Megaloblastic anemias are caused by impaired DNA synthesis due to vitamin B12 or folate deficiency. The summary examines megaloblastic anemias, including causes such as vitamin B12 or folate metabolism defects, clinical features like pallor and neurological symptoms, investigation findings in peripheral blood and bone marrow showing megaloblasts and macroovalocytes, and treatment involving vitamin B12 or folate supplementation.

1. Megaloblastic Anaemias
Dr. Raghav Kakar
M.D. Paeds

2. Definition
Megaloblastic anemia is a general term used to
describe a group of anemias caused by impaired
DNA synthesis. It is characterized by abnormal
findings in peripheral blood smear
(macroovalocytes) and bone marrow samples
(megaloblastic hyperplasia). Megaloblasts, the
hallmark of these anaemias, are caused by
asynchronous maturation between the nucleus
and the cytoplasm due to DNA synthesis
impairment

3. Aetiology
 Vitamin B₁₂ deficiency or defective metabolism
 Folate deficiency or defective metabolism
 Drug induced ( Purine and Pyramidine analogs)
 Others:
• Pernicious
anaemia(autoimmune)
• Surgeries
• Nitrous oxide
• Oroticaciduria
• Lesch-Nyhan syndrome
• Sideroblastic anemia
• Aplastic anemia
• Thiamine responsive
megaloblastic anemia.
• Alcohol excess

4. • Vitamin B₁₂ Deficiency

5. Structure of vit B₁₂

6. Dietary Sources of vitamin B₁₂

7. Vit B₁₂ Metabolism
• Once ingested, vit B₁₂ is bound to R
protein in the stomach.
• Pancreatic proteases release vitamin
B₁₂ from the R proteins in the small
intestine, where it binds to intrinsic
factor (IF).
• Bound to IF, which is produced by
gastric parietal cells, vitamin B₁₂ is
transported to the terminal ileum.
• Enterocytes in the terminal ileum
absorb vit B₁₂ , break the vitamin B₁₂ -
IF complex, and release vitamin B₁₂
into the portal circulation bound to
transcobalamin II.

8. • Vitamin B₁₂ is then transported to different
tissues throughout the body.
• Humans recycle vitamin B₁₂ via the
enterohepatic circulation; it is excreted in bile
and reabsorbed in the terminal ileum.
• The liver stores 2 to 3 mg of vitamin B₁₂ .
• The primary role of vitamin B₁₂ is serving as a
cofactor for two major metabolic reactions.

10. • Metabolically active forms
1. 5-deoxyadenosyl cobalamin - liver
2. Methylcobalamin - plasma
3. Hydroxocobalamin - contains cobalt ion in its oxidised
form(co III)- Main dietary form
4. Cyanocobalamin - stable synthetic form

11. Absorption of vit B₁₂
2 mechanisms
Active (75%) – requires the presence of intrinsic factor ( a
glycoprotein produced by gastric mucosa)
Passive – absorption occurs by diffusion and works when
pharmacological doses of vitamin B12 are ingested (buccal,
duodenal and ileum).

12.  Proteins involved in active absorption are :
• Intrinsic factor { a glycoprotein }.
• Transcobalamin I (TC I). Also known as haptocorrin or R protein. It is found in mature
granulocytes and monocytes. TC I binds to 70% of cobalamin and protects it from the
acid environment of the digestive system.
• Transcobalamin II (TC II). TC II is synthesized by epithe-ial and endothelial cells,
monocytes and fibroblasts. It binds around 30% of circulating cobalamin and is its only
real carrier, transporting it to target cells where it will be used.
• Transcobalamin III (TC III). TC III is found in neutrophils; it has no known action.
• Cubilin. Endothelial B₁₂ receptor that mediates cobalamin absorption in the ileum

13.  Liver is the principal storage organ.
 Other storage sites : Kidney, Heart and Brain
 Cobalamin enters portal blood after 6 hrs of oral ingestion.
• Amount recirculated in bile 0.5 - 5µg.
• Only traces are excreted in urine; in pharmacological
doses large part is excreted in urine.

14. Age RDA
0-6 months 0.4µg
7-12 months 0.5µg
1-3 years 0.9µg
4-8 years 1.2µg
9-13 years 1.8µg
14+ years 2.4µg

15. Causes of Vit B₁₂ Deficiency
Stage of Cobalamin
metabolism
Cause of Cobalamin deficiency
Food ingestion Strict vegetarianism without fortification & supplementation,
Exclusively breast fed babies.
Digestion Gastrectomy, Gastric atrophy, H. pylori infection, Use of antacids
(H2 receptor blockers or proton pump inhibitors etc.)
Absorption Ileal resection, malabsorption syndromes, pernicious anemia, fish
tapeworm infestation, pancreatic exocrine failure, drugs
interfering absorption ( metformin, colchicine, neomycin, ethanol
etc.)
Transportation Congenital deficiency or defect in transcobalamin II (C776G,
G1196A polymorphisms)
Intracellular
metabolism
Congenital deficiency in various intracellular enzymes required for
conversion to its active forms

16. Causes of vitamin B₁₂ deficiency
 Inadequate Intake
• Dietary – Food Fads, veganism, malnutrition
• Maternal deficiency causing B₁₂ deficiency in breastmilk
 Failure to secrete IF
• Congenital deficiency of IF
• Juvenile pernicious anaemia a/w
- IgA deficiency
- Gastric Atrophy ( Autoimmune )
• Gastric mucosal disease
- Gastrectomy
- Corrosives

17.  Transport defects of vitamin B₁₂
- R-binder ( Transcobalamin-1) deficiency
- Deficiency of TC II
 Failure of absorption in small intestine
A) Malabsorption of vit B₁₂
- Abnormal IF
- Imerslund-Grasbeck Syndrome
- Chelating agents (EDTA, Phytates)
B) Generalized Malabsorption Disorders
- Intestinal resection
- Crohn’s disease
- Zollinger-Ellison Syndrome
- Tb of terminal ileum
- Pancreatic insufficiency

18. C) Competition for vitamin B₁₂
- Small bowel bacterial overgrowth
- Giardia Lamblia, Diphyllobothrium latum
 Vitamin B₁₂ metabolism defects
A) Congenital
- Adenosylcobalamin deficiency
- Deficiency of methylmalonyl mutase co-A
- Methylcobalamin deficiency
B) Acquired
- Hepatic Pathology
- PEM
- Drugs induced ( p-aminosalicylic acid, ethanol,
colchicines, neomycin )

19. Clinical features
• Non specific manifestations : weakness, lethargy, FTT,
feeding difficulties.
• Infants may have regression of milestones and ITS.
• On examination, most patients have pallor, sallow yellow
complexion.
• Hyperpigmentation of knuckles and nail bed.
• Neurologic symptoms may include paresthesia, sensory
deficits, hypotonia, seizures, and neuropsychiatric changes.

20. Folic Acid Deficiency

21. • Folic acid derivatives (i.e. "folates") are acceptors and donors of
one-carbon units for all oxidation levels of carbon.
• The active coenzyme form is tetrahydrofolate (THF).
• In folate deficiency, THF production is depleted causing slowing of
DNA synthesis, resulting in pancytopenia due to defective
haematopoiesis.
• The cells that are produced have immature nuclei compared with
the degree of maturation of cytoplasm due to arrest of nuclear
maturity
• The biosynthetic pathways of methionine, homocysteine, purines,
and thymine rely on one-carbon units being provided by THF.

23. Absorption and Transport
• Normally absorbed from duodenum and upper
jejunum and to a lesser extent from lower jejunum and
ileum.
• It exists in nature as folates in polyglutamate form.
(conjugated folates)
• Polyglutamate form is first cleaved to mono and di
glutamates, which further undergo reduction in the
mucosal cells to form tetrahydrofolate (THF), a
monoglutamate.

24. Functions
• Folate acts as a co-enzyme for 2 important
biochemical reactions involving transfer of 1-
carbon units to form other compounds.
• These reactions are:
1.Thymidylate Synthetase Reaction
2.Methylation of Homocystiene to Methionine.

25. THF
DHFMethylene THF
Methyl THFIntestinal cell
Dietary folates
Dihidrofolate
Redutase
MethionineHomocysteine
Thymidylate Synthase
DNASynthesis
dTMPdUMP
Role of Vitamin B12 and Folate in DNA synthesis
VitB12 (Methylcobalamin)

26. The Folate Trap
• The conversion of methylene-THF into methyl-THF,
which is catalysed by MTHFR, is irreversible.
• The only way to make further use of methyl-THF and to
maintain the folate cycle consists in the vitamin-B12-
dependent remethylation of homocysteine to
methionine (regenerating THF).
• The methyl group transfer is therefore greatly
dependent on methyl-THF and the availability
of vitamin-B12.

27. • In cases of vitamin-B12 deficiency, it is possible
that, in spite of sufficient availability of folates
(and methyl-THF), an intracellular deficiency of
biologically active THF arises.
• This situation is called a ‘folate trap’ (or methyl
group trap) because, on the one hand, the
concentration of methyl-THF continues to rise
but, on the other hand, due to it being prevented
from releasing methyl groups, a ‘metabolic dead-
end situation’ develops, which leads to the
inevitable blockage of the methylation cycle.

28. • Hence, the principal problem is the decreasing
activity of methionine synthase under vitamin-
B12 deficiency with secondary disorders
affecting the folate metabolism and insufficient
de-novo synthesis of purines and pyrimidines.
• The deficiency in active folic acids first affects
the quickly dividing and highly proliferating
haematopoiesis cells in the bone marrow and
can even lead to pancytopenia.

29. 29
Age RDA
0-6months 65µg DFE
7-12months 80µg DFE
1-3 years 150µg DFE
4-8 years 200µg DFE
9-13 years 300µg DFE
14-18 years 400µg DFE
19+ years 400µg DFE

30. Causes of folate deficiency
1. Inadequate intake
-Poverty,
-Food fads,
-Goat milk,
-Sustained boiling losses upto 50-95% folate,
-Post bone marrow transplant ( Heat sterilized food )
2. Increased requirements
- Chronic haemolytic anemia
- Rapid growth ( prematurity, pregnancy )
- Dyserythropoetic anemias
- Hypermetabolic states ( Hyperthyroidism, infection )

31. - Malignancies ( Lymphomas, Leukemias )
- Post Bone marrow transplant (Regeneration of bone
marrow and epithelial cells
- Cirrhosis
3. Defective absorption
A) Congenital, isolated defect of folate alabsorption
B) Acquired
- Idiopathic steatorrhea
- Tropical sprue
- Gastrectomy
- Jejunal resection
- Broad spectrum antibiotics
- Post bone marrow transplant [ Total body
irradiation, intestinal GVHD ]

32. 4. Disorders of Metabolism
A) Congenital (Enzyme deficiencies)
- Methylenetetrahydrofolate reductase
- Dihydrofolate reductase
- Glutamate formimino transferase
- Methylene tetrahydofolate cyclohydrolase
B) Acquired
- Drugs – Methotrexate, pyrimethamine)
- Vitamin B12 deficiency
- Hepatic pathology
- Alcohol consumption
- Defective utilization of folate

33. Clinical features
• Older children present with pallor, easy fatigability,
irritability, chronic diarrhea or poor weight gain.
• Hemorrhages from thrombocytopenia may occur
in advanced cases.
• Congenital folate mal-absorption may be further
a/w hypogammaglobulinemia, severe infections,
Failure to thrive, neurological abnormalities and
cognitive delays

34. Investigations
The goal is to confirm the diagnosis of
megaloblastic anemia, distinguish between
folate or cobalamin or combined deficiency,
and to determine the underlying cause—
dietary, sociocultural or disease related.

35. 1. Peripheral Blood Examination
a) Haemoglobin : Decreased
b) Red cells : Characteristic macrocytosis is seen.
Marked anisocytosis, poikilocytosis with presence of
macroovalocytosis. Basophilic stippling may be seen.
c) Retic count : Low to normal
d) Indices : Elevated MCV ( >120fl ), elevated MCH (
>50pg)
e) Leucocytes : May be reduced. Presence of
Hypersegmented Neutrophil is characteristic.
f) Platelets : Moderately reduced

37. 2. Bone Marrow Examination
a) Marrow cellularity : Marrow is Hypercellular with a
decreased myeloid : erythroid ratio (from 3:1 to 1:1)
b) Erythropoiesis : Erythroid Hyperplasia is due to
characteristic megaloblastic erythropoeisis.
c) Orthochromatic features : sieve like nucleus and
haemoglobinized cytoplasm and mitotic figures seen
d) Dyserythropoiesis : nuclear remnants, bi- and
trinucleated cells and dying cells

38. Coarse grainy sieve like nuclei

39. e) Other cells : Howell Jolly bodies, Band cells, Giant
metamyelocytes, Megakaryocytes may be increased
with pseudohyperdiploidy and agranular cytoplasm.
f) Marrow Iron : Increase in number and size of iron
granules in erythroid precursors. Ringed sideroblasts
are rare.
g) Chromosomes : Random chromosomal abnormalities
such as chromosomal breaks or centromere spreading
may be seen.

40. Giant Band Form

41. 3. Vitamin B12 Assessment
• Serum Homocysteine and Serum Methylmalonic acid
levels are raised.
• Both are sensitive indicators of vit b12 deficiency and
corelate with clinical abnormalities and therapeutic
response. Specificity is low.
• Excessive excretion of methylmalonic acid in urine
(normal = 0 to 3.5mg/day) is a reliable and sensitive
index of vitamin b12 deficiency
• Serum vit b12 levels : Normal value : 200-800 pg/ml
Deficiency levels : less than 80 pg/ml

42. 4. Folate Assessment
• Only Homocysteine is raised with normal methylmalonic
acid levels.
• RBC folate assessment is the best measure of metabolically
active folate and included 5-MTHF in the assay.
• Serum folate measures the circulating pool of folate but
does not accurately reflect the amount of THF present in
tissues
• Serum Folate Levels : Normal : 5-20ng/ml
• Deficiency levels : < 3 ng/ml
• RBC Folate Normal levels : 150-600ng/ml

43. 5. Serum and Urine Assessment
a) Serum LDH is elevated to range of 2,000-5,000 . It
reflects increaded turnover of cells in the marrow due to
ineffective erythropoiesis.
b) Measurement of IF and urinary proteins detects
Imerslund-Grasbeck syndrome
c) Urinary excreation of Orotic acid to rule out
oroticaciduria
d) Schilling test and Formiminoglutamic acid (FIGLU) test
were used in the past to diagnose vit b12 assay and folic
acid deficiency. However these tests are not used
currently

45. Treatment
Cobalamin Deficiency
– Daily dose of 25-100mcg may be used to start the
therapy.
– Monthly IM injection in a dose of 200-1000mcg can
be started as maintainance therapy.
Conventional therapy :
1000 mcg of cyanoCbl or Hydroxy Cbl IM daily for 1 week.
Followed by 100 mcg of cyanoCbl weekly for 1 month.
Followed by 100 mcg of cyanoCbl monthly.

46. – In patients with TC II deficiency, doses of 1000mcg IM, 2
or 3 times weekly are required to maintain adequate
control.
– Patients with methylmalonic aciduria, may require 1-2
mg vit b12 parenterally daily.
Congenital methylmalonic aciduria may be diagnosed in utero
by measurements of methylmalonate in amniotic fluid or
maternal urine.
– The cause of vit b12 deficiency must ultimately dictate
treatment dosage as well as the duration of therapy.
– Dose adjustmenst should be made in response to clinical
status and lab values.

47. Response to b12 therapy :
 1-2 days : Reduction of serum iron, indirect bilirubin and
LDH. Normal haemopoiesis
 3-4 days : Reticulocytosis
 10 days : Hb starts to increase, MCV reduces.
 14 days : Disappearance of Hypersegmented neutrophils.
 2 months : Resolution of anemia.
 3-12 months : Resolution of neuropathy.

48. Folate deficiency
– Once estabilished, a dose of 0.5 to 1 mg/day may be
administered orally or parenterally.
– If diagnosis is in doubt, a smaller dose of 0.1 mg/day
may be used for 1 week as a diagnostic test.
– Doses of folate > 0.1mg/day can correct the anemia
of vitamin b12 deficiency but may aggravate any
associated neurological abnormality

49. – Folic acid therapy should be continued for 3-4
week until a definite haematological response has
occurred.
– Maintainance therapy with a multivitamin
(containing 0.2 mg of folate ) is adequate.

50. Juvenile Pernicious Anemia

52. Oroticaciduria
• Rare autosomal recessive disorder.
• Appears in 1st year of life.
• C/F : Growth failure, developmental retardation,
megaloblastic anemia and increased urinary
excreation of Orotic acid.
• Most common metabolic error in the de novo
synthesis of pyrimidine

53. Diagnosis :
• Presence of severe megaloblastic anemia.
• Normal b12 and folate levels.
• No evidence of TC deficiency.
• Presumptive diagnosis : Increased Urinary Orotic acid
• Confirmation : Assay of the transferase and
decarboxylase enzymes in RBCs
Treatment :
• Uridine administration. It is refractory to Vit b12 or
folate administration

54. Thiamine responsive megaloblastic anemia
• Very rare autosomal recessive disorder.
• Characterised by :
• Megaloblastic anemia
• Sensorineural hearing loss
• Diabetes mellitus
• Presents in Infancy
• Ringed sideroblasts with megaloblasts seen in bone
marrow.
• T/t : Continuous Thiamine supplementation. (Does not cure
hearing disability

55. Lesch Nihan Syndrome
• Also known as juvenile gout, is a
rare inherited disorder caused by a deficiency
of the enzyme HGPRT.

56. Imerslund Grasback Syndrome
• Autosomal recessive disorder
• Selective vitamin b12 malabsorption in the terminal ileum
• Appears by 6 years of life.
• C/F : Megaloblastic anemia along with neurological defects
and/or Proteinuria
• Mutation is seen in CUBN or AMN gene, which are also a
key receptor for protein reabsorption in kidney

57. • T/t : 1 mg of IM Hydroxycobalamin daily until
reticulocyte recovery after which the dosing
was spaced to once a week.
• The disease can be fatal if left untreated.

58. Thank you