Classification of Anaemia: Microcytic Hypochromic Anaemia

Chapter 2

Prepared by Noor Izzah Abd Rahman

Classification of Anaemia Microcytic & Hypochromic

Normochromic & Normocytic Macrocytic

MCV<RR MCH<RR

Defects in haem

synthesis

Defects in globin

synthesis

•Iron deficiency •ACD •Sideroblastic (congenital)

•Thalassaemia •Haemoglobinopathies

MCV within RR MCH within RR

Acute blood loss Haemolysis ACD Marrow infiltration

MCV>RR

Megaloblastic Non-megaloblastic

B12/Folate deficiency Liver disease Drug induced MDS

Iron Regulation

Normal Iron Absorption and Metabolism

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

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

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

Ferroportin

• Transmembrane protein

• Found on the surface of most cells: • Enterocytes

• Hepatocytes

• RE system

• Regulates iron release from those tissues (iron exporter)

• ‘Hepcidin receptor’

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

Causes of Iron deficiency

Major causes of IDA in Western

Society

Blood loss: •GIT •Urinary

Increased demand: •Growth

•Pregnancy

Inadequate intake •Infants

•vegetarian

Iron sequestration at inaccessible sites (pulmonary

haemosiderosis)

Malabsorption

Haemolysis

Major causes of IDA in developing

countries

Parasitic infection

Malnutrition

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)

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

• 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

• 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

Diagnosis - FBE

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

Diagnosis- Iron studies

Ferritin Serum Iron

Transferrin Tf Saturation

TIBC TfR

Results in IDA

Differential diagnoses

• Thalassaemias/ Haemoglobinopathies

– Not all hbpathies are microcytic and hypochromic

• Anaemia of chronic disease

• Congenital sideroblastic anaemia

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

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

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

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

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

STRUCTURE OF HAEMOGLOBIN

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

STRUCTURE OF HAEM

• Haem structure: the iron (Fe)at the centre enables oxygen to bind

Development of Haemoglobin

Stages of Haemoglobin Development

• Embryonic haemoglobin – Hb Gower 1 z2e2

– Hb Portland a2g2

– Hb Gower 2 a2e2

• Foetal Haemoglobin – Hb F a2g2 Foetus 100% Adult <1%

• Adult haemoglobins – Hb A2 a2d2 Adult 1.8-3.6%

– Hb A a2b2 Adult 96-98%

– The globin genes are arranged on the chromosomes in order of expression

Inherited defects of globin sythesis

• These are due to:

1. Synthesis of an abnormal haemoglobin eg haemoglobinopathies

2. Reduced rate of synthesis of α or β chains: thalassaemia

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

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

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)

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)

Signs & Symptoms of Thalassaemia

• Severe anaemia first detected in early chilhood

• Jaundice, hepatosplenomegaly, marked bone changes (frontal bossing)

• Microcytic hypochromic anaemia

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)

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

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 %

Comparison of a normal blood film with b-thal major

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

HbH Disease

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

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?