case history of 9 month old infant Paediatric Clinical Approach to this case examination workup at blood centre HPLC screening laboratory findings screening of father mother prominent facial features PBF and bone marrow findings usg abdomen xray skull prbc transfusion therapy in thalassemia major classification of thalassemia national burden in india pathogenesis- anemia skull bone iron overload world thalassemia day

1. An Integrated Teaching Session on-
A CASE OF
9 months old male patient with h/o splenomegaly,
severe anemia & Caput quadratum (diagnosed as
Thalassemia Major)
In integration with Dept. of Paediatrics
Presented by- Dr. Shiny K. Kajal
(JR-1, Dept of IHBT)
Moderator- Dr. Anshul Gupta

2. RELEVANT CASE HISTORY
• A 9-month-old infant presented to paediatric OPD on 06/03/2023
with chief complaints of-
 Noisy breathing since birth
 Fever for 1 week
• He was admitted and treatment was started for the same under
the paediatric department

3. Paediatric Clinical Approach to this case

4. • Birth History- LSCS/CIAB/2.2kg/Term
- LSCS done in view of Oligohydramnios
• Immunization History- Complete till date
• Dietary History- Exclusively Breastfed till 6 months.
• Antenatal History- 1st child, ANC card made, iron and calcium
supplements were taken, viral markers negative, Hospital delivery

5. RELEVANT PAST HISTORY
• No history of Blood transfusion
• No History of previous hospital admission
• No history of any surgical procedure
• No history of NICU admission

6. EXAMINATION
• General Examination: The patient was irritable,
Pulse- 142 bpm, RR- 32/min, Temperature- 99 degrees F
Pallor present
Head circumference- 43 cm
Depression in Anterior fontanelle
Prominent Parietal bones (Caput Quadratum)
• CVS – S1, S2 present
• RS - Bilateral Air entry equal
No added sounds

7. • Per Abdomen examination
Inspection- Symmetrical, No Visible scar, pulsations,
Umbilicus normal
Palpation- Liver palpable 6.5 cm below the subcostal margin
Liver span 11.5cm
Spleen palpable 6 cm below the subcostal margin

8. Workup at Blood Centre
• 2ml Blood sample was received at Blood Centre for Blood grouping.
• A requisition for 1 unit of PRBC was received in view of anemia.
• Another 2 ml blood sample was received for requisition of HPLC screening
in view of Chronic Anemia, prominent facial features and
hepatosplenomegaly.
 The Blood sample for HPLC screening was taken before the transfusion as
Post-transfusion sample does not give valid HPLC Screening results.

9. Workup at Blood Centre
• Blood Group- O Positive
• 1 PRBC unit, 100 ml, was issued from Blood Centre on 6/3/23
TESTS 06/03 07/03
TLC 34,440 36,200
Hb 5.7g/dl 6.5g/dl
Platelets 1,40,000 90,000

10. Lab findings before HPLC Screening
(based on blood sample received at Blood Centre)
Hb- 6.5 g/dL
MCV- 72.8 fl
MCH- 20.2 pg
RBC count- 3.21 x 106 /ul
Mentzer’s Index- 22.7

11. HPLC Screening:
• 2 ml blood sample was
processed.
• Following findings were
observed on the chromatogram-
LA1c/CHb-1 - 75.7
(eluted with HbF)
Hb A2 - 2.3
Hb Ao - 1.7

12. Diagnosis on HPLC Screening

13. HPLC Screening of Father

14. HPLC Screening of Mother

15. WHAT IS HPLC SCREENING?
Cation exchange High Performance Liquid Chromatography
is the method of choice for
• the initial screening of Hemoglobin variants like HbS, HbD,
HbE and HbC
• for accurate quantification of HbA2 and HbF concentrations
• A 2 mL blood sample is processed and report is given in form of
chromatogram where different peaks are identified in defined
windows with relevant information like retention time, relative
percentage and area

16. A NORMAL HPLC CHROMATOGRAM

17. AN OVERVIEW OF HPLC EVALUATION

19. OTHER INVESTIGATIONS
SUPPORTING
DIAGNOSIS

20. Lab findings on presentation
• LDH- Very high
• Raised Bilirubin
• Raised SGOT

21. Prominent Facial Features

22. PBF & Bone Marrow Aspiration Findings
IMPRESSION-
PBF- Microcytic hypochromic
anemia
BM- Marked erythroid
hyperplasia, possibly due to
hemolytic process

23. USG Abdomen
IMPRESSION-
• Liver is enlarged in size
measuring 8.2 cm
• Spleen is enlarged measuring
8.7 cm
Hepatosplenomegaly

24. Xray Skull

25. Current Management
• The patient is now registered with dept of IHBT and Paediatrics and gets
regular blood tranfusions, once in 3-4 weeks.
• The patient has received 8 cycles of blood transfusion till now.
• Last Hb levels- 9.2g/dl
• Genetic testing of the patient and the parents has been done at PGI
chandigarh and the reports are awaited.

26. • Generally, Patients are transfused with 11 to 14 ml of red cells per kg every 3
to 4 weeks according to the “Hypertransfusion programme”, usually started
at the age of 6 months, to maintain the haemoglobin concentration
constantly above 9.5 to 10.0 g/dl according to AABB standard guidelines.
• Immediate post transfusion hemoglobin levels typically average 12-14 g/dL,
returning to baseline after 3 to 6 weeks.
• Iron chelation therapy is usually started at the age of 3 years. An iron chelator
is given along with vitamin C to promote iron excretion.

27. • If the haematocrit of the red cell concentrate used is 0.65, then 3-4 ml/kg will
raise the Hb by 1 g/dl in the absence of hypersplenism.
• Generally, in a single transfusion, an attempt is made to raise the Hb by 4
g/dl if transfusions are scheduled at 3-5 weekly intervals.

28. • In addition, the marrow suppressive effect of hypertransfusion prevents the
release of the deformed, short-lived thalassemic erythrocytes and delays the
onset of hypersplenic changes, permitting splenectomy to be postponed
safely until an older age.
• The bony abnormalities are now entirely preventable.
• The definitive analysis for Thalassemias remains DNA
Analysis, HPLC being a screening test.

29. Thalassemia Major

30. Inherited Disorders of Hemoglobin
These disorders are divided into three broad groups:
• Haemoglobinopathies
• Thalassaemias
• Hereditary persistence of foetal haemoglobin (HPFH)

31. Thalassemias
• Autosomal recessive inherited group of disorders of hemoglobin synthesis.
• reduced or absent production of one or more of the globin chains due to
molecular lesions
• They are the commonest, most prevalent of all known single gene disorders
in the world.

32. CLASSIFICATION
The classification of thalassaemias is based on
(1) the type of globin chain that is deficiently synthesized
(2) clinical expression of the disease
(3) Number of globin chains involved
(4) In association with other disease- eg.
Sickle cell β thalassaemia (Micro drepanocytic disease)
α
ᵦ
Major
Minor
Gene deletions
Point mutations
Inter
media
Both- Homozygous
Eg- Cooleys anemia (Homo-beta)
Haemoglobin Bart’s Hydrops Foetalis
Syndrome (Homo- Ao)
Single- Heterozygous
(Trait)

33. Prevalance
• β thalassaemias are prevalent in “β thalassaemia belt” which covers
Mediterranean region, Africa, Middle East, some areas of India, Pakistan,
and Southeast Asia.
• In India, β thalassaemia is more common in certain communities of North
India such as Punjabis, Lohanas, Sindhis, Bengalis, Gujaratis, Bhanushalis,
and Jains
• Thalassemia is common in malaria endemic areas because the defective red
cells do not provide favourable environment to the malarial parasite to
complete its life cycle.

35. • Huge burden in India with an estimated 100,000 patients with a β
thalassemia syndrome, approximately 1.2 per 1000 live births.
• 3-4 % β thalassemia carriers- 35 to 45 million carriers in population of 1.21
billion people.
• Estimated that 2 million units of packed red cells would be needed for
transfusion of thalassemia patients in our country
• Punjab state has a population of 29 million with 1.5 million carriers with beta
thalassemia (3.96%)
National Burden of disease

36. Pathogenesis
1. Anemia
• In β thalassaemia major, the underlying genetic defect is responsible for
inability of erythroid cells to synthesise adequate amounts of β globin chains.
• This causes excessive accumulation of free α chains since there are no
complementary β chains to form a tetramer.
• The unbound α chains precipitate within erythroblasts and red cells. These α
chain inclusions damage the cell membrane leading to the lysis of
erythroblasts and red cells in the bone marrow, i.e. ineffective erythropoiesis

37. Anemia continued..
• in addition to intramedullary destruction, red cells are also destroyed
peripherally in the spleen (haemolysis)
• Reduced synthesis of haemoglobin due to lack of β globin production leads
to the formation of microcytic hypochromic red cells
• Excessive peripheral destruction of red cells invariably leads to
splenomegaly.
• Haemoglobin F is the predominant in β thalassaemia major and is due to
increased proliferation of cells capable of synthesising γ chains. HbF does
not release oxygen as readily to the tissues as HbA since it poorly binds 2,3-
diphosphoglycerate and thus exacerbates tissue hypoxia.

38. 2. Skeletal changes
Severe anaemia and tissue hypoxia stimulate erythropoietic drive and cause
extreme bone marrow hyperplasia.
Expansion of hyperactive bone marrow causes weakening and deformities of
skull and of facial bones. Thinning of cortex may lead to pathological fractures.

39. 3. Iron overload
Iron absorption from the intestine is increased in β thalassaemia major due to
ineffective erythropoiesis.
Chronic regular blood transfusion therapy markedly increases the iron
accumulation and causes iron overload.
Iron overload may damage parenchymal cells of various organs such as
pancreas (diabetes mellitus), liver (cirrhosis), gonads (infertility), and heart
(arrhythmias and heart failure).

40. Molecular Basis for the Thalassemia Syndromes
• More than 200 distinct thalassemia mutations are now known.

41. Laboratory Features
1. Peripheral blood examination:
 Patient presents with Severe Anaemia
 Haemoglobin concentration between 2 and 6 g/dl
 Anaemia is typically microcytic and hypochromic
 On peripheral blood smear examination, red blood cells show marked
anisopoikilocytosis, severe hypochromia, plenty of target cells, Howell-Jolly
bodies,basophilic stippling, and nucleated red cells
 Reticulocytosis is modest (5—15%) but is less as compared to the degree of
anaemia. The reason for this is ineffective erythropoiesis.
 Leucocytes and platelets are usually unremarkable.

42. 2. Haemoglobin electrophoresis: This characteristically shows elevated HbF
(10–98%). HbA2 may be normal or increased.In homozygous β0 thalassaemia,
HbA is completely lacking, while in β0/β+ and β+/β+ thalassaemias, some
amount of HbA is present.

43. 3. Other investigations:
• Bone marrow examination shows severe erythroid hyperplasia.
• Storage iron is increased
• Acid elution test reveals heterogeneous distribution of HbF in red cells.
• Unconjugated serum bilirubin and serum iron are increased while osmotic
fragility of red cells is decreased.
• HPLC findings in thalassaemia major and minor as discussed

44. Clinical features
 Typically children with β thalassaemia major present with severe anaemia,
failure to thrive, retarded growth, hepatosplenomegaly, and skeletal and
facial changes.
 Frontal bossing and overgrowth of maxilla produce thalassemic or
“chipmunk” facies.
 Chronic transfusion therapy is also associated with risk of transmission of
viral infections such as human immunodeficiency virus (HIV), and hepatitis Β
and C viruses (HBV and HCV).

45. Clinical features contd..
• These children suffer from
– repeated infections
– raised serum iron level that favors bacterial proliferation has been implicated
– Folate deficiency commonly develops due to increased erythroid turnover.
• Untreated patients usually die from anemia or infections before five years of
age.

46. • Early institution of regular blood transfusion therapy is associated with normal
growth and development during the first decade of life.
• Iron overload gradually develops during adolescence. Most of the patients fight
with life by 3rd decade of life due to refractory heart failure and arrhythmias.

47. Strategies for Prevention of Thalassaemias
1. Health Education- World Thalassemia Day is a global healthcare event
celebrated every year on May 8th to raise awareness about the condition

48. 2. Carrier screening and genetic counselling:
i. Screening involves identification of heterozygous individuals
ii. Heterozygous persons should not marry another heterozygote for the
same gene due to the risk of having affected children.
iii. In genetic counselling couples at risk (i.e. when both partners are genetic
carriers) are explained various options available such as prenatal
diagnosis followed by selective termination of pregnancy and alternative
methods for having a child such as artificial insemination, adoption, etc.

49. 3. Prenatal diagnosis:
Prenatal diagnosis of affected foetus and counselling for selective termination
of pregnancy.
• Prenatal diagnosis is carried out in couples who are already having an
affected child (retrospective diagnosis)
• or in couples who are identified as carriers by screening (prospective
diagnosis).

50. Additional Management Process
1. Splenectomy: Indication for splenectomy is transfusion requirement
exceeding 180 to 200 ml of packed red cells/kg/year
2. Haematopoietic stem cell transplantation: Although Regular transfusions
and iron chelation therapy have improved the quality of life in thalassaemia,
bone marrow transplantation is the only form of therapy that can cure the
disease but is associated with significant morbidity and mortality.
3. Enhancement of HbF production: Currently hydroxyurea and butyrate are
being tried to increase HbF production that will improve haemoglobin level
and reduce ineffective erythropoiesis
4. Gene therapy: This consists of introduction of normal gene in stem cells to
replace the abnormal gene. Research is in progress for gene therapy.

51. • The Dept. of Pediatrics along with Dept. of Immunohematology & Blood
Transfusion &Dept. of Cardiology provide free of cost treatment at Adesh
Thalassemia Day care centre to registered Thalassemia patients providing
 Leukodepleted Blood Transfusions
 Iron Chelation Therapy
 Lab investigations
• Dept. of IHBT also performs 3-cell panel and 11-cell panel Antibody
Screening to detect alloantibodies in Thalassemia Children.

52. References
1. AABB Standards of Transfusion Medicine
2. Rossi’s Principles of Transfusion Medicine
3. Essentials of Hematology, Kawathalkar
4. Prevelance and management of thalassemia in India by S.S Yadav
5. National Health Mission guidelines on Thalassemia
6. Biorad Thalassemia Manual
7. DGHS Transfusion Medicine Manual

53. If I may please take the privilege of
few minutes, before I conclude my
presentation