Year : 2010 | Volume
: 53 | Issue : 1 | Page : 57--62
Detection of Hb variants and hemoglobinopathies in Indian population using HPLC: Report of 2600 cases
Ritesh Sachdev, Arpita R Dam, Gaurav Tyagi
SRL Ranbaxy Pvt Ltd, Clinical Reference Lab, Sector-18, Udyog Vihar, Gurgaon, Haryana, India
A 803, Plot 7A, Navrattan Apartments, Sector 23, Dwarka, New Delhi - 110 075
Background: Inherited abnormalities of hemoglobin synthesis include a myriad of disorders ranging from thalassemia syndromes to structurally abnormal hemoglobin variants. Identification of these disorders is immensely important epidemiologically and aid in prevention of more serious hemoglobin disorders. Aims: High performance liquid chromatography (HPLC) forms an important tool for accurate and speedy diagnosis of various hemoglobin disorders. About 2600 cases have been studied for identification of various hemoglobin disorders in Indian population. Material and Methods: The study was performed on BIORAD VARIANT using beta thalassemia short program. Results and conclusion: Abnormal hemoglobin fractions on HPLC were seen in 327 of the 2,600 cases displayed. Of this, the beta thalassemia trait was the predominant abnormality with a total of 232 cases (8.9%). There were 15(0.6%) cases of beta thalassemia major and 16 of thalassemia intermedia. The rest comprised of Hb D Punjab (13 cases; 0.5%), Elevated Hb F (25 cases; 0.9%), Hb E (seven cases including two Hb E homozygous and five Hb E heterozygous), Double heterozygous Hb E-beta thal trait (six cases), Hb Q India (five cases), Double heterozygous Hb Q India -beta thal trait (two cases), Hb S (total cases three including one Hb S homozygous; two Hb S -beta thal trait) and one case each of Hb J Meerut, Hb D-Iran and Hb Lepore trait. Detection of this abnormal hemoglobin, particularly keeping in mind a high prevalence of Hb A2, will help in prevention of more serious hemoglobinopathies including beta thalassemia major. HPLC forms a rapid and accurate tool in early detection and management of various hemoglobin disorders.
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Sachdev R, Dam AR, Tyagi G. Detection of Hb variants and hemoglobinopathies in Indian population using HPLC: Report of 2600 cases.Indian J Pathol Microbiol 2010;53:57-62
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Sachdev R, Dam AR, Tyagi G. Detection of Hb variants and hemoglobinopathies in Indian population using HPLC: Report of 2600 cases. Indian J Pathol Microbiol [serial online] 2010 [cited 2020 Jan 20 ];53:57-62
Available from: http://www.ijpmonline.org/text.asp?2010/53/1/57/59185
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.
Accurate and timely detection of various Hb variants including beta thalassemia trait can prevent occurrence of more serious disorders like thalassemia major in newborns. 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. Cation exchange HPLC offers a reliable tool for early, accurate detection thereby aiding in prevention and management of various hemoglobinopathies.
Material and Methods
A total of 2600 cases received from January till August 2008 for Hb variant analysis were studied for various hemoglobinopathies and variants. The geographical distribution of all the cases predominantly included parts of northern India (New Delhi, Haryana, U.P, and Jammu and Kashmir) and some from Nepal. The tests were performed on an instrument manufactured by BIO RAD laboratories, USA. The instrument, known as BIO RAD 'VARIANT' (beta thalassemia short program) utilizes the principle of high performance liquid chromatography (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-one to three million. Sample ratio was increased in case of low total area and vice versa. Samples were also run on LH 500 analyzer (BECKMAN COULTER) beforehand to obtain the Hemoglobin values and indices. Sickling test was also performed (using sodium meta bisulphite) when S- window was eluted in the sample.
A total of 2600 cases were studied. Of these, 327 cases displayed abnormal hemoglobin fractions on HPLC. The major abnormality observed was of high Hb A2. A cut-off of over 3.9% was taken for diagnosis of beta thalassemia trait.  A total of 232 cases (8.9%) of beta thalassemia trait were diagnosed. The retention time for Hb A2 ranged between 3.59-3.67 minutes. Predominant peripheral blood findings were microcytosis and hypochromia with raised RBC counts.
There were 15(0.6%) cases of beta thalassemia major and 16 cases of thalassemia intermedia. Cases diagnosed with beta thalassemia intermedia had variable degree of anemia with anisopoikilocytosis and microcytic hypochromic (MCHC) blood picture. Hb F levels were raised with a variable reduction in Hb A.
Cases diagnosed with thalassemia major presented within the first two years of life. Marked anemia, anisopoikilocytosis, microcytic hypochromic blood picture with many nucleated RBCs dominated the blood picture. All the cases had Hb F more than 90%.
Hb D- Punjab heterozygous constituted 13 (0.5%) cases. HPLC displayed a D Window with variant percentage ranging from 33-39% and retention times of 4.09-4.14 minutes. The blood counts were essentially normal [Figure 1]a.
Twenty five adult cases had isolated Hb F elevation with normal blood counts. A possibility of hereditary persistence of fetal hemoglobin was raised in such cases with a recommendation for molecular confirmation.
Hb E variant included Hb E homozygous (two cases), Hb E heterozygous (five cases) and Hb E -Beta Thalassemia trait double heterozygous (6 cases). Hb E presents as raised peak in the A2 region with retention times ranging from 3.68-3.79 minutes. Hb E heterozygotes have on an average raised Hb E of 30% (usually less than 40%). Hemoglobin is slightly decreased to normal with microcytic hypochromic indices. Hb E homozygous had high Hb E of 84.2 % (Hb 4.6 gm/dL) and 75 % (Hb 7.6 gm/dL). Peripheral blood picture was of microcytic hypochromic anemia with target cells [Figure 1]b. There were six cases of Hb E-beta thal trait double heterozygous. Characteristic features included high Hb E ranging from 40-75%, high Hb F ranging from 13-40%, low hemoglobin ranging from 5-7 gm/dL and MCHC blood picture [Figure 2]a. A study of the family of parents in three cases confirmed the diagnosis. Hb Lepore constituted one case. Hb A2 was raised to 12.1% with mild anemia [Figure 2]b. Hb Q-India heterozygous constituted five cases. The characteristic findings include an unknown peak (range 11-20%) on HPLC with a typical retention time of 4.77 plus/minus 0.01 min. Hematological parameters were essentially within normal limits [Figure 3]a. Two cases had an Hb Q-India peak along with raised Hb A2. These were diagnosed as double heterozygous for Hb Q India and beta thalassemia trait. Hematological profile included mild anemia with MCHC indices [Figure 3]b.
Hb S homozygous (1 case) presented with a variant S-Window of 76.4% and retention time 4.42 minutes [Figure 4]a. Sickling test was positive. Hb was 7.2 gm/dL with target cells and few irreversibly sickled cells in the peripheral smear. Hb F was raised to 17.7%. Two cases had high Hb A2 along with high Hb S, MCHC blood picture with target cells. These were diagnosed as double heterozygous for Hb S-beta thalassemia trait [Figure 4]b.
One case was diagnosed as Hb D-Iran. It presented with raised A2 peak of 44.9%, retention time of 3.58 minutes and normal hematological parameters [Figure 5]a. Compared with Hb E heterozygous, Hb D Iran tends to have Hb A2 more than 40% whereas Hb A/E has percentage of abnormal hemoglobin less than 40%. Family studies along with molecular studies were recommended for a definite diagnosis.
One case was diagnosed as having Hb J. Elevated P3 window was observed with an abnormal hemoglobin peak of 25.4%, retention time 1.81 minutes. Hematological profile was normal [Figure 5]b. Possible diagnosis of Hb J Meerut was made with a recommendation for molecular studies. 
Cation exchange HPLC is emerging as one of the best methods for screening and detection of various hemoglobinopathies with rapid, reproducible and precise results.  Our study included predominantly population of northern India. There were in total 12.5% hemoglobin variants detected. Beta thalassemia trait formed the largest sub group of abnormal hemoglobin (8.9%). The characteristic hematological findings in a typical case of beta thal trait include microcytosis with raised RBC counts. Hemoglobin may be normal or slightly reduced. The mutations common in an Indian setting include IVS1-5(G-C), 619 bp deletion, IVS 1-1(G-T), CD8/9(+G), CD41/42(-CTTT), CD15 (G-A), CD30 (G-C).  Hb A2 levels >7% are usually seen with 619bp deletion. 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. Careful evaluation of indices with iron profile will usually help in such cases. Similarly milder forms of thalassemia or a co inheritance of delta thalassemia may lead to borderline A2 levels. Genetic studies should be advised in such cases for a conclusive opinion.
Thalassemia major and intermedia constituted approximately 0.6% of cases. Children with thalassemia tend to present within two years of life and are dependent on regular blood transfusions. History of recent blood transfusion must be sought along with correct age so as to aid in an accurate diagnosis. Hb D-Punjab tends to have a normal phenotypic presentation. There is a mutation in the beta chain at b121 Glu ®Gln (GAA-CAA).  We had detected Hb D- Punjab in 0.5% of cases. 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 D-Homozygous has Hb D values between 70-90%. Patients with coexistent Hb D and thalassemia trait tend to have mild anemia and are asymptomatic. Double heterozygosity of Hb D with Hb S leads to anemia which is moderately severe. 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 hemoglobin variant in South East Asia and second most prevalent worldwide. Hb E homozygous presents with Hb E values between 70-90%. Clinical features include anemia, microcytosis, and hypochromia with target cells. Hb E Heterozygous individuals are normal; Hb E levels are usually less than 40%. Double heterozygous HbE -beta thalassemia trait are important and symptoms resemble those of thalassemia major. We had 6 such cases, presenting with severe anemia, marked anisopoikilocytosis, microcytosis, target cells and basophilic stippling.
Hemoglobin Q-India is a rare alpha-chain structural variant caused by the mutation AAG-->GAG (Asp-->His) in the position of codon 64 of the alpha1 gene.  On CE HPLC, abnormal hemoglobin with retention window of 4.77 plus/minus 0.01 min is seen. We had five cases of Hb Q-India heterozygous, with normal hematological profile. Electrophoresis at alkaline pH reveals migration hemoglobin to S/D/G position. Hb Q India with thalassemia trait shows features of thalassemia trait.
Hb S homozygous presents as an S-Window with abnormal hemoglobin ranging from 70-90%. Values of Hb F generally are raised in parts of central India and Orissa.  Sickling test was positive. Two cases with high Hb A2 levels were diagnosed as double heterozygous for Hb S -beta thalassemia trait.
Hb D-Iran in India is mainly seen in northwestern region. The mutation is in beta chain (b22, Glu®Gln; GAA®CAA).  On HPLC, abnormal hemoglobin elutes in the A2 window. Differentiation with Hb E on HPLC relies mainly on the fact that, in heterozygous states, Hb D Iran is usually more than 40% whereas Hb E is less than 40%. Alkaline electrophoresis shows migration of abnormal Hb in the S/D/G position whereas Hb E migrates in the C/E/O position. Final confirmation is by molecular diagnosis. Phenotypic presentation is normal.
Hb J presents as elevated P3 peak on HPLC. Important is to note that a P3 peak up to six per cent is usually normal. Values six to 12% indicates sub optimal specimen. Values more than 15% -25% indicate Hb J. Our case had a retention time of 1.81 minutes and the possibility of HbJ was considered.  Hb J is usually asymptomatic or patients may present with mild anemia. Hemoglobin electrophoresis at alkaline pH shows a fast moving band ahead of Hb A. The mutations can be seen either in alpha or beta chains. Hb J Meerut is an alpha chain variant with retention time of 1.88 minutes.
A limitation of this technique is that possibility of alpha thalassemia, normal A2 beta thalassemia or other hemoglobinopathies that elute with similar retention values on HPLC cannot be ruled out by CE HPLC. A disclaimer should always accompany the reports.
To conclude, HPLC forms a rapid, accurate and reproducible tool for early detection and management of hemoglobinopathies and variants. This is especially important in view of high incidence of beta thalassemia trait in the Indian subcontinent. Our study had nine per cent beta thalassemia trait. Early detection of traits will prevent occurrence of thalassemia major in offspring. Detection of other variants becomes important due to complex interactions in cases with double heterozygous and homozygous states, which may lead to severe hematological abnormalities. Findings must be supplemented by hemogram findings, family/sibling studies, hemoglobin electrophoresis, other confirmatory techniques and molecular studies based on HPLC findings and on a case-to-case basis.
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