Accidental detection of clinically silent compound heterozygous Hb D Punjab/Hb Q India while analyzing HbA1c by high performance liquid chromatography

HbA1c is routinely prescribed investigations for diagnosing and monitoring diabetes and high-performance liquid chromatography (HPLC) is preferred method which is also able to identify presence of hemoglobin variant. A case was encountered where presence of variant hemoglobin was indicated. On further investigation with three different instruments, diagnosis of compound heterozygous Hb D Punjab/Hb Q India was made. The chromatogram on Bio-Rad D10 showed Hb D Punjab (ααββHbD Punjab)-29.89% at 3.96 minutes retention time (RT), Hb Q India (ααHbQ Indiaββ) -9.5% with 4.45 minutes RT, hybrid of HbQ India/Hb D Punjab (ααHbQ IndiaββHbD Punjab)-6% with 4.66 minutes RT, Hb A2 (ααδδ) was 2.5% and Hb A (ααββ) was 52.2%. Analysis done on Bio-Rad variant V-II confirmed these findings. Analysis done on Sebia capillary electrophoresis revealed major peak of 50.9% in zone 9/Z(A) constituted by Hb A, second peak of 39.8% in zone 6/Z(D) constituted by co-elution of Hb D and Hb Q India, third peak of 8.8% in zone 3-4/Z(A2-C) constituted by co-elution of Hb A2 and hybrid of Hb D Punjab/Hb Q India and a fourth peak of 0.5% in zone 1 representing Hb A2HbQ India (ααHbQ Indiaδδ). Ideally variants detected while analyzing HbA1c should be further investigated for confirmation and result of which should be shared, discussed and the patient should be encouraged for screening of available family members for relevant variant hemoglobin. Combination of cation exchange HPLC and capillary electrophoresis in this case was sufficient to arrive at conclusion.

A Novel Association of Hbq India Trait with Sickle Cell Anemia: A New Insight in Hemoglobinopathies

HbQ India is a rare α-chain structural hemoglobinopathy usually asymptomatic and presents in the heterozygous form or co-inherited with β-thalassemia trait. Herein, we are reporting the third case of novel association of HbQ India with HbS trait hemoglobinopathy in a 30-years-old young male presented with chief complaints of yellowish discoloration of sclera since 5-years with raised serum bilirubin levels along with pedigree analysis of the family.

HBQ-India: an uncommon hemoglobin variant

Background: HbQ-India is a rare alpha chain variant. It is an important member of the hemoglobin Q family molecularly characterized by replacement of aspartic acid by histidine. It usually presents in the heterozygous state. It becomes symptomatic only in the homozygous state and when present in association with other conditions like beta-thalassaemia, alpha thalassaemia, HbE and HbH. The Sindhi is one of the largest linguistic communities, migrated about 65 years back from the Sindh province of west Pakistan to India. They are a high-risk community for beta thalassaemia gene in India with a carrier frequency ranging from 5 to 12 % with a distinct regional variability.Methods: A total 343 cases were screened for hemoglobinopathies in Sindhi population. Detail history was taken from each patient and pertinent physical finding were noted. CBC, Peripheral smear and HPLC were performed. During screening we observed that few samples showed an unknown peak at a retention time of 4.7 min on HPLC and comparison with reference chromatograms indicated them to be HbQ India and it is confirmed by amplification restriction mutation system polymerase chain reaction (ARMS-PCR).Results: We found 13 cases, 12 cases of HbQ India and 1 case of HbQ-beta thalassaemia in Sindhi population of Aurangabad in Maharashtra.Conclusions: India is known as a country with a high prevalence of different types of hemoglobinopathy. Now a days HPLC, IEF, ARMS-PCR, DNA sequencing are the methods available for the diagnosis of the abnormal Hb like HbQ-lndia. HPLC is a cheaper alternative to ARMS-PCR in the detection of rare hemoglobinopathies.

Hemoglobin Patterns in Sickle Cell Hemoglobinopathies- A Large Prospective Study in North Maharashtra

Introduction: Sickle cell haemoglobinopathy is an inherited hemoglobinopathy resulting from a mutation occurring in betaglobin gene, on chromosome 11. The gene is prevalent in some tribes of North Maharashtra. The main aim of the study is to determine haemoglobin patterns in cases with sickle cell hemoglobinoathies in North Maharashtra using HPLC testing system. Material and Methods: This is a prospective study done over a period of 6 years. 10081 patients having positive solubility test or negative solubility test but having clinical suspicion of Sickle cell hemoglobinopathies were studied in detail and all samples were subjected for HPLC testing. Results: Prevalence of sickle cell hemoglobinopathy in this study was 70.36%. Most common pattern of haemoglobin observed was SA (89.72%). A slight female preponderance (54%) was noted. Predominant age group was paediatric (39.96%), followed by12-20yrs (33.97%). Oldest case for HbSS was 55yrs male. Predominant category affected was ST (82.05%). Conclusion: A very high prevalence of Sickle cell hemoglobinopathy was noted in this study. This is because the study was done in areas where Pawara and Bhill community resides who have a high frequency of HbS gene. Solubility test was found to be cost effective and easy screening test (Sensitivity being70.36%). HPLC found to be Rapid and accurate test for diagnosis of hemoglobinopathy and had helped in diagnosis of some rare heterozygous disorders like SA-HBQ India, SA-Hereditary persistence of foetal haemoglobin, HBD-SA. This is one of the largest and first of its kind prospective study which will help in prevention and cost effective management in targeted population.

HbD Punjab/HbQ India compound heterozygosity: An unusual association.

Background: Haemoglobinopathies are the commonest hereditary disorders in India and pose a major health problem. Both beta thalassaemia and structural haemoglobin variants are relatively common in north western India. Here we report a 29 year old Sindhi female who was referred to us for a haemoglobinopathy work up and genetic counseling since her spouse was a classical beta thalassaemia carrier. Method: A complete blood count was done on an automated cell counter. Haemoglobin analysis was carried out using HPLC Variant Haemoglobin Testing System. The cellulose acetate electrophoresis was carried out [pH 8.9]. Confirmation of mutations was done by automated DNA sequencing. Results: HPLC analysis showed four major peaks, HbA0, a peak in the HbD window, an unknown peak [retention time 4.74 minutes] and a peak in the HbC window. The HbA2 level was 2.2% and the HbF level was 0.7%.Cellulose acetate electrophoresis at alkaline pH, a slow moving band was seen at the HbS/D position along with a prominent band at the HbA2 position. DNA sequencing of the β and α genes showed presence of the 2 hemoglobin variants :Hb D [b 121GAA à CAA] and Hb Q [a 64 AAG à GAG]. The δ globin gene was normal. The additional peak in the HbC window was due to the formation of a heterodimer hybrid. Conclusion: Both HbD Punjab and HbQ India are relatively common in India but their co-inheritance has not been described in the country. This is the second report of compound heterozygosity for HbQ India/HbD Punjab haemoglobinopathy globally, and the first one from India.