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Page 1: Hemoglobinopathies - Lab diagnosis

Hemoglobinopathies:approach and lab diagnosis

Dr. Ankit RaiyaniHematology department

SSH

Page 2: Hemoglobinopathies - Lab diagnosis

The haemoglobin molecule•2 pairs of globin chains each with a haem group•Seven different globin chains•Transient embryonic haemoglobins- Hb Gower 1(ζ2ε2), Hb Gower 2(α2ε2), Hb Portland 1(ζ2γ2) and Hb Portland 2•Hb F(α2γ2) -predominant haemoglobin of fetal life•Hb A(α2β2) -major haemoglobin found in children and adults• In normal adults 96 – 98% of hemoglobin is HbA, Hb A2 (2 – 3%) and HbF (<1%)

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•There are many naturally occurring, genetically determined variants of human haemoglobin (>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’.

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HaemoglobinopathiesThey can be grouped into three main categories:1. Those owing to structural variants of haemoglobin,

such as Hb S.2. Those owing to failure to synthesize one or more of

the globin chains of haemoglobin at a normal rate, as in the thalassaemias.

3. 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

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Structural variants •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.

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Hb with reduced solubility•HbS•HbC

•Hb D Punjab

•Hb O Arab

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Unstable Haemoglobins• 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 the congenital 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 state• Hb Köln is the most common variant in this rare group of disorders

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Unstable Haemoglobins

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Haemoglobins with Altered Oxygen Affinity•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.

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Haemoglobins with Altered Oxygen Affinity

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Hb M• 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).

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Hb M

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• 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

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•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

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• 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.

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Laboratory detection of hemoglobin variants 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 microcytosis

14. Molecular techniques 15. Procedures for use in

under-resourced laboratories

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Suggested scheme of investigation for structural variants

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CBC• The blood count, including Hb and red cell indices, provides valuable information useful in the diagnosis of both α and β thalassaemia interactions with structural variants• Thalassaemias are typically microcytic and hypochromic anemia's.• Thalassemia causes a uniform microcytosis without increase in RDW•Hb H and Δβ thal however can cause an increased RDW.

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Peripheral smear• 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 haemoglobin•Discriminant functions using various formulae have been proposed as a basis for further testing for thalassaemia• Mentzer index- MCV / RBC count in million• >13 s/o iron deficiency anaemia• <13 s/o thalassemia

•NOT ADVISABLE as it may lead to individuals who have both iron deficiency and thalassaemia trait not being tested promptly

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β-thal heterozygote

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β-thal homozygote

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Hb E trait

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HbE/β-thal

400x 1000x

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Haemoglobin E homozygote

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Hb H disease

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Hb H disease

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Haemoglobin C homozygote

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HbSC disease

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Sickle cell homozygote

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Collection of Blood and Preparation of Haemolysates

• EDTA is the most convenient anticoagulant because it is used for the initial full blood count and film• Although samples taken into any anticoagulant are satisfactory.• Cells freed from clotted blood can also be used.

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Preparation of Haemolysate for Qualitative Haemoglobin

Electrophoresis• Centrifuge EDTA blood at 3000-5000 rpm and remove

plasma• Wash packed red cell with NS for three time and remove

supernatant as much as possible at the last washing round• Lyse 2 volumes of washed packed cells in 1 volume of

distilled water and then add 1 volume of carbon tetrachloride (CCl4). • Alternatively, lyse by freezing and thawing, then add 2

volumes of CCl4. • Shake the tubes vigorously for approximately 1 min,

then centrifuge at 1200 g (3000 rev/min) for 30 min at 4°C. • Transfer the supernatant to a clean sample container

and adjust the Hb to 100 ± 10 g/l with water. • If an unstable Hb is suspected organic solvents should

be avoided.

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Control Samples• Interpretation of migration patterns of test samples is undertaken by comparison to migration and separation of known abnormal haemoglobins used as control materials. • Ideally, a mixture of Hbs A, F, S and C should be included on each electrophoretic separation.

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Preparation of controls

• The control can be made from either• the combination of a sickle cell trait sample (Hb

A + Hb S) combined with a Hb C trait sample (Hb A + Hb C) and normal cord blood (Hb F + Hb A) or

• the combination of normal cord blood with a sample from a person with Hb SC disease (Hb S + Hb C).

• Prepare lysates by the method given for a purified haemolysate.•Mix equal volumes of the lysates together and add a few drops of 0.3 mol/l KCN (20 g/l).• Analyse samples by electrophoresis to assess quality.• Aliquot and store frozen.

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Quality Assurance•Haemoglobinopathies are inherited conditions, some of which carry considerable clinical and genetic implications, precise documentation and record-keeping are of paramount importance.• The use of cumulative records when reviewing a patient’s data is very useful because it of itself constitutes an aspect of quality assurance. • In some situations, repeat sampling, family studies or both may be required to elucidate the nature of the abnormality in an individual.

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• It is necessary to use reference standards and control materials in each of the analyses undertaken and in some cases to use duplicate analysis to demonstrate precision. • There are international standards for Hb F and Hb A2• These are extremely valuable because the target values have been established by collaborative studies.• Control materials can be prepared in-house or obtained commercially. • Samples stored as whole blood at 4°C can be used reliably for several weeks.

Quality Assurance

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Cellulose Acetate Electrophoresis at Alkaline pH

•Haemoglobin electrophoresis at pH 8.4–8.6 using cellulose acetate membrane is simple, reliable and rapid.

• It is satisfactory for the detection of most common clinically important haemoglobin variants

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Principle •At alkaline pH, haemoglobin is a negatively charged protein and when subjected to electrophoresis will migrate toward the anode (+). •Structural variants that have a change in the charge on the surface of the molecule at alkaline pH will separate from Hb A. •Haemoglobin variants that have an amino acid substitution that is internally sited may not separate and those that have an amino acid substitution that has no effect on overall charge will not separate by electrophoresis.

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•Hemolysate in wells• Sample applicator dipped and applied on soaked cellulose acetate plate• Place cellulose acetate, face-down, in electrophoretic chamber.• Run elctophoresis at 300 volts for 10-20 min.• Stained with Ponceau S• Interpretation

Procedure

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Interpretation• Satisfactory separation of Hbs C, S, F, A and J is obtained• In general, Hbs S, D and G migrate closely together, as do Hbs C, E and OArab. •Differentiation between these haemoglobins can be obtained by using acid agarose gels, citrate agar electrophoresis, HPLC or IEF• Lepore Hbs and Hb DPunjab migrate slightly anodal to Hb S (i.e. they are slightly faster than S); •Hb C migrates slightly cathodal to Hb E (i.e. it is slightly slower than E).

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• In general, Hb S, D and G migrate closely together, as do Hb C, E and OArab.

• Lepore Hbs and Hb DPunjab migrate slightly anodal to Hb S (i.e. they are slightly faster than S)

•Hb C migrates slightly cathodal to Hb E (i.e. it is slightly slower than E).

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Relative mobilities of some abnormal haemoglobins. Cellulose acetate, pH 8.5

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• All samples showing a single band in either the S or C position should be analysed further using acid agarose or citrate agar gel electrophoresis, HPLC or IEF to exclude the possibility of a compound heterozygote such as SD, SG, CE or COArab

•When an abnormal haemoglobin is found, it may be of diagnostic importance to measure the percentage of the variant; this can be done by the electrophoresis with elution procedure for Hb A2 estimation

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• The quality of separation resulting from this procedure is affected primarily by both the amount of haemoglobin applied and the positioning of the origin. •Delays between application of the sample and commencement of the electrophoresis, delay in staining after electrophoresis or inadequate blotting of the acetate prior to application will cause poor results. • This technique is sensitive enough to separate Hb F from Hb A and to detect Hb A2 variants.

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Citrate Agar Electrophoresis at pH 6.0•Hemolysate in wells• Sample applicator dipped and applied on agar gel plate• Place agar gel plate, face-down, in electrophoretic chamber.• Run electrophoresis at 50 volts for 60 min.• Apply the Dianisidine stain solution for 10 min•Wash in three changes of 3% acetic acid, float gels onto the hydrophilic side of a piece of Gel Bond and leave to dry• Interpretation

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Interpretation

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Agarose Gel Electrophoresis• Agarose gels are commercially available as substitutes for both alkaline and acid separation systems.• They are simple to use and particularly useful in laboratories that process small numbers of samples.•With acid agarose systems, the principle of the test is the same as that of citrate agar electrophoresis at the same pH, but it should be noted that there are significant differences in mobility of some variant haemoglobins. •With alkaline systems, in general the same separation patterns are obtained, but where individual application notes are available these should be used for reference.

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Electrophoresis Advantages

• Commercial, widely available, rapid methods used for many years.•Gives an estimate of HbA2 level.• Identifies some variant haemoglobins which are well characterized

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Electrophoresis Disadvantages

• Labor-intensive.• Inaccurate in quantification of low-concentration variants (HbA2) and in detection of fast variants (HbH, Hb Barts).• The precision and accuracy for Hb A2 using scanning of electrophoretic gels is poor (in comparison to HPLC).

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Will be covered in subsequent session

Automated High-Performance Liquid Chromatography

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Isoelectric Focusing• IEF utilizes a matrix containing carrier ampholytes of low molecular weight and varying isoelectric points (pIs). • These molecules migrate to their respective pIs when a current is applied, resulting in a pH gradient being formed• For haemoglobin analysis, a pH gradient of 6–8 is usually used• Haemoglobin molecules migrate through the gel until they reach the point at which their individual pIs equal the corresponding pH on the gel.• At this point, the charge on the haemoglobin is neutral and migration ceases.• The pH gradient counteracts diffusion and the haemoglobin variant forms a discrete narrow band

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Interpretation • IEF is satisfactory for analysis of haemolysates, whole blood samples or dried blood spots. • The use of dried blood spots is suitable for samples that have to be transported long distances and where only a few drops of blood can be obtained. •Whereas IEF has the advantage that it separates more variants than cellulose acetate, it also has the disadvantage that it separates haemoglobin into its post-translational derivatives. For instance, Hb F separates into F1 (acetylated F) and F11; Hb A can produce five bands – A0, A1, A(αmet), A(βmet) and A(αβmet) – and similarly for other haemoglobins. • This makes interpretation more difficult. Identification of variants is still only provisional using IEF, and second-line methods should be used for further analysis.

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Relative mobilities of some abnormal haemoglobins in IEF

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Tests for Hb S•Tests to detect the presence of Hb S depend on the decreased solubility of this haemoglobin at low oxygen tensions.

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• The sickling phenomenon may be demonstrated in a thin wet film of blood (sealed with a petroleum jelly/paraffin wax mixture or with nail varnish). • If Hb S is present, the red cells lose their smooth, round shape and become sickled.• This process may take up to 12 h in Hb S trait, whereas changes are apparent in homozygotes and compound heterozygotes after 1 h at 37°C. • These changes can be hastened by the addition of a reducing agent such as sodium metabisulphide or sodium dithionite• A test on a positive control of Hb A plus Hb S must be performed at the same time.

Sickling in Whole Blood

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Hb S Solubility Test•Principle-• Sickle cell haemoglobin is insoluble in the deoxygenated state in a high molarity phosphate buffer. • The crystals that form refract light and cause the solution to be turbid

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Interpretation• A positive solubility or sickling test indicates the presence of Hb S and as such is useful in the differential diagnosis of Hbs D and G, which migrate with Hb S on cellulose acetate electrophoresis at alkaline pH.• Positive results are also obtained on samples containing the rare haemoglobins that have both the Hb S mutation and an additional mutation in the β chain. • A positive solubility test merely indicates the presence of a sickling haemoglobin and does not differentiate between homozygotes, compound heterozygotes and heterozygotes

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Tests for HbS• False-positive results • in severe leucocytosis; in hyperproteinaemia (such as

multiple myeloma); and in the presence of an unstable haemoglobin, especially after splenectomy

• False-negative results• in patients with a low Hb. • old or outdated reagents are used and if the

dithionite/buffer mixture is not freshly made. • False-negative results are likely to be found in infants

younger than age 6 months and in other situations (e.g. post-transfusion), in which the Hb S level is <20%

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Detection of an unstable haemoglobin

•Haemoglobin variants exhibit a wide range of instability but the clinically unstable haemoglobins can be detected by both the heat stability test and the isopropanol test.• Samples analysed should be as fresh as possible and certainly less than 1 week old. • Controls should be of the same age as the test sample; a normal cord blood sample can be used as a positive control. • The isopropanol test uses chemically prepared controls.

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Heat Stability Test• Principle-• When haemoglobin in solution is heated, the hydrophobic

van der Waals bonds are weakened and the stability of the molecule is decreased.• Under controlled conditions, unstable haemoglobins

precipitate, whereas stable haemoglobins remain in solution.•Method• Add 0.2 ml of lysate, to a tube containing 1.8 ml of buffer.

The negative control is obtained from a fresh normal sample.• Place the tubes in a waterbath at 50°C. Examine the tubes

at 60, 90 and 120 min for precipitation.• Interpretation and Comments• A major unstable haemoglobin will have undergone marked

precipitation at 60 min and profuse flocculation at 120 min. The normal control may show some (fine) precipitation at 60 min, but this should be minimal.

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Isopropanol Stability Test

• Principle- • When haemoglobin is dissolved in a solvent such as

isopropanol, which is less polar than water, the hydrophobic van der Waals bonds are weakened and the stability of the molecule is decreased.

• Advantage –• Does not require a 37°C waterbath • Positive controls can be made by modification of the reagent

buffer• Interpretation• A normal sample will remain clear until 30 min, when a

slight cloudiness may appear.• Some unstable haemoglobins will show clearly observable

precipitation even after 5 min incubation, whereas milder variants will not show precipitation until 20 min.

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Detection of Hb Ms•Methaemoglobin (Hi) has iron present in the ferric form. • Inherited variants of haemoglobin that undergo oxidation to methaemoglobin more readily than Hb A are referred to as Hb Ms• One of the causes of a very rare condition, congenital methaemoglobinaemia. (Other cause is methaemoglobin reductase deficiency)•Methaemoglobin levels vary, but may be as high as 40% of the total haemoglobin. •Methaemoglobinaemia per se may also be caused by oxidant chemicals.•Methaemoglobin variants may be detected by haemoglobin electrophoresis at pH 7

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•Methaemoglobin variants can be distinguished from methaemoglobin A (Hi A) by their absorption spectra. Each methaemoglobin has its own distinct absorption spectrum. •Hi A has two absorption peaks at 502 nm and 632 nm, whereas the peak absorbances for the variant Hb Ms are at different wavelengths• Method

• Lyse washed red cells from a blood sample of known Hb A and of the test sample with water to give haemoglobin concentration of about 1 g/l.

• Convert the haemoglobin to Hi by the addition of 5 μl of potassium ferricyanide solution to each ml of haemolysate.

• Leave for 10 min at room temperature.• Record the spectrum of Hi A using an automatic scanning spectrometer.• Compare to the spectrum of Hi in the test sample.

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Absorption maxima of methaemoglobins in the range of 450–650 nm.

Normal methaemoglobin is shown by a solid line; Hb M Saskatoon is shown by a dotted line.

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Detection of altered affinity haemoglobins

• Electrophoretic and chromatographic techniques are not useful because the amino acid substitution often does not involve a change in charge.• The most informative investigation is the measurement of the oxygen dissociation curve• The most significant finding is a decreased Hill’s constant (‘n’ value) because this can only come about by a change in the structure of the haemoglobin. • The p50 may be either increased (low-affinity haemoglobin) or decreased (high-affinity haemoglobin). •High-affinity haemoglobins result in an increase in Hb level, whereas low-affinity haemoglobins result in a decrease in Hb level.

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• Determination of the oxygen dissociation curve depends on two measurements: pO2 with which the blood is equilibrated and the proportion of Hb that is saturated with oxygen. Methods for determining the dissociation curve fall into three main groups:

1. The pO2 is set by the experimental conditions and the percentage saturation of Hb is measured.

2. The percentage saturation is predetermined by mixing known proportions of oxygenated and deoxygenated blood and the pO2 is measured.

3. The change in oxygen content of the blood is plotted continuously against pO2 during oxygenation or deoxygenation and the percentage saturation is calculated.

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Differential diagnosis of common haemoglobin variants

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Comparison of the relative mobilities of some abnormal haemoglobins by different methods

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Investigation of suspected thalassaemia

• Full blood count with red cell indices and blood film and, in selected cases, reticulocyte count

• Hb A2 measurement by cellulose acetate electrophoresis with elution

• Hb A2 measurement of microcolumn chromatography

• Automated HPLC • Quantitation of Hb F• Assessment of the distribution of Hb F• Assessment of iron status • Demonstration of red cell inclusion bodies• DNA analysis

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Quantitation of Hb A2

• Estimations may be made by elution after cellulose acetate electrophoresis or by chromatography, either microcolumn or HPLC.

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Measurement of Hb A2 by Elution from Cellulose Acetate

•Principle•Haemolysate is separated into its component fractions by alkaline electrophoresis on cellulose acetate membrane. • The relative proportions of the separated fractions are quantitated by spectrometry of the eluates of the separated fractions

•Duplicate values obtained should be within 0.2%. • This method is inaccurate in the presence of Hb C, Hb E and Hb OArab because they do not separate from Hb A2

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Measurement of Hb A2 by Microcolumn Chromatography

• Principle-• It depends on the interchange of charged groups on the

ion exchange cellulose with charged groups on the haemoglobin molecule. • When a mixture of haemoglobins is adsorbed onto the

cellulose, a particular haemoglobin component may be eluted from the column using a buffer (developer) with a specific pH and/or ionic strength, • whereas other components (either a single haemoglobin or

a mixture of haemoglobins) may be eluted by changing the pH or ionic strength of the developer

• Anion exchanger diethylaminoethyl (DEAE) cellulose (Whatman DE-52 microgranular pre-swollen), with Tris-HCl developers or glycine-KCN developers

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Microcolumn chromatography Principle

+

++

+

+

++

++

+

+

Hb A

HbA2

Hb F

Hb E

Hb H

Hb Bart’s

Cl-

Cl-Cl- Cl-

Cl-

Cl-

Cl-

Cl-

Cl-

Cl-

Cl-

Cl-

Cl-

Cl-

Cl-

Cl-

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Interpretation of HbA2 values• Hb A2 values should be interpreted in relation to a

reference range established in each individual laboratory using blood samples from the local population with a normal Hb and red cell indices

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Quantitation of Hb F•Hb F may be estimated by several methods based on its resistance to denaturation at alkaline pH, by HPLC or by an immunological method

•Modified Betke Method•Method of Jonxis and Visser• Radial immunodiffusion method

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Assessment of the intracellular distribution of Hb F•Differences in the intracellular distribution of Hb F • heterozygotes for δβ thalassaemia -- heterocellular

distribution • Classical African type of HPFH -- pancellular

distribution

• Immunofluorescent Method

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Interpretation of Hb F values

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Demonstration of Hb H Inclusion Bodies• Staining solution. 1.0% brilliant cresyl blue or New methylene blue •Method• Mix 2 volumes of fresh blood (within 24 h of

collection) with 1 volume of staining solution.• Incubate at 37°C for 2 h or at room temperature for 4

h.• Resuspend the cells and spread a thin blood film.• Examine the film as for a reticulocyte count. • The inclusion bodies appear as multiple greenish-blue

dots, like the pitted pattern on a golf ball• They can be readily distinguished from reticulocytes,

which exhibit uneven reticular material or infrequent fine dots.

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HbH preparation interpretation• In α+ thalassaemia trait, only a very occasional H body (1:1000 to 1:10 000) is usually seen• This test is most useful in Hb H disease, where inclusions are usually found in more than 30% of red cells.

•Number of cells developing inclusions does not correlate with genotype• Absence of inclusion does not preclude a diagnosis of α thalassaemia trait

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Thank you!!