Causes of megaloblastic anaemia

1. Folic acid deficiency a. Inadequate diet b. Alcoholism c. Steatorrhea or sprue d. Other causes of malabsorption, including partial gastrectomy e. Pregnancy and lactation f. Leukaemia, myelofibrosis, and chronic haemolytic anaemia 2. Vitamin B12 deficiency a. Pernicious anaemia b. Gastrectomy c. Sprue d. Long-term dietary deficiency e. Parasites (Diphyllobothrium latum
3. Drug induced megaloblastic anaemia 4. Congenital disorders (very rare) 5. Leukaemia 6. Di Guglielmos syndrome

Source of folic acid > Green vegetables, liver, yeast > Destroyed by prolonged boiling & acid pH

Source of vitamin B12 > Synthesized by bacteria form large bowel > Liver & kidney > Dairy products, fish, shellfish

LABORATORY DIAGNOSIS OF MACROCYTIC ANAEMIA

LABORATORY TEST Peripheral smear

INTERPRETATION
Macrocytic anaemia

Bone marrow examination

Megaloblastic changes

No megaloblastic changes

Reticulocyte count

Low

High

Low

Therapeutic response

Responds to vit B12

Responds to folic acid

Diagnosis

Vit. B12 deficiency

Folic acid deficiency

Probable haemolytic anaemia

Possible liver disease

ABSORPTION OF FOLIC ACID AND VIT. B12

Terminal ileum

Blood Tissue cells

Absorption of Vit. B12

Absorption of Vit. B12 and folate

A Normal Healthy Individual

PGA
5-m-THF (Serum Folate) Vit.B12 Polyglutamate (RBC folate)
Polyglutamate is stored in RBCs

THF

DNA

LAB RESULTS FOR NORMAL HEALTHY PERSON Serum Folate RBC Folate Serum Vit. B12 Normal Normal Normal

Normal Red Blood Cell

Folate Deficiency
PGA Low 5-m-THF (Serum Folate)

Normal Vit.B12

Low THF

Low Polyglutamate (RBC folate)

Polyglutamate is stored in RBCs is low

Low DNA

LAB RESULTS FOR FOLATE DEFICIENCY Serum Folate RBC Folate Serum B12 Low Low Normal

Macrocytic Cells

Vit. B12 deficiency

PGA Normal or High 5-m-THF (Serum Folate)

Low Vit.B12

Low THF

Low or Normal Polyglutamate (RBC folate)

Polyglutamate is stored in RBCs is low

Low DNA

LAB RESULTS FOR Vit. B12 DEFICIENCY Serum Folate RBC Folate Serum B12 Normal or High Low or Normal Low

Macrocytic Cells

Vit.B12 and Folate Deficiency

PGA Low 5mTHF (Serum Folate)
With low Vit.B12 and low folate present, 5-m-THF is not converted to THF

Low Vit.B12

Low THF

Low Polyglutamate (RBC folate)

Polyglutamate is stored in RBCs is low
As with folate or Vit.B12 deficiency, the RBCs are macrocytic

Low DNA

LAB RESULTS FOR Vit.B12 and FOLATE DEFICIENCY Serum Folate RBC Folate Serum Vit.B12 Low Low Low

Macrocytic Cells

Deficiency

Folic acid Serum RBC

Serum vit. B12 N

Folic acid Vit B12 N/

Folic acid & B12

Contraindications : - patients on critical ill

THERAPEUTIC TRIALS
Usual diet

- angina pectoris - congestive heart disease - thrombocytopenia with bleeding

0,2 mg folic acid oral

1 week

reticulocyte response + + 1 - 2 mg Vit. B12

reticulocyte response

GENETIC DEFECTS OF HAEMOGLOBIN (Haemoglobinopathy)

Reduced or abnormal synthesis of normal globin Reduced Abnormal Thalassaemia Hb - abnormalities Hb varian Fraction of normal haemoglobin in :

adult
HbA2 HbF < 3.5% < 1.0%

full-term infant
< 1% 50 - 85%

Mediterania, Afrika, Timur Tengah, India, Burma, Asia Tenggara meliputi : China selatan, Malaysia Indonesia

The geographycal distribution of the thlassaemia and haemoglobin abnormalities

Occur in tropical = sub tropical area Protection against Falciparum malaria

CLASSIFICATION CLINICAL - Thalassaemia major - Thalassaemia intermedia

- Thalassaemia minor
- Silent carrier

GENETIC

The globin gene clusters on chromosom 16 and 11 in embrionic, foetal and adult

Prenatal age (weeks)

Postnatal age (weeks)

Synthesis of individual globin chain in prenatal and postnatal life

PATHOPHYSIOLOGIC a-THALASSAEMIA
Foetus and neonates g chain 2 genes g4 Hb Barts a2 g2 HbF Thalassaemia-a Hydrops foetalis a chain 4 genes a2 b 2 HbA Child and adult b chain 2 genes b4 HbH Inclusion HbH HbH disease Heinz bodies precipitation Bone marrow Ineffective Erythropoiesis Peripheral blood MICROCYTIC HYPOCHROMIC ANAEMIA Increased erythropoiesis Intramedullary Bone changes Extramedullary Hepatosplenomegaly

DEATH Organ dysfunction Iron overload Iron absorption

Multiple Transfusion

1. HYDROPS FETALIS
-- / - 4 gen deletion Suppress a chain synthesis Failure of HbF synthesis

Death in utero
(Hydrop fetalis)

 Laboratory Hb MCV MCH Anisocytosis, poikilocytosis 3 - 10 g/dL 110 - 119 fl,

Retikulocytosis, NRBC +++

Electrophoresis : Hb Barts (g4) 80-90% Hb Portland + Hb A absent

a thalassaemia : four a gene deletion (hydrops foetalis)

2. HbH DISEASE

3 genes deletion
HbH (b4), unstable, termolabile Clinic :

Normal birth
Anaemia after one year Hepatosplenomegaly & icterus
Normal HbH
b thal. HbE

Normal b thal. trait b thal. trait

Haemoglobin electrophoresis HbH inclusion bodies HbH disease

2. HbH DISEASE

Laboratory
1. Hb 8-10 g/dL Microcytic hypochromic anaemia

Target cells ++, NRBC +, poikilocytosis and anisocytosis

2. Reticulocytosis, HbH inclusion bodies +++

3. Hb electrophoresis
adult Hb Barts HbH HbA2 2-40% neonates 25%

HbF

 HbH maybe found in 1. a thalassaemia : HbH disease, a thal 1 trait 2. Myeloproliferative disorders ALL, AML &

sideroblastic anaemia
Defect a gene transcription Deficiency a RNA Deficiency a chain HbH (5-70%)

3. a THALASSAEMIA 1 TRAIT (MINOR)

2 genes deletion a - / ahomozygous a thal 2

-- / aa
Clinic : none Laboratory :

heterozygous a thal 1

1. Hb 10 - 12 g/dL Microcytic hypochromic anaemia VER 60 - 70 fl HER 20 - 25 pg Anisocytosis

3. a THALASSAEMIA 1 TRAIT (MINOR) Laboratory 2. Hb electrophoresis Cord blood : Hb Bart 5-6% after 1-3 month

Hb Bart 0%
Adult : Microcytic hypochromic without anaemia

Ferritin N /
Electrophoresis Hb : Hb Bart 0% HbH inclusion bodies 1/1000 1/10.000 RBC

4. a THALASSAEMIA 2 TRAIT (SILENT CARRIER) -a / aa Clinic : normal Laboratory Cord blood : HbH < 1%

PATHOPHYSIOLOGIC b-THALASSAEMIA
Foetus and neonates Child and adult

a chain (4 genes) b chain (2 genes)

HbA a2 b 2

g chain (2 genes)
HbF a2 g2

d chain (2 genes)
HbF a2 g2 HbA a2 b 2 HbA2 a2 d2

b Thalassaemia major Heinz bodies Bone marrow Ineffective Erythropoiesis

b Thalassaemia minor MICROCYTIC HYPOCHROMIC

Iron overload

Iron absorption

MICROCYTIC HYPOCHROMIC

Transfusion

ANAEMIA
Increased erythropoiesis

ORGAN DYSFUNCTION

Intramedullary haematopoiesis Bone changes

Extramedullary haematopoiesis Haepatomegaly Splenomegaly Hipersplenism

homozygote b thalassaemia (major) post splenectomy

homozygote b thalassaemia (major)

heterozygote b thalassaemia (minor)

b Thlassaemia major

Clinic :
Severe anaemia becomes apparent at 3 - 6 month after birth Hepatomegaly Splenomegaly (increased RBC destruction in pooling), expansion the plasma volume Expansion of bone (thalassaemic facies) Iron overload (endocrine organs &

myocardium)
Pneumococcal & meningococcal, hepatitis B, hepatitis C, HIV infection

b Thlassaemia major

Laboratory :
Microcytic hypochromic anaemia, reticulocyte (%), normoblasts, target cells & basophilic stippling Haemoglobin electrophoresis : HbA2 normal, HbF HbA absence or almost complete absent or

Ferritin 1000 - 1500 mg/L

Ferritin is raised in viral hepatitis & inflamatory disorders

Mongoloid facies

Hair on end appearance

b thalassaemia trait (minor) Clinic : Symptomless abnormality Laboratory : Mild or no anaemia (Hb 10-15 g/dL) RBC microcytic hypochromic Haemoglobin electrophoresis :

HbA2
HbF

> 3.5%
N/