3. INTRODUCTION
Megaloblastic anaemias are a heterogeneous group of
disorders that result from problems in DNA synthesis.
o These anaemias share common morphologic characteristics, i.e
large cells with an arrest in nuclear maturation.
o These incompletely developed cells are called megaloblasts.
A megaloblast is an erythroid cell whose morphological
hallmark is nuclear - cytoplasm asynchrony/dissociation.
o This means that there is failure of nuclear maturation whereas
there is continuous maturation of the cytoplasm.
o These megaloblastic changes are most apparent in rapidly
dividing cells such as blood cells.
o In the peripheral blood, macrocytosis, immature nuclei, and
hypersegmented neutrophils may be seen.
o Since these cells are underdeveloped, they also have a
shortened life expectancy.
4. CAUSES OF MEGALOBLASTIC ANAEMIAS
The two most common causes of megaloblastic anaemias are:
o Folic acid (Vitamin B9) deficiency
o Vitamin B12 deficiency
Other causes can include:
o Nitrous oxide poisoning
o Drugs:
• Folic acid antagonists
Methotrexate, Proguanil, Pyrimethamine
• Purine synthesis antagonists
6-Mercaptopurine, 6-Thioguanine
• Pyrimidine antagonists
5-Fluororacil, Zidovudine
• Ribonucleotide reductase inhibitors
Cytosine arabinoside, Hydroxyurea
5. CAUSES OF MEGALOBLASTIC ANEMIA
• Phenytoin
o Alcohol abuse
o Direct interference of DNA synthesis by HIV infections
and myelodysplastic disorders.
o Erythroleukemia
o Inborn errors of metabolism:
• Lesch-Nyhan syndrome
• Inherited deficiency of Intrinsic factor (IF)
• Inherited deficiency of cubulin (Grasbeck Imerslung)
disease
• Inherited deficiency of methylenetetrahydrofolate reductase
(MTHFR).
• Inherited deficiency of transcobalamin (TC) II.
6. 6
LABORATORY INVESTIGATIONS
Full blood count:
o ↓ HB ↓ PCV ↓ RCC
o → ↓ WBC (T)
o → ↓ Diff
o ↓ PLT count
o ↓ Retic count
o RCI
• MCV ↑ MCHC → MCH ↑
7. LABORATORY INVESTIGATIONS contd
Peripheral blood film:
o RBCs
• There is anisocytosis and poikilocytosis
• Normochromic, macrocytic ovalocytes
• Nucleated RBCs
• Howell-Jolly bodies
o WBCs
• Normal or reduced on film
• Neutrophils have multisegmented nuclei (hypersegmented
> 6 lobes)
o PLTS
• Reduced on film.
9. LABORATORY INVESTIGATIONS contd
Bone marrow examination:
o Hypercellular, the erythropoiesis is usually active but
maturation is megaloblastic.
o More mature RBC precursors are destroyed in the BM prior to
entering the blood stream (intramedullary haemolysis) making
erythropoiesis ineffective.
o Myelopoiesis may be normal or slighly reduced.
o Myeloid : Erythroid ratio normally 2:3 becomes 1:1
o There is usually megaloblastic features seen in the myeloid
cells and also in megakaryocytic cells.
Biochemical tests:
o Increased bilirubin
o Increased lactate dehydrogenase
10. VITAMIN B12 DEFICIENCY
o PATHOPHYSIOLOGY
o AETIOLOGY OF B12 DEFICIENCY
o CLINICAL FEATURES
o DIAGNOSIS OF VITAMIN B12 DEFICIENCY
o DIAGNOSIS OF THE CAUSE OF VITAMIN B12
11. PATHOPHYSIOLOGY OF VITAMIN B12
DEFICIENCY
Vitamin B12 is a complex molecule at the core of which is a
corrin ring: a tetrapyrrole structurally homologous to haem but
with a cobalt atom, rather than iron, at its center.
Vitamin B12 is known to participate in only 2 enzymatic
reactions in humans.
o First, it is a cofactor for methionine synthase in the conversion
of homocysteine to methionine.
• Methionine is activated to S-adenosyl methionine, which aids
in purine and thymidine synthesis, myelin production,
protein/neurotransmitters/fatty acid/phospholipid production
and DNA methylation.
• 5-Methyl tetrahydrofolate provides a methyl group, which is
released to the reaction with homocysteine, resulting in
methionine.
12. PATHOPHYSIOLOGY OF VITAMIN B12
DEFICIENCY contd
• This reaction requires Vitamin B12 as a cofactor.
• The creation of 5-methyl tetrahydrofolate is an irreversible
reaction.
• If Vitamin B12 is absent, the forward reaction of homocysteine to
methionine does not occur, and the replenishment of
tetrahydrofolate stops.
• The failure of methionine synthesis due to cobalamin deficiency
may either:
1. Lead to an accumulation of 5-methyl-tetrahydrofolate, trapping
folate in a chemical form unusable in purine synthesis (the
“methylfolate trap" hypothesis).
2. Impair methylation reactions needed to produce formyl-
tetrahydrofolate, a precursor to 5,10-methylenetetrahydrofolate.
13. PATHOPHYSIOLOGY OF VITAMIN B12
DEFICIENCY contd
o Secondly, it is essential for the conversion of methylmalonyl-
CoA to succinyl-CoA.
• Deficiency leads to tissue accumulation of methylmalonyl acid
and results in methylmalonic acidaemia as well as
methylmalonic aciduria.
14. AETIOLOGY OF VITAMIN B12
DEFICIENCY
The major causes of Vitamin B12 deficiency are due to
malabsorption, very rarely is it due to reduced intake or
increased demand.
INADEQUATE DIETARY INTAKE OF VITAMIN B12
o Vegetarians who doesn't eat enough eggs or dairy products to
meet their Vitamin B12 needs.
o Adults who do not consume adequate amounts of meat,
poultry and fish.
o Children are at a higher risk for Vitamin B12 deficiency due to
inadequate dietary intake, as they have fewer vitamin stores
and a relatively larger vitamin need per calorie of food intake.
o Babies born to mothers who are vegetarians may also not get
enough Vitamin B12
o Heavy drinking
15. AETIOLOGY OF VITAMIN B12
DEFICIENCY
MALABSORPTION OF VITAMIN B12
o Gastric causes
• Atrophic gastritis
This is a process of chronic inflammation of the stomach
mucosa, leading to loss of gastric glandular cells and their
eventual replacement by fibrous tissues.
As a result, the stomach's secretion of essential substances such
as HCL, pepsin, and IF is impaired.
It can be caused by persistent infection with Helicobacter
pylori, or can be autoimmune.
• Gastrectomy
Surgical removal of the stomach or a portion of the stomach
(total or partial gastrectomy), can be due to bleeding ulcers, or
carcinoma of the stomach.
16. AETIOLOGY OF VITAMIN B12
DEFICIENCY
• Pernicious anaemia
This is an autoimmune gastritis resulting from the destruction
of gastric parietal cells and the associated lack of intrinsic
factor to bind ingested vitamin B12 .
Unavailabilty of IF is usually as a result of either:
1. Immunological destruction of the gastric parietal cells so they
can not produce IF.
• Which can be due to lymphocytes that are capable of
destroying the parietal cells.
• Generation of antibodies which combine directly with the
parietal cells and destroy them.
17. AETIOLOGY OF VITAMIN B12
DEFICIENCY contd
2. Lack of IF due to antibodies produced against the IF
o Two types:
• Type I –Blocking antibodies
Antibodies bind to IF and does not allow B12 to bind to it.
• Type 2–Binding antibodies
Antibodies bind to B12 - IF complex and so it is not available
for absorption at the ileal surface.
• Drugs
Forms of achlorhydria (artificially induced by drugs such as
proton pump inhibitors and histamine 2 receptor antagonists)
can cause Vitamin B12 malabsorption from foods, since acid is
needed to split B12 from food proteins and bind it to salivary
binding proteins.
18. AETIOLOGY OF VITAMIN B12
DEFICIENCY contd
o Intestinal causes
• Intestinal inflammatory diseases, such as Crohn's disease,
Coeliac disease.
• Biological competition for vitamin B12 by the marine parasite
Diphyllobothrium latum (fish tapeworm)
• Surgical removal of the small bowel for example in Crohn's
disease) such that the patient presents with short bowel
syndrome.
• Blind loop syndrome:
This is a condition where the intestinal mucosal wall has lost
its motility, and therefore food is unable to be propelled down
the intestinal tract.
This leads to bacterial overgrowth in parts of the small bowel
and the organisms consuming all Vitamin B12.
19. AETIOLOGY OF VITAMIN B12
DEFICIENCY contd
Can be due to:
- Surgical blind loop
- Scleroderma
- Amyloidosis
• Pancreatic Diseases
Failure of the exocrine pancreas with reduced amounts of
pancreatic peptidases will not allow the degradation and
dissociation of R binders Vitamin B12
Pancreatic gastrinoma can also induce the Zollinger Ellison
syndrome:
- Pancreatic gastrinoma
- Peptic ulcer
- Vitamin B12 deficiency leading to megaloblastic anaemia.
20. CLINICAL FEATURES
The commonest presentation is anaemia which tends to
develop slowly.
o The clinical features of the patient will depend upon severity
o They can be divided into 2:
Clinical features due to hypoxia caused by the anaemia
Clinical features due to lack of Vitamin B12 in the tissues
21. CLINICAL FEATURES contd
Clinical features due to hypoxia caused by the anaemia:
o Weakness, tiredness, or light-headedness
o Rapid heartbeat and breathing
o Pale skin
o Sore tongue
o Easy bruising or bleeding, including bleeding gums
o Stomach upset and weight loss
o Diarrhea or constipation
22. CLINICAL FEATURES contd
Clinical features due to lack of Vitamin B12 in the tissues:
o Vitamin B-12 deficiency affects the nervous system, leading to
a variety of symptoms:
• Paresthesias – numbness-abnormal sensation in fingers and
toes.
• Loss of vibration sense particularly to high frequency vibration
waves at 256 mHz whereas the sense at lower vibration 128
mHz is preserved.
• Loss of joint position sense
• Subacute combined degeneration of spinal cord
Demyelination of the fibres of the dorsal and lateral columns
of SC.
Spastic ataxia.
23. CLINICAL FEATURES contd
• Dementia similar to Alzheimers disease
• Depressive illness or schizophrenia
• Frank psychoses-Megaloblastic madness
• In infants symptoms include irritability, failure to thrive,
apathy, anorexia, and developmental regression.
Patients with megaloblastic anaemia as a result of pernicious
anemia sometimes have other autoimmune diseases such as
Graves' disease, Hashimoto's thyroiditis and vitiligo
(depigmentation or blanching of skin areas).
o They may present with premature greying of the hair
o Loss of tongue papillae
o Patients occasionally have hyperpigmentation.
24.
25. DIAGNOSIS OF VITAMIN B12
DEFICIENCY
The patient’s history may include symptoms of anaemia,
underlying disorders causing malabsorption, and neurologic
symptoms.
o The most common neurologic symptoms are symmetric
paresthesias or numbness and gait problems.
o The physical examination may reveal pallor, oedema,
pigmentary changes in the skin, jaundice, or neurologic defects
such as impaired vibration sense, impaired position and
cutaneous sensation, ataxia, and weakness.
o Laboratory investigations - FBC, PBF.
o The first test performed to confirm the diagnosis of Vitamin
B12 deficiency is generally measurement of the serum Vitamin
B12 level, which would be low.
26. DIAGNOSIS OF VITAMIN B12
DEFICIENCY contd
o Serum homocysteine and methylmalonic acid levels are high
o Methylmalonic acid levels in urine would be high.
o Therapeutic trial:
• Physiological amounts of Vitamin B12 are given and the
response monitored.
Reticulocyte response begins after 2-3 days and peaks at 7
days.
Hb should rise by 1.5 -2 g/dl every 3 weeks, white cell and
platelet counts become normal within 7-10 days
Reversal of megaloblastic haemopoesis to normoblastic
features haemopoesis in 48 hours.
Reversal of neutrophil hypersegmentation after 2 weeks.
27. DIAGNOSIS OF THE CAUSE OF VITAMIN
B12
If the patient consumes sufficient amounts of Vitamin B12 and
has clinically confirmed Vitamin B12 deficiency, then
malabsorption must be present.
o Barium studies
• To detect lack of gastric mucosa
• To detect intestinal immobility or the blind loop syndrome
o Chronic atrophic gastritis can be diagnosed on the basis of an
elevated fasting serum gastrin level and a low level of serum
pepsinogen.
o Endoscopy to confirm gastritis and rule out gastric carcinoma
o Stool microscopy - Ova and parasites
28. DIAGNOSIS OF THE CAUSE OF VITAMIN
B12
o The Schilling test
• Testing the absorption of B12 using radioactive vitamin B12
o Antiparietal cell and intrinsic factor antibodies.
29. FOLIC ACID DEFICIENCY
o PATHOPHYSIOLOGY
o AETIOLOGY OF FOLIC ACID DEFICIENCY
o CLINICAL FEATURES
o DIAGNOSIS OF FOLIC ACID DEFICIENCY
30. PATHOPHYSIOLOGY OF FOLIC ACID
DEFICIENCY
Folic acid absorbed from the diet must be activated to produce
active tetrahydrofolic acid (THF).
o Without adequate levels of biologically active THF, the ability
to repair and replicate DNA is decreased.
o Vitamin B12 is a cofactor for the activation of folic acid in a
step that also converts homocysteine to methionine.
o In the case of inadequate folic acid intake, THF production is
depleted, and DNA synthesis is slowed.
The effect on haematopoiesis is to reduce the rate of cell
production.
o The cells that are produced have immature nuclei compared
with the degree of maturation of the cytoplasm.
31. AETIOLOGY
There are several causes of folic acid deficiency, including the
following:
o Inadequate dietary intake due to:
• Limited consumption of fresh, minimally cooked food.
• Chronic alcoholism
• Long-term need for intravenous nutrition (total parenteral
nutrition)
• Old age
• Famine
o Malabsorption due to:
• Decrease in the intestine
Inflammatory bowel disease such as Coeliac disease,
Crohns disease
• Pancreatic disease
32. AETIOLOGY contd
o Increased demand due to:
• Chronic haemolytic states
SCD, thalassemias, G6PD deficiency, PNH
• Aquired haemolytic states
Immune haemolytic anaemia
Increased demand for the vitamin
Hyperthyroidism
Extensive psoriasis
Malignancy
Tb
Malaria
Pregnancy
Lactation
Growing children
34. CLINICAL FEATURES
Symptoms of folic acid deficiency are often subtle.
o Clinical features due to hypoxia caused by the anaemia:
• Persistent fatigue
• Pale skin
• Irritability
• Diarrhea
• Feel lightheaded.
• Loss of appetite
• Mouth sores
• Tongue swelling
• Growth problems
35. CLINICAL FEATURES contd
o Clinical features due to lack of folic acid in the tissues:
• Deficiency of folic acid will cause impairment of cell
multiplication in the growing foetus which can lead in a
number of congenital abnormalities:
Neural tube defects
- Spina bifida
- Meningocoele
Abnormalities of BM formation
- Abnormalities of myocardial formation
Vasculopathy and arteriosclerosis due to raised plasma
homocysteine which causes endothelial cell damage.
36. DIAGNOSIS OF FOLIC ACID
DEFICIENCY
A proper history must be taken.
A good physical examination must also be done.
The plasma folic acid must be assayed.
The red cell folate must be assayed.
NB
o In folic acid deficiency both plasma and red cell folate will be
reduced, whereas in Vitamin B12 deficiency the red cell folate
would be high.
Therapeutic trial:
o Physiological amounts of folic acid are given and the response
monitored.