Skip to main content

Advertisement

SpringerLink
Log in

Laboratory Approach to Hemolytic Anemia

  • Review Article
  • Published:
The Indian Journal of Pediatrics Aims and scope Submit manuscript

Abstract

Hemolytic anemias are a group of disorders with varied clinical and molecular heterogeneity. They are characterized by decreased levels of circulating erythrocytes in blood. The pathognomic finding is a reduced red cell life span with severe anemia or, compensated hemolysis accompanied by reticulocytosis. The diagnostic workup or laboratory approach for hemolytic anemias is based on methodical step-wise testing which includes red blood cell morphology, hematological indices with increased reticulocyte count along with clinical features of hemolytic anemias. If conventional laboratory tests are unable to detect the underlying cause of hemolysis, genetic testing is recommended. Sanger sequencing along with conventional testing is the most efficient way to diagnose the underlying genetic causes, especially in thalassemias/hemoglobinopathies, if required. However, hemolytic anemias being highly heterogeneous disorders, next-generation sequencing-based screening is rapidly becoming an efficient way to decipher the etiologies where common causes have been excluded.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  1. Siddon AJ, Tormey CA. Chapter Six: the chemical and laboratory investigation of hemolysis. Adv Clin Chem. 2019;89:215–58.

    PubMed  Google Scholar 

  2. Rets A, Clayton AL, Christensen RD, Agarwal AM. Molecular diagnostic update in hereditary hemolytic anemia and neonatal hyperbilirubinemia. Int J Lab Hematol. 2019;41:95–101.

    PubMed  Google Scholar 

  3. Sachdeva MUS, Varma N, Chandra D, Bose P, Malhotra P, Varma S. Multiparameter FLAER-based flow cytometry for screening of paroxysmal nocturnal hemoglobinuria enhances detection rates in patients with aplastic anemia. Ann Hematol. 2015;94:721–8.

    CAS  PubMed  Google Scholar 

  4. Higgs DR. The molecular basis of α-thalassemia. Cold Spring Harb Perspect Med. 2013;3:a011718.

    PubMed  PubMed Central  Google Scholar 

  5. Sharma P, Das R. The pursuit of rare hemoglobins. Indian J Pathol Microbiol. 2016;59:102–3.

    PubMed  Google Scholar 

  6. Sharma P, Das R, Trehan A, et al. Impact of iron deficiency on hemoglobin A2% in obligate β-thalassemia heterozygotes. Int J Lab Hematol. 2015;37:105–11.

  7. Sharma P, Das R. Cation-exchange high-performance liquid chromatography for variant hemoglobins and HbF/A2: what must hematopathologists know about methodology? World J Methodol. 2016;6:20–4.

    PubMed  PubMed Central  Google Scholar 

  8. Cappellini MD, Fiorelli G. Glucose-6-phosphate dehydrogenase deficiency. Lancet. 2008;371:64–74.

    CAS  PubMed  Google Scholar 

  9. Brandt O, Rieger A, Geusau A, Stingl G. Peas, beans, and the pythagorean theorem - the relevance of glucose-6-phosphate dehydrogenase deficiency in dermatology. J Dtsch Dermatol Ges. 2008;6:534–9.

    PubMed  Google Scholar 

  10. World Health Organisation. Glucose-6-phosphate dehydrogenase deficiency. WHO Working Group. Bull World Health Organ. 1989;67:601–11.

  11. Beutler E, Kuhl W, Gelbart T, Forman L. DNA sequence abnormalities of human glucose-6-phosphate dehydrogenase variants. J Biol Chem. 1991;266:4145–50.

    CAS  PubMed  Google Scholar 

  12. Canu G, De Bonis M, Minucci A, Capoluongo E. Red blood cell PK deficiency: an update of PK-LR gene mutation database. Blood Cells Mol Dis. 2016;57:100–9.

    CAS  PubMed  Google Scholar 

  13. Manco L, Ribeiro ML, Almeida H, Freitas O, Abade A, Tamagnini G. PK-LR gene mutations in pyruvate kinase deficient portuguese patients. Br J Haematol. 1999;105:591–5.

    CAS  PubMed  Google Scholar 

  14. Fermo E, Bianchi P, Chiarelli LR, et al. Red cell pyruvate kinase deficiency: 17 new mutations of the PK-LR gene. Br J Haematol. 2005;129:839–46.

    CAS  PubMed  Google Scholar 

  15. Zanella A, Fermo E, Bianchi P, Valentini G. Red cell pyruvate kinase deficiency: molecular and clinical aspects. Br J Haematol. 2005;130:11–25.

    CAS  PubMed  Google Scholar 

  16. Marcello AP, Vercellati C, Fermo E, et al. A case of congenital red cell pyruvate kinase deficiency associated with hereditary stomatocytosis. Blood Cells Mol Dis. 2008;41:261–2.

    CAS  PubMed  Google Scholar 

  17. Jamwal M, Aggarwal A, Das A, et al. Next-generation sequencing unravels homozygous mutation in glucose-6-phosphate isomerase, GPIc.1040G>A (p.Arg347His) causing hemolysis in an Indian infant. Clin Chim Acta. 2017;468:81–4.

    CAS  Google Scholar 

  18. Kedar PS, Gupta V, Dongerdiye R, Chiddarwar A, Warang P, Madkaikar MR. Molecular diagnosis of unexplained haemolytic anaemia using targeted next-generation sequencing panel revealed (p.Ala337Thr) novel mutation in GPI gene in two Indian patients. J Clin Pathol. 2019;72:81–5.

    CAS  PubMed  Google Scholar 

  19. Jamwal M, Aggarwal A, Palodi A, et al. A nonsense variant in the hexokinase 1 gene (HK1) causing severe non-spherocytic haemolytic anaemia: genetic analysis exemplifies ambiguity due to multiple isoforms. Br J Haematol. 2019;186:e142–5.

    PubMed  Google Scholar 

  20. Koralkova P, Van Solinge WW, Van Wijk R. Rare hereditary red blood cell enzymopathies associated with hemolytic anemia - pathophysiology, clinical aspects, and laboratory diagnosis. Int J Lab Hematol. 2014;36:388–97.

    CAS  PubMed  Google Scholar 

  21. Bolton-Maggs PHB, Langer JC, Iolascon A, Tittensor P, King MJ. Guidelines for the diagnosis and management of hereditary spherocytosis - 2011 update. Br J Haematol. 2012;156:37–49.

    PubMed  Google Scholar 

  22. Bianchi P, Fermo E, Vercellati C, et al. Diagnostic power of laboratory tests for hereditary spherocytosis: a comparison study in 150 patients grouped according to molecular and clinical characteristics. Haematologica. 2012;97:516–23.

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Joshi P, Aggarwal A, Jamwal M, et al. A comparative evaluation of eosin-5′-maleimide flow cytometry reveals a high diagnostic efficacy for hereditary spherocytosis. Int J Lab Hematol. 2016;38:520–6.

    CAS  PubMed  Google Scholar 

  24. King M-J, Telfer P, MacKinnon H, et al. Using the eosin-5-maleimide binding test in the differential diagnosis of hereditary spherocytosis and hereditary pyropoikilocytosis. Clin Cytom. 2008;74B:244–50.

    CAS  Google Scholar 

  25. Suemori S, Wada H, Nakanishi H, Tsujioka T, Sugihara T, Tohyama K. Analysis of hereditary elliptocytosis with decreased binding of eosin-5-maleimide to red blood cells. Biomed Res Int. 2015;2015:451861.

    PubMed  PubMed Central  Google Scholar 

  26. King MJ, Behrens J, Rogers C, Flynn C, Greenwood D, Chambers K. Rapid flow cytometric test for the diagnosis of membrane cytoskeleton-associated haemolytic anaemia. Br J Haematol. 2000;111:924–33.

    CAS  PubMed  Google Scholar 

  27. Jamwal M, Aggarwal A, Kumar V, et al. Disease-modifying influences of coexistent G6PD-deficiency, gilbert syndrome and deletional alpha thalassemia in hereditary spherocytosis: a report of three cases. Clin Chim Acta. 2016;458:51–4.

    CAS  PubMed  Google Scholar 

  28. Hunt L, Greenwood D, Heimpel H, Noel N, Whiteway A, King MJ. Toward the harmonization of result presentation for the eosin-5′-maleimide binding test in the diagnosis of hereditary spherocytosis. Cytometry B Clin Cytom. 2015;88B:50–7.

  29. Yawata Y. Characteristics of red cell membrane disorders in the Japanese population. Rinsho Byori. 1997;45:367–76.

    CAS  PubMed  Google Scholar 

  30. Facer CA. Erythrocytes carrying mutations in spectrin and protein 4.1 show differing sensitivities to invasion by plasmodium falciparum. Parasitol Res. 1995;81:52–7.

    CAS  PubMed  Google Scholar 

  31. Gallagher PG. Abnormalities of the erythrocyte membrane. Pediatr Clin N Am. 2013;60:1349–62.

    Google Scholar 

  32. King MJ, Garçon L, Hoyer JD, et al. ICSH guidelines for the laboratory diagnosis of nonimmune hereditary red cell membrane disorders. Int J Lab Hematol. 2015;37:304–25.

    PubMed  Google Scholar 

  33. Kedar PS, Colah RB, Kulkarni S, Ghosh K, Mohanty D. Experience with eosin-5′-maleimide as a diagnostic tool for red cell membrane cytoskeleton disorders. Clin Lab Haematol. 2003;25:373–6.

    CAS  PubMed  Google Scholar 

  34. King MJ, Zanella A. Hereditary red cell membrane disorders and laboratory diagnostic testing. Int J Lab Hematol. 2013;35:237–43.

    PubMed  Google Scholar 

  35. King MJ, Jepson MA, Guest A, Mushens R. Detection of hereditary pyropoikilocytosis by the eosin-5-maleimide (EMA)-binding test is attributable to a marked reduction in EMA-reactive transmembrane proteins. Int J Lab Hematol. 2011;33:205–11.

    PubMed  Google Scholar 

  36. Wrong O, Bruce LJ, Unwin RJ, Toye AM, Tanner MJA. Band 3 mutations, distal renal tubular acidosis, and southeast Asian ovalocytosis. Kidney Int. 2002;62:10–9.

    CAS  PubMed  Google Scholar 

  37. Reardon DM, Seymour CA, Cox TM, Pinder JC, Schofield AE, Tanner MJA. Hereditary ovalocytosis with compensated haemolysis. Br J Haematol. 1993;85:197–9.

    CAS  PubMed  Google Scholar 

  38. Rasool J, Manzoor F, Bhat S, Bashir N, Geelani S. Hereditary stomatocytosis: first case report from valley of Kashmir. Med J Dr DY Patil Univ. 2015;8:347–9.

    Google Scholar 

  39. Jamwal M, Aggarwal A, Sachdeva MUS, et al. Overhydrated stomatocytosis associated with a complex RHAG genotype ancluding a novel de novo mutation. J Clin Pathol. 2018;71:648–52.

    CAS  PubMed  Google Scholar 

  40. Stewart GW, Amess JA, Eber SW, et al. Thrombo-embolic disease after splenectomy for hereditary stomatocytosis. Br J Haematol. 1996;93:303–10.

    CAS  PubMed  Google Scholar 

  41. Rees DC, Iolascon A, Carella M, et al. Stomatocytic haemolysis and macrothrombocytopenia (mediterranean stomatocytosis/macrothrombocytopenia) is the haematological presentation of phytosterolaemia. Br J Haematol. 2005;130:297–309.

    CAS  PubMed  Google Scholar 

  42. Jamwal M, Aggarwal A, Maitra A, et al. First report of mediterranean stomatocytosis/macrothrombocytopenia in an Indian family: a diagnostic dilemma. Pathology. 2017;49:811–5.

    PubMed  Google Scholar 

  43. Aggarwal A, Jamwal M, Das R. Molecular genetics of inherited red cell membrane disorders. In: Saxena R, Pati H, editors. Hematopathology. Singapore: Springer Singapore; 2019. p. 77–90.

    Google Scholar 

  44. Stitziel NO, Kiezun A, Sunyaev S. Computational and statistical approaches to analyzing variants identified by exome sequencing. Genome Biol. 2011;12:227.

    PubMed  PubMed Central  Google Scholar 

  45. Biesecker LG, Burke W, Kohane I, Plon SE, Zimmern R. Next-generation sequencing in the clinic: are we ready? Nat Rev Genet. 2012;13:818–24.

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Agarwal AM, Nussenzveig RH, Reading NS, et al. Clinical utility of next-generation sequencing in the diagnosis of hereditary haemolytic anaemias. Br J Haematol. 2016;174:806–14.

    CAS  PubMed  Google Scholar 

  47. Russo R, Andolfo I, Manna F, et al. Multi-gene panel testing improves diagnosis and management of patients with hereditary anemias. Am J Hematol. 2018;93:672–82.

    CAS  PubMed  Google Scholar 

  48. Roy NBA, Wilson EA, Henderson S, et al. A novel 33-gene targeted resequencing panel provides accurate, clinical-grade diagnosis and improves patient management for rare inherited anaemias. Br J Haematol. 2016;175:318–30.

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Sun Y, Ruivenkamp CAL, Hoffer MJV, et al. Next-generation diagnostics: gene panel, exome, or whole genome? Hum Mutat. 2015;36:648–55.

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The authors thank the clinical and laboratory staff involved in patient management and laboratory diagnosis. They thank the PGIMER, Chandigarh; Department of Biotechnology (Ministry of Science and Technology, Government of India), New Delhi and Indian Council for Medical Research, New Delhi for various institutional and extramural research grants.

Author information

Authors and Affiliations

  1. Department of Hematology, Post Graduate Institute of Medical Education and Research, Chandigarh, 160012, India

    Manu Jamwal, Prashant Sharma & Reena Das

Authors
  1. Manu Jamwal
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Prashant Sharma
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Reena Das
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

MJ, PS and RD have written the manuscript and all authors approve the final version of the article. RD is the gauarantor for this paper.

Corresponding author

Correspondence to Reena Das.

Ethics declarations

Conflict of Interest

None.

Source of Funding

The authors thank the PGIMER, Chandigarh; Department of Biotechnology (Ministry of Science and Technology, Government of India), New Delhi and Indian Council for Medical Research, New Delhi for various institutional and extramural research grants.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Jamwal, M., Sharma, P. & Das, R. Laboratory Approach to Hemolytic Anemia. Indian J Pediatr 87, 66–74 (2020). https://doi.org/10.1007/s12098-019-03119-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12098-019-03119-8

Keywords

Search

Navigation