Biochemical Genetics

, Volume 54, Issue 6, pp 816–825 | Cite as

Mass Spectrometry-Based Diagnosis of Hemoglobinopathies: A Potential Tool for the Screening of Genetic Disorder

  • Rajdeep Das
  • Gopa Mitra
  • Boby Mathew
  • Vijay Bhat
  • Cecil Ross
  • Debnath Pal
  • Amit Kumar MandalEmail author
Original Article


Hemoglobinopathies are caused by point mutation in globin gene that results in structural variant of hemoglobin. While 7 % of world populations are carrier of hemoglobinopathies, the prevalence of the disease varies between 3 to 17 % across different population groups in India. In a diagnostic laboratory, alkaline gel electrophoresis and cation exchange-based HPLC (CE-HPLC) are most widely used techniques for characterization of hemoglobin variants. In the above methods, the differential surface charge of hemoglobin molecule in variants is exploited for their characterization. Sometime, co-migration of variants in gel electrophoresis and co-elution or elution with unknown retention time in automated CE-HPLC might lead to ambiguity in the analysis of hemoglobinopathies. Under such circumstances, it is necessary to use other analytical methods that provide unambiguous results. Mass spectrometry-based proteomics approach and DNA sequence analysis are examples of such alternative methods. In the present study, liquid chromatography coupled to mass spectrometry has been used for three commonly observed variants in India, e.g., HbE, HbQ India and HbD Punjab that appeared with inappropriate results in the conventional analysis. A customized hemoglobin variant database has been used in the mass spectrometry-based analysis of those three variants. Mass spectrometry-based proteomics approach was used to analyze above variant sample accurately.


Hemoglobinopathy Mass spectrometry HbE HbQ India HbD Punjab 



We acknowledge DST (Government of India) for funding mass spectrometry facility at St. John’s Research Institute. We acknowledge DBT (Government of India) (BT/PR13926/MED/31/97/2010) for partially funding the study. Patients are acknowledged for providing sample. Rajdeep Das was supported with Senior Research Fellowship by ICMR (Government of India).

Compliance with Ethical Standards

Conflict of Interest

There is no conflict of interest among authors.


  1. Agarwal S, Gupta UR, Kohli N, Verma N, Agarwal SS (1989) Prevalence of haemoglobin D in Uttar Pradesh. Indian J Med Res 90:39–43PubMedGoogle Scholar
  2. Balgir SR (2000) The burden of haemoglobinopathies in India and the challenges ahead. Curr Sci 79:1536–1547Google Scholar
  3. Das R, Mitra G, Mathew B, Ross C, Bhat V, Mandal AK (2013) Automated analysis of hemoglobin variants using nanoLC − MS and customized databases. J Proteome Res 12:3215–3222CrossRefPubMedGoogle Scholar
  4. Das R, Muralidharan M, Mitra G, Bhat V, Mathew B, Pal D, Ross C, Mandal AK (2015) Mass spectrometry based characterization of Hb Beckman variant in a falsely elevated HbA1c sample. Anal Biochem 489:53–56CrossRefPubMedGoogle Scholar
  5. Deka R, Reddy AP, Mukherjee BN, Das BM, Banerjee S, Roy M, Dey B, Malhotra KC, Walter H (1988) Hemoglobin E distribution in ten endogamous population groups of Assam, India. Hum Hered 38:261–266CrossRefPubMedGoogle Scholar
  6. Dorn-Beineke A, Frietsch T (2002) Sickle cell disease—pathophysiology, clinical and diagnostic implications. Clin Chem Lab Med 40:1075–1084CrossRefPubMedGoogle Scholar
  7. Estey MP, Clarke G, Sia W, Toor E, Higgins TN (2015) A mother and newborn with brown blood. Clin Chem 61:466–470CrossRefPubMedGoogle Scholar
  8. Forget BG, Higgs DR, Steinberg M, Nagel RL (2001) Disorders of hemoglobin: genetics, pathophysiology and clinical management. Cambridge University Press, CambridgeGoogle Scholar
  9. Hardison RC, Chui DH, Riemer CR, Miller W, Carver M, Molchanova T, Efremov GD, Huisman TH (1998) Access to a syllabus of human hemoglobin variants (1996) via the World Wide Web. Hemoglobin 22:113–127CrossRefPubMedGoogle Scholar
  10. HbVar: a database of human hemoglobin variants and thalassemias. Available at: Accessed July 2015
  11. Joutovsky A, Hadzi-Nesic J, Nardi MA (2004) HPLC retention time as a diagnostic tool for hemoglobin variants and hemoglobinopathies: a study of 60,000 samples in a clinical diagnostic laboratory. Clin Chem 50:1736–1747CrossRefPubMedGoogle Scholar
  12. Lukens JN (1998) The abnormal hemoglobins: general principles. In: Lee GR, Foerster J, Lukens J, Paraskevas F, Greer JP, Rodgers GM (eds) Wintrobe’s clinical hematology, 10th edn. Lippincott Williams & Wilkins, Baltimore, pp 1329–1345Google Scholar
  13. Mandal AK, Bisht S, Bhat SV, Krishnaswamy PR, Balaram P (2008) Electrospray mass spectrometric characterization of hemoglobin Q (Hb Q-India) and a double mutant hemoglobin S/D in clinical samples. Clin Biochem 41:75–81CrossRefPubMedGoogle Scholar
  14. Mathew B, Bhat V, Mandal AK (2011) Analysis of hemoglobin variants using nondenaturing gel electrophoresis and matrix-assisted laser desorption ionization mass spectrometry. Anal Biochem 416:135–137CrossRefPubMedGoogle Scholar
  15. Old J, Harteveld CL, Traeger-Synodinos J, Petrou M, Angastiniotis M, Galanello R (2012) Prevention of thalassaemias and other haemoglobin disorders: volume 2: laboratory protocols, 2nd edn. Thalassaemia International Federation, Nicosia, CyprusGoogle Scholar
  16. Panigrahi I, Bajaj J, Chatterjee T, Saxena R, Mahapatra M, Pati HP (2005) Hb Q India: is it always benign? Am J Hematol 78:245–246CrossRefPubMedGoogle Scholar
  17. Patel AP, Naik MR, Shah NM, Sharma NP, Parmar PH (2012) Prevalence of common hemoglobinpathies in Gujarat: an analysis of a large population screening program. Natl J Comm Med 3:112–116Google Scholar
  18. Piplani S (2000) Hemoglobin E disorders in North East India. J Assoc Phys India 48:1082–1084Google Scholar
  19. Riou J, Godart C, Hurtrel D, Mathis M, Bimet C, Bardakdjian-Michau J, Préhu C, Wajcman H, Galactéros F (1997) Cation-exchange HPLC evaluated for presumptive identification of hemoglobin variants. Clin Chem 43:34–39PubMedGoogle Scholar
  20. Steinberg MH, Adams JG 3rd (1991) Hemoglobin A2: origin, evolution, and aftermath. Blood 78:2165–2177PubMedGoogle Scholar
  21. Wajcman H, Préhu C, Bardakdjian-Michau J, Promé D, Riou J, Godart C, Mathis M, Hurtrel D, Galactéros F (2001) Abnormal hemoglobins: laboratory methods. Hemoglobin 25:169–181CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Rajdeep Das
    • 1
  • Gopa Mitra
    • 1
  • Boby Mathew
    • 1
  • Vijay Bhat
    • 2
  • Cecil Ross
    • 3
  • Debnath Pal
    • 4
  • Amit Kumar Mandal
    • 1
    Email author
  1. 1.Clinical Proteomics Unit, Division of Molecular Medicine, St. John’s Research InstituteSt. John’s National Academy of Health SciencesBangaloreIndia
  2. 2.Manipal HospitalBangaloreIndia
  3. 3.Department of Medicine, St. John’s Medical College and HospitalSt. John’s National Academy of Health SciencesBangaloreIndia
  4. 4.Supercomputer Education and Research CentreIndian Institute of ScienceBangaloreIndia

Personalised recommendations