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Macrocytic anaemia

Introduction and epidemiology

Macrocytic anaemia is a form of anaemia characterised by macrocytosis (MCV > 100 fL). Megaloblastic anaemia is a subtype of macrocytic anaemia characterised by decreased DNA synthesis in haematopoietic stem cells, causing RBCs to be larger, oval, and blast-like, and neutrophils to be hypersegmented. The most common cause of both macrocytic and megaloblastic anaemia is folate and B12 deficiency.

Etiology

• Megaloblastic anaemia
  • B12 deficiency
    • Insufficient intake
    • Pernicious anaemia
    • Crohn disease
    • Small bowel resection
  • Folate deficiency
    • Insufficient intake
    • Alcoholism
  • Drugs that block DNA synthesis (methotrexate)
  • Myelodysplasia
• Non-megaloblastic macrocytic anaemia
  • Liver disease
  • Alcoholism

Pernicious anaemia is an autoimmune disease where autoantibodies are produced against intrinsic factor or the parietal cells which produce IF. These antibodies will either destroy the parietal cells or block B12 from binding to IF, both of which result in B12 malabsorption.

Clinical features

General features of anaemia are present.

In severe B12 deficiency, cell lines other than the RBC may be affected as well, potentially causing pancytopaenia. It may also lead to demyelination of the spinal cord, also called funicular myelosis or subacute combined degeneration of the spinal cord, which may lead to ataxia and decreased proprioception and vibration sense.

Diagnosis and evaluation

In case of macrocytic anaemia, a blood smear should be made to look for megaloblastic changes.

If the macrocytic anaemia is megaloblastic, serum homocysteine and methylmalonic acid (MMA) should be measured to differentiate between B12 and folate deficiency:

• B12 deficiency: Elevated homocysteine and methylmalonic acid levels,
• Folate deficiency: Elevated homocysteine levels, normal methylmalonic acid

If there is deficiency, the underlying cause should be sought. Testing for anti-parietal cell or anti-IF antibodies can diagnose pernicious anaemia.

Treatment

Treatment is targeted at the underlying cause, if possible. If not, supplements might necessary. B12 deficiency is ideally treated with intramuscular injections rather than oral therapy, as oral absorption of B12 supplements is poor.

If a patient is suffering from B12 deficiency but given folate supplements, the surplus folate can normalise the megaloblastic anaemia, giving a false impression that they had folate deficiency. However, folate does not stop the progression of and can even worsen B12 deficiency-induced spinal cord degeneration, which can be irreversible. It’s therefore important to confirm that B12 levels are normal before administering folate supplements.

Haemolytic anaemias

Introduction and epidemiology

Haemolytic anaemias are a form of anaemia characterized by the pathologically increased breakdown of RBCs, reducing their to less than the normal 120 days. To compensate for increased turnover of RBCs the bone marrow can increase the output of RBCs 6 – 8-fold. Anaemia only manifests if the rate of destruction exceeds this increased production rate.

There exist many types of haemolytic anaemia, both congenital and acquired.

Types

Here are some of the most important:

Congenital haemolytic anaemias:

• Haemoglobinopathies – characterised by defects of haemoglobin. These defects often cause haemoglobin to precipitate in the cell, which alters its shape.
  • Sickle cell anaemia
  • Thalassaemia
• Membranopathies – diseases characterised by abnormal RBC membrane
  • Hereditary spherocytosis
  • Paroxysmal nocturnal haemoglobinuria
• Enzymopathies
  • Glucose 6-phosphate dehydrogenase deficiency
  • Pyruvate kinase deficiency

Acquired haemolytic anaemias:

• Immune-mediated haemolytic anaemias
  • Warm antibodies (idiopathic or secondary to autoimmune disease or haematological malignancy)
  • Cold antibodies (idiopathic or secondary to autoimmune disease or haematological malignancy)
  • ABO/Rhesus incompatibility (a problem of neonates)
• Non-immune-mediated haemolytic anaemias
  • Microangiopathic haemolytic anaemias (TTP/HUS)
  • Macroangiopathic haemolytic anaemias
    • Prosthetic heart valves
    • Severe aortic stenosis
  • Drugs
  • Infection
  • Paroxysmal nocturnal haemoglobinuria

Corpuscular and extracorpuscular haemolysis:

We can classify haemolytic anaemias as corpuscular and extracorpuscular based on whether the cause of haemolysis is inside or outside the RBCs.

• Corpuscular haemolytic anaemias
  • RBC membrane defects
  • Enzymopathies
  • Haemoglobinopathies
• Extracorpuscular haemolytic anaemias
  • Immune-mediated haemolytic anaemias
  • Non-immune-mediated haemolytic anaemias

Pathomechanism

RBCs are broken down by macrophages of the mononuclear phagocyte system (MPS), also called the reticuloendothelial system (RES). The majority of the breakdown occurs in the spleen. Haemoglobin is split into the globin part and the heme part. The globin part is broken down to amino acids while the heme part is converted to indirect bilirubin. Indirect bilirubin is then converted to direct bilirubin in the liver and so on.

We distinguish between intravascular haemolysis and extravascular haemolysis depending on where the haemolysis happens. In intravascular haemolysis pathological haemolysis occurs in vessels while in extravascular haemolysis the physiological haemolysis in the MPS is pathologically increased.

Clinical features

In addition to features of anaemia, haemolysis may cause jaundice and gallstones. Some types of haemolytic anaemia may cause specific symptoms. Haemolysis may occur continuously or intermittently.

Diagnosis and evaluation

A protein called haptoglobin is important in the diagnosis of haemolysis. Haptoglobin is a plasma protein which binds to free haemoglobin in the plasma. When there is haemolysis, more haemoglobin is released into the plasma. Haptoglobin in the plasma will bind to the released haemoglobin. This decreases the amount of free circulating haptoglobin, which is what’s measured in the lab.

• Laboratory tests
  • In both types of haemolysis
    • Haptoglobin ↓
    • Lactate dehydrogenase ↑
    • Indirect bilirubin ↑
    • Reticulocytes ↑ – due to increased erythropoiesis
    • Urinary urobilinogen ↑
  • Only in intravascular haemolysis
    • Free haemoglobin in plasma ↑ (only in severe cases)
    • Brown-coloured urine – due to haemoglobinuria or haemosiderinuria
• Peripheral blood smear
  • Spherocytes – Small, spherical RBCs with no central pallor

It can be difficult to differentiate intravascular and extravascular haemolysis on a lab test as no one parameter is different in the two. In extravascular haptoglobin can be normal, and there is rarely free haemoglobin in the plasma.

The Coombs test is essential in the diagnosis of antibody-mediated anaemias. There are two types of Coombs test, the direct type and the indirect type. The direct Coombs test is positive if there are autoantibodies against the patient’s own RBCs bound to the RBCs in the patient’s blood. The direct Coombs test is positive in immune-mediated haemolytic anaemias.

The indirect Coombs test is positive if there are autoantibodies against foreign (not the patient’s) RBCs in the patient’s blood. The indirect Coombs is used to check if the patient’s blood contains anti-D antibodies, which would cause haemolytic disease of the newborn due to ABO or Rh incompatibility.

Sickle cell anaemia

Introduction and epidemiology

Sickle cell disease is the most common intrinsic haemolytic anaemia, i.e. the most common cause of haemolytic anaemia due to intrinsic defects of the RBCs. It’s most common in African and Mediterranean populations.

It’s caused by a point mutation in the beta globin gene causes haemoglobin to precipitate into a sickle-shape when deoxygenized. This leads to microvascular occlusion and haemolysis.

Sickle cell disease causes moderately severe anaemia with clinically detectable jaundice due to haemolysis. It also causes hyposplenism, which leads to increased susceptibility to severe infections by encapsulated bacteria, like osteomyelitis and sepsis.

Pathophysiology

Sickle cell anaemia is caused by a point mutation in the β-globin gene, causing a glutamate to be replaced by a valine. The mutated β-globin gene causes an abnormal form of haemoglobin called haemoglobin S (HbS) to form.

People who only have the mutation on one allele are heterozygotes and is said to have sickle cell trait. In these people haemoglobin S accounts for only 40% on all haemoglobin, which is often not enough to be symptomatic. People who have the mutation of both alleles are homozygotes and have manifest sickle cell anaemia. These people have 75 – 95% haemoglobin S, the remaining being foetal haemoglobin (HbF).

The problem with HbS is that, when deoxygenated, the HbS molecules polymerize and form a gelatinous network inside the RBC. This causes the RBC to change shape to the characteristic sickle-shape, forming a stiff, viscous sickle cell. Several factors can induce this sickling, like:

• Hypoxia
• Low pH
• Fever
• Infection
• Exercise
• Dehydration
• Abrupt temperature changes
• Stress

The sickle cells cause two problems. Because they are stiff, they can’t circulate properly through capillaries, causing capillary occlusion with microinfarctions and ischaemia, especially of the spleen. Second, they’re more prone to haemolysis in the spleen. The increased RBC turnover that occurs with the haemolysis may increase the demand for folate, causing macrocytic anaemia.

Clinical features

People with sickle cell disease have continuous anaemia, sometimes with jaundice. Most people with sickle cell anaemia experience symptoms within the first years of life.

Acute complications can also occur. Acute splenic sequestration crises can occur, where a large number of sickled cells accumulate in the spleen, causing sudden drop in blood volume and splenomegaly. Acute aplastic crisis occurs in case of parvovirus B19 infection, in which the virus causes complete but temporary cessation of RBC production. Vaso-occlusive crises is a consequence of sudden vaso-occlusion, leading to episodes of severe bone pain and dactylitis, as well as priapism.

Due to chronic microinfarctions of the spleen these patients lose splenic function early. This makes them more susceptible to infections by encapsulated bacteria, like S. pneumoniae, H. influenzae and salmonella. Chronic microinfarctions also cause chronic pain and can cause infarction of virtually any organ.

Diagnosis and evaluation

• Laboratory results corresponding to haemolytic anaemia
• Peripheral blood smear
  • Sickle cells
  • Target cells
  • Howell-Jolly bodies
• Haemoglobin electrophoresis – for confirmation of diagnosis

Treatment

• Avoiding painful crises
  • Avoid triggers
  • Pneumococcal and H. flu vaccines
  • Antibiotic prophylaxis during invasive procedures
  • Vigorous oral hydration during or in anticipation of periods of exercise, stress
  • Treatment with hydroxyurea – increases the amount of foetal haemoglobin which reduces the proportion of HbS
• Management of acute crises
  • Vigorous hydration
  • Treatment of underlying cause
  • Analgesia
  • Blood transfusion
  • Bone marrow transplant
    • The only curative treatment is allogenic haematopoietic stem cell transplantation. This is only indicated in severe cases and only in children.

Thalassaemia

Introduction and epidemiology

Thalassaemia is a group of diseases characterised by one or more defective globin genes. Like sickle cell anaemia it is more common in Africa and the Mediterranean, but also south-east Asia.

Pathophysiology

We distinguish alpha and beta thalassaemia, based on whether the alpha or beta globin genes are defective. Alpha globin chain production is controlled by two genes, so there are four alleles that can be defective. Beta globin chain production is controlled by only one gene so there are only two alleles that can be defective.

Clinical features

The clinical features of each type depend on how many alleles are defective. In alpha thalassaemia, one or more of the four genes for the alpha globin chain are deleted. In beta thalassaemia, one or both of the genes for the beta globin chain are defective.

Severity of the anaemia increases with increased number of affected genes, and ranges from asymptomatic to mild haemolytic anaemia to severe haemolytic anaemia. If all four alpha genes are deleted, the condition is incompatible with life (Hb Barts -> hydrops foetalis).

Diagnosis and evaluation

• Laboratory results
  • Corresponding to haemolytic microcytic anaemia
  • RDW normal or ↑
• Peripheral blood smear
  • Target cells
  • Teardrop cells
• Bone marrow biopsy
  • Reactive hyperplasia
• Haemoglobin electrophoresis – to confirm diagnosis
• DNA analysis (PCR) – to confirm diagnosis

Treatment

In mild thalassaemias, no treatment is necessary. In severe ones, lifelong regular blood transfusions are necessary. Treatment with iron chelators is necessary to prevent iron overload. Haematopoietic stem cell transplantation could be curative in severe cases.

Enzymopathies

Glucose 6-phosphate dehydrogenase deficiency

Glucose 6-phosphate dehydrogenase (G6PD) deficiency is a deficiency of the rate-limiting enzyme of the pentose phosphate pathway, which is essential for preventing oxidative damage to RBCs. Deficient RBCs are susceptible to haemolysis when exposed to oxidants. It’s an X-linked condition.

Like the haemoglobinopathies it’s most common in Africa, Asia and the Mediterranean. The enzyme G6PD is important to regenerate glutathione, an important antioxidant.

Most patients are asymptomatic until they’re exposed to factor which precipitates oxidative injury and resulting haemolysis. This can occur due to infection, fava beans, and certain drugs like sulphonamides. During these haemolytic crises, Hb drops rapidly, possible falling below 50 g/L over 1 – 2 days. Symptoms of crisis include jaundice, pallor, dark urine, and abdominal or back pain.

G6PD deficiency causes normocytic haemolytic anaemia. On blood smear, bite cells and Heinz bodies may be present. Measurement of the G6PD enzyme activity in RBCs confirms the diagnosis. Management involves avoiding triggers of haemolytic crises.

Pyruvate kinase deficiency

RBCs only produce energy by glycolysis, which depends on pyruvate kinase (PK). When PK is deficient, RBCs will be deficient in ATP, which can lead to haemolysis. In contrast with G6PD deficiency, PK deficiency causes chronic haemolytic anaemia.

Autoimmune haemolytic anaemia

Autoimmune haemolytic anaemia (AHIA) is a haemolytic anaemia that occurs due to the body producing autoantibodies against its own RBCs. We distinguish two types of AHIA based on which temperature the autoantibodies best bind to the RBCs. In warm AIHA, also called warm agglutinin disease, the autoantibodies bind more strongly to the RBCs at body temperature (37°C). In cold AIHA, also called cold agglutinin disease, the autoantibodies bind to the RBCs more strongly in lower temperatures. The autoantibodies are called warm agglutinins and cold agglutinins, respectively.

Cold agglutinin disease

Cold agglutinin disease can be idiopathic (especially in elderly women) or it can be secondary to lymphoma, CLL or mycoplasma infection. The cold agglutinins are of IgM type and bind most strongly to RBCs at 4°C, but they bind at higher temperatures too. The warmest temperature the antibodies will bind to RBCs at varies from person to person; most clinically significant cold agglutinins bind to RBCs at around 28 degrees.

The periphery of the body can easily reach 28 degrees and so patients can experience haemolysis all the time. Cold obviously makes the symptoms worse as more autoantibodies bind to RBCs.

Warm agglutinin disease

Warm agglutinin disease can be idiopathic but is secondary in 2/3 of cases. It can be secondary to lymphoid neoplasms, solid tumors, autoimmune diseases and certain drugs, especially methyldopa. The warm agglutinins are of IgG type.

Pathophysiology

When autoantibodies bind to the RBCs the complement system is activated. This forms the membrane attack complex (MAC), which causes intravascular haemolysis. The opsonized RBCs are haemolysed in the spleen, which causes extravascular haemolysis. Intravascular haemolysis is only characteristic for cold agglutinin disease while extravascular haemolysis occurs in both.

Clinical features

Cold agglutinin disease:

• Acrocyanosis
• Splenomegaly
• Features of anaemia, especially pallor

Warm agglutinin disease:

• Features of anaemia, especially pallor
• Jaundice
• Splenomegaly

Diagnosis and evaluation

• Laboratory findings
  • Signs of haemolysis
• Peripheral blood smear
  • Normochromic anaemia
  • Anisocytosis
  • Autoagglutination
  • Polychromasia (RBCs that stain both basophilic and eosinophilic)
• Positive direct Coombs test
• Detection of cold agglutinins
  • Only in cold agglutinin disease of course

Treatment

The underlying cause, if any, should be treated. Corticosteroids or rituximab can improve the symptoms. People with cold agglutinin disease should avoid low temperatures.

Macroangiopathic haemolytic anaemias

Macroangiopathic haemolytic anaemias are characterised by the intravascular haemolysis of RBCs due to mechanical forces in the large blood vessels, hence the name. The most important causes are severe aortic stenosis and prosthetic heart valves.

In severe aortic stenosis the lumen of the aortic valve is so narrow that the blood that flows through it reaches very high velocities. This rapid, turbulent transvalvular blood flow damages the RBC membrane, causing them to haemolyse. Turbulent blood flow through prosthetic heart valves causes similar damage to the RBCs.

The peripheral blood smear will show fragmented red blood cells (schistocytes).

Paroxysmal nocturnal haemoglobinuria

Introduction and epidemiology

Paroxysmal nocturnal haemoglobinuria (PNH) has an unfortunate name. Its name implies that the main clinical feature is the presence of haemoglobin in the urine in the night, but this symptom isn’t present in everyone with PNH. Also, the name doesn’t explain the most important clinical features of the disease: Haemolysis, venous thrombosis, and pancytopaenia. It’s a rare disease.

Pathophysiology

All cells of the blood have a so-called membrane bound glycosylphosphatidylinositol (GPI) anchor. Many proteins are connected to this anchor, the most important being CD55 and CD59. The function of CD55 and CD59 is to act as a shield against complement-mediated haemolysis, protecting the blood cells, especially the RBCs, from getting destroyed. This anchor is encoded by the PIGA gene.

In PNH a haematopoietic stem cell acquires a mutation in PIGA, which causes them to lose the GPI anchor. As such they can’t express CD55 and CD59 either, making them susceptible to complement-mediated haemolysis. This mutated stem cell, and all the cells which will derive from it, are known as PNH clones. The RBCs derived from the PNH clone are not protected against complement-mediated haemolysis and are therefore constantly haemolysed.

The severity of the PNH depends on how much of the bone marrow is comprised of PNH clones. Many healthy people have some PNH clones in their bone marrow but are asymptomatic (subclinical PNH). However, if a person with a few PNH clones in their bone marrow develops aplastic anaemia the PNH clone will have more space to proliferate. As such there is a hypothesis that clinical PNH arises from the expansion of a PNH clone in an injured bone marrow.

Approximately half of all patients with PNH also have aplastic anaemia, and approximately half of all patients with aplastic anaemia have PNH.

Venous thrombosis and vasoconstriction are important features of PNH. The current hypothesis is that free haemoglobin produced by constant haemolysis scavenges and inactivates NO in the serum, and that free haemoglobin activates the endothelium. The loss of the potent vasodilator and the activation of the endothelium causes thrombosis.

Classification

We distinguish three types of PNH, based on the context under which it was diagnosed:

• Classic PNH – established PNH and symptoms but without another bone marrow disorder
• PNH in the setting of another bone marrow disorder – established PNH and symptoms and with aplastic anaemia, MDS, etc.
• Subclinical PNH – presence of PNH clones but without clinical symptoms

Clinical features

• Haemolytic anaemia
• Pancytopaenia – due to the high occurrence of simultaneous aplastic anaemia
• Venous thrombosis
  • Often in unusual locations, like hepatic, cerebral or abdominal veins
  • Can cause Budd-Chiari syndrome
• Abdominal pain
• Dark urine in the morning – due to increased haemolysis during the night and concentration of urine during the night
• Renal failure – due to chronic haemoglobinuria
• Pulmonary hypertension – due to vasoconstriction in the pulmonary circulation

Diagnosis and evaluation

• Laboratory
  • Haemolytic anaemia
  • Pancytopaenia
• Negative Coombs test (as the haemolysis is mediated by complement and not antibodies)
• Flow cytometry – absence of CD55 and CD59 on blood cells

Treatment

There are only two established therapies for PNH. The first is eculizumab, an anti-C5 antibody, which inhibits complement-mediated haemolysis. The second is allogenic stem cell transplantation.