1. LABORATORY TESTS/ DIAGNOSIS FOR THALASSEMIA
DR SALMAN MOHD SAHID
PATHOLOGY DEPT, HTAA
7/12/2007

2. INTRODUCTION
1925: A Severe disease was described in North America by Thomas Benton Cooley;
 Italian children with the characteristic anemia and bone deformities.

3. Later : the termed, "thalassa anaemia" was introduced "anemia by the sea“; because of recognized links to the Mediterranean region.

4. WHAT IS THALASSEMIA ?
Inherited disease of blood that reduces the amount of haemoglobin body can make and so can cause anaemia.
GENETIC DISEASE

5. Inherited autosomal recessive disorders characterised by reduced or absent synthesis of one or more Globin chain type.
The imbalanced globin chain synthesis leads to ineffective haemoglobin formation and shortened red cell life span.
WHAT ???? IS THALASSEMIA ?

6. The hemoglobin molecule
Resides in RBCs, responsible for carrying and transporting O2
Is a tetramer, consist of four polypeptide groups, 2 α and 2 β chains (α2β2)
Up to 4 O2 molecules can bind to the 4 heme groups.

8. TYPE OF HAEMOGLOBIN - determined by the globin chains that comprise it
EMBRYO & FETUS
Gower 1 (ζ2ε2).
Portland (ζ2γ2).
Gower 2 (α2ε2).
Fetus (α2γ2).
ADULT :
Hb A (α2β2)
- 96 – 98%.
Hb F (α2γ2)
- 0.5 – 0.8%.
Hb A2 (α2δ2)
- 1.5 – 3.2%.

9. GLOBIN GENES Genes for globin chain occur in two clusters:
Chromosome 11 – ε, δ, β.
Chromosome 16 – ζ, α.

10. GLOBIN SYNTHESIS - Start in nucleus.
- All globin genes: 3 exons (coding) & 2 introns.
- Initial RNA is transcribed from both introns & exons.
- RNA derived from introns is removed by process called splicing.
- RNA then capped by additional structure at 5’ end and polyadenylated at 3’ end.
- mRNA enters cytoplasm & to ribosomes where translation take place until terminal codon is reach & release of complete globin chain.

11. GLOBIN GENE EXPRESSION
ATA
ATG
TAA
AATAAA
DNA
TRANSCRIPTION
mRNA
PROCESSING
5’Cap
Poly A-3’
Nucleus
SPLICING
5’Cap
Poly A-3’
Cytoplasm
TRANSLATION
Ribosome

12. Haem synthesis occur largely in the mitochondria by a series of biochemical reactions commencing with the condensation of glycine and succinyl coenzyme A under the action of rate limiting enzyme delta aminolevulinic acid(ALA) synthase.
Pyridoxal phosphate (vit B6) is a coenzyme for this reaction which is stimulated by erythropoietin.
Ultimately protoporphyrin combines with iron in the ferrous (fe2+) state to form haem. Each molecule combines with a globin chain made on the polyribosomes.
A tetramer of four globin chains each with its own haem group in a “pocket” is then formed to make up a hemoglobin molecule.

13. SWITCH FROM FETAL TO ADULT HB
Beta globin gene is expressed at low level in early fetal life.
Main switch to adult Hb –
3 – 8 months of life
gamma chain is largely replaced by beta.

14. THALASSEMIA (HAEMOGLOBINOPATHY)
Pathogenesis:
Reduced rate of syntesis of normal globin chains.
Synthesis of abnormal Hb.
Failure to complete neonatal switch (HPFH).
Find

15. THALASSEMIA (HAEMOGLOBINOPATHY) CLASSIFICATION
According to 3 criteria:
The affected globin gene(s) e.g , ,  etc.
Whether the reduction in the rate of synthesis of the affected globin is partial e.g + or absolute o
The genotype e.g homozygous o

16. ALPHA THALASSEMIA
Lack of Alpha Chain
More than 95% of  thal are due to deletion of one or both of the tandem  globin genes located on chromosome 16.

17. ALPHA THALASSEMIA Gene Deletion forms
Deletions of both  genes complex. Some are complete abolition of the  globin synthesis
0 kb
10 kb
30 kb 2
2 1
-- FIL
-- SEA
-- MED
 Thalassemia gene result in 0

18. ALPHA THALASSEMIA
Gene Deletion forms – leave one  globin gene intact.
 1
- 4.2
- 3.7
 Thalassemia gene result in +

19. ALPHA THALASSEMIA / + heterozygote -/ +homozygote -/-
5 possible genotypes:
Type
Genotype
Normal
/
+ heterozygote
-/
+homozygote
-/-
o heterozygote
--/ 
ohomozygote
--/--
o +double hetero
--/-

20. Characteristics of α-thalassaemia
Result in either reduce (α+) or absent (α0) of α-globin chains.
α+-thal trait = α-thalassaemia-2
α0-thal trait = α-thalassaemia-1
HbH disease
Hb-Bart’s hydrops fetalis
Asymptomatic
symptomatic

21. BETA THALASSEMIA Lack of beta chain
Most of  thalassemia result from point mutation within or close to the  globin gene complex.
Each mutation result in reduction or abolition of  globin chain function.

22. Characteristic of β -thalassaemia
Very heterogenous at molecular level
> 200 point mutations and deletions.
Degree of severity corresponds to magnitude of the defective β-globin gene.
Classified into severe, mild, silent
Variable severity of clinical and haemotological phenotypes.

23. MUTANT GENE Globin gene protein sequence
CCT l GAT l GCT l GTT l ATG l GGC l AAC l
Substitution of nucleotide produces mutant globin gene, which affect the production of globin chain.

24. ABNORMAL SPLICING CAGCTACCA CAGGTACCA 5’ Splicing site 3’
Introns
Normal mRNA
Mutant splicing
CAGGTACCA
Exons
Mutant mRNA

25. BETA THALASSEMIA CLINICAL SYNDROME
Thalassemia Minor or Thalassemia Trait (ß+/ßa) or (ßo/ßa)
Thalassemia Intermedia (ß+/ß+) or (ß+/ßo)
Thalassemia Major or Cooley's Anemia (ßo/ßo)

26. SYNTESIS OF ABNORMAL HB
MANY NATURALLY OCCURING GENETICALLY DETERMINED VARIANTS OF HUMAN HB > 750.
MANY HARMLESS BUT SOME SERIOUS CLINICAL SYNDROME.
IN MALAYSIA – HB E, HB CONSTANT SPRING, HB S (SICKLE CELL ANAEMIA).
Find

27. THALASSAEMIA A medically debilitating disease
Life-long blood transfusion
Expensive medications
8-10 hours injection daily for 5-6 days/week
Multi-organ complications
Burden to healthcare
Cost of treatment will rise exponentially & not sustainable for any country
If new births are not prevented or number drastically reduced
Prevention & control program critical for success

28. THALASSAEMIA IN MALAYSIA
Commonest inherited blood disorder in Malaysia, considered “high” and a major public health problem that requires intervention.
Estimated about 3 – 5 % of the population are carriers ( 600, million)
About babies born are transfusion dependant thalassaemia yearly
At any time there are about 2,500 patients (mostly children) who are transfusion-dependent for survival.

29. Why do we need a programme?
Burden to patients
Burden to families
Burden to the government
Preventable
Cost effective
Annual prevention costs are constant while annual treatment costs rise year-on-year
The cost effectiveness of a prevention programme increases with every year it is in place
An effective prevention programme is essential in order to meet the cost of treating existing patients
A right to be informed of genetic risk

30. OBJECTIVES OF THE NATIONAL THALASSAEMIA PREVENTION AND CONTROL PROGRAM.
To reduce morbidity and mortality among the thalassaemia cases, by providing optimal clinical care.
To reduce the impact of thalassaemia on individual, family and community.
To reduce the prevalence of blood transfusion dependent thalassaemia cases.
To provide safe and adequate blood supply.
To create awareness regarding thalassaemia in the community.

31. Initial Screening Full Blood Count (FBC) Red cell indices – MCV, MCH,
MCHC,
RDW – in thalassemia trait uncomplicated by iron deficiency is normal.

32. RED CELLS INDICES RDW – N<15% MCV = PCV / RBC (femtolitres)
N = 80 – 100 fl
MCH = Hb / RBC (picograms)
N = 27 – 34 pg
MCHC = Hb / PCV
N = g/dl

33. HAEMOGRAM ITS CLINICAL SIGNIFICANCE
17 y/o Male
Asymptomatic

34. LAB INVESTIGATIONS Screening Test Diagnostic test 1. Hb Analysis
1. Full Blood Picture.
2. Reticulocyte count.
3. Test that determine iron status.
Diagnostic test
1. Hb Analysis

35. INVESTIGATIONS FBP - smear
Thalassemia trait
Thalassemia major
Immune haemolytic A

36. INVESTIGATIONS Reticulocytes count
- are juvenile red cells.
- Number of reticulocytes in periperal blood is a fairly accurate reflection of erythropoietic activity.
Range of retic count in health =
50-100x109/l
(0.5 – 2.5%)

37. Iron (Fe) status determination
Methods include:
Ferritin
Serum iron
Transferrin saturation (Iron/Total Iron Binding Capacity)
Zinc protoporphyrin (ZnPP)

38. INVESTIGATIONS Serum ferritin
- Only small fraction of total body ferritin circulates in serum but it is related to total iron stores.
- differentiate iron deficiency & AOCD.
- Assess Iron overload in Thalassemia Major.
In few situations may not true reflection of iron stores –
Acute & chr Leukaemia, some malignancies (eg neuroblastoma), acute hepatic failure & infection.

39. NORMAL RANGE Ferritin– M (16 – 323 ųg/l), F (7 – 282 ųg/l)
(Normal values in child similar to adult F, higher in infant 1-3 months ie up to 350 ug/I).
Iron – M (11 – 28 ųg/i), F (7 – 26 ųg/l)

40. Iron (Fe) status interpretation
IDA Thal Trait AOCD
S. Ferritin L N N / H
S. Iron L N L
TIBC H N L
TS L N N / L
ZnPP H N L

41. Iron (Fe) status determination: Interpretation
Measurement of iron status in samples with hypochromic, microcytic and normal HbA2 and HbF values is useful to distinguish between:
cases of uncomplicated iron deficiency with possible α thalassaemia trait or
silent α thalassaemia trait
AVOID unnecessary investigation and
inappropriate iron therapy

42. Iron (Fe) status determination: Interpretation
However, it is important to note that iron deficiency can co-exist with the thalassaemias misinterpreted.
It is sometimes necessary to recommend repeating the haematology screen after correction of iron deficiency

43. DIAGNOSTIC TEST HB ANALYSIS
Traditionally this consist of:
1.screening for abnormal Hb
2. Quantitation of A2 & F.

44. HB ANALYSIS - screening of abnormal Hb
Techniques :
i. Cellulose acetate electrophoresis at pH 8.6 & 6.5
ii. Automated Agarose Gel Electrophoresis at alkaline and acid pH
iii. Citrate agar electrophoresis at pH 6.0

45. HB ELECTROPHORESIS
i. Cellulose acetate electrophoresis at pH 8.5 & 6.5
ii. Automated Agarose Gel Electrophoresis at alkaline and acid pH
Electrophoresis on alkaline buffered gels (pH 8.5) - For the separation of the normal hemoglobins (Hb A and Hb A2) and for the detection of the major hemoglobin variants: S or D and C or E.
Electrophoresis on acidic gel, should follow to confirm the identification of hemoglobin variants, in particular to differentiate hemoglobins S from D and E from C.

46. Cellulose acetate

47. HB ELECTROPHORESIS i. Citrate agar electrophoresis at pH 6.0
An alternative technique to confirm the identity of a variant hemoglobin

48. HB ANALYSIS – Quantitation of A2 & F
Can be done with:
Microcolumn chromatography for A2.
Alkaline Denaturation for HbF
High Performance Liquid Chromatography (HPLC).

49. Microcolumn chromatography for A2. Alkaline Denaturation for HbF.
-Reliable when performed by competent technologist
-Labor intensive , slower TAT

50. High Performance Liquid Chromatography (HPLC).
Quantification Of Hb A 2 and F
Detection of variant hemoglobins.
Quick, less labour intensive, gave good precision and reproducibility with rapid TAT

51. HPLC Method

52. High Performance Liquid Chromatography (HPLC
Interpretation of the chromatograms requires expertise.
Useful for large scale screening.

53. Supplementary Tests H-inclusion Kleihaur Test Sickle Test
Sickle Solubility Test
Functional or Stability Tests

54. H-inclusion
Used to differentiate α-thal carriers from ß-thal carriers.
Intra-erythroyte inclusion bodies induced by precipitating Hb H with basic dyes (brilliant cresyl blue/ new methalene blue).
Hb H inclusion bodies are (ß4) scattered through rbc.
(Heinz bodies are larger than H inclusion & lie close to margin of rbc membrane & may occur singly).

55. HbH inclusion body detection
Supravital stains (brilliant cresyl blue or methylene blue).
Interpretation:
HbH inclusion bodies - α thalassaemia, especially HbH disease

56. Heinz bodies

57. Kleihaur Test (acid elution technique)
To distinguish between  thalassaemia trait and hereditary persistence of fetal haemoglobin (HPFH).
In HPFH, distribution of Hb F is homogenous, while in  thalassaemia trait it is heterogenous.
Limitation:
-The haemoglobin elution step is sensitive to pH,
time and temperature.
-Subjective interpretation of the stained blood film.
-Require experience of the scientist/technician
performing the test.

58. Sickle Test
Detection of Hemoglobin S. Sickle cell formation is induced when blood is deoxygenated (e.g. by the addition of a reducing agent, sodium dithionite or sodium metabisulphite )
Sickle test is to be performed only when electrophoresis at alkaline pH shows a variant band at the position of Hb S and / or HPLC shows the characteristic of Hb S.

59. Sickle Solubility Test
Detection of Hemoglobin S. This test depend on the decreased solubility of deoxygenated Hb S in concentrated phosphate buffer solution. The mixture of Hb S in a reducing solution give a turbid suspension which can be easily visualized
It is proposed to send specimen to the IMR for the sickle solubility test where the sickling test is negative.

60. Functional or stability tests
Hb stability test
Hb E detection by DCIP test
Oxygen dissociation curve

61. Recommendation
HPLC is recommended as primary screening tool in all laboratories due to its many advantages .
Hb electrophoresis should be used to further verify abnormal peaks identified with HPLC or detection of variants not detectable by HPLC.
Supplementary tests may be utilise whenever necessary.
For cases where diagnosis cannot be made by the methods mentioned above, molecular techniques shall be employed for confirmation.

62. Confirmatory Diagnosis
Currently molecular diagnosis available in
Malaysia at:
IMR
HUKM
UMMC
Some private labs outsource to Singapore or Australia
HKL to commence next year

63. ALGORITHM: Screening for thalassaemia in Malaysia
Full blood count / RBC indices
HPLC: Hb Constant Spring
MCH >27 pg
MCV >80 fl
MCH < 27 pg
MCV < 80 fl
Low iron level
Normal Hb A2
Hb subtypes: Hb A2, Hb F
Iron status
Normal iron
Increased Hb A2
Normal iron
Normal / Low Hb A2
Hb A2 > 4-9%
Hb A2 > 10%
Iron deficiency anaemia (IDA)
Treat & repeat Hb A2
Hb variant: Hb E / Lepore
PCR: -thal (thal 2 / 1 trait)
-thal trait
HPLC & DCIP
-thal trait
(Hb F raised 5-15%)
If positive: For family study
DCIP –ve = Hb Lepore
DCIP +ve = Hb E
Courtesy of Prof. Dr. E. George

64. Workflow for Thalassaemia screening Action   
Collect 2.5ml blood in EDTA MO / MA / SN
FBC MLT at Health Centre
   Review red cell indices MLT at Health Centre
MCH
    
> < 27
To sent : MLT at Health Centre
1.PBF smear
2.FBC result
3.EDTA blood
4.PER PAT form
Sent to State Hospital 
Hb analysis MLT & Pathologist
Results to Health Centre SN
Counselling MO / Counselor
No further
action

65. HAEMOGRAM OF TRAIT ITS CLINICAL SIGNIFICANCE
17 y/o Male
Asymp– family screening
Hb A %
Hb A2/E – 31.2 %
Hb F – 0.7 %
Diagnosis:
Haemoglobin E trait.

66. Additional Information…

67. Molecular diagnosis
Methods are based on the polymerase chain reaction.
dot blot analysis,
reverse dot blot analysis,
the amplification refractory mutation system (ARMS),
denaturing gradient gel electrophoresis,
mutagenically separated PCR,
gap-PCR and
Restriction endonuclease analysis.

68. PRECISE DIAGNOSIS ß thal
Screening for DNA sequence (unknown mutation) – Electrophoretic tech. eg
single strand conformation polymorphism (SSCP), Denature gradient gel electrophoresis (DGGE) etc.
THEN confirmed by direct gene sequencing.

69. PRECISE DIAGNOSIS ß thal
Screening for DNA sequence (known mutation) –
Allele specific oligomer hybridization with either dot blot analysis or reverse dot blot anaysis.

70. PRECISE DIAGNOSIS α thal
PCR based direct target sequence analysis (non radioactive).
Gene mapping (radioactive)

71.  THALASSEMIA SYNDROMES
α-thal 2 , 3.7 del
SEA α0

72. THANK YOU