This document provides information on hemoglobinopathies and thalassemias. It begins by defining hemoglobinopathies as genetically determined abnormalities of the hemoglobin molecule, associated with globin chains. It then discusses the different types of globin chains and normal hemoglobin development. The document classifies hemoglobinopathies into 5 major classes including structural abnormalities, thalassemia syndromes, thalassemic hemoglobin variants, hereditary persistence of fetal hemoglobin, and acquired hemoglobinopathies. It provides detailed information on beta thalassemia syndromes, genetics, clinical features, hematologic findings, bone marrow findings, laboratory tests, and complications/management of thalassemia major. It also briefly discusses

1. HEMOGLOBINOPATHIES
DR.ANAMIKA

2. INTRODUCTION
• Genetically determined abnormality of the structure or synthesis of the
haemoglobin molecule are called haemoglobinopathies.
• The abnormality is associated with globin chains, heme portion will be
normal.
• Range in severity is from asymptomatic laboratory anomalies to death in
utero.

3. GLOBIN
• GLOBIN – Different types of Hbs are formed depending on composition
of associated globin chain tetrads; responsible for different physical
properties of Hbs.
• α like chains – α & ζ chains
• Β Like chains – β, γ, ε, δ chains
• Globin chains – Held together by non-covalent bonds in a tetrahedral
array, giving the Hb, a spherical shape.

4. HEMOGLOBIN
• Tetramer of globin polypeptide chains:
a pair of α-like chains and a pair of β-like chains
• 6 Wks – Hb Portland(ζ 2γ2), Hb Gower I(ζ 2ε2) and Hb
Gower II(α2 ε2).
• 10-11 Wks – Hb F(α2 γ2)
• 35 Wks – Hb A2(α2 δ2)
• 38 Wks – Hb A(α2 β2)
• New born – Hb F-50-85%
• 1 Yr – Hb A is the major Hb, HbF < 2%,
HbA2 - 1.8-3.5%

6. HEMOGLOBIN

7. CLASSIFICATION OF HEMOGLOBINOPATHIES
5 Major classes
I. STRUCTURAL – Qualitative abnormality.
 Decreased solubility – Sickle cell anemia (Hb S), Hb C, Hb
SC, Hb D.
 Decreased stability – Congenital Heinz body anemia.
 Altered O2 Affinity – High- Polycythemia.
Low- Anemia, cyanosis.
II. THALASSEMIA SYNDROMES – Deficient globin chain
biosynthesis.
 α – Thalassemia.
 β – Thalassemia.
 δβ Thal, γδβ Thal, αβ Thal.

8. III. THALASSEMIC Hb VARIANTS –
– HbS/β-Thal.
– HbS/α-Thal.
– Hb E.
– Hb Constant Spring.
– Hb Lepore.
IV. HEREDITARY PERSISTENCE OF FETAL Hb-(
HPFH) – High levels of Hb F in adult life.
V. ACQUIRED HEMOGLOBINOPATHIES –
Modifications of normal Hb by toxins.

9. THALASSEMIA

10.  “Thalassa” – (Gk.) Sea.(referring to Mediterranean)
 Grp of congenital anemias that have deficient synthesis of one or more
of the globin subunits of the normal Hb.
 Thomas Cooley identified it.
 It is quantitative hemoglobinopathy since no structurally abnormal
hemoglobin is synthesized.

11. CLASSIFICATION OF THALASSEMIAS
Normally α : β - 1:1
Degree of imbalance is related to the clinical expression.
A. Impaired α–chain synthesis – α-Thal
B. Impaired β–chain synthesis – β-Thal
C. Misc. Thalassemic syndromes –HbS Thal, HbD Thal, HbE Thal, δβ-

12. BETA -
THALASSEMIA

13. ß THALASSEMIA SYNDROMES
• Thalassemia Major – Most severe form, homozygous state
• Thalassemia Intermedia – Double heterozygous state, milder form
• Thalassemia Minor/Trait(BTT) – Heterozygous state, asymptomatic with little or no
anemia
• Thalassemia Minima – Clinically undetectable

14. GENETICS
• Autosomal Recessive disorders
• 2 β- Thalassemia genes (βThal)–
• β˚ - No production of β-Chains.
• β+ - Some but still subnormal production.
• Any combination of normal β-genes and βThal genes are possible –
various phenotypes.
• Point mutation on globin gene cluster; i.e, single nucleotide substitution
– Splicing mutation – Most common cause of β+ Thalassemia
– Promoter region mutation - β+ Thalassemia
– Chain terminator mutation - β0 Thalassemia

15. BETA -THALASSEMIA MAJOR
• Also K/A Cooleys anemia
• Homozygous form of β0 / β0 or β+ / β+ or double heterozygous β0 /β+
• Infants well at birth
• Later by 6 months , develop moderate to severe anemia, failure to thrive,
hepatosplenomegaly and bone changes
• Transfusion dependent

17. CLINICAL FEATURES
Irritable, pale infant, failure to thrive, diarrhea, fever
and enlarged abdomen.
Severe anemia → Cardiac failure.
Chronic Hemolysis → Gout, Gall stones, Icterus
Thrombotic complications.
Massive Splenomegaly.
Flattened nose, wide set eyes, frontal bossing of the skull, prominent
cheek bones, and overgrowth of zygomatic bones– “Chipmunk Facies” /
thalassemic facies/ mongoloid facies.
Enlarged marrow cavities d/t hyperplastic marrow.
Hair-on-end appearance on X-Ray skull.

19. HEMATOLOGIC FINDINGS
• Anemia – moderate to severe when first diagnosed ; 3-8gm%
• MCV – 50-70fl
• MCH -12-20pg
• MCHC - 22-32%
• PERIPHERAL SMEAR-
– RBCs are microcytic and hypochromia is marked and red cells are thin
– Moderate to marked degree of aniso-poikilocytosis
– Many target cells. Central puddle of Hb may be eccentric
– Basophilic stippling – constant feature
– Nucleated RBCs (mainly late normoblasts )- 5 - 40/100 WBC
– Presence of tear drop , elliptical, fragmented red cells and occasional red cell with Howel Jolly
body
• Aggregates of free α chains can be seen by phase contrast microscopy on supravital
staining with methyl violet
• Reticulocyte count <2 % because of ineffective erythropoiesis & does not correspond to
severity of anemia

21. IRON STATUS
• S. ferritin >1000 µg/L ( 50-150)
• Transferrin saturation ↑’d 55-90% (30-35%)
• Total iron binding capacity ↓ ‘d 250-300 µg% (320-360)
• S. Iron is increased

22. BONE MARROW
• Markedly hypercellular
• Erythroid hyperplasia is marked
• M:E ratio is reversed to 1:1 to 1:2
• Erythropoiesis is normoblastic
• Some normoblasts demonstrates features of
dyserythropoiesis like irregular nuclear and
cytoplasmic borders.
• Normoblasts demonstrate pink inclusions of
free α chains and basophilic stippling in
intermediate and late normoblasts
• Few gaucher like cells may be seen
• Myelopoiesis and megakaryopoiesis - normal
• Abundance of iron ( Prussian blue stain)

23. LABORATORY TESTS FOR DIAGNOSIS
1. Acid elution test / kleihauer’s cytochemical method
• Hb F levels are high 30%-90%; being higher in β0 Thalassemia than in β+ Thalassemia
• In the red cells, Hb F can be demonstrated by acid elution test
• Procedure : blood smear fixed with ethyl alcohol and incubated in prewarmed citric
acid phosphate buffer solution for 5 mins and then stained with hematoxylin and
erythrosine B
• Result:
– Red cells containing Hb F  stained pink (resistant to acid elution)
– Red cells containing Hb A  appear as GHOST cells

25. 2. Hb Electrophoresis
– Done on starch agarose/ cellulose acetate membrane
– Shows bands of both Hb A and Hb F in β+ Thalassemia
– In β0 Thalassemia – Hb F >90%
3. HPLC
4. Globin Chain synthesis analysis
– Due to lack of synthesis of ß chains,α:ß ratio is altered to 2-30:1 (normal 1:1).
– In transfused patients – Globin chain synthesis analysis is done by separating peripheral
blood reticulocytes
– Done by incubation of red cells with a radioactive tracer such as H-leucine
5. Mutation studies by DNA analysis, DNA Scanning and DNA Sequencing

26. HPLC

28. THALASSEMIA INTERMEDIA
• Double heterozygote for mild β+ Thalassemia alleles
• It’s a clinical spectrum intermediate between thalassemia major and minor
• Pts are anemic but not transfusion dependent
• Hematological features are of moderate severity
• Hb- 7-10gm/dl
• Hb F – 10-30%

29. THALASSEMIA MINOR/ ß THALASSEMIA TRAIT
(BTT)
• Heterozygotes for thalassemia gene
• Carriers are asymptomatic
• Women during pregnancy are diagnosed as trait cases in thalassemia screening
programmes.
• Hematologic findings
– Hb- 10-12gm/dl
– RBC count > 5.2 million /cumm
– MCV -65-80fl
– MCH – 20-25pg
– MCHC is nearly normal – 29-33%
– Hb A -90-93%
– HbA2 – 3.6-8% (normal < 3.5 %)

31. • If Hb A2 is 3.3 -3.7%  iron status to be studied because it can be BTT with associated
IDA
• Peripheral smear – mild degree of aniso-poikilocytosis with microcytosis, hypochromic
, few target cells and low RDW compared to IDA
• Serum iron and serum ferritin are normal to increased
• NESTROF test
– Naked eye single tube red cell osmotic fragility test
– Method : 5 ml of 0.35% saline solution is taken in two test tube. To the tubes is added 0.02
ml of blood of a normal person( control) and patients blood ( test )
– After half an hour put a white paper with dark black line behind both tubes
– In control tube , black line is clearly seen.
– In test tube, line is not clearly seen in positive cases since microcytic hypochromic red cells
of thalassemia are more resistant to lysis than normal normocytic normochromic red cells
– Used in screening programmes
– If +ve  do HbA2 estimation to detect BTT
– False positives : IDA, Hb E thalassemia , Hb D thalassemia

33. THALASSEMIA VS IRON DEFICIENCY
Differential diagnosis of BTT – iron deficiency anemia
BTT – high RBC count , target cells, and stippled cells, HbA2
> 3.5%
IDA – RDW increased and MCHC is low, Hb A2 – 1-3.5%
Thal Minor Fe Def Anemia
RDW N ↑
RBC N or ↑ ↓↓
MCV ↓↓ ↓
MCH ↓↓ ↓
MCHC ↓ ↓↓
HbA2
↑ N
FEP N ↑

34. Management of Thalassemia Major
• Regular blood transfusions at an early age – to prevent
hyperstimulation of the bone marrow.
• “Supertransfusion Regimen”
• Bone marrow transplantation
• Cord blood transplantation
• In-utero Stem cell transplantation
• Gene Therapy – to correct defects at molecular level.
• HbF reactivation - 5-Azacytidine, Hydroxyurea, Cytarabine,
Vinblastine, Butyrates.
• Splenectomy – when excessive destruction of RBCs.

35. Complications of Treatment
• Transfusion Related–
• Infections – HBV, HCV, HGV.
• Hemosiderosis –Each unit BT contains 200mg Iron.
PATHOGENESIS –tissue damage d/t- Iron-induced peroxidative injury
to the phospholipids of lysosomes and mitochondria; free radical
formation.
Myocardial Iron Toxicity
Hepatic fibrosis and cirrhosis.
Endocrinal
Pulmonary insufficiency
CHELATION – Desferrioxamine(DFO) , Deferiprone(DPO) and
Deferasirox

36. • BM Transplantation Related
• Chronic Graft-Vs-Host Disease.
• Short stature and fertility related problems.

37. PREVENTION OF THALASSEMIA
MAJOR
• All mothers during first antenatal check up screened for Hb, MCV, MCH, MCHC, PS,
and NESTROF test.
• Pts with MCV<70, MCH <23 and +ve NESTROF test Assess HbA2 level of mother ,if
3.6-8% , then evaluate father also.
• If positive for trait  if < 12 weeks – chorionic villous biopsy sampling
12-20 weeks – amniotic fluid sampling
• Then PCR analysis on fetal DNA is done to detect point mutations

38. MISC. THALASSEMIC SYNDROMES
1. HbS/β-Thalassemia(βS/βThal)
• Double heterozygous state of HbS & β thalassemia.
• Severity depends upon inheritance of β-Thal gene i.e β˚ or β+
CLINICAL FEATURES
•Hepatosplenomegaly.
•Lymphadenopathy.
•Vaso-occlusive crisis
•Mild episodes of skeletal pain and fever

39. LABORATORY FEATURES –
Similar to SCA to Heterozygous Thal
 Microcytic hypochromic anemia (Clue to the presence of Thalassemia)
MCV, MCH, MCHC
 Hb – 5-10g/dL
 PS – Anisocytosis, Poikilocytosis, Target cells, basophilic stippling
 Increase in HbF and Hb S levels
• Hb Electrophoresis –
- In HbS/β˚Thal – HbA – Absent
HbS – 70%
HbA2 & HbF – Increased
- In HbS/β+Thal – HbA – 50%
HbS – 50%
HbA2 & HbF - Increased

40. 2. Hb E THALASSEMIA
– Patient inherits ßthal gene from one parent and HbE gene from another parent
– HPLC and Hb electrophoresis  HbA , HbF and HbE inß+/E
– No HbA in ß 0 /E
3. Hb D THALASSEMIA
– HbA , HbF and HbD
4. δß THALASSEMIA
– Reduced or absent production of both δ and ß chains and an increase in γ chain synthesis
– 2 types – δß + & (δß) 0
– Δß + production of Hb Lepore  has normal α chains and δß chains instead of ß chain
– HbF 10-20%
– HPLC – shows humps on downward slope

42. • 5. HEREDITARY PERSISTENCE OF FETAL HEMOGLOBIN
• Group of heterogenous disorders in which the absence of δ and β-
chains synthesis is compensated for by increased γ -chain production
into adult life.
• Deletion / inactivity of the β and δ-structural gene complex.
• No β and δ-Chains production → Excess of α–chains → Combine with
γ – chains to form HbF.
• Typical finding – PANCELLULAR distribution Hb F
• HPLC- near total absence of HbA and HbF >90%

45. A. ALPHA THALASSEMIA
Normal Hb α-Chain Production
• α chains of globin are not / partly synthesized.
•most frequently by deletions of DNA that involve one or
more α-genes.
•Less common causes are point mutations and presence of
an abnormal α-gene.

48. HYDROPS FETALIS/ HB BARTS
• Deletion of all the four α genes
• Intra-uterine death; if born , dies in 2 hours
• Baby is pale and bloated
• Placenta is edematous
• Moderate to massive hepatomegaly
• Barts has high affinity for oxygen, therefore oxygen does not dissociate from γ4  severe tissue hypoxia and
fetal death
• Hb Electrophoresis
• Hb Bart’s – 80-90%
• Hb H & Hb Portland – 10-20%
• TREATMENT
• No effective therapy.
• IU transfusions, In-utero stem cell transplants attempted unsuccessfully.
• Early termination of at-risk pregnancy.

49. Hb H DISEASE–(α-Thal-1/α-Thal-2)Or(–,–/–,α)
•Most frequent in South-East Asia.
•Excess of β-Chains form tetramers(β4) – Hb H.
β
ββ
β
β
ββ
β
Hb H
PATHOPHYSIOLOGY
•HbH – An unstable thermolabile protein with high O2
affinity.
•Form intracellular inclusions Membrane Damage

50. • Shortened RBC life span → Chronic Hemolytic Anemia.
• Ineffective Erythropoesis – Not very severe.
CLINICAL FEATURES –
• Mild to severe anemia.
• Worsening of anemia during pregnancy, infections, oxidant
drugs intake.
• Splenomegaly.
• Respiratory infections, leg ulcers, gall stones, Jaundice
• Moderate skeletal changes.
LABORATORY FEATURES
• Hb – 6-10 g/dL.
• Reticulocytosis – 5-10%
• MCV, MCH, MCHC – Reduced
• Erythrocytosis.

51. • PS – Microcytic Hypochromic BP.
– Variable poikilocytosis, anisocytosis.
HbH inclusions when stained with supravital stain.
Brilliant cresyl blue;40X
– Blue globules, Many per cell, require time for formation
– Gives a golf ball appearance
• BM – Erythroid hyperplasia with normoblasts having scant
supply of Hb.
•Hb Electrophoresis –
•HbH – Up to 40%
•HbA2 – Decreased •HbF - Normal

52. • α-THALASSEMIA TRAIT
 Common in Mediterranean Area, West Africa and in South-East
Asia.
 Asymptomatic or mild anemia.
 Hb – 10-12 g/dL.
 MCV – 60-70 fL, MCH – 20-25 pg.
 PS – Microcytosis, Hypochromia
– Occasionally HbH inclusions
– Target cells, Basophilic stippling

53. SILENT CARRIER – (α-Thal-2/Normal)
• One α-gene is deleted.
• Adequate normal Hb synthesis.
• Definitive diagnosis by Gene Mapping.
• Asymptomatic

54. REFERENCES
• Tejinder singh. Atlas and text of hematology. 3rd edition.
• Kumar, Abbas, Fausto. Robbins and Cotran Pathologic Basis of Disease.9th ed.
• Internet sources