|Year : 2013 | Volume
| Issue : 3 | Page : 104-109
Study of hemoglobinopathies and Hb variants in population of Western India using HPLC: A report of 7,000 cases
Atul Shrivastav1, Umang Patel2, Jayesh R Joshi1, Amarjeet Kaur2, Ashok S Agnihotri1
1 Department of Pathology, C. U. Shah Medical College, Surendranagar, Gujarat, India
2 Department of Hematology, Green Cross Pathology and Molecular Laboratory, Ahmedabad, Gujarat, India
|Date of Web Publication||19-Dec-2013|
Department of Pathology, C. U. Shah Medical College, Dudhrej Road, Surendranagar, Gujarat 363 001
Source of Support: None, Conflict of Interest: None
Context : Various hemoglobinopathies are one of the major public health problems of India.
Aims: High performance liquid chromatography (HPLC) is a speedy and accurate tool for diagnosis of various hemoglobinopathies. In present study about 7,000 cases have been studied for identification of various hemoglobin (Hb) disorders in western India.
Settings and Design : Study is conducted from May 2010 till April 2013 for various hemoglobinopathies and variants. The geographical distribution of all cases included states of western India.
Materials and Methods: Complete blood count (CBC) is done on CELL DYN 3700 analyzer and then HPLC is performed on BIO-RAD 'VARIANT II' (beta thalassemia short program) in samples received in our laboratory. Simple Statistical analysis is done with help of Microsoft Office Excel 2007.
Results: A total of 7,261 cases were included in our study, out of these 1,615 (22.24%) cases showed abnormal Hb fractions. The major abnormality observed was of high HbA2, a cutoff value of > 3.9% was considered for diagnosis of beta thalassemia trait (BTT). A total of 839 cases (11.55%) of BTT were diagnosed. Other hemoglobinopathies were also identified in varying proportions.
Conclusions: HPLC is simple, accurate, and superior technique combined with complete automation makes it an ideal method for diagnosis of hemoglobinopathies.
Keywords: Hemoglobinopathies, high performance liquid chromatography, India, thalassemia
|How to cite this article:|
Shrivastav A, Patel U, Joshi JR, Kaur A, Agnihotri AS. Study of hemoglobinopathies and Hb variants in population of Western India using HPLC: A report of 7,000 cases. J Appl Hematol 2013;4:104-9
|How to cite this URL:|
Shrivastav A, Patel U, Joshi JR, Kaur A, Agnihotri AS. Study of hemoglobinopathies and Hb variants in population of Western India using HPLC: A report of 7,000 cases. J Appl Hematol [serial online] 2013 [cited 2019 Mar 18];4:104-9. Available from: http://www.jahjournal.org/text.asp?2013/4/3/104/123308
| Introduction|| |
Abnormalities of hemoglobin (Hb) synthesis are among the most common inherited disorders of man and can be quantitative (thalassemia syndrome) or qualitative (variant Hbs). Of these, thalassemia syndromes particularly beta thalassemia major and certain alpha thalassemia are serious and a major cause of morbidity.  WHO figures estimate that 5% of the world population is carrier for Hb disorders.  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 constitute 10% of the total numbers in the world.  India spends nearly Rs. 1,000 crore per annum in the treatment of thalassemia patients. Majority of the centers in India use conventional methods for diagnosis of hemoglobinopathies, which includes clinical and family history, red cell indices, complete blood counts (CBC), HbA2, HbF estimation, sickling test, and Hb electrophoresis. The limitations of these methods include identification of Hb variants with same electrophoretic mobility as in S/D/G/Q/Lepore and A2/E/C and diagnosing certain compound heterozygous states (Hb S + β thal, Hb S + Hb D, Hb D + Hb E, Hb E + β thal, Hb D + β thal).  Potential interactions between various Hb variants in heterozygous state may lead to serious homozygous Hb variants in the offspring. Double heterozygous states between certain variants can also lead to hematological defects. The use of cation-exchange high performance liquid chromatography (CE-HPLC) to separate and quantify various normal and abnormal Hb fractions has been increasing.  It offers a reliable tool for early, accurate detection; thereby aiding in prevention and management of various hemoglobinopathies. 
| Materials and Methods|| |
A total of 7,261 cases received from May 2010 till April 2013 for Hb variant analysis were studied for various hemoglobinopathies and variants. The geographical distribution of all cases included states of western India (predominantly from Gujarat and also from Rajasthan and Madhya Pradesh). Conventional Naked Eye Single Tube Red cell Osmotic Fragility Test (NESTROFT) was performed by placing a drop of blood in 0.36% buffered saline solution and observing for hemolysis, (as described by Mehta  ) in all blood samples. Samples were run on CELL DYN 3700 analyzer (Abott) before performing HPLC to obtain the Hb values and red blood cell (RBC) indices. The tests were performed on an instrument manufactured by BIO-RAD laboratories, USA. The instrument, known as BIO-RAD 'VARIANT II' (beta thalassemia short program) utilizes the principle of HPLC. An Hb A2/F calibrator and two levels of controls (BIO-RAD) were analyzed at the beginning of each run. The total area acceptable was between 1 and 3 million. History of blood transfusion and relevant family history were taken in all cases. The software delivers a printed report showing the chromatogram, with all the hemoglobin fractions eluted. The integrated peaks are assigned to manufacturer-defined "windows" derived from specific retention time (RT).  This RT is the time that elapses from the sample injection to the apex of the elution peak, of normal hemoglobin fraction and common variants  [Table 1]. The "windows" are established ranges in which common variants have been observed to elute using the variant beta-thalassemia short program. The printed chromatogram shows all the hemoglobin fractions eluted, the RT, the areas of the peaks, and the values (%) of different hemoglobin components [Figure 1] and [Figure 2]. If a peak elutes at a RT that is not predefined, it is labeled as an unknown. Each analytical cycle, from sampling to printing of results takes about 6.5 min.
|Figure 1: (a) Chromatogram of beta thalassemia trait. (b) Chromatogram of beta thalassemia major|
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|Figure 2: (a) Chromatogram of Hb S homozygous (Hb SS). (b) Chromatogram of Hb D-Punjab heterozygous|
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Prior approval is taken by institutional ethical committee for this study.
| Results|| |
Of the total 7,261 samples tested 3,412 (46.99%) were male and 3,849 (53%) were female. The age range of patients was from 1 month to 82 years. Of the total cases 1,615 (22.24%) showed abnormal Hb fractions. The major abnormality observed was of high Hb A2. A cutoff of over 3.9% was taken for diagnosis of beta thalassemia trait (BTT). A total of 839 (11.55%) cases of BTT were diagnosed [Figure 1]a. The RT for Hb A2 was between 3.61 and 3.68 min. [Table 1] shows manufacturer assigned windows for BIO-RAD variant HPLC system.
Various laboratory parameters are shown in [Table 2]. We observe that, in present study, predominant blood findings were microcytosis and hypochromia with raised RBC counts.
|Table 2: Relevant laboratory parameters in relation to normal and abnormal hemoglobin variants |
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There were 292 (4.02%) cases of beta thalassemia major [Figure 1]b and 16 (0.22%) cases of thalassemia intermedia. Cases diagnosed with thalassemia major presented within the first 2 years of life. Marked anemia, anisopoikilocytosis, microcytic hypochromic blood picture with many nucleated RBCs dominated the blood picture.
Hb S homozygous presents as separate S-Window with abnormal Hb ranging from 60 to 90% [Figure 2]a. Sickling test was positive in all such cases. It constituted 85 (1.17%) cases. Hb S heterozygous presents as an S-Window with abnormal Hb ranging from 30 to 40% constituting 214 (2.95%). There were 52 (0.72%) cases diagnosed as double heterozygous for Hb S-BTT.
Hb D-Punjab heterozygous [Figure 2]b and homozygous constituted 47 (0.65%) and 11 (0.15%) cases, respectively. HPLC displayed a D Window with RT of 4.07-4.16 min.
Hb E variant included Hb E homozygous (10 cases), Hb E heterozygous (11 cases), and Hb E-BTT double heterozygous (four cases). Hb E presents as raised peak in the A2 region with RT ranging from 3.56 to 3.68 min.
Eight adult cases had isolated Hb F elevation with normal blood counts. A possibility of hereditary persistence of fetal Hb was raised in such cases with a recommendation for molecular confirmation. Hb Q-India heterozygous constituted four cases. The characteristic findings include an unknown peak (range 11-20%) on HPLC with a typical RT of 4.77 min. Hematological parameters were essentially within normal limits. Hb Lepore constituted two cases. Hb A2 was raised to 16.3% with mild anemia. [Figure 3] shows distribution of various hemoglobinopathies in our study [Table 3].
|Figure 3: Bar diagram showing distribution of hemoglobinopathies; majority of cases are from beta thalassemia trait and major followed by sickle cell anemia homozygous and heterozygous (n = 1,615)|
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| Discussion|| |
Hemoglobinopathies and thalassemia both are common disorders and exert significant burden on various developed and developing countries of world including India. Adequate measures and screening procedures should be adopted to reduce this burden. The laboratory diagnosis of hemoglobinopathies and thalassemia may be required (a) to confirm a provisional diagnosis, such as significant sickling disorders or thalassemia major; (b) to explain a hematologic abnormality such as anemia, microcytosis, or polycythemia; (c) to identify an abnormality in the presymptomatic phase, as in neonatal screening; (d) to predict serious disorders of globin-chain synthesis in the fetus and offer the option of termination of pregnancy; (e) to permit genetic counseling of prospective parents; and (f) to allow preoperative screening for the presence of sickle cell Hb. 
There are quite a few reports ,,, stating reliability of NESTROFT and RBC indices in diagnosis of hemoglobinopathies, Chakrabarti et al.,  suggest NESTROFT as screening test, another study conducted by Gorakshakar and Cola  shows that RBC indices are helpful in diagnosis of thalassemias, but the result must be supported by other test for confirmation, Dangi et al.,  stated that NESTROFT is an efficient test but they also said that next confirmatory test is also advised for confirmation. In their study, Yousafzai et al.,  said that NESTROFT is easy and simple, but there is a lack of harmony between different laboratory procedures of NESTROFT; and RBC indices are machine dependent and with variable sensitivity and specificity. In our study we found that sensitivity and specificity of NESTROFT test is 37.5 and 70%, respectively and sensitivity and specificity of Mentzer's index are 56.2 and 94%, respectively. Both of these (NESTROFT and RBC indices) could not be considered as diagnostic test because of their low sensitivity and specificity, another thing is that NESTROFT is very difficult to standardize and there is direct effect of other hematological disorders and recent transfusion on RBC indices measurement. As a result of this, some unfortunate couples meet with a catastrophe when first declared as negative for thalassemia by NESTROFT screening test and then get a thalassemic baby.
The most common investigative tools for confirmation of hemoglobinopathies in the clinical laboratory are alkaline and acid electrophoresis for Hb variants and hemoglobinopathies, Hb A2 quantification by ion-exchange column chromatography, and Hb F quantification by alkali denaturation or radial immunodiffusion for thalassemia.
Electrophoresis of Hb variants with similar mobility has inherent limitations. The identification of variants is dependent on the technical performance of electrophoresis, which has many variables. These variables can affect the quality of separation and relative positioning of the bands. Variants that migrate identically or similarly would be very difficult, if not impossible, to evaluate without the unknown sample being electrophoresed directly adjacent to the reference Hb mixture or adjacent to several known stored specimens.  The compound heterozygous disorders (Hb S D-Punjab, Hb S E, and Hb S-β thalassemia) or unusual variants (Hb Q India, Hb D Punjab, and Hb D Iran) are all clinically significant with varying degree of severity, making precise identification important. , None of these can be conclusively identified by a single electrophoretic technique.  Definite identification usually requires DNA analysis or amino acid sequencing. HPLC has been shown to be a sensitive, specific, and reproducible alternative to electrophoresis. 
In our study we found that total 1,615 (22.24%) person have Hb variants. BTT formed the largest subgroup of abnormal Hb 839 (11.55%) persons. The high incidence of traits underscores the need for antenatal screening for prevention of thalassemia major in offspring. Conditions with borderline Hb A2 need careful interpretation. Iron deficiency may lead a low Hb A2 and hence may mask a thalassemia trait, whereas B12/folate deficiency may lead to slightly raised Hb A2 leading to a false diagnosis of a trait. So, apart from iron deficiency anemia(IDA), megaloblastic anemia will also result in borderline HbA2 levels, the latter returning to normal after adequate therapy. ,,,
Thalassemia major and intermedia constituted approximately 4.02 and 0.22% of cases, respectively. History of recent blood transfusion must be sought along with correct age so as to aid in an accurate diagnosis. Hb S homozygous presents in 1.17% as an S-window with abnormal Hb ranging from 60 to 90%. Sickling test was positive in all such cases, whereas Hb S heterozygous presents in 2.95% as an S-window with abnormal Hb ranging from 30 to 40%. Double heterozygopus Hb S + β thal constituted 52 cases with 0.71%. Homozygous D and heterozygous D constituted of 0.15 and 0.65%, respectively. On CE-HPLC, it elutes in the D-window, separate from Hb S peak. On alkaline electrophoresis, it migrates in the S/D region.
Hb E results from a beta chain mutation (b26 Glu ® Lys)  and tends to elute in A2 window on HPLC. It is the most common Hb variant in southeast Asia and second most prevalent worldwide.  Hb E homozygous presents with Hb E values between 70 and 90% and Hb E heterozygous presents with Hb E values <40%.
In this large prospective study by using HPLC we demonstrate that HPLC is an excellent, powerful diagnostic tool for the direct identification of Hb variants with a high degree for precision in the quantification of major and minor, normal and abnormal Hb fractions. With the integration of proper algorithms involving RT, %Hb, and RBC indices; a clinical laboratory is capable of identifying about 75% of the common variants encountered without the need for confirmatory studies such as alkaline and acid electrophoresis.  More importantly, identification of the common Hb variants (i.e., Hb D-Punjab, HbE, and β thalassemia) that in combination with Hb S lead to clinically significant sickling disorder which can be quickly and accurately accomplished by HPLC, without the need for confirmatory testing. HPLC has been shown to have a high degree of reproducibility and precision. The simplicity of sample preparation, accurate quantification of Hb concentration combined with complete automation, makes HPLC an ideal methodology for the routine diagnosis of Hb disorders.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3]