Analysis of Double Heterozygous Haemoglobinopathies from a Tertiary Care Center in North India

Background: Haemoglobinopathies and thalassemias are inherited conditions, being diagnosed with increasing prevalence in India. The double heterozygosity for α and β chain variants leads to the formation of abnormal heterodimer hybrids, which can lead to diagnostic dilemmas. Hematological parameters of double heterozygous conditions have not been analysed much in the literature. Methods: This study is a retrospective analysis of haemoglobin High Performance Liquid Chromatography (HPLC) from January 2006 to August 2014. Hematological parameters of these patients were also analysed and correlated with respective haemoglobin HPLC findings. Family screening of cases was also done wherever possible. Result: Out of 6180 cases, 14 cases were found to be of double heterozygous with 10 cases of Hb E-β thalassemias, and 4 cases of Hb S-β thalassemias. In Hb E-β thalassemias, significant negative correlation was noted between haemoglobin and Red cell Distribution Width (RDW) and also between RDW and Red Blood Cell (RBC) count. In Hb S-β thalassemias, significant negative correlation was seen between Hb A2 level and RBC count. Conclusion: Although, haemoglobin chain disorders require combination of techniques, HPLC is a cost effective and powerful tool for characterization of these disorders. This study also highlights the importance of hematological parameters (Hb, RDW and RBC count) in elucidation of double heterozygous haemoglobinopathies from much commoner variants of haemoglobinopathies, particularly in under resourced areas. No similar studies correlating HPLC findings and RBC indices have been found in the literature.

Pearson correlation between RBC count and RDW showed a significant negative correlation (p value < 0.05). Pearson correlation between Hb level and RDW also showed a significant negative correlation (p value < 0.05).
Hb S-β thalassemia: Patient showing a peak at the S-window with increased Hb A2 (3.5-5.5%) was labelled as a double heterozygous state of Hb-S and β-thalassemia. [4] In the present study, 4 (0.06%) patients were diagnosed as Hb S-β thalassemias.

Discussion
The coinheritance of two different haemoglobinopathies is a rare occurrence. Although, they superficially resemble the non-coinherited variants, they show subtle variations in clinical, laboratory and management aspects. Among the laboratory investigations, HPLC is useful in presumptive diagnosis of various haemoglobinopathies. Although, DNA based techniques are needed for better delineation of these disorders, HPLC analysis in conjunction with clinical profile and different haematological parameters is pertinent for evaluation of double heterozygous haemoglobinopathies.
The cumulative frequency of haemoglobinopathies in India has been found to be 4.2% in a previous study. [5] A comparable frequency of haemoglobin chain disorders (4.02%) was observed in our study.
Hb E-β Thalassemia: Patients having double heterozygous state of Hb E and β-thalassemia is characterized by marked clinical variability ranging from mild and asymptomatic anaemia to a life-threatening disorder requiring transfusions from infancy. [6] This is different from other double heterozygous states for structural β-chain variants and β-thalassemia. A study by Mohanty D et al [7] reported a prevalence of 0.19% which is similar to the present study (0.16%). Higher prevalence (2.14%) noted in the study by Baruah MK et al [8] is due to regional variation of haemoglobin disorders in India (Table 2). Mean values of HbA2 and Hb F in this study were similar to another study by Baruah MK et al [8] (Table 3). In addition, there was a negative correlation between RDW and Hb and also between RDW and RBC count (p value < 0.05). However, no studies correlating HPLC findings and RBC indices have been found in the literature. Family study of two cases in the same family has been depicted in figure 2. Similar family study for diagnosis of double heterozygous conditions has also been used in other studies. [1,9] Hb S-β Thalassemia : Hb S-β thalassemia is a double heterozygous state of Hb S and β-thalassemia. The clinical course is similar to that of Hb SS, however anaemia is comparatively milder than in sickle cell anaemia [4] . A study by Mohanty D et al [7] reported a prevalence of 0.02% which compares favourable with our study (0.06%).
Higher prevalence (0.6%) noted in the study by Baruah MK et al [8] is due to regional variation of haemoglobin  (Table 2). Previous study showed mean values of MCV for Hb S-β + thalassemias and Hb S-β 0 thalassemias to be 72 fl and 67.8 fl respectively [4] and a comparable mean value of MCV of 69.4 fl was noted in our study. Mean values of Hb S, HbA2 and Hb F in this study were similar to another study by Baruah MK et al [8] (Table  3). There was a negative correlation between Hb A2 and RBC count as well as between Hb A2 and HCT (p value < 0.05). No similar studies correlating these parameters have been found in the literature.

Conclusion
In conclusion, rarity of these disorders makes the diagnosis challenging. Haemoglobin chain disorders require a combination of techniques including HPLC, which is one of the most important diagnostic modality for elucidation of double heterozygous cases. Diagnostic difficulties are encountered by overlapping laboratory findings, prompting the search for newer parameters. Hematological parameters like haemoglobin level, RBC indices, and Hb A2 level and their correlation are important for characterization of double heterozygous state. This study highlights the significant negative correlation between RDW and Hb as well as RDW and RBC count in double heterozygous cases of Hb E-β thalassemia (p value <0.05), which helps particularly in elucidation of challenging cases. Role of family screening in a suspected case is also crucial. To the best of our knowledge, correlation of different hematological parameters for characterization of double heterozygous haemoglobinopathies has not been reported so far in the Indian literature. However, further studies including larger number of cases are required to validate the statistical correlation. This may unmask significant haematological parameters, which lead to better diagnostic evaluation and understanding of these disorders particularly in under resourced areas.