Hemoglobinopathies - Lab diagnosis

1. Hemoglobinopathies:
approach and lab diagnosis

2. The haemoglobin molecule
embryonic
fetal

3. haemoglobinopathies

4. Haemoglobinopathies
structural variants
failure to synthesize
failure to
switch

5. Structural variants

6. Hb with reduced solubility

7. Unstable Haemoglobins

8. Unstable Haemoglobins

9. Haemoglobins with Altered Oxygen
Affinity

10. Haemoglobins with Altered Oxygen
Affinity

11. Hb M

12. Hb M

13. Investigation of patients with a suspected
haemoglobinopathy

16. Laboratory detection of hemoglobin
variants

19. Peripheral smear

20. β-thal heterozygote

21. β-thal homozygote

22. Hb E trait

23. HbE/β-thal
400x 1000x

24. Haemoglobin E homozygote

25. Hb H disease

26. Hb H disease

27. Haemoglobin C homozygote

28. HbSC disease

29. Sickle cell
homozygote

30. Collection of Blood and Preparation of
Haemolysates
• EDTA

31. Preparation of Haemolysate for Qualitative
Haemoglobin Electrophoresis

32. Control Samples

33. Preparation of controls

34. Quality Assurance

35. Quality Assurance

36. Cellulose Acetate Electrophoresis at
Alkaline pH

37. Principle

38. Procedure

40. Interpretation

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

45. Citrate Agar Electrophoresis at pH 6.0

46. Interpretation

47. Agarose Gel Electrophoresis

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 covered in subsequent session
Automated High-Performance Liquid
Chromatography

51. Isoelectric Focusing

52. Interpretation
still only provisional

53. Relative mobilities of some abnormal
haemoglobins in IEF

54. Tests for Hb S

55. • 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 HbA plus Hb S must be
performed at the same time.
Sickling inWhole Blood

57. Hb S SolubilityTest

58. Interpretation

59. Tests for HbS

60. Detection of an unstable haemoglobin

61. Heat StabilityTest

62. Isopropanol StabilityTest

63. Detection of Hb Ms

66. Detection of altered affinity haemoglobins

68. Differential diagnosis of common
haemoglobin variants

69. Comparison of the relative 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

72. Quantitation of Hb A2

73. 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 HbA2

74. Measurement of Hb A2 by Microcolumn
Chromatography

75. 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-

76. 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

77. 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 andVisser
• Radial immunodiffusion method

78. Assessment of the intracellular
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 Hb F values

80. 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.

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

82. Thank you!!