THALASSAEMIA A group of chronic, inherited anemias characterised by defective Hemoglobin (Hb) synthesis and ineffective erythropoiesis, particularly common in persons of Mediterranean, African and Southeast Asian ancestry. Thalassaemia results from unbalanced Hb synthesis caused by production of at least one globin polypeptide chain (α, β, γ, δ). There are 2 forms of thalassaemia Thalassaemia major Thalassaemia minor (trait)

THALASSAEMIA MAJOR Thalassaemia major is sometimes known as Cooleys Anaemia, Homozygous, Bete Thalassaemia or Mediterranean Anaemia. Is a serious inherited childhood anaemia. Children with Thalassaemia major cannot make enough haemoglobin. Because of this, their bone marrow cannot produce enough red blood cells. The red blood cells that are produced are nearly empty.

THALASSAEMIA MINOR People with Thalassaemia Minor, sometimes known as Trait, carry Thalassaemia but they are not ill. They are completely healthy and normal but some of them have slight anaemia. Most people with Thalassaemia Minor do not even know that they have it. It is only discovered if the person has a special blood test or if they have a child with Thalassaemia Major. It is important to know if you have Thalassaemia Minor later in life. The reason for this is that it may cause some problems if the person and their partner wants to start a family. Thalassaemia minors red blood cell are also different from normal blood cells.

CLINICAL FEATURES MINOR - clinically asymptomatic MAJOR Severe anemia Expanded marrow space Jaundice Leg ulcers Cholelithiasis Splenomegaly Bone changes and fractures Growth failure

HAEMOGLOBIN Hb is most commonly measured to detect anaemia. Low Hb indicates anaemia (various types) and blood loss. In some relatively rare genetic diseases, the alterations in the structure of the Hb molecule can be detected by electrophoresis.

HAEMOGLOBIN cont.. Hb 120g/L 130-180 LOW Hb electrophoresis Normal Hb A2 4.8% 1.8-3.5 HIGH Hb F 1.5% 0.5-0.8 HIGH Hb inclusions not seen in red cells

HAEMOGLOBIN cont… HbA2 - The haemoglobin is composed of two alpha chains and two delta chains. HbA2 is usually raised in the β thalassaemias, and in unstable haemoglobinopathies where the amino acid substitution is on the β chain. HbF - is the primary hemoglobin produced by the fetus during gestation. The determination of fetal hemoglobin is an aid in evaluating low concentrations of hemoglobin in the blood (anemia). HbF may be raised in various haematological conditions among which are the thalassemias. HbH inclusions - Performed as part of a haemoglobinopathy/thalassaemia screen. For diagnosis of alpha thalassaemia.

Mean cell volume (MCV) - the average volume of a single red cell. Terms such as `microcytic' and `macrocytic' are descriptive of low and high MCV, respectively. LOW MCV- Microcytic/hypochromic anaemia Iron deficiency Thalassaemia Anaemia of chronic disease (uncommonly microcytic) Sideroblastic anaemia (uncommon: acquired forms more often macrocytic) Lead Poisoning (uncommon) Hb E trait or disease MCV 65 f/L 80-100 LOW

Could The Near Normal Hb and Low MCV be Due to Other Conditions? Hb 120g/L (130-180) MCV 65fL (80-100) - Blood film shows mild hypochromic, microcytic cells with occasional target cells and basophilic stippling

Potential Causes of Anaemia 1) Blood loss 2) Reduced red cell life span (haemolytic anaemia) - consequent on an intrinsic abnormality of red cells either inherited or acquired - consequent on extrinsic factors 3) Inadequate production of red cells - deficiency in iron, vitamin B12 or folic acid; aplastic or hypoplastic anaemias; ineffective red cell production; bone marrow infiltration by malignant cells; bone marrow fibrosis 4) Low-affinity haemoglobin 5) Abnormal distribution of red cells within the vasculature - hypersplenism

Causes of Microcytic Anaemia Microcytosis is a highly prevalent finding during blood examination and is detected in ~3% of patients admitted to hospital Microcytosis occurs when the number of abnormally small RBCs in the blood is such that the mean corpuscular volume (MCV) drops to below the normal range The investigation of microcytosis/microcytic anaemia is often a challenge for the clinician and in most cases is related to impaired hemoglobin synthesis

Causes of Microcytic Anaemia cont… Non-thalassemic causes of microcytic anaemia include 1) Iron deficiency anaemia 2) Anaemia of chronic disease / inflammation 3) Congenital sideroblastic anaemia

Iron Deficiency Anaemia Iron deficiency is the primary cause of microcytosis and therefore a complete iron-status analysis should always be undertaken. In severe cases the MCV can be in the range of 50-60fL A blood smear will typically show erythrocytes that are microcytic and hypochromic as well as poikilocytes and occasional target cells

Iron Deficiency Anaemia cont. Low serum ferritin (indication of iron stores) and serum iron (indication of iron available for Hb synthesis) as well as an increased TIBC are consistent with this diagnosis Potential sources of blood loss should be investigated including occult bleeding, worm infestation and gum disease Once the primary cause of the deficiency has been corrected, treatment may involve 6-12 months of oral iron supplementation to replenish iron stores and should involve regular Hb monitoring

Anaemia of Chronic Inflammation Associated with conditions such as rheumatoid arthritis and malignancy is most often normochromic-normocytic with low serum iron and adequate reticulo-endothelial stores May progress to become hypochromic-microcytic in nature with MCV frequently in the range of 70-80fL A decrease in serum iron and TIBC is typical although serum ferritin remains normal An inflammatory state should be looked for in those patients with microcytosis and normal iron status whom belong to ethnic groups with a low incidence of inherited hemoglobinopathies. If an inflammatory state is present it should be treated and MCV monitored.

Sideroblastic Anaemias Hereditary forms show a hypochromic-microcytic picture but serum iron is raised and TIBC saturated In primary acquired forms the peripheral blood film is characteristically dimorphic with some hypochromic cells and a raised MCV Bone marrow examination is essential and may show erythroid hyperplasia, excess iron and a large number of sideroblasts Treatment may involve the exclusion of precipitating drugs and toxins, pyridoxine and/or folic acid supplementation as well as blood transfusions in refractory cases. Tranfusions have the potential to increase the already significant serum iron burden

What is basophilic stippling?

HOW DOES IT OCCUR? Beta thalassemia is caused by mutations in the in haemoglobin beta gene which prevents the production of the beta chains in haemoglobin. This causes an increased alpha to beta chain ratio and precipitation of alpha ribosomal DNA resulting coarse basophilic stippling. The stippling effect is aggregations of the ribosomal DNA or protein granulations in the cytoplasm of erythrocytes

IDENTIFICATION The granulations are usually variable in size and number. An identification technique named the Ramanowsky stain is used to confirm basophilic stippling as it stains the particles a violet blue colour. The RNA is not blue, it is only the stain required for identification that causes it to appear this way

CONDITIONS CAUSING STIPPLING Coarse stippling is characteristic of lead poisoning and thalassemia. In thalassemia it is most commonly stippling in the cytoplasm of younger cells. Lead poisoning often presents as stippling in the cytoplasm of nucleated red blood cells

LEAD POISONING

Beta Thalassemia minor Haematological Profile Hypochromasia + Anisocytosis ++(microcytes) Polikilocytosis Target cells + Immature Forms Coarse basophilic stippling

Beta Thalassemia major Haematological Profile: Hypochromasia) +++ Anisocytosis +++ (Macrocytes, Microcytes Poikilocytosis Target Cells +++ Tear Drops Schistocytes Acanthocytes, Howell Jolly Bodies, Target Cells (post splenectomy Immature Forms Polychromasia ++ Nucleated RBC +++ (bone marrow response)

BETA THALASSEMIA MINOR The usual haematological characteristics of beta thalassemia minor include -hypochromia -microcytosis -diminished osmotic fragility -erythrocytes with basophilic stippling

STIPPLING IN THALASSEMIA Basophilic stippling is not a definitive technique to distinguish between the different forms of thalassemia. It has been found to occur in both beta thalassemia minor and major, although it may be more prevalent in minor.

TECHNIQUES More reliable identification techniques for the various thalassemias include: -Complete blood count (CBC) - a measurement of the size, number and maturity of different blood cells in a specific volume of blood. -Haemoglobin electrophoresis with A2 quantitation - a lab procedure that differentiates the types of haemoglobin present.

BLOOD SMEAR DIAGNOSIS Previous profiles show basophilic stippling is not the only abnormality present on a blood smear Merck manual states that a blood smear is almost diagnostic for beta thalassemia major due to many nucleated erythroblasts, target cells, small pale RBC and punctuate and diffuse basophilia. Basophilic stippling alone is not enough to diagnose and differentiate between the types of thalassemias.

β-Thalassaemia The globin part of haemoglobin A1 has 4 protein sections, two of these are alpha and two are beta-chains. In β-thalassaemia minor you have only one normal β-chain instead of two. Β-thalassaemia major occurs when you have no functional beta chains

α-thalassaemia The heterozygous form of α-thalassaemia means there is only one alpha chain whereas the homozygous form occurs when there is no functional alpha chain. Usually the synthesis of alpha and beta globins is very tightly regulated to produce equal numbers in both.

Beta Thalassaemia In the case of beta thalassaemia with the decrease in β-globin it means that the haemoglobin molecules contain more alpha globin than beta globin.

Pathology of β-thalassaemia Iron levels are normal The concentration of HbA2 is increased HbF is increased both are trying to compensate for the decrease in HbA1 Low MCV, hypochromic, microcytosis and basophillic stippling

Pathology of α-thalassaemia HbA2 and HbF are normal in most cases Decreased or absent alpha-chains production results in excess gamma chains during foetal life and excess beta chains later. This causes the tetramers, Hb Barts and Hb H which precipitate in red cells and decrease red cell survival. Tests for Hb H inclusions are used to detect it DNA analysis may be required for definitive diagnosis of alpha thalassaemia

Case E FBC Results Ferritin 55µg/L 30-330 Hb electrophoresis Normal Hb A2 4.8% 1.8-3.5 Hb F 1.5% 0.5-0.8 Hb H inclusions not seen in red cells