Thalassemia and various structural hemoglobin variants are very common in India 1. If they are detected at an early age or before marriage- various untoward consequences can be prevented, especially in next generation. There are multiple hemoglobin variants including Hemoglobin E- which is very common in South East Asia. Interaction between various hemoglobin variants or Thalassemia may lead to formation of compound heterozygous state, which may be lethal in some instances. High-Performance Liquid Chromatography (HPLC) is a very rapid, accurate and reliable instrument for screening and early detection of Thalassemia and various structural hemoglobin variants. In this study, we wanted to get a picture of incidence of Thalassemia and various structural hemoglobin variant in tertiary care centre in Eastern India. Special emphasis including parental study has been given in cases of rarer variants.

This study was done in a tertiary care centre in Eastern India for 1 year period from May,2024 to April,2025 . Study population came from adjoining area of that institute mostly. Children referred from Pediatric Department, subjects for Antenatal Check up (ANC), prospective couple undergoing HPLC prior to marriage constituted the bulk of cases. Total number of the samples were 900.They were analyzed by Cation Exchange HPLC machine. Lifotronic Hemoglobin analyser(HPLC) machine, Model H9]. Here allowed range of total area is between 50,000 to 300,000. Ethnicity of the subjects, their symptom profile (whether asymptomatic or not), presence of splenomegaly or any other relevant clinical findings were analysed in details. For each patient, 5 ml of intravenous blood collection were done in EDTA containers. Complete blood count(CBC) with RBC indices were analyzed in automated hematology analyser(Sysmex KX 21).Peripheral blood smear(PBS) were evaluated in every cases. Reticulocyte staining was done in some cases. When Hemoglobin was eluted in S – window, Sickling test and Solubility tests were also done. Kleihauer-Bekte test was done in selected cases.

Total number of samples were 900. Abnormalities were noted in 150(16.66%) cases. Most common abnormality was β thalassemia trait (Hemoglobin A2 level ≥4%) Total 90(10%) cases showed Hemoglobin A2 level between 4-7%. Retention time was 3.0- 3.33 minute(180-200 sec). All cases were asymptomatic. Most of the cases showed normal or mildly reduced Hemoglobin, elevated RBC count with normal Red cell distribution width( RDW). PBS showed uniform microcytosis with few target cells in most of the cases. β thalassemia homozygous state was observed in 6 cases(0.6%). Among them, four were transfusion dependent since early childhood. Evaluation revealed severe anemia, anisopoikilocytosis, target cells, basophillic stippling and increased nucleated RBC in peripheral smear. Value of Hemoglobin F was≥70% in all cases. Other 2 cases showed history of blood transfusion, but not on regular basis. They revealed moderate anemia, microcytic hypochromic RBC in peripheral smear. Hemoglobin F value was elevated. Out of 6 cases, Parental study was possible in 3 cases. All showed β thalassemia trait in both the parents. High Hemoglobin E value was noted in 30 cases(3.33%). Among them, 25 cases showe isolated additional peak in Hemoglobin E window. They were asymptomatic, Hemoglobin was slightly reduced with microcytic hypochromic RBCs with few target cell in peripheral smear. Hemoglobin E was between 25-30% in HPLC in all the cases. They were Hemoglobin E trait(heterozygous). 2 cases showed Hemoglobin E value 80% and 85% respectively. They have moderate anemia with similar peripheral blood finding like Hemoglobin E trait. They were diagnosed as Hemoglobin E homozygous. 3 cases reveled high Hemoglobin F value(≥ 10%), other than elevated Hemoglobin E value(≥ 50%). They showed severe anemia (Hemoglobin less than 7 gm/dl) with no history of transfusion. Parental study showed both had β thalassemia trait and Hemoglobin E trait in one of the parents. They were diagnosed as compound heterozygous state for β thalassemia and Hemoglobin E. Elevated peak in S window was seen in four cases. Hemoglobin S value was between 20- 30%. Sickling study was done and was positive in all four cases of heterozygous Hemoglobin S. 1 case ( student of Africa studying here) showed high Hemoglobin C level(≥ 25%),with no complaint. That was a case of Hemoglobin C heterozygous. 19 cases showed isolated elevation of Hemoglobin F in HPLC. Retention time of Hemoglobin F is 25-35 seconds.CBC with peripheral smear study could not yield any abnormality. They were diagnosed as cases of heterozygous state of Hereditary Persistance of fetal Hemoglobin (HPFH). One case from Pediatric Department (1 yr 3 months) with pallor and jaundice with splenomegaly showed 100% HB F on HPLC. Peripheral smear showed microcytic hypochromic cells with anisopoikilocytosis, target cells. Reticulocyte count was 1.8%.Serum Bilirubin was 3.6 mg/dl( Indirect bilirubin was 3.2 mg/dl). Kleihauer-Bekte test showed pan- cellular pattern. There was history of consanguinous marriage in parents. They were asymptomatic. Their CBC with peripheral smear were normal other than mild microcytic hypochromic cells. HPLC showed HB F between 15-20% with normal HB A2 levels. Kleihauer-Bekte test showed heterocellular pattern. Parents were cases of δβ Thalassemia trait whereas child was case of δβ Thalassemia homozygous. The child was advised mutation analysis but party could not afford it. Four cases were diagnosed as HB E disease with HB E value of 80-85% . Among them, three were ANC cases. They have anemia (HB- 8.6 to 9.2). Peripheral smear showed microcytic hypochromic cells with increased RBC count. Parental study was possible in one case. HB E value was 26-30%. Peripheral smear showed microcytic hypochromic cells with increased RBC count in both the parents. Both have normal hemoglobin level according to their age and gender. One patient with mild pallor with jaundice showed additional peak in D window .His HB D value was 81%. His CBC revealed microcytosis, hypochromia with increased RBC count. . Peripheral smear showed microcytic hypochromic cells with numerous target cells. Parental study was done. CBC with peripheral smear was normal other than few target cells. HB D values were between 32-35% in both the parents. A 10 yr boy with pallor and knee pain referred for evaluation. His hemoglobin was 8.4. Peripheral smear showed reduced density with some curved cells with pointed ends. Sickling and solubility tests were positive. HPLC reveled additional peak in S window. His HB S value was 81%, HB F value was 11%, with no HB A. He was diagnosed as a case of Sickle cell disease. Parents were apparently asymptomtic .CBC were normal except reduced RBC count. HB S value was 31% and 35% in both the parents. One case of HB J Meerut trait was found with abnormal peak in P3 window. Later, her mother was found to be a case of HB J Meerut trait also. Table 1(I)- Distribution of cases according to source of referral: Source of cases Number of cases(n=900) β thal trait HB E trait Others From Pediatric department 287(31.89%) 54 1 19 ANC check up cases 306 (34%) 15 11 11 Premarital screening 98(10.89%) 9 6 7 Preconceptional check up 85(9.45%) 4 4 9 Others 124(13.77%) 8 3 8 Table 2(II)- Distribution of cases along with multiple hematological parameters: Disease category Number(n=900) Hb (g/dl) RBC Count(million/cmm) MCV(fl) MCH (pg) MCHC (g/dl) Normal 734(81.55%) 11.2± 1.8 4.4±1.7 83±7.2 27.2±3.2 32.1± 1.7 β thal trait 90(10%) 10.1± 1.2 5.2± 0.9 69±5.4 21.8± 2.6 30.2± 1.5 β thal major 6(0.66%) 6.3±3.9 2.8±1.3 71.2± 11.5 22.2± 5.6 29.4± 1.7 HB E trait 25 (2.77%) 10.9± 1.5 4.8± 0.8 76.9± 5.7 25.2± 2.7 31.9± 1.6 HB E disease 4 (0.44%) 9.1± 1.1 4.6±1.2 63.6± 5.8 22.6± 2.9 30.7± 1.8 Eβ Thalassemia 3 (0.33%) 6.5± 1.6 3.4± 0.8 65.4± 4.9 23.7± 2.6 28.6± 1.4 HB S trait 3 (0.33%) 10.7± 0.8 4.4± 0.7 81.4± 6.1 26.8± 2.3 30.2± 1.5 HB S disease 1(0.11%) 8.4 3.5 80.4 25.3 29.7 HB D trait 5 (0.55%) 11.1± 1.2 4.6± 0.4 83.5± 4.5 28.3± 2.4 31.5± 1.2 HB D Disease 4 (0.44%) 10.6± 1.1 4.5± 0.8 69.3± 6.2 24.2± 3.1 29.2± 1.6 Sβ Thalassemia 1 (0.11%) 8.5 3.8 72.5 26.4 30.9 HPFH trait 19 (2.11%) 11.1±0.7 4.1± 0.7 82± 4.2 27.2± 2.7 31.3± 1.4 δβ Thalassemia (homozygous) 1(0.11%) 8.2 3.4 75.8 24.5 29.7 HB J Meerut 1 (0.11%) 10.2 4.2 76.8 26.8 30.4 Others 3 (0.33%) 10.6± 0.7 3.9± 09 80.2± 4.7 27.4± 2.8 30.3± 1.4

Among the inherited genetic disorder, Hemoglobinopathy is the commonest in India 1. There are multiple variants of it in our country. Our study also detected multiple variants also. This is possibly due to difference in ethnicity, migration and mixing of population. There are multiple diagnostic tools for diagnose Thalassemia and Hemoglobinopathy . Acid and alkaline electrophoresis, ion-exchange column chromatography for HB A2 estimation ,Alkali denaturation for HB F quantification .Problem with electrophoresis is that it fails to separate different variants from each other. HPLC has advantage over them and they can detect multiple fractions of hemoglobin in single step. HPLC is a sensitive and specific diagnostic tool which is used for reliable quantification of different hemoglobin fraction and diagnose. As per literature, prevalence of β Thalassemia varies from 3.5% to 15% in general population.2 In our study, prevalence of β Thalassemia trait is 10%. Sachdev et al 3, in their study in North India, found abnormalities in hemoglobin in 327 cases out of 2600 samples examined. 8.9% of cases were β Thalassemia trait and 0.6% were β Thalassemia Major. According to Colah et al 4, 1.5% of world’s population are carrier of β Thalassemia trait.In their study of 55,413 cases, Dolai et al5 found β Thalassemia trait in 10.38% of cases, HbE trait in 4.30% of cases, Sickle cell trait in 1.12% cases, HbD trait in 0.37% cases. Jain et al 6 reported 29.3% of cases involved by hemoglobinopathies. They found 55.84% of the abnormal hemoglobin is due to β Thalassemia trait and 15.70% by HbE trait. Mondal et al 7 reported in their study that 4.29% of cases were involved by β Thalassemia trait, Sickle cell trait 0.46%, HbE trait in 2.68% . In our study, HbE trait is 2.77%, Sickle cell trait is 0.33%, Sickle cell disease is 0.11%, HbD trait is 0.55%, HbD disease is 0.44% of cases. Ghosh et al 8 found 29 cases with HbE trait and 4 cases with β Thalassemia trait among 188 antenatal mother in their study. In our study 15 cases showed β Thalassemia trait and 11 cases showed HbE trait among 306 ANC cases. Our study also found one J Meerut cases with abnormal hemoglobin peak in P3 window like their study. In another study in West Bengal, Sur et al 9 reported prevalence of carrier state of β Thalassemia is 5.95% among antenatal mother. HbE trait or disease may not be clinically symptomatic usually. But when they interact with β Thalassemia or other abnormal hemoglobin variant , they may produce lethal consequences.10,11 In our study ,all of our HbE trait or disease were asymptomatic .But, all the 3 cases of Eβ Thalassemia were presented with pallor with jaundice and gave history of regular transfusion. Parental study revealed one of parent positive for β Thalassemia trait and other with HbE trait. All of the parents are clinically normal apparently and never required tansfusion. Iron deficiency anemia is very much prevalent in India as well as in our study population. Various studies showed that Iron deficiency anemia has made its impact on HB A2 level(12,13,14) . Denic et al 14 suggested that concurrent Iron deficiency anemia must be ruled out in cases with microcytic hypochromic blood picture with borderline HB A2. In these cases, iron study or parental study recommended. Genetic counseling with DNA analysis recommended if they are not contributory. In some cases, microcytic hypochromic anemia which is refractory to management, found with HB A2 level showing normal or reduced or increased. They should be worked up for association of of alpha thalassemia. Coinheritance with beta thalassemia gene should be ruled out because Alpha thalassemia has high prevalence in India.15 Molecular genotype testing for alpha thalassemia diagnose these refractory cases and unwanted iron therapy can be avoided.16

HPLC is a very sensitive, specific, rapid method for diagnosis of Thalassemia and other hemoglobin variant, which are very prevalent in our country. It’s use make diagnosis early and prevent fatal consequences in offsprings. Findings of HPLC should be judiciously interpreted with other investigation. Parental study, Iron study is helpful in some cases to reach appropriate diagnosis. However, some cases still require DNA analysis, which is a challenge in resource poor environment. CONFLICT OF INTEREST: None AUTHOR CONTRIBUTION: Conceptualization: Dr.Sankha Chatterjee Project administration & draft prepration: Dr.Ashmita Chakraborty Writing Original Draft: Dr.Mrinal Sikder Supervision: Dr.Rajashree Pradhan, Dr. Sajeeb Mondal.

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