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Hemoglobinopathies - Lab diagnosis
- 1. Hemoglobinopathies: approach and labdiagnosis
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- 4. Haemoglobinopathies structural variants failure tosynthesize failure to switch
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- 6. Hb with reducedsolubility
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- 9. Haemoglobins with AlteredOxygen Affinity
- 10. Haemoglobins with AlteredOxygen Affinity
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- 13. Investigation of patientswith a suspected haemoglobinopathy
- 16. Laboratory detection ofhemoglobin variants
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- 30. Collection of Bloodand Preparation of Haemolysates • EDTA
- 31. Preparation of Haemolysatefor Qualitative Haemoglobin Electrophoresis
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- 36. Cellulose Acetate Electrophoresisat Alkaline pH
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- 42. Relative mobilities ofsome abnormal haemoglobins. Cellulose acetate, pH 8.5
- 45. Citrate Agar Electrophoresisat pH 6.0
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- 48. 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
- 49. 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).
- 50. Will be coveredin subsequent session Automated High-Performance Liquid Chromatography
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- 53. Relative mobilities ofsome abnormal haemoglobins in IEF
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- 55. • The sicklingphenomenon 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 HbA plus Hb S must be performed at the same time. Sickling inWhole Blood
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- 60. Detection of anunstable haemoglobin
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- 66. Detection of alteredaffinity haemoglobins
- 68. Differential diagnosis ofcommon haemoglobin variants
- 69. Comparison of therelative mobilities of some abnormal haemoglobins by different methods
- 70. Investigation of suspected thalassaemia •Full blood count with red cell indices and blood film and, in selected cases, reticulocyte count • HbA2 measurement by cellulose acetate electrophoresis with elution • HbA2 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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- 73. Measurement of HbA2 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 HbA2
- 74. Measurement of HbA2 by Microcolumn Chromatography
- 75. Microcolumn chromatography Principle + + + + + + + + + + + Hb A HbA2 HbF Hb E Hb H Hb Bart’s Cl- Cl-Cl- Cl- Cl- Cl- Cl- Cl- Cl- Cl- Cl- Cl- Cl- Cl- Cl- Cl-
- 76. Interpretation of HbA2values • 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
- 77. Quantitation of HbF • 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 andVisser • Radial immunodiffusion method
- 78. Assessment of theintracellular distribution of Hb F • Differences in the intracellular distribution of Hb F • heterozygotes for δβ thalassaemia -- heterocellular distribution • ClassicalAfrican type of HPFH -- pancellular distribution • Immunofluorescent Method
- 79. Interpretation of HbF values
- 80. Demonstration of HbH 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.
- 81. 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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