1. Microcytic hypochromic anaemia can be classified based on mean corpuscular volume (MCV) and mean corpuscular haemoglobin (MCH) levels. Defects in iron metabolism, haemoglobin synthesis or blood loss can cause microcytic hypochromic anaemia. 2. Normocytic normochromic anaemia is caused by acute blood loss or bone marrow failure/suppression. Macrocytic anaemia results from vitamin B12 or folate deficiency or liver disease. 3. Diagnosis of the type of anaemia involves a full blood examination and iron studies to measure ferritin, serum iron, transferrin

1. Classification of Anaemia:
Microcytic Hypochromic Anaemia

2. Classification of Anaemia
Microcytic & Normochromic
Hypochromic & Normocytic Macrocytic
MCV<RR MCV within RR
MCH within RR MCV>RR
MCH<RR
Defects in Acute blood loss Megaloblastic Non-megaloblastic
Defects in
haem globin Haemolysis
synthesis synthesis ACD
Marrow infiltration
•Thalassaemia Liver
•Iron
•Haemoglobinopathies B12/Folate deficiency disease
deficiency
•ACD Drug
•Sideroblastic induced
(congenital) MDS

3. Iron Regulation

4. Normal Iron Absorption and
Metabolism

5. Ferritin
• Iron storage protein
• Produced by all living organisms including bacteria, algae, &
higher plants and animals
• In humans, it acts as a buffer against iron deficiency and
iron overload
• Consists of:
• Apoferritin – protein component
• Core- ferric, hydroxyl ions and oxygen
• Largest amount of ferritin-bound iron is found in:
– Liver hepatocytes (majority of the stores)
– BM
– Spleen
• Excess dietary iron induces increased ferritin production
• Partially digested ferritin= HAEMOSIDERIN- insoluble and
can be detected in tissues (hepatocytes) using Perl’s
Prussian blue stain

6. Transferrin (Tf)
• Transports iron from palsma to erythroblast
• Mainly synthesized in the liver
• Fe3+ (ferric) couples to Tf
• Apotransferrin = Tf without iron
• Contains sites for max 2 iron molecules
• The amount of diferric Tf changes with iron status
– Levels decreased when cellular iron demand is
increased
– Increased levels lead to increase hepcidin production
that decreases iron absorption

7. Transferrin Receptor (TfR)
• Provides transferrin- bound iron access into cell
• Control of TfR synthesis is one of major
mechanisms for regulation of iron metabolism
• Cells maintain appropriate iron levels by altering TfR
expression and synthesis
• Increased by iron deficiency
• Located on all cells except mature RBC
• Can bind up to 2 Tf
• apoTf is not recognized by TfR

8. Ferroportin
• Transmembrane protein
• Found on the surface of most cells:
• Enterocytes
• Hepatocytes
• RE system
• Regulates iron release from those tissues (iron
exporter)
• ‘Hepcidin receptor’

9. Hepcidin
• Is an antimicrobial peptide produced in the liver
• Act as a negative regulator of intestinal iron absorption
& release from macrophages
• Hepcidin binds to the ferroportin receptor & cause
degradation of ferroportin, resulting in trapping of iron
in the intestinal cells
• As a result, iron absorption & mobilization of storage
iron from the liver & macrophage are lowered
• Increased synthesis of hepcidin occurs when transferrin
saturation is high and decreased synthesis when iron
saturation is low

11. Causes of Iron deficiency
Increased demand:
•Growth
Blood loss: •Pregnancy
•GIT
•Urinary Inadequate intake
•Infants
•vegetarian
Major causes of
IDA in Western
Malabsorption Society
Iron sequestration at
inaccessible sites (pulmonary
Haemolysis haemosiderosis)
Major causes of IDA Parasitic infection
in developing
countries
Malnutrition

12. Symptoms of Iron Deficiency
• Mainly attributed to anaemia
– Fatigue
– Pallor
– Shortness of breath
– Tachycardia
– Failure to thrive
• More specific features (only apparent in severe
IDA ):
– Koilonychia
– Glossitis
– Unusual dietary cravings (pica)

13. Stages of Iron Deficiency
• 3 stages
• Stage 1
• Characterized by a progressive loss of storage
iron
• Body’s reserve iron is sufficient to maintain
transport and functional compartments through
this phase, so RBC development is normal
• No evidence of iron deficiency in peripheral blood
and patient experiences no symptoms

14. • Stage 2
• Defined by exhaustion of the storage pool of
iron
• For a time, RBC production is normal relying
on the iron available in transport
compartment
• Anaemia may not be present but Hb level
starts to drop
• Serum iron, ferritin and Tf saturation
decreased
• Increased TIBC, Tf and TfR

15. • Stage 3
• Microcytic hypochromic anaemia
• Having thoroughly depleted storage iron and
diminished transport iron, developing RBCs
are unable to develop normally
• The result is first smaller cells with adequate
[Hb], although these cannot be filled with Hb
leading to cells becoming microcytic &
hypochromic
• FBE parameters & iron studies all outside RR

16. Diagnosis - FBE
• Hb or borderline
• RBC
• Hct/PCV
• MCV
• MCH
• MCHC
• RDW
• +/- thrombocytosis
• Elongated cells
• Target cells (severe IDA)

18. Diagnosis- Iron studies
Ferritin Serum Transferrin Tf TIBC TfR
Iron Saturation
Results in
IDA

19. Differential diagnoses
• Thalassaemias/ Haemoglobinopathies
– Not all hbpathies are microcytic and hypochromic
• Anaemia of chronic disease
• Congenital sideroblastic anaemia

21. Treatment of Iron Deficiency
• Treatment of underlying cause (ulcers)
• Dietary supplementation
– Oral supplements
• Transfusion
– If anaemia is symptomatic and life threatening
– No prompt response to treatment
• Dimorphic blood film is present in treated IDA
– With oral supplements-newly produced cells are
normochromic normocytic
– Transfused cells are normochromic and normocytic

22. Anaemia of Chronic Disease
• Anaemia of chronic inflammation
• Usually normochromic normocytic; microcytosis &
hypochromia develop as the disease progress
• Iron stores abundant, but iron is NOT available for
erythropoiesis
• There are several proposed mechanism for abnormal
iron haemostasis in ACD:
• Lactoferrin competes with transferrin for iron
– RBC don’t have lactoferrin receptors
• Ferritin increases
• Cytokines inhibit erythropoieis
• HEPCIDIN

23. ACD- Role of Hepcidin
• Increase in hepcidin:
– Levels can be increased up to 100 times in ACD
– Release from liver after stimulation by IL-6
– Acute phase reactant
• Binds to ferroportin
– Decreases iron absorption and export from cells

24. Diagnosis & Treatment
• Identification of the disease
• CRP & IL 6
• Measurement of hepcidin levels via ELISA, HPLC
or LCMS
• Iron studies to distinguish from IDA
• Failure to respond to iron supplementation
Tx:
• Maintaining normal Hb is challenging
• EPO administration + IV iron
• Anti-inflammatory therapy

25. Sideroblastic anaemia
• Can either be inherited or acquired
• Rare condition
• Most common mutation is in ALA synthase gene
(ALAS2) located on X chromosome
• Abnormal haem synthesis & presence of ringed
sideroblasts in erythroid precursors (visible if
stained with Perls Prussian Blue)
• Microcytic hypochromic anaemia
– Ineffective erythropoiesis
– Systemic iron overload

26. STRUCTURE OF HAEMOGLOBIN
Polypeptides are made up of 2a chains and 2B chains, a2B2. Haem groups bind
oxygen.

27. STRUCTURE OF HAEM
• Haem structure: the iron (Fe)at the centre
enables oxygen to bind

28. Development of Haemoglobin

29. Stages of Haemoglobin Development
• Embryonic haemoglobin
– Hb Gower 1 2 2
– Hb Portland 2 2
– Hb Gower 2 2 2
• Foetal Haemoglobin
– Hb F 2 2 Foetus 100% Adult <1%
• Adult haemoglobins
– Hb A2 2 2 Adult 1.8-3.6%
– Hb A 2 2 Adult 96-98%
– The globin genes are arranged on the chromosomes in order of
expression

30. Inherited defects of globin synthesis
• These are due to:
1. Synthesis of an abnormal haemoglobin eg
haemoglobinopathies
2. Reduced rate of synthesis of α or β chains:
thalassaemia

31. Β- Thalassaemia
• Caused by defective B globin chain synthesis
• Due to mutations in the B globin gene
• The unpaired α chain precipitate in the
developing cells leading to damage to the RBCs
surface ~ leading to removal of RBCc by
macrophages
• Leads to ineffective erythopoiesis
• The more α chain in excess, the more haemolysis
occurs
• Can be divided into B-thal minor and B-thal major

32. B-thal minor
• Results when 1 of the 2 gene that produces B-
chain is defective (heterozygous)
• Usually present as a mild asymptomatic
anaemia
• Hepatomegaly and splenomegaly are seen in
some patients

33. B-thal major
• Characterized by severe anaemia first
detected in early childhood as σ to β switch
takes place
• Patient presents with jaundice,
hepatosplenomegaly, marked bone changes
(frontal bossing)

34. α thalassaemia
• Due to large deletions in the α globin genes
• Notation for the normal α gene complex or
haplotype is expressed as α α, signifying 2
normal genes on chr 11
• There are 4 clinical syndromes of α
thalassaemias; silent carrier, α-thal
minor/trait, HbH disease (due to
accumulation of unpaired B chain,
homozygous α-thal (hydrops foetalis)

36. Signs & Symptoms of Thalassaemia
• Severe anaemia first detected in early
chilhood
• Jaundice, hepatosplenomegaly, marked bone
changes (frontal bossing)
• Microcytic hypochromic anaemia

37. Laboratory Findings
• Most thalassaemias are microcytic & hypochromic
• Hb and PCV, MCV
• RCC
• Poikilocytosis, target
cells, elliptocytes, polychromasia, nRBCs, basophilic
stippling
• Bone marrow – hypercellylar with extreme erythroid
hyperplasia
• Electrophoresis- decresead % of Hb A
• Supravital stain to detect α thalassaemia major (HbH)

38. Treatment
1. Transfusion
2. Iron chelation therapy- desferrioxamine
3. BM transplantation
4. Hydroxyurea- to increase Hb F levels enough to
eliminate transfusion requirements for patients
with thalassaemia major

39. Hb 107 120-
160g/L
RCC 5.50 3.80-
5.401012/L
MCV 61 80-100 fL
MCH 19.5 27-32 pg
Hb A2 5.0 1.8-3.5 %
Hb F <0.1 0.0-1.0 %

40. Comparison of a normal blood film
with b-thal major
Normal Blood Film Intermittently transfused -thal
HbF>90%
Bain B. ‘Blood Cells. A practical guide’2006 Free chains form Heinz bodies and inclusions
Marked haemolysis
reticulocytosis
Basophilic stippling and Pappenheimer bodies

41. HbH Disease

42. Study Questions
• What are the main causes of IDA?
• Draw a diagram that explains how iron
haemostasis is maintained in the body
• Discuss different stages of development of IDA
• How would you differentiate between
different microcytic and hypochromic
anaemia?
• Explain the involvement of iron regulatory
proteins in ACD

43. Study Questions
• Describe how you would approach the investigation of a patient who has been
diagnosed with mild microcytic hypochromic anaemia. In your answer include the
tests, expected results and how they would help you differentiate the disorders to
make a final diagnosis.
• Are thalassaemias & haemoglobinpathies the same? Why?
• Why do patients with iron deficiency and a suspected thalassaemia need to
receive iron replacement therapy before Hb electrophoresis and HPLC can be
performed? How does iron deficiency influence these tests and the results
obtained?
• Describing the principle and rationale, explain why Hb electrophoresis and HPLC
can be used to diagnose these disorders. Are there any analytical errors that could
lead to inaccurate results?
• What role does prenatal diagnosis & genetic counseling have in this group of
disorders?