Distribution of hemoglobinopathies in patients presenting for electrophoresis and comparison of result with High performance liquid chromatography

Correspondence: Dr. Runa Jha, MBBS, MD Department of Pathology Tribhuvan University Teaching Hospital, Maharajgunj, Kathmandu, Nepal Email: runa75jha@yahoo.com Background: Nearly 226 million carriers of thalassemias and abnormal hemoglobin are present worldwide according to the World Health Organization (WHO). The laboratory plays an important role in the investigation of the thalassemias and hemoglobinopathies. Cellulose acetate electrophoresis at alkaline pH and diagnosis based mainly on visual impression of thickness of band may miss the thalassemic trait patients. The aim of this study was to find out different hemoglobinopathies and thalassemia presenting in our hospital and to compare electrophoresis results with HPLC.


INTRODUCTION
The World Health Organisation (WHO) reports that the frequency of thalassemias and hemoglobinpathies carriers is 5.1% with nearly 226 million carriers worldwide. 1r the most severe cases, the only curative treatment is bone marrow transplantation with a human leukocyte antigen (HLA) compatible sibling.For the majority of patients, therefore, treatment remains supportive and consists of lifelong transfusion/chelation and management of acute and progressive organ damage. 2 Thus management of these diseases pose a significant burden on the healthcare system and family.
In several countries; there are screening programs with the aim of identifying carriers of hemoglobin disorders in order to assess the risk of a couple having a severely affected child and to provide information on the options available to avoid such an eventuality.Nepal doesn't have any such programme and there has been no population based study to find out the prevalence of thalassemia and hemoglobinopathy in Nepal.However there are some published data about such disorders in Nepalese population. 3,4e aim of this study was to find out different hemoglobinopathies and thalassemia presenting in our hospital and to compare electrophoresis results with HPLC.

MATERIAL AND METHODS
This study was performed in the Hematopathology section of Department of Pathology of Tribhuvan University Teaching Hospital (TUTH) on cases sent for electrophoresis, during 18 months period from October 2013 to March 2015 with the aim to identify differerent types of hemoglobinopathies and thalassemias presenting to TUTH, the ethnicity and hemogram findings of such patients and to compares electrophoresis and high performance liquid chromatography results.All electrophoresis performed in the department during the study period was evaluated.Before electrophoresis was performed, a complete blood count (CBC) by automated hematology analyser and peripheral smear examination was performed on all cases.Our laboratory only has facility of cellulose acetate electrophoresis at alkaline pH and our diagnosis is based mainly on visual impression of thickness of band seen.Some cases where hemogram findings were suspicious of thalassemia trait but electrophoresis did not show prominent band at A2 position were also sent for high performance liquid chromatography (HPLC).Some other cases were also randomly selected and sent for HPLC at     and HPLC is shown in table 4 Electrophoresis was not able to detect 7 cases of beta thalassemia trait.The figure may be higher because not all such cases were sent for HPLC.
There was an eight month old child with anemia and microcytic hypochromic blood picture who had strong band at HbA2 position and Hb F position, Hb F was 40% , Band at Hb A position was faint .This was considered as compound heterozygous for Hb E -beta thalassemia by electrophoresis as hemogram findings and family study was suggestive.One of the parent was Hb E trait and one was Beta thalassemia trait.But no opinion was made in HPLC, may be considering the age of patient.HbA here was 5.8% and hemoglobin eluting at HbA 2 position was 46.8% in HPLC in this case.Hb J variant showed peak in HPLC however impression of HbH was made by presence of peak before Hb F. HbH can be seen better as fast moving band in electrophoresis and by HbH inclusion in supravital stain.In electrophoresis, in Hb M, strong band was seen and Hb F position but Hb F percentage calculated by alkali denaturation method was only 1%.So a conclusion was drawn that it was a band comigrating with HbF.
Percentage of different hemoglobins detected by HPLC in various conditions are given in table 6.This includes only cases in whom HPLC findings were available Mean HbF level in different abnormal hemoglobin is shown in table 5. Highest mean HbF was seen in thalassemia major (81.9%) followed by compound heterozygous for HbE beta thalassemia.

DISCUSSION
The prevalence of thalassemias and hemoglobinopathies varies with geographic locations.In Southeast Asia α-thalassaemia, β-thalassaemia, hemoglobin (Hb) E and Hb Constant Spring (CS) are prevalent.Hb E is the hallmark of Southeast Asia attaining a frequency of 50-60 per cent at the junction of Thailand, Laos and Cambodia.Hb CS gene frequencies vary between 1 and 8 per cent. 5e disorders of Hb frequently encountered in India include beta thalassemia, HbE -beta thalassemia, HbE, HbD and sickle cell anemia.In study by Mondal et al beta thalassemia trait was the most common abnormality found , followed by HbE trait and then E-beta thalassemia followed by beta thalassemia major/intermedia.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. 6In study of Goswami et al it was found that Hb E trait was the most common hemoglobinopathies (34.4%) followed by homozygous E (25.3%), beta-thalassemia trait (17.8%),E-β-thalassemia (15.1%), β-thalassemia major (1.5%), sickle cell-β-thalassemia (3.4%), sickle cell trait. 7Study done by Mehandi et al in Saudi population found Beta Thalassemia trait to be the most common hemoglobinopathy detected followed by Sickle cell trait and sickle cell alpha Thalassemia trait.The Hb variant E and D, which are more prevalent in Southeast Asia were rarely found among Saudis. 8In study done by Patel U et al in population of Gujarat, beta Thalassemia trait was most common hemoglobinopathy, followed by Thalassemia major, sickle cell anemia and sickle cell trait. 9In our study, beta thalassemia trait was most common, followed sickle cell anemia and then different variants of alpha thaassemia.
Other variants detected included compound heterozygous for HbE beta Thalassemia, thalassemia major, sickle cell beta Thalassemia, Hb E trait, and one case each of delta beta thalassemia, HbD and HbM.
HbE occurs at an extremely high frequency in many countries in Asia.Because there is also a high frequency of different beta-thalassemia alleles in these populations, the coinheritance of HbE and beta thalassemia, HbE beta  thalassemia, occurs very frequently. 10In our study also 9.3% abnormal hemoglobins were HbE beta Thalassemia.Although molecular analysis was not done in these cases, diagnosis was made by combination of electrophoresis findings and by screening of parents.These patients had absent HbA band and increase HbF and HbA2.One parent of these patients was Beta thalassemia trait and other was Hb E trait.These patients had lower mean hemoglobin and red cell indices than HbE homozygous and HbE trait.Since this study selected cases with abnormal electrophoresis findings, this may be the reason of low number of HbE homozygous and HbE trait.Since these patients are asymptomatic may not have presented to hospital or may not have been referred for electrophoresis.The Hb E trait included in our study were also asymptomatic patients, their electrophoresis being run as part of family screening of patients having abnormal electrophoresis.
Fifity three percent were male and 47% were females in study of Manan et al. 11 Similar data was also found earlier by Yagnik and Balgir and reported 65.5, 56 and 62.1% of male patients, respectively. 12,13In our study also 66% patients were males.As suggested by Manan et al this might be due to the gender bias among the parents of these ill children who seek medical care and are ready to spend more for their male children only. 11rtain communities in India like Sindhis, Gujratis, Punjabis, and Bengalis are more commonly affected with beta thalassemia, the incidence varying from 1 to 17%.Some population groups from the north eastern regions have a high prevalence of HbE. 14 In study of Goswami et al occurrence of hemoglobinopathies was highest (72.1%) among Rajbanshis followed by Muslims (54.9%).In tribes like "Santal" and "Oraowo" and in Bengali Hindu and Marwari/Behari approximately equal percentage (34%) was observed while least belonged to mongoloid like "Nepalis" and other 'Hill men' population (17.5%). 15In our study also, maximum number of patients belonged to Tharu community (37.1%).Although abnormal hemoglobins were also found in other variable number of castes, most of them belonged to Terai region.Study done in Nepal on sickle cells anemia by Shrestha A and Karki S also showed that sickle cell anemia was most common in the Tharu community. 5 study of Mehdi SR et al, MCV and MCH were significantly low (P < 0.001) in cases of thalassemias presenting microcytic hypochromic picture on peripheral blood smear, however, these values were within the normal limits in sickle cell disorders.The red cell count was increased in cases of thalassemias while it was not much affected in sickle cell disorders.The indices were lower in sickle cell α thalassemia trait. 9In another study, Mehadi et al also concluded that moderate degree of microcytosis (MCV ≤ 78fl) and hypochromia (MCH ≤ 27pg) was a feature of β thalassemia trait and homozygous α-thalassemias.However, microcytosis was more marked in β thalassemia trait compared to heterozygous α-thalassemias. 16 our study, the mean hemoglobin as well as RBC count was lowest for beta thalassemia major (4.4 gms/dl,), followed by HbE beta thlassemia ( 6gm/dl).Sickle cell anemia patients had lower mean hemoglobin level than beta thalassemia traits (7.9 gm/dl vs 10.6 gm/dl).While RBC count was normal in sickle cell anemia ( mean 3.4 milliom/cumm),it was elevated in case of beta thalassemia trait(mean 5.2 milliom/cumm).Like their study, in our study also MCV and MCH were low in thalassemia.The beta thalassemia Many investigators have used different mathematical indices to distinguish beta thalassemia trait from iron deficiency anemia, using only a complete blood count.This process helps to select appropriate individuals for a more detailed examination; however, no study has found 100% specificity or sensitivity for any of these RBC indices.Vehapoglu A et al compared different mathematical indices and found that MCV and RBC counts and their related indices (Mentzer index and Ehsani index), have good discrimination ability in diagnosing beta thalassemia trait. 17In Mentezer index, if the quotient of the mean corpuscular volume (MCV, in fL) divided by the red blood cell count (RBC, in Millions per microlitre) is less than 13, thalassemia is said to be more likely.If the result is greater than 13, then iron-deficiency anemia is said to be more likely.In a lot of cases, the index may fall in between 11 and 13, such cases a peripheral blood smear and iron studies would help to differentiate iron deficiency from thalassemia. 18However another study suggests Srivastava formula to be more reliable. 19e HbA2 analysis is considered the gold standard for diagnosing thalassemia.Several studies have shown that iron deficiency directly affects the rates of HbA2 synthesis in bone marrow; therefore, 16-20 weeks of iron therapy should be instituted, after which a repeat serum iron with electrophoresis should be done to confirm improvement in the HbA2 levels. 20The most common problem is the presence of microcytosis with HbA2 and HbF concentrations within the reference range.This may be due to iron deficiency or α-thalassemia trait.Iron deficiency anemia produces a wide range of red cell abnormalities (reduction of MCV, MCH and hemoglobin levels and normal or lowered RBC) depending on the severity at the time of hematological analysis.For this reason iron deficiency anemia can be easily mistaken for some forms of heterozygous Thalassemia.Raised RBC with low MCV and MCH is more consistent with α thalassemia trait. 21The Hb A2 level may be modified by many factors.
The most frequent problem is the co-existence of an iron deficiency which may even normalize the Hb A2 level requiring a novel Hb assay after iron deficiency treatment.
In beta thalassemia carriers presenting with a normal Hb A2 level, the most frequent cause is a co-inherited delta globin abnormality.Increased levels of Hb A2 may result from the co-existence of a variant with electrophoretic or chromatographic properties close to that of Hb A2.As a rule, this situation has to be verified when a level of Hb A2 higher than 8 per cent is observed.It is always better to perform hemoglobin electrophoresis before any blood transfusion because though single transfusion does not affect hemoglobin pattern in electrophoresis but multiple transfusion shows significant difference.Quantitation of HbF is more important than HbA2 in beta thalassemia major.A2 percentage is normal in Thalassemia major due to Intermittently transfused β-thalassemic major patients showed both 'A' and 'F' band thicker and prominent, A band being thicker than F band in most cases.Whereas regularly transfused patients showed A band mainly, HbF band seen only in few cases in study by Paunipagar et al. 22 Six patients in our study showed strong A and F band and no other band.Two of these were delta beta thalassemia who were asymptomatic and had never received transfusion whereas the other four were transfused Thalassemia major.
The highest adult levels of HbF are seen in beta and delta beta thalassemia, or hereditary persistence of fetal hemoglobin (HPFH), in which HbF can constitute up to 100% of the hemoglobin.In sickle cell disease, HbF usually constitutes only between 5% and 20% of the total hemoglobin.In the presence of some hemoglobin variants (HbS, HbC, Hb Lepore and some unstable hemoglobins) and in association with the beta -thalassemia trait, a slight increase (1 to 5%) of HbF is found in the heterozygotes, while higher levels can be found in the homozygous.HbF levels are variable (10-80%) in presence of HbE and beta thalassemia, the important determinants being the age, the presence of alpha-thalassemia and of genetic determinants of gamma chain synthesis.Increase in HbF keeps HbS more soluble in the deoxygenated state, and the illness is thus less severe. 23In our study highest level of HbF was found in beta Thalassemia major (mean 84.9%, range 64% to 95%).This was followed by compound heterozygous for HbE /beta thalassemia ( 10-55% Differentiation of HbE disease beta thalassemia from homozygous HbE in samples containing HbA2/E > 75% and HbF < 15% is difficult.In places where the molecular analysis is not available, HbF > 5% in combination with MCV < 55 fL and hemoglobin < 100 g/ could be used for screening of beta-thalassemia/HbE disease. 26Mean HbF in HbE beta Thalassemia cases in our study was 26% ( range 5-55%).These patients had hemoglobin ranging from 4.1 to 7.9 gm/dl (mean 5.9 gm/dl)and MCV ranging from 55.2 to 64 fl( mean 60.8 fl).Although molecular analysis is not used for diagnosis in our study, these patients were symptomatic, had moderate to severe anemia and their one parent had beta thalassemia trait.
History of recent blood transfusion must be sought along with correct age so as to aid in an accurate diagnosis.Conditions with borderline Hb A2 need careful interpretation.Iron deficiency may lead to 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. 27moglobin electrophoresis is a labor intensive and time consuming method and is not that efficient when quantifying low concentrations of HbA2 and HbF.The HPLC method is a sensitive and precise method and has become the preferred method for thalassemia screening because of its simplicity, superior resolution, rapid assay time and accurate quantification of Hb fraction.

CONCLUSION
Beta Thalassemia trait and sickle cell anemia both are common in Nepal , along with some other hemoglobinopathies A sharp peak of hemoglobinopathies and thalassemias are seen in Tharu community though other communities are also effected.These abnormal hemoglobins and thalassemias are mainly seen in Terai region.Lowest hemoglobin was seen in thalassemia major followed by compound heterozygous for HbE beta thalassemia.Electrophoresis is time consuming and labour intensive method that fails to quantify hemoglobin percentage and thus is not appropriate test in beta thalassemia screening which is diagnosed by elevated HA2 level.As identification of traits is necessary to reduce the birth of thalassemia major cases, it should be used only in conjuction with more advanced techniques like HPLC or others.This study is only hospital based and diagnosis is mainly based on combination of different findings and not on genetic analysis.This study provides only a glimpse of different abnormal hemoglobins and their ethnic distribution.To know the exact burden of thalassemias and hemoglobinopathies and their ethnic and geographic distribution, community based studies are required and molecular methods should be used for mutation identification

Figure 1 :
Figure 1: Frequency of different hemoglobinopathies and thalassemias

Figure 2 :
Figure 2: Caste distribution of cases

Figure3:
Figure3: a)E beta thalssemia showing strong band at HbF abd HbA2 position b)normal control showing strong band at HbA position c)Beta thalassemia trait showing strong band at HbA and visible band at HbA2 position which is stronger than that of normal control d) Hb E trait showing strong band at Hb A and HbA2 position

Table 1 : Age distribution in different hemoglobinopathies and thalassemias Age group in years
1

Table 2 : Sex distribution in different hemoglobinopathies and thalassemias Diagnosis Female Male Total Percentage
the cost of investigator.Screening of parents was advised in all cases though it could not be done in all cases due to unavailability of one or both parents.So finally this study included cases with hemoglobinopathy diagnosed by either electrophoresis and/or HPLC.Those cases with suspected hemoglobinopathy but normal electrophoresis or HPLC findings were excluded Cellulose acetate Electrophoresis at alkaline pH was performed and interpreted according to standard protocol.disease and homozygous HbE presented with microcytic hypochromic anemia whereas sickle cell disease and HbE trait had normocytic normochromic or mild microcytic hypochromic blood picture.RDW was highest for thalassemia major and compound heterozygous for HbE beta thalassemia.Correlation between result of electrophoresis

Table 5 : Mean HbF % in different hemoglobin variants
24 Sickle cell anemia in our study had HbF upto 25% (mean 9.6%).Average Hb F was 19.62% in sickle cell disease in study of Shrikhande AV et al .Increase need for erythropoiesis because of chronic hemolysis or hematuria and pregnancy can precipitate Vitamin B12 and folic acid deficiency in Sickle cell disease leading to macrocytosis.24Howevermacrocytosiswasnotseen in any sickle cell anemia patients in our study.The MCV in sickle cell anemia ranged from 63 to 89 fl in our study(mean 74.7 fl)Hemoglobin levels in HbE beta thalassemia range widely between the different phenotypes, from 3 g/dl or less to as high as 11 g/dl.Mean level of HbF can range from 10-50%.The heterozygous state for HbE is characterized by minimal morphological abnormalities of the red cells and normal red cell indices; HbE constitutes 25%-30% of the hemoglobin.Homozygotes for HbE have hypochromic microcytic red cells with significant morphological abnormalities including increased numbers of target cells They are mildly anemic and the overall hematological findings are very similar to those of heterozygous βbeta thalassemia.25Inour study hemoglobin in HbE beta thalassemia ranged from 4.3 to 7.9gm/dl.Mean HbF in these cases was 30.9% ( range 16% to 42.8%)in our study.The red cell indices were normal to slightly microcytic hypochromic and Red cell distribution width (RDW) ranged from 12.4 t0 16.5 ( Mean 14.1).HbF was also not elevated.There was only one case of HbE homozygous in our study.This patient had hemoglobin 7.1 gm/dl but showed marked anisocytosis and low MCV and MCH.This patient's RDW was 27.