Hemoglobinopathies - Lab diagnosis

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Dr. Ankit RaiyaniHematology departmentSSH

2 pairs of globin chains each with a haem groupSeven different globin chainsTransient embryonic haemoglobins- Hb Gower 1(22), Hb Gower 2(22), Hb Portland 1(22) and Hb Portland 2Hb F(22) -predominant haemoglobin of fetal lifeHb A(22) -major haemoglobin found in children and adultsIn normal adults 96 98% of hemoglobin is HbA, Hb A2 (2 3%) and HbF (1000) Many are harmless, while some have serious clinical effects. Collectively, the clinical syndromes resulting from disorders of haemoglobin synthesis are referred to as haemoglobinopathies.

They can be grouped into three main categories:Those owing to structural variants of haemoglobin, such as Hb S.Those owing to failure to synthesize one or more of the globin chains of haemoglobin at a normal rate, as in the thalassaemias.Those owing to failure to complete the normal neonatal switch from fetal haemoglobin (Hb F) to adult haemoglobin (Hb A). Referred to as hereditary persistence of fetal haemoglobin (HPFH).

An individual can also have a combination of two or more of these abnormalities

Usually brought about by point mutations affecting one or, in some cases, two or more bases, coding for amino acids of the globin chains.Less commonly, structural change is caused by shortening or lengthening of the globin chain.Many variant haemoglobins are haematologically and clinically silent because the underlying mutation causes no alteration in the function, solubility or stability of the haemoglobin molecule. Many of these variants are separated using electrophoresis or chromatography, but some are not and remain undetected.


Hb D PunjabHb O Arab

Amino acid substitutions close to the haem group, or at the points of contact between globin chains, can affect protein stability and result in intracellular precipitation of globin chains.The precipitated globin chains attach to the red cell membrane giving rise to Heinz bodies, and the associated clinical syndromes were originally called thecongenital Heinz body haemolytic anaemias.Changes in membrane properties may lead to haemolysis, often aggravated by oxidant drugs.Heterogeneity- Many are almost silent and are detected only by specific tests, whereas others are severe, causing haemolytic anaemia in the heterozygous stateHb Kln is the most common variant in this rare group of disorders

Haemoglobin variants with altered oxygen affinity are a rare group of variants that result in increased or reduced oxygen affinity.Mutations that increase oxygen affinity are generally associated with benign lifelong erythrocytosis.Haemoglobin variants with decreased oxygen affinity are usually associated with mild anaemia and cyanosis. However, owing to the reduced oxygen affinity, these patients are not functionally anaemic despite the reduced Hb.

Such haemoglobins have a propensity to form methaemoglobin, generated by the oxidation of ferrous iron in haem to ferric iron, which is incapable of binding oxygen.Despite marked cyanosis, there are few clinical problems. Most are associated with substitutions that disrupt the normal six-ligand state of haem iron.

Methaemoglobinaemia is also found in congenital NADH methaemoglobin reductase deficiency, as well as after exposure to oxidant drugs and chemicals (nitrates, nitrites, quinones, chlorates, phenacetin, dapsone and many others).

Investigation of a person at risk of a haemoglobinopathy encompasses the confirmation or exclusion of the presence of a structural variant, thalassaemia trait or both.If a structural haemoglobin variant is present, it is necessary to ascertain the clinical significance of the particular variant so that the patient is appropriately managed. If it is confirmed that thalassaemia trait is present, it is not usually necessary to determine the precise mutation present because the clinical significance is usually negligible. Investigation of patients with a suspected haemoglobinopathy

The exception to this is an antenatal patient whose partner has also been found to have thalassaemia trait. If prenatal diagnosis is being considered, it may be necessary to undertake mutation analysis to predict fetal risk accurately and to facilitate prenatal diagnosis

In the majority of patients, the presence of a haemoglobinopathy can be diagnosed with sufficient accuracy for clinical purposes from knowledge of the patients ethnic origin and clinical history (including family history) and the results of physical examination combined with relatively simple haematological tests

Initial investigations should include determination of haemoglobin concentration and red cell indices. A detailed examination of a well-stained blood film should be carried out.In some instances, a reticulocyte count and a search for red cell inclusions give valuable information. Assessment of iron status by estimation of serum iron and total iron binding capacity and/or serum ferritin is sometimes necessary to exclude iron deficiency.

Other important basic tests are haemoglobin electrophoresis or high-performance liquid chromatography (HPLC), a sickle solubility test and measurement of Hb A2and Hb F percentage.

In cases of common haemoglobin variants and classicalthalassaemia trait, accurate data from these tests will facilitate a reliable diagnosis without the need for more sophisticated investigations. However, definitive diagnosis of some thalassaemia syndromes can only be obtained using DNA technologySimilarly, in particular situations, haemoglobin variants will require unequivocal identification by the use of DNA technology or protein analysis by mass spectrometry

Laboratory investigation of a suspected haemoglobinopathy should follow a defined protocol, which should be devised to suit individual local requirements. The data obtained from the clinical findings, blood picture and electrophoresis or HPLC will usually indicate in which direction to proceed.

1. Blood count and film examination2. Collection of blood and preparation of haemolysates 3. Cellulose acetate electrophoresis, Tris buffer, pH 8.5 4. Citrate agar or acid agarose gel electrophoresis, pH 6.0 5. Automated HPLC 6. IEF 7. Tests for Hb S 8. Detection of unstable haemoglobins

9. Detection of Hb Ms 10. Detection of altered affinity haemoglobins 11. Differentiation of common structural variants 12. Neonatal screening 13. Tests, such as zinc protoporphyrin estimation, to exclude iron deficiency as a cause of microcytosis14. Molecular techniques 15. Procedures for use in under-resourced laboratories

Suggested scheme of investigation for structural variants

Blood Count and FilmHemoglobinLow in thalassemia disease Hb < 6 g% in thal majorHb 6-10 g% in thal intermediaHb 10-12 g% in thal minor or thal traitNormal or slightly low in heterozygote ( Male ~15 g%, Female ~ 13 g%)MCV/MCHCut-off level : MCV 80 fl, MCH 20 pgLow in thalassemia diseases and thalassemia traitNormal or slightly low in a-thal-2 trait, HbE trait, Hb CS trait

RBC MorphologyThalassemia disease : Hypochromia, Anisocytosis, Poikilocytosis, Polychromasia, Target cells, Basophilic Stippling, NRBCThalassemia Trait and Homo E : Modest change in RBC morphology

CBCThe blood count, including Hb and red cell indices, provides valuable information useful in the diagnosis of bothandthalassaemia interactions with structural variantsThalassaemias are typically microcytic and hypochromic anemia's.Thalassemia causes a uniform microcytosis without increase in RDWHb H and thal however can cause an increased RDW.

PBS examination may reveal characteristic red cell changes such as target cells in Hb C trait, sickle cells in sickle cell disease and irregularly contracted cells in the presence of an unstable haemoglobinDiscriminant functions using various formulae have been proposed as a basis for further testing for thalassaemiaMentzer index- MCV / RBC count in million>13 s/o iron deficiency anaemia