Introduction

Haemoglobinopathies are amongst the most common genetic disorders. These genetic disorders cause defect in the beta globin chain of haemoglobin. Haemoglobinopathies cause a wide spectrum of clinical presentation ranging from mild anemia to life threatening anemia requiring blood transfusion for survival and being an inherited disease needs early diagnosis and management and genetic counselling, to diagnose these disorders most commonly test and technique used is haemoglobin variant study by high performance liquid chromatography or HPLC method. The 2 most common haemoglobin variant seen are Beta Thalassemia and Sickle cell anemia.

Haemoglobinopathies are relatively common genetic disorders and according to the World Health Organization (WHO), around 5% of human population have this genetic defect [1].

In India, about 15000 babies are born every year with thalassemia major [2], the prevalence of beta thalassemia carrier is around 3-4% [3-5]. Haemoglobin S is seen in tribal population of central, west and southern India and may reach upto 48% of the population [6]. Haemoglobin E is common in north eastern states where as Haemoglobin D is common in Punjab. Haemoglobin variants in India are relatively common as compared with worldwide data, and similarly it is the most common genetic disorder seen in India [3].

Haemoglobin variants are diagnosed with the help of clinical features along with investigative modalities like blood counts, indices and Haemoglobin separation studies like electrophoresis and HPLC methods.

The Haemoglobin variants in our centre was studied by HPLC method of Haemoglobin separation which is a common alternative to electrophoresis [7, 8].

The aim of the study is to determine the prevalence of haemoglobinopathies from the haemoglobin variant test conducted over 3 years at our tertiary care centre. This will in turn reveal the magnitude of the problem and thus allow early diagnosis and genetic counselling.

The limitation of the study is the sample size of the test conducted and a larger sample size study will provide better prevalence rate of hemoglobinopathies.

Materials and Methods

The study was conducted over 3 years from beginning of 2020 to end of 2022 and involved 100 patients. Patients having history of blood transfusion in previous 3 months were excluded from the study.

Clinical history, family history, and complete blood count with red blood cell indices and peripheral blood smears were analysed for each patient. 3 ml blood samples for estimating blood counts and red blood cell indices were collected in EDTA vacutainer and analysed by fully automated 5-part differential coulter for complete blood count and analysed by Bio-Rad D10 dual mode fully automated cation exchange HPLC analyser, peripheral blood smear was stained with Leishman stain. The samples were run after running and approval of quality control materials.

The haemoglobin variant analysis by HPLC generated a chromatogram with different area peaks demonstrating individual fractions of haemoglobin separation which were identified using retention time. If the peak was not identified, it was labelled as unknown peak by the software.

Diagnosis was made using the chromatogram interpretation in co-relation with the clinical, family history, and haematological profile of the patients.

Haemoglobin electrophoresis is a similar, but old technique that can be used to study the various haemoglobin variants. However, since more than a decade HPLC method has become the investigation of choice for studying the variants as it provides good separation and has a large databank to identify unknown variants.

Statistical analysis was done using SPSS software and Microsoft Excel. Data were expressed as mean and SD.   

Results and Discussion

The Haemoglobin variant study was done on 100 patients of which 73 were having normal haemoglobin variant and 27 were having haemoglobinopathy. The age group ranged from 7 months to 84 years and mean age being 25 years. In the normal haemoglobin variant group, the male: female ratio was 20:53, where as in the abnormal haemoglobin variant group the male: female ratio was 17:10. A chromatogram is considered normal if the total area was between 1-5 million and had majorly HaemoglobinA₀ and HaemoglobinA₂ was less than 3.3% and Haemoglobin F < 0.8%. All abnormal Haemoglobin variants along with their haematological findings are shown in Table 1 and along with chromatographic concentrations of haemoglobin fractions based on globin chain is summarized in Table 2.

In our study, 77 cases were having normal haemoglobin fractions (77%). Most common abnormal haemoglobin variant seen was beta thalassemia trait 17 cases (17%) of mean age 26 years and having mean haemoglobin of 7.5 g/dl, the red blood cell indices, and PBS findings suggested microcytic hypochromic anemia and clinically asymptomatic. The only haemoglobin fraction elevated were HaemoglobinA₂, having a mean value of 4.8% (Figure 1).

Haemoglobin S was seen in 8 cases of which 4 were sickle trait and 4 were sickle beta thalassemia heterozygous. Sickle cell trait Haemoglobin AS were asymptomatic with mean Haemoglobin S fraction of 31.7%, mild anemia (Haemoglobin – 9.3 g/dl), and having normal red blood cell indices and PBS findings (Figure 2).

Sickle beta thalassemia cases were clinically symptomatic, with mean Haemoglobin S fraction of 60.9%, mean Haemoglobin F was 21.18 % few cases had history of blood transfusions, mean Haemoglobin was 6.7 g/dl and red blood cell indices, and PBS findings suggested microcytic hypochromic anaemia (Figure 3).

We had 1 case of Beta thalassemia major who was a diagnosed case with history of blood transfusions having mean Haemoglobin F 97.6%, significant anemia (Haemoglobin–6.2 g/dl) with red blood cell indices and PBS showing microcytic hypochromic anemia (Figure 4).

1 case of Haemoglobin E trait was seen having mean Haemoglobin A₂ – 26.4%, which based on the retention time and technical limitation of the machine Haemoglobin E coelutes with Haemoglobin A₂ and having normal clinical and haematological profile a diagnosis of Haemoglobin E trait was made (Figure 5).

In this study, Haemoglobin variant was seen in 27 patients and the most common variant was beta thalassemia trait seen in 17 out of 27 patients. However, a wide range of abnormal haemoglobin variant ranging from 35 to 60% has been reported from various part of India [9, 10]. Beta thalassemia trait was in seen in 17% of total cases study and was the commonest variant seen, other studies have also found similar findings in their institution or regions [11-17]. The cases of beta thalassemia trait were characteristically asymptomatic with mild microcytic hypochromic with only abnormality noted in chromatograms were of HaemoglobinA₂ between 4-8%. Slightly raised A₂ levels as the only finding is also seen in vitamin B12/Folic acid deficiency, but it was differentiated from beta thalassemia based on red blood cell indices and vit B12/Folic acid estimation levels.

Haemoglobin S or sickle Haemoglobin (Haemoglobin S) was seen in total 8 cases (8%) out of which 4 cases presented as sickle cell trait and 4 cases as sickle beta thalassemia. The prevalence of Haemoglobin S was lower as compared to other studies like Dash et al. [10] (29%) and Balgair et al. [9] (39.3%). However, the most common type of Haemoglobin S seen was sickle cell trait Haemoglobin AS which was similar to other studies. Cases of sickle beta thalassemia were anaemic with PBS picture of microcytic hypochromic anaemia, whereas sickle cell trait was asymptomatic.

One case of beta thalassemia major was seen which was having significant anaemia and haemoglobin of 6.2 g/dl on repeated blood transfusion with typical chromatogram of Haemoglobin F as most prominent band.

One case of Haemoglobin E trait was identified which was clinically asymptomatic and haematologically normal with only prominent finding of HaemoglobinA₂ 26.4% and based on retention time diagnosis of Haemoglobin E was made.

Conclusion

Beta thalassemia trait is very common in most parts of India, which was also seen in this study. The condition although is asymptomatic but holds significant clinical outcome if both parents are beta thalassemia trait and the child becomes beta thalassemia major, a significant clinical, mental, and financial burden to the whole family. Hence, the identification of beta thalassemia trait, evaluation, and genetic counselling can help and guide the patient and family, similarly this is true for all haemoglobinopathies. The novelty of the study is that it addresses to an extent the prevalence of haemoglobinopathy amongst the local population accessing the tertiary care centre and help provide necessary services.

HPLC is a simple, sensitive, and accurate method to identify Haemoglobin variant in an individual, but the chromatogram data should always be interpreted with clinical findings and family history of the patient.

Acknowledgements

The authors would like to acknowledge the institute and all concerned departments and staffs for sharing the clinical and laboratory data and their support.

Disclosure Statement

No potential conflict of interest was reported by the authors.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Authors' Contributions

All authors contributed to data analysis, drafting, and revising of the paper and agreed to be responsible for all the aspects of this work.

ORCID

Pradnya Phalak

https://orcid.org/0000-0003-4747-4302

Abhijit Pratap

https://orcid.org/0000-0001-8265-7687

Ethics approval and consent to participate

All clinical investigations must be conducted according to the Declaration of Helsinki principles. The authors must comply with the guidelines of the International Committee of Medical Journal Editors (www.icmje.org) with regard to the patient’s consent for research or participation in a study. Patients' names, initials, or hospital numbers must not be mentioned anywhere in the manuscript (including figures). Editors may request that authors provide documentation of the formal review and recommendation from the institutional review board or ethics committee responsible for oversight of the study.

HOW TO CITE THIS ARTICLE

Pradnya Phalak, Abhijit Pratap. Haemoglobin Variant Study by HPLC Method at a Tertiary Care Centre. J. Med. Chem. Sci., 2023, 6(11) 2841-2848.

DOI: https://doi.org/10.26655/JMCHEMSCI.2023.11.29

URL: https://www.jmchemsci.com/article_176193.html