 4% of cases with sickle cell disease.
One case of Hb E trait identified in our study was clinically normal, but peripheral blood smear revealed microcytosis and few target cells. Hb E trait is more common in ethnic groups from Assam. A multicenter study revealed that Hb E trait was mainly seen in Dibrugarh in Assam (23.9%) and Kolkata in West Bengal (3.92%) among six ethnic groups from Assam, the prevalence of Hb E trait varied from 41.1% to 66.7%.  Detection of this variant is very important because when combined with thalassemia or Hb S, it gives rise to moderate to severe disease. In our study, we found one case of Hb SE disease.
One case of homozygous Hb D was identified. The patient had presented with mild hemolytic anemia and splenomegaly. Hb D Punjab is one of the common hemoglobinopathy in India, with the prevalence of 1-3% of population in North-West India especially Sikhs in Punjab, followed by Jammu and Kashmir and Gujarat.  Hb D was identified by a peak in the D window with a retention time of 4.06 min.(3.90-4.30). These were differentiated from Hb S peak (retention time 4.30-4.70 min). The other studies have shown the prevalence of Hb D as 31-45% and Hb D Punjab as 0.25%. 
We found 13 cases with borderline HbA2. Different authors have established different cutoff values for HbA2 for diagnosis of beta thalassemia trait, which ranges from 3.5% to 4%. It has been recommended that each laboratory needs to establish their own normal ranges.  Our range for borderline HbA2 was 3.5-3.9%. Rangan et al. used the term borderline with HbA2 levels of 3.0-4.0% and found mutations in 32% people with HbA2 3.4-3.9%. , Similar findings were described by Colah et al.  These cases were carefully evaluated as Iron deficiency may lead to low HbA2 levels, masking the thalassemia trait. Vitamin B12 deficiency may give rise to increased HbA2, giving a false positive diagnosis of thalassemia trait. Such patients should be treated for anemia and Hb electrophoresis should be repeated after treatment for definitive diagnosis.
HPLC also helped us in detecting various heterozygous states. Though these abnormal variants have less clinical significance, when combined with other variants, they can give rise to severe disease. This once again highlights the significance of mass screening of the population. Maximum number of cases was of a combination of Hb S with thalassemia trait. Although these states can be diagnosed both on HPLC and gel electrophoresis, use of HPLC has an added advantage of further sub-classifying these syndromes based on identification and quantification of various Hbs depending on their retention time. Hb D Punjab and Hb D Iran exhibit identical electrophoretic mobilities at alkaline pH, but their mean retention time in HPLC is unique and significantly different. This differentiation is in fact quite significant especially in a double heterozygous state with Hb S, as Hb SD-Punjab is a significant sickling disorder whereas Hb SD-Iran is clinically benign. , All the cases of heterozygous states should undergo proper genetic counseling to avoid severe hemoglobinopathies in offsprings.
We found elevated Hb F in three cases, all of which were <1 year of age. A group of conditions with Hb F production persisting beyond infancy without significant hematologic abnormalities is known as HPFH; hence they were advised to repeat Hb electrophoresis after the age of 1 year.
The major limitation of HPLC is high cost and requirement of knowledge, skill, and experience for interpretation of results. Alpha thalassemia, normal HbA2 beta thalassemia, and other rare hemoglobinopathies that elute with similar retention values on HPLC cannot be ruled out. Hence, the reports must carry a note to interpret the findings with reference to hemogram, family/sibling studies, other confirmatory techniques, and molecular studies when necessary.
| Conclusion|| |
To conclude, HPLC is an ideal method for routine diagnosis of hemoglobinopathies. We found the relatively lower incidence of different hemoglobinopathies in and around Pune district. Continuous awareness programs, mass screening of the population especially childbearing age and school going children will help in early detection of heterozygous states. This can in turn with proper genetic counseling help in reducing the morbidity and mortality. Pune being an educational hub with a mixture of the population from different ethnic groups, so there is a change in the demographic profile of the disease and needs to be studied in detail.
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Conflicts of interest
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
[Table 1], [Table 2]