Methods for hemoglobin analysis and hemoglobinopathy testing
- Carolyn Hoppe, MD
Carolyn Hoppe, MD
- Associate Hematologist/Oncologist, UCSF Benioff Children's Hospital Oakland
- Associate Scientist, Children's Hospital Oakland Research Institute
- Director, Hemoglobinopathy Reference Laboratory
Hemoglobinopathies are inherited disorders in which a genetic abnormality affects the amount or structure of one or more of the globin chains in hemoglobin (Hb), the major oxygen transport protein in red blood cells (RBCs). The most common hemoglobinopathies such as thalassemia and sickle cell disease (SCD) are relatively straightforward to diagnose. However, there are thousands of other genetic abnormalities in the globin genes, the majority of which are clinically silent; for many of these, characterizing the abnormality and its clinical implications is more challenging. It is important to identify clinically significant hemoglobinopathies causing SCD and thalassemia to inform optimal management in affected individuals and to offer genetic counseling and reproductive options in carriers.
This topic reviews methods for hemoglobinopathy testing and their appropriate use. Additional information about the diagnosis of specific disorders and an overview of prenatal hemoglobinopathy testing are presented separately:
●Sickle cell trait and SCD – (See "Diagnosis of sickle cell disorders".)
●Alpha and beta thalassemias – (See "Clinical manifestations and diagnosis of the thalassemias".)
●Unstable hemoglobin variants – (See "Unstable hemoglobin variants".)
- Weatherall DJ. The New Genetics and Clinical Practice, Oxford University Press, Oxford 1991.
- Natarajan K, Townes TM, Kutlar A. Disorders of hemoglobin structure: sickle cell anemia and related abnormalities. In: Williams Hematology, 8th ed, Kaushansky K, Lichtman MA, Beutler E, et al. (Eds), McGraw-Hill, 2010. p.ch.48.
- Huisman TH. The structure and function of normal and abnormal haemoglobins. In: Bailliere's Clinical Haematology, Higgs DR, Weatherall DJ (Eds), W.B. Saunders, London 1993. p.1.
- Trent RJ. Diagnosis of the haemoglobinopathies. Clin Biochem Rev 2006; 27:27.
- https://www.cdc.gov/ncbddd/sicklecell/documents/nbs_hemoglobinopathy-testing_122015.pdf (Accessed on May 31, 2016).
- Kanter J, Telen MJ, Hoppe C, et al. Validation of a novel point of care testing device for sickle cell disease. BMC Med 2015; 13:225.
- McGann PT, Schaefer BA, Paniagua M, et al. Characteristics of a rapid, point-of-care lateral flow immunoassay for the diagnosis of sickle cell disease. Am J Hematol 2016; 91:205.
- Quinn CT, Paniagua MC, DiNello RK, et al. A rapid, inexpensive and disposable point-of-care blood test for sickle cell disease using novel, highly specific monoclonal antibodies. Br J Haematol 2016; 175:724.
- Lorey F, Cunningham G, Vichinsky EP, et al. Universal newborn screening for Hb H disease in California. Genet Test 2001; 5:93.
- Harthoorn-Lasthuizen EJ, Lindemans J, Langenhuijsen MM. Influence of iron deficiency anaemia on haemoglobin A2 levels: possible consequences for beta-thalassaemia screening. Scand J Clin Lab Invest 1999; 59:65.
- El-Agouza I, Abu Shahla A, Sirdah M. The effect of iron deficiency anaemia on the levels of haemoglobin subtypes: possible consequences for clinical diagnosis. Clin Lab Haematol 2002; 24:285.
- Mosca A, Paleari R, Ivaldi G, et al. The role of haemoglobin A(2) testing in the diagnosis of thalassaemias and related haemoglobinopathies. J Clin Pathol 2009; 62:13.
- Lawrie D, Glencross DK. Use of the Mentzer index will assist in early diagnosis of iron deficiency in South African children. S Afr Med J 2015; 105:702.
- Bruccoleri F, Zepponi E, Balucanti F, et al. [Determination of the diagnostic value of erythrocyte discrimination indices (Mentzer and Srivastava) and of glycerol lysis time (GLT 50) in microcytosis]. Quad Sclavo Diagn 1982; 18:67.
- Demir A, Yarali N, Fisgin T, et al. Most reliable indices in differentiation between thalassemia trait and iron deficiency anemia. Pediatr Int 2002; 44:612.
- Beyan C, Kaptan K, Ifran A. Predictive value of discrimination indices in differential diagnosis of iron deficiency anemia and beta-thalassemia trait. Eur J Haematol 2007; 78:524.
- Mentzer WC Jr. Differentiation of iron deficiency from thalassaemia trait. Lancet 1973; 1:882.
- Kutlar F. Diagnostic approach to hemoglobinopathies. Hemoglobin 2007; 31:243.
- Huisman TH. Introduction and review of standard methodology for the detection of hemoglobin abnormalities. In: The Hemoglobinopathies, Huisman TH (Ed), Churchill Livingstone, Edinburgh 1986. p.1.
- HEMOGLOBIN BIOLOGY
- Normal hemoglobins
- Types of abnormalities
- AVAILABLE TESTING METHODS
- Overview of methods
- Protein chemistry methods
- - Overview of protein chemistry methods
- - HPLC
- - Isoelectric focusing
- - Capillary electrophoresis
- - Gel electrophoresis
- Molecular (DNA-based) methods
- - Overview of DNA-based methods
- - Traditional DNA sequencing and allele-specific PCR
- - Gap-PCR and MLPA
- - Array CGH
- - NGS
- Point-of-care assays
- Other disease-specific tests
- - Sickle cell prep
- - Heinz body prep
- - Hb H staining
- INITIAL EVALUATION
- Indications for hemoglobin analysis
- Overview of approach
- Clues from the CBC
- Common clinical scenarios
- - Population screening (eg, routine newborn screen)
- - Prenatal testing
- - Patient with suspected sickle cell disorder
- - Patient with suspected thalassemia
- - Patient with unknown variant
- REFERRAL TO A SPECIALIZED LABORATORY
- SOCIETY GUIDELINE LINKS
- SUMMARY AND RECOMMENDATIONS