Megaloblastic anaemia

1. MEGALOBLASTIC ANAEMIAS

2. Introduction ..
⚫Characterized by defective synthesis of
deoxyribonucleic acid (DNA) in all proliferating cells
⚫Most commonly result from lack of folic acid or
vitamin B12

3. MEGALOBLASTIC ANAEMIA
[ VITAMIN B12 DEFICIENCY ]

4. Normal Vitamin B12 Metabolism
⚫Vitamin B12 is composed of
⚫A corrin nucleus which has 4 pyrrole rings bound to
a central cobalt atom
⚫A 5,6 dimethylbenzimidazole group which is
attached to the corrin ring and to the central cobalt
atom
⚫Important cobalamins that are distinguished
according to the ligand attached to the central
cobalt atom are : cyanocobalamin,
hydroxocobalmin, adenosylcobalamin and
methylcobalamin

5. Sources
⚫Liver, dairy products and seafish are major
sources
⚫Although bacteria in the large intestine synthesize
vitamin B12 it cannot be absorbed from this site
⚫Minimum amount required for an adult is 1 to 4 µg
per day

6. Absorption of Vitamin B12
⚫2 mechanism
⚫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

7. Vitamin B12 in food
R-Binder
B12-R-Binder complex
IF-B12 complex + Freed R-Binder
Intrinsic Factor (IF)
B12-TCII
Circulation
Receptor-IF-B12
TCII
IF Receptor
Degradation
Epithelial cell of
terminal iIeum
Stomach
Duodenum

8. Transport of Vitamin B12
⚫Following absorption by the ileal mucosal cells,
vitamin B12 is carried in the plasma by various
transporting proteins:
Transcobalamin I
Transcobalamin II
Transcobalamin III

9. Transcobalamin I (TC I) is an alpha-globulin
produced by granulocytes. It functions as a
circulating reserve store of B12. TC I carries mostly
methylcobalamin.
Transcobalamin II (TC II) is a beta-globulin formed
in the liver and is the dominant carrier of B12
immediately after absorption. It is the main agent
for rapid transport of B12 to the body cells.
Transcobalamin III (TC III) is an alpha-globulin.
TC III may act as a defence mechanism by
depriving pathogens of B12 at sites of infection

10. Storage sites
⚫Total amount of vitamin in body is 2-5 mg (
adequate for 3 years )
⚫Major site : liver
⚫Excreted through the bile and shedding of
intestinal epithelial cells
⚫Most of the excreted vitamin B12 is again
absorbed in the intestine (enterohepatic
circulation)

11. Functions of Vitamin B12
⚫Synthesis of methionine from homocysteine
⚫Conversion of methyl malonyl CoA to succinyl
CoA

12. FH4
FH2
Methylene FH4
Methyl FH4
Intestinal cell
Dietary folates
Dihidrofolate
Redutase
Methionine
Homocysteine
Thymidylate Synthase
DNA Synthesis
dTMP
dUMP
Role of Vitamin B12 and Folate in DNA synthesis
VitB12 (Methylcobalamin)

13. General Morphological Features Of
Megaloblastic Anemia

14. PERIPHERAL BLOOD FINDINGS
1. Hemoglobin – decreased
2. Hematocrit – decreased
3. RBC count – decreased/normal
4. MCV - >100fl ( normal 82-98fl)
5. MCH –increased
6. MCHC – NORMAL
7. Reticulocytopenia.
8. Total WBC count – normal / low
9. Platelet count – normal/ low
10. Pancytopenia, especially if anaemia is severe.

15. PERIPHERAL SMEAR
⚫RBC:
- Macro ovalocytes (macrocytic normochromic)
[ macrocytosis is the earliest sign in Vit B12 deficiency
and can be detected even before the onset of anaemia
]
-In severe anaemia in addition to macrocytosis,
marked anisopoikilocytosis, basophilic stippling, howell
jolly bodies, Cabot’s rings may be found

16. ⚫Late or intermediate erythroblast with fine, open
nuclear chromatin (megaloblast) may be seen in
peripheral blood in severe anaemia

17. Marked macro-ovalocytosis (MCV 134 fl) in the peripheral blood smear of a
patient with vitamin B12 deficiency.

19. PERIPHERAL SMEAR
⚫WBC
⚫Normal count or reduced count
⚫Hypersegmented neutrophils is one of the earliest sign of
megaloblastic haematopoiesis and can be detected even
in the absence of anaemia (when more than 5% of
neutrophils show ≥ 5 lobes; 1% neutrophils with ≥ 6 lobes)
⚫PLATELETS:
⚫Normal or decreased (severe anaemia)
⚫Giant platelet can occur

22. BONE MARROW
⚫Markedly hypercellular
⚫Myeloid : erythroid ratio decreased or reversed.
(Normally, there are three myeloid precursors
for each erythroid precursor resulting in a 3:1
ratio, known as the M:E (myeloid to erythroid)
ratio)
⚫Erythropoiesis : MEGALOBLASTIC

23. MEGALOBLAST
1. Cell and nuclear size and amount of cytoplasm
(deeply basophilic royal blue) are increased
2. Nuclear chromatin is sieve like or stippled
(open)
3. Nuclear cytoplasmic asynchrony/dissociation
4. Abnormally large precursor (promegaloblast
and early megaloblast) are increased in BM –
Maturation arrest
5. Abnormal mitoses (increased)

26. ⚫Granulocytic series also display megaloblastic
changes
⚫Most prominent change – giant metamyelocyte with
horseshoe shaped nuclei and finer nuclear chromatin,
and in band forms
⚫Megakaryocytes are often large with multiple nuclear
lobes and paucity of cytoplasmic granules

28. BIOCHEMICAL FINDINGS
⚫Increase in serum unconjugated bilirubin-
because of ineffective erythropoiesis
⚫Increase is LDH
⚫Normal serum iron and ferritin

29. Causes of Vit B12 deficiency
⚫Insufficient dietary intake (very rare)
⚫Strict vegetarians
⚫ Deficient absorption
⚫Pernicious anaemia
⚫Total or partial gastrectomy
⚫Prolonged use of PPI or H2 blockers
⚫Diseases of small intestine
⚫Fish tapeworm infestation

30. PERNICIOUS ANEMIA
⚫ThomasAddison (1849)
⚫Disease of elderly – 5th to 8th decades (median
age at diagnosis – 60 years)
⚫Genetic predisposition
⚫T
endency to form antibodies against multiple self
antigens

31. PATHOGENESIS
⚫ Immunologically mediated, autoimmune
destruction of gastric mucosa
⚫ CHRONIC ATROPHIC GASTRITIS – marked loss
of parietal cells
⚫ Three types of antibodies:
a) Type I antibody- 75% - blocks vitamin B12 and IF
binding
b) Type II antibody – prevents binding of IF-B12
complex with ileal receptors
c) Type III antibody – 85-90% patients – against
specific structures in the parietal cell

32. ⚫Pathological changes are infiltration by mononuclear
cells in submucosa and lamina propria of fundus and
body of the stomach, progressive loss of parietal and
chief cells, and their replacement by intestinal type
mucous cells

33. ⚫Associated with other autoimmune disorders
like Hashimoto’s, Graves’, vitiligo, diabetics
mellitus, primary hyperparathyroidism,
Addison’s and Myasthenia gravis
⚫Patients with pernicious anaemia have
increase risk of gastric cancer

34. DIAGNOSTIC FEATURES
1. Moderate to severe megaloblastic anemia
2. Leucopenia with hypersegmented neutrophils
3. Mild to moderate thrombocytopenia
4. Mild jaundice due to ineffective erythropoiesis and
peripheral hemolysis
5. Neurologic changes
6. Low levels of serum B12

35. 7. Elevated levels of homocysteine
8. Striking reticulocytosis after parenteral
administration of vitamin B12
9. Serum antibodies to intrinsic factor (specific)
and anti parietal cell antibodies in serum
10. Abnormal Schilling test, pentagastrin-fast
achlorhydria

36. GASTRECTOMY
⚫Total gastrectomy :
⚫Secondary to Vit B12 deficiency as it removes the
site of synthesis of intrinsic factor
⚫Prophylactic vitamin B12 after surgery
⚫Partial gastrectomy
⚫Regular follow up after surgery for early detection of
deficiency

37. DISEASES OF SMALL INTESTINE
⚫Tuberculosis, whipple’s disease, blind loop syndrome or
resection of small intestine may interfere with absorption
that occurs in the terminal ileum
⚫Blind loop syndrome –
⚫ stasis of small intestine contents (diverticulum / stricture)
may predispose to bacterial colonization and proliferation
⚫Utilization of most of the ingested Vit B12 by bacteria may
lead to reduced or non avail of Vit for absorption

38. INFESTATION BY FISH
TAPEWORM
⚫Diphyllobothrium latum (inadequately cooked fish)
⚫Vitamin deficiency by competing with the host for
vitamin in food
⚫Diagnosis made by demonstration of ova in stool

39. CLINICAL FEATURES
⚫Anaemia, mild jaundice and sometimes
neurological involvement
⚫Neurological involvement in the form of
⚫Peripheral neuropathy
⚫Subacute combined degeneration of spinal cord
⚫Cerebral changes (personality changes, dementia &
psychosis)
⚫Patients can present with only neurological
abnormalities without megaloblastic anaemia

40. LABORATORY FEATURES
1. Morpholgical changes of megaloblastic anaemia in
PS and BM
2. Serum vitamin B12 assays
3. Methylmalonic acid (MMA) and homocysteine in
serum
4. Schilling test
5. Intrinsic factor antibodies in serum

41. 1. SERUM VITAMIN B12 ASSAYS
Various methods are available, e.g.
microbiological methods using
Lactobacillus leichmannii or radio-isotope
techniques (RIA) using 57CoB12, coated
charcoal and IF.

42. RADIO-ISOTOPE DILUTION ASSAY:
A known amount of radioactive (hot) B12 is
diluted with the non-radioactive (cold) B12
in the test serum, released from serum
proteins by heat or chemical means.
A measured volume of the hot and cold
mixture is bound to intrinsic factor (IF)
which is added in an amount insufficient
to bind all the hot B12. The bound B12 is
separated from the free and its
radioactivity counted.

43. The count is inversely proportional to the
B12 concentration in the test serum.
The higher the serum B12 the greater will
be the dilution of the radioactive B12 and
thus less radioactivity attached to the IF.
By comparison with standards of known
B12 content, the B12 content of the test
serum can be calculated.

44. ⚫In Vitamin B12 deficiency ,
⚫Serum Vitamin B12 and red cell folate are depressed
⚫Serum folate is normal or increased ( accumulation of
5-methyl tetrahydrofolate ) [folate trap]

45. 2. Methylmalonic acid (MMA) and
homocysteine in serum
⚫Recent reports of S.methylmalonic acid and
S.homocysteine are more sensitive for detection of
Vitamin B12 than estimation of Vitamin B12
⚫Raised early in tissue deficiency even before
appearance of hematological changes

46. 3. SCHILLING TEST
⚫For evaluation of absorption of vitamin B12 in the
GIT
⚫Performed in 2 parts – part 1 and part 2
⚫Part 1 :
⚫0.5 to 1 µg of radiolabelled vitamin B12 is given
orally
⚫ After 2 hrs IM dose (1000 µg) of unlabelled vitamin
B12 is given [ saturates binding sites of TC I and TC
II and displaces any bound radiolabelled vitamin B12
(thus permitting urinary excretion of absorbed
radiolabelled vitamin B )

47. ⚫Radioactivity is measured in subsequently
collected 24 hr urine sample and expressed as a
% of total oral dose
⚫In normal persons, > 7% of the oral dose of
vitamin B12 is excreted in urine
⚫If excretion is less than normal it indicates impaired
absorption, which may be due to either lack of IF or
small intestinal malabsorption
⚫Part 2 performed if part 1 of test is abnormal

48. ⚫Part 2 : patient is orally administered
radiolabelled vitamin B12 along with IF while
remainder of test is carried out out as in part 1
⚫Excretion becomes normal – lack of IF
⚫Excretion remains below normal – defective
absorption in small intestine

49. 4. INTRINSIC FACTOR ANTIBODIES IN
SERUM
⚫Detection of anti-IF antibodies in serum is diagnostic
of pernicious anemia

50. MANAGEMENT OF B12 DEFICIENCY
When B12 deficiency is suspected a trial of B12 is
essential. Failure of response can only be
determined after careful follow-up over a period of
several months, particularly if the patient is non-
anaemic.
Standard therapy for all cases of B12 deficiency is
by regular intramuscular injections of B12, usually
in the form of hydroxycobalamin. In patients with
inadequate dietary intake supplements may be
given by mouth. Underlying conditions should be

51. ⚫After initiation of therapy, reticulocyte count begins to
increase around 3rd day – peak by 6th or 7th day –
gradually returns to normal by end of 3rd week
⚫Hematocrit steadily rises and normalises in about 1-
2 months
⚫Blood transfusion is indicated in severely anaemic
symptomatic patients or in patients with CCF

52. NOTE:
Both B12 and folate are given to patients if
B12 deficiency has not been excluded.
This is to prevent neurological damage, e.g.
subacute combined degeneration of the
spinal cord.