Scheme for investigation of Haemoglobinopathies

Fig: Suggested scheme of investigation for structural variants.

Fig: Scheme of investigation for thalassemia

LABORATORY DIAGNOSIS OF HAEMOGLOBINOPATHIES

LABORATORY DIAGNOSIS OF HAEMOGLOBINOPATHIES

The 1978 International Committee for Standardization in Hematology expert panel on abnormal Hbs has prepared recommendations for laboratory investigation of these conditions.

1.      A complete blood count (CBC)

2.      Electrophoresis at pH 9.2

3.      Test for solubility and sickling

4.      Quantification of HbA₂ and HbF were recommended.

If abnormal Hb was found in these further test including electrophoresis at pH 6.2, globin chain separation and isoelectric focusing were recommended. If the presence of an unstable Hb or Hb with altered oxygen affinity was suspected then heat and isopropanol stability test were recommended. In addition to these test the iron status of patient should be known either by measuring ferritin or by iron/TIBC.

Complete Blood Count:

Thalassemia – Decreased Hb, low MCV <76 fL (normal 90-100 fL), low MCH <27 pg (normal 26-35 pg), High RBC count or count in URL (in IDA RBC count is low and Hb is low), in iron repleted patient suggest diagnosis of thalassemia. These low MCV, low MCH, low Hb, high RBC are the thalassemic indices.

Electrophoresis: 

Electrophoresis under alkaline pH is the common initial screening method for detection and preliminary identification of hemoglobinopatheis. It is done in cellulose acetate or agarose in Tris EDTA borate buffer or barbital buffer in pH 9.2. Visualization of electrophoretogram is done by protein stain like Amido Black or Ponceau S. Electrophoresis at pH 6.0 using citrate buffer is preformed when an abnormal band is noted on alkaline Hb electrophoresis. In acid electrophoresis the overlapping band seen in alkaline pH are further resolved and appear at different position.

Other advanced procedure are IEF, Capillary electrophoresis, HPLC, ESI-MS, DNA analysis, ASO-PCR, etc.

Specific test:

Determining HbH: 

HbH is insoluble tetramer of β₄ and arises in α-thalassemia. If these tetramers are oxidized precipitation occurs that can be viewed microscopically. In laboratory oxidation is achieved by staining unfixed cells with methylene blue or brilliant cresyl blue at 37⁰C. Golf ball like inclusions can be seen in red cells. In α-thalassemia minor 1 cell in 1000 to 10,000 may contain inclusions.

Sickling test: 

This is useful in confirming the presence of HbS in sample following initial electrophoresis at alkaline pH. When fully oxygenated HbS is fully soluble but when deoxygenated, polymerization occurs, forming insoluble fibers that deform red cells giving rigid sickle shape. In lab, deoxygenation and lysis of the RBC is achieved by use of solution of sodium metabisulfite in a phosphate buffer. For this few drops of sodium metabisulfite is added to a drop of anticoagulated blood on a slide. Seal between the slide and cover glass with petroleum jelly/paraffin wax mixture, or with nail varnish. Sickling takes place almost immediately in sickle cell anaemia and should be obvious in sickle cell trait within 1h. After this the slide is observed in microscope to see the sickling phenomena. 

Solubility testing: 

Solubility testing is based on the reduced solubility of deoxy HbS in presence of reducing agent, e.g. sodium dithionite. Here reagent containing sodium dithionite is added to packed red cell and turbidity compared with positive and negative control. In positive, the sample is centrifuged. A positive test will show a dark red band at the top while the solution below will be pink or colorless. Positive test indicates HbS and help in differential diagnosis of Hbs D and G which migrate with HbS.

Test for Unstable Hemoglobins: 

These tests use either heat or isopropanol to precipitate the unstable Hb and must be performed on fresh blood. There are more than 100 unstable Hbs resulting mainly from the interchange of nonpolar amino acid for polar amino acid residue in either α or β chain associated with the heme cleft.

Isopropanol stability test: 

Non polar isopropanol weakens the internal bonds within Hb decreasing the stability of Hb molecules. Normal HbA precipitates within 40 minutes at 37⁰C in presence of 17% isopropanol in pH 7.4 TRIS buffer. Unstable Hbs usually precipitates and gives turbidity within 5-20 minutes under these conditions.

Heat stability test:

  

Positive, negative and test samples are heated to 50⁰C for 2 hr. Normal Hb is stable when heated to 50⁰C. However unstable Hbs precipitates and flocculates to varying extent when similarly treated.

Globin chain analysis: 

Here the globin chains and heme in red cell lysate are first dissociated using urea and dithiothreitol. The dissociated globin chains are then separated electrophoretically at both an alkaline and acid pH. 

OTHER STRUCTURAL HEMOGLOBIN VARIANTS

OTHER  STRUCTURAL HEMOGLOBIN VARIANTS

Carboxyhemoglobin: 

It is formed by preferential attachment of CO over oxygen to Hb. Carboxyhemoglobin saturation levels of 15-25% are assocaited with dizziness, headaches and >50% is life threatening.

Methemoglobin: 

Under alkaline conditions, the ferrous iron is oxidized to ferric state by toxic agents like nitrates, aniline dyes, drugs like quinones, sulfonamides, etc. This oxidation convertes heme to hematin and Hb to methemoglobin. MetHb is unable to reversibly bind oxygen thus produces cyanosis. MetHb in cell is reduced to ferrous state by NADH-cytochrome reductase system. MetHbenemia is treated by administration of ascorbic acid or methylene blue.

Sulfhemoglobin: 

It is produced by reaction of sulfur containing compounds with heme to form an irreversible oxidation of Hb by introducing sulfur in porphyring rings. It also cannot carry oxygen.

All the hemogloginopathies results in unstable hemoglobins. The unstable hemoglobin variants results from neutral substitutions affecting residues that contact the haem group and generally present as a congenital Heinz body haemolytic anaemia. Amino acid susbtitution in both chains in vicinity of haem group can alter oxygen affinity or propensity to methaemoglobin formation. The diagnosis is made by heat denaturation or isopropanol precipitation test and identification of the globin mutation by protein or DNA analysis. 

Other Hemoglobins like HbC, HbD Punjab, Hb Lepore and HbE

Hemoglobin C: 

Hb C arises from substitution of lysine for glutamic acid at position 6 of β-globin chain. This may be homozygous or heterozygous state. It is the second most commonly studies after HbS of all Hb variants. There is large band in C position. HbF is variable.

Hemoglobin D Punjab: 

It is an Hb variant in which glutamic acid at position 121 of β-globin chain is replaced with glutamine. There is normal or marginally raised HbF and HbA₂ with large band in HbD. There is a band in S position which migrates to A position in acid electrophoresis. There is mild decrease in Hb levels, MCV and MCH with target cell observed.

Hemoglobin Lepore: 

Hb lepore is classified as δβ hybrid Hb variant on the basis that the non-α-chain is a hybrid of δ and β globin chains. It is unique in that it is the only hemoglobinopathy named after the family name of the index case. It arise because there are deletions of part of 3’ portion of the δ globin gene are in 5’ portion of the β-globin chain with resultant formation of a δβ-fusion gene. There is greatly raised HbA₂ with marginally reduced HbA. The HbA₂ concentration is usually >10% of total Hb. 

There is band in S position or between A and S position. At acid pH there is a single band in A position for all Hb lepore variants. There is greatly reduced level of Hb, MCV, MCH. The similarity of the hematology in Hb Lepore and β-thalassemia makes the careful review between these two conditions. The two key pieces of evidence to distinguish between two are the finding of greatly raised HbA₂ and small band in S position on electrophoresis at alkaline pH in Hb Lepore.

Hemoglobin E (β26 Glu → Lys):

It is also the most prevalent hemoglobin variant, and mainly found in south-east Asia. HbE has electrophoretic mobility similar to HbA₂ or C at pH 8.6 but can be differentiated by citrate agar electrophoresis at acid pH as it migrates to HbA position. Such Hb are unstable.

Elongation Hemoglobins: 

These results from lengthening of either C or N terminus of either globin chain. E.g. Hb Constant Spring. In this variant the C-terminal TAA codon in α-chain gene is changed to CAA and there is an addition of 31 amino acid sequences at the C terminal end to give an α-globin chain of 173 amino acid residues rather than normal 142 residue. This increase in length results in instability of Hb variant, and synthesis of this elongated globin chain is reduced. In electrophoresis this variant migrates cathodally to the application point may be seen. This electrophoretic mobility is unique is that it is the only Hb variant that moves towards the cathode rather than anode.

Hemoglobin S:

Hemoglobin S: 

It is the most widespread Hb variants and arise from substitution of valine for glutamic acid at position 6 in the A helix of the β-globin chain. The widespread distribution of single point gene mutation responsible for the synthesis of HbS in areas where P. falciparum malaria is endemic is due to protection of HbS heterozygotes from manifestation of malaria. is one of the commonest genetic disorders with estimated 60 million carriers worldwide. It was the first human disorder to be understood at molecular level and is the first hemoglobinopathy to be identified. 

Sickle haemoglobin polymerizes on deoxygenation to produce the classic sickle-shaped erythrocytes that give the disease its name. This leads to microvascular occlusion and shortened red cell survival producing anaemia, endothelial damage, organ damage susceptibility to infection due to hyposplenism. There is single base change (CAG to CTG) in the sixth codon of the β globin gene that leads to the substitution of valine for glutamic acid resulting in HbS.

The primary pathophysiological event is sickling is intracellular polymerization of deoxy HbS. The β6 valine substitution alters the surface charge of Hb, resulting in interaction between Hb tetramers and the formation of 14-stranded polymers. These forms fibre bundles. The formation of polymer fibers is affected by four variables: oxygen tension, HbS concentration, temperature and presence of non-sickling Hb. Small increases in Hb concentration as occur with cellular dehydration, may act as trigger for sickling process.

Polymer formation make RBC less deformable and fragile and less flexible. This compromises oxygen delivery leading to further sickling during deoxy state. Deformed cells adhere or accumulate in intravascular space occluding blood flow. There is loss of potassium due to deformed membrane exceeding sodium gain, resulting in loss of cell water and increased concentration of intracellular Hb. This is accompanied by an up to four fold increase in intracellular calcium.

Homozygous HbSS: 

Here a valine for glutamic acid substitution occurs on both β-globin chains due to inheritance of mutated β-globin chain genes from both parents. This condition is described as sickle cell anemia or sickle cell disease because of sickle shaped RBC. In electrophoresis there is no HbA, but small HbA₂. There is single large band in HbS with small bands at HbA₂ and HbF (raised HbF) position. HbS forms 85% to 90% of total Hb. The sickle cell screen test is positive.

CBC analysis shows moderate to major decrease in Hb (6-10 g/dL) with normal or raised MCV, MCH. In PBS there are sickle red cells, target cells and howell jolly bodies, boat shaped RBC, etc.

Heterozygous Hemoglobin S (HbS Trait): 

There is increased HbA and HbS. HbF concentration is variable. Electrophoresis at both alkaline and acid pH shows bands in A and S position. CBC analysis shows slightly decreased Hb, typically sickle cells are not seen in PBS.

STRUCTURAL HEMOGLOBIN VARIANTS

STRUCTURAL HEMOGLOBIN VARIANTS (Haemoglobinopathies)

Hemoglobinopathies, the most common single gene disorder and are structural Hb variants arising from mutations in globin genes, which results in alteration in normal amino acid residues in one or more globin chains.

Over 900 qualitative variants have been identified in which the hemoglobin molecule is structurally and in some case functionally altered. These arises most commonly due to single amino acid substitutions, but also insertion or deletion of amino acids and polypeptide fusion as  result of recombination between globin genes, e.g. Hb Lepore. The most common structural variants are haemoglobins S, C, D^punjab and E which affect worldwide.

Hb variants are named using letter (Hbs S, D, E, etc), the family name of the index case (Hb Lepore), the place of discovery of variant or place or origin (Hb Edmonton) or the name of the river (Hb Saale). 

Classification of Hemoglobin variants: 

Hb variants are classified according to the type of mutation.

·         Single point mutations in α-globin chain give rise to a suubstitution of one amino acid residue. E.g. substitution of valine for glutamic acid at position 6 in the A helix of the β-globin chain resulting HbS (sickle cell disease).

·         Deletion of Hb variants arises from the deletion of one to five amino acid residues in the globin chain. E.g Hb Vicksburg (β75(E19)^Leuà0) is an example where Hb having a deletion of leucine in position of 75 of the β-chain.

·         Insertion Hbs arise from an insertion of one to three amino acid residue into the globin chain. Hb Grady is an example having an insertion of 3 amino acid residue (Glutamine-phenylalanine-threonine) between position 118 and 119 of α-chain.

·         Deletion-insertion Hbs arise from the deletion of a portion of normal amino acid residue sequence and the insertion of another sequence which resultant lengthening or shortening of the globin chian. E.g. Hb Monteral in which the 3 nomal amino acid residues between positions 72 and 76 of the β-globin chain are replaced with a four amino acid residue.

·         Elongation Hbs results from a single base pair mutation or frameshift at the 3’ end of exon 3 or 5’ end of exon 1 of the α₂ or the β-globin chain. The elongation Hb, Hb Constant spring (found in constant spring district of jamaica), has an additional 31 amino acid residue joined at position 142 (the carboxy terminal) of the α-chain.

·         Fusion Hbs result from the fusion of either α or β-globin chain with a portion of another globin chain. Hb Lepore-Hollandia results from the fusion of the first 22 amino acid residue of the δ-chain, with tha amino acid sequence from position 50 onward of normal β-globin. 

Types of Hemoglobin variants: 

 In α-chain variants, the variant usually forms <25% of total Hb since the mutation occurs only in one of the four genes coding α-globin chain. For β-chain variants in heterozygous state, the variant forms 25%-50% of total Hb. This is used to categorized an unknown Hb variant as either an α- or β-globin chain variant. 

β-THALASSAEMIA

β-THALASSAEMIA

This is prevalent throughout tropical and north Africa, Mediterranean, Middle East and South-east Asia, including India. Over 200 different mutations of the β globin gene or its promoter are known to cause β thalassaemia, the majority being single nucleotide substitution (point mutations). Each mutation is linked to specific pattern of RFLPs within β globin gene cluster (or haplotype). In severe forms of β thalassemia (β thalassemia major) there is severe impairment or absence of β chain production. As a consequence excess α chain accumulates and precipitates in red cell precursors leading to their destruction within bone marrow leading to ineffective erythropoiesis. Also there is hemolysis in mature red cells due to inclusions. 

Red cell containing fetal Hb survives preferentially since there is less globin chain imbalance. The anaemia produces tissue hypoxia, which stimulates erythropoietin production and massive expansion of erythropoiesis in bone marrow and extramedullary sites.

If untreated, β thalassemia major leads to severe anaemia, wasting and growth retardation, with death in early childhood. As a result of expansion in erythropoiesis there are bone deformities and enlargement of liver and spleen. Widening of the dipole of the facial bones and skull gives rise to a characteristic thalassaemic facies, splaying of the teeth and frontal bossing.  

The treatment of β thalassemia major is regular blood transfusion to maintain Hb between 10-14 g/dL. But inevitable complication of multiple transfusions is iron overloading. If untreated this leads to haemosiderosis which causes multisystem dysfunction due to abnormal accumulation of iron in tissues. Vitamin C enhances the urinary excretion of iron, so patients receiving iron chelating agents like desferrioxamine to see the efficacy of chelation and is done by measuring ferritin.

Allogenic bone marrow transplantation from HLA matched siblings has been used with considerable success for treatment of β thalassemia major with disease free survival rates up to 95% in younger children.

Mutations that severely disrupts or abolish β globin synthesis (β⁰ thalassaemia) includes those that prevent normal splicing of mRNA (splice junction or splice site mutations) or generate a non-functional mRNA by premature translation termination (nonsense mutations), and mutation that partially disrupts or reduced β globin synthesis (β⁺, β⁺⁺ thalassemia) includes small nucleotide deletions or insertions leading to frameshift mutations.

β-thalassaemia can be co-inherited with α-thalassemia and leads to imbalance in globin chain synthesis and results in more ineffective erythropoiesis, and hereditary persistence of fetal hemoglobin (HPFH). HPFH can be linked to β globin cluster, and includes large deletions and point mutations in promoter regions of the γ globin genes. The heterozygous (carrier) state for β thalassaemia (β thalassaemia trait) is usually without harmful effects. 

Most individuals have slightly reduced Hb concentrations and elevated red cell count. A raised HbA₂ concentration is an important diagnostic marker and distinguishes β thalassaemia trait from α thalassemia, in which HbA₂ is normal or low. This reflects during β thalassemia there is high output from δ globin gene. Similarly HbF levels are frequently slightly raised in β thalassemia heterozygotes.  

Genotype		Heterozygote			Homozygote
	MCH & MCV	% HbA2	%HbF	%HbA	%HbF	Clinical status
β0	Reduced	Raised	<4	0	>94% and remainder is HbA2	Thalassaemia major
β+	Reduced	Raised	<4	5-50%	50-90	Thalassaemia major
β++	Slightly reduced	Raised or borderline	<2	50-90%	10-50	Thalassemia intermedia

β⁰ Thalassemia major: 

Sometimes called Cooley’s anaemia after the physician who in 1925 first described the condition in children of Italian and Greek immigrants in New York. This condition results from mutation that interferes with translation, in about 50% of all mutations. There is also frame shift or non sense mutation that prematurely terminates β-globin chain.

Clinical presentation is usually at <1 year of age with features that includes failure to grow, abdominal girth expansion, and failure to thrive. There is frontal bossing (rounded eminence on forehead), pallor, etc. These features are due to marrow expansion caused by ineffective erythropoiesis with production of highly unstable α-globin tetramers leading to increased plasma volume and formation of extramedullary erythropoietic tissue.

Typical CBC results include severe anemia with Hb between 3-6.5 g/dL, MCV <72 fL, and MCHC <320 g/L. There is microcytosis, target cells, polychromasia, nucleated red cells, anisocytosis. There is major HbF band with absence of HbA band and variable HbA₂ (1-6%). 

Electrophoresis at alkaline and acid pH shows dominant band in F position.

β-thalassemia (β-thalassemia Intermedia): 

There is reduced production of β-globin chain with subsequent reduction in quantity of HbA. There is large HbF band with reduced HbA. HbA₂ is above reference interval. Bands in A and F positions are seen on electrophoresis. The Hb is significantly reduced (6-10 g/dL).

β-thalassemia minor (β-thalassemia trait): 

The CBC shows low normal or decreased Hb and hematocrit, decreased MCV (<72 fL) and MCH (<27 pg). PBS has occasional hypochromia, poikilocytosis, and target cells. The diagnosis of this minor condition with appropriate indices in CBC, is dependent on finding of raised HbA₂ concentration (>3.5%). Iron deplete individuals should become iron replete before a definitive diagnosis as HbA₂ may be falsely low in iron deficiency individual. HbF will be raised (>1%).

δβ-Thalassemia: 

There is deletion of δ- and β-gene. There is increase in HbA with reduced HbA₂ and raised HbF. Hb lepore is sometimes classified as this type of thalassemia due to reduction in production of both delta and β globin chain or abnormal Hb chain. Thalassemic indices on CBC includes low MCV, MCH, normal RDW.

Hereditary persistence of Fetal Hemoglobin (HPHF): 

This describes the group of genetic conditions in which the concentration of HbF is increased because of reduction of β-globin synthesis and compensatory increase in δ-globin synthesis. Here Hb, MCV, MCH are within reference intervals. 

α-Thalassemia major

Fig: Cellulose acetate electrophoresis at pH 8.6.

1.      F, A, Hb Bart, Albumin in newborn.

2.      A, HbH, Albumin in an adult

3.      HbA and HbJ in adult

α-Thalassemia major: 

(α-Thalassemia major is the result of a two α-chain gene deletion. These deletions may be on same gene (-/αα, α⁰-thalassemia) described as cis-deletion or on different genes (-α/-α, α⁺-thalassemia) described as trans deletion. The CBC shows mildly reduced Hb level with low MCV and MCH. HbF and HbA₂ within reference level. The presence of thalassemic indices in patient with normal HbA₂ and HbF is often the basis for diagnosis of α-thalassemia major, particularly if family history is positive.

Thalassemia trait: 

There is a single α-globin chain deletion (-α/αα). A CBC shows normal or marginally reduced Hb, MCV and MCH. 

GENETIC CONTROL OF HEMOGLOBIN SYNTHESIS

GENETIC CONTROL OF HEMOGLOBIN SYNTHESIS

Human globins are encoded by the genes of α and β gene clusters located on chromosome 16 and 11 respectively. The α-like genes include an embryonic gene (ζ) and two adult genes (α₂ and α₁). The β-like genes comprise an embryonic gene (ε), duplicated fetal (γ) and adult (δ and β) genes. 

Adult                                      

HbA₁ = 97%

HbA₂ = 2-3.3%

HbF = 0.1 – 1%

Newborn

HbA₁ = 25%

HbA₂ = <1%

HbF = 75%

Fig: Synthesis of Globin chains variants during prenatal and postnatal life