B12/Folate Anaemia

• haemolytic anaemia is a result of an increased rate of red cell destruction - causing a shortening of the red blood cell life span (from 120 days to 110/100 or even as low as 15 days)
• EPO in bone marrow can expand by up to 8-fold (8 mill.) to compensate for accelerated red cell destruction = compensated haemolytic process
• anaemia results when the rate of red cell destruction exceeds the rate of red production
• there are 2 types of haemolysis:

  1. intravascular = destruction of red cells within the blood stream
  2. extravascular = destruction of red cells by macrophages in the liver, spleen and marrow
• in intravascular haemolysis, when Hb is released into plasma and binds to haptoglobin, haemopexin and albumin to form methaemalbumin. excess free Hb is filtered by glomerulus but its reabsorptive is reached, Hb enters urine and iron is released causing haemoglobinuria and haemosiderinuria
• intravascular haemolysis occurs by the red cell becoming haemolysed or burst
• free Hb means that there is free iron which is toxic to the body
• extravascular haemolysis is an extension of a normal process and is the main type of haemolysis
• when globin is broken down to amino acids and reutilised (within the AA pool) iron is released and binds to transferrin and is reused and protoporphyrin is broken down to unconjugated bilirubin and after conjugation, in the liver, it is excreted as urobilinogen and stercobilinogen
• jaundice occurs when the liver cannot keep up with conjugation of bilirubin
• clinical features of haemolytic anaemia include:

  • pallor - anaemia
  • jaundice - increased unconjugated bilirubin (liver can't keep up)
  • splenomegaly - enlarged spleen
  • dark urine - increase in urobilinogen
  • faeces - increase in stercobilinogen
  • pigment gallstones - made of the breakdown products of RBC
  • ulceration - sickle cell anaemia (v rigid and can block blood vessels )
  • CVD/respiratory problems
• there are 2 main categories of haemolytic anaemias:

  1. Inherited = meaning there is an intrinsic defect in the red cell
  2. acquired = the defect is extrinsic to the red cell
• inherited haemolytic anaemia can be:

  • red cell membrane disorder
  • metabolic disorder - key enzyme deficiency
  • haemoglobin disorder - globin focused
• the main functions of the red cell membrane are:

  • separate cell contents in cytoplasm from plasma
  • maintain biconcave shape - maximising surface area, and flexibility and increasing gas exchange
  • regulate intracellular cation concentration (sodium-potassium pump)
  • interference between cell and its environment via membrane surface receptors (blood group antigens)
• the red cell membrane is composed of 50% protein 40% lipid (cholesterol and free fatty acids) and 10% carbohydrate (glycoproteins and glycolipids)
• protein band 3 goes through the lipid bilayer and into the membrane cytoskeleton and is a key attachment within the red cell membrane
• spectrin is a major protein in the cytoskeleton. it is a heterodimer of alpha and beta subunits which form oligomers with branching radial structures - it associates with ankyrin actin and protein 4.1
• actin binds to the tail end of alpha and beta spectrin units. the lattice of spectrin tetramers are held by actin filaments with teh lattice attatched to the lipid layeer by protein band 3
• ankyrin anchors the assembled spectrin molecules to the lipid bilayer
• protein band 4.1 stabilises spectrin binding to actin and binds directly to glycophorins A and C, protein band 3 and phosphatidylserine. it strengthens the binding between lipid bilayers and protein cytoskeleton
• hereditary spherocytosis (HS) is a spectrin deficiency that affects 1:5 Caucasians. it causes defective spectrin-protein 4.1 binding due to either an ankyrin or protein 4.1 deficiency or abnormality
• deficiency or abnormality of proteins causes the decreased density of skeletal proteins leading to lipid bilayer destabilisation and lipid loss in the form of microvesicles. this causes a reduced surface area resulting in a spheroidal shape
• spherocytes have impaired deformability due to the weakening of the cytoskeleton-lipid bilayer interactions.
• the splenic chords are a network of reticulum cells lines with macrophages which link up to the venous sinuses via small fenestrations - the size of these fenestrations is smaller than the size of the red cell so the red cell membrane must have some degree of deformability to allow the cell to pass through
• hereditary spherocyte cells are unable to negotiate their passage through splenic chords because of their lack of deformability and become trapped - most of the trapped cells are removed by the macrophages
• the spleen is a hostile environment for red cells due to haemoconcentration and stasis, acidic pH and exposure to macrophage induced membrane injury
• exposure to the conditions of the spleen worsens the loss of membrane and further decreases deformability - this is known as splenic conditioning and prepares the red cell for destruction upon the next pass through the spleen
• splenic conditioning results in a cumulative worsening of the spheroidal deformity and accelerates the rate of red cell destruction
• the lifespan of spherocytes can be as little as 15 days in the most severe forms of heredity spherocytosis
• splenectomy will return the red cell life span back to normal but will have no effect on membrane loss and spheroidal deformity and will make the patient immunocompromised
• secondary metabolic changes take place in spherocytes as they allow the influx of sodium ions but there is an increased efflux under the action of sodium-potassium pump which is ATP dependant
• spherocytes cause an increased RBC glycolysis and increased requirement for glucose
• definitive test for spherocytes is the red cell osmotic fragility test - susceptibility of cells to lysis when placed in a gradient of hypotonic solutions
• the lifespan of a spherocyte is roughly 15 days and the main site of destruction is the spleen - a splenectomy invariably returns the RBC lifespan to normal but will compromise the immune system and spherocytes will still be formed in other parts of the reticuloendothelial system
• hereditary elliptocytosis (HE) is spectrin abnormalities in the alpha and beta chains - not a deficiency but a change in the way they bind. it can be caused by protein4.1 abnormality or deficiency or by an abnormality in protein 3
• hereditary elliptocytosis is usually asymptomatic as red cells are not trapped in the spleen and only 15% of patients develop haemolysis with features similar to hereditary spherocytosis
• anaerobic metabolic pathways (very rare) in red cells include:

  • glycolysis
  • pentose phosphate pathway
  • 2,3 diphosphoglycerate shunt
  • glutathione metabolism
  • pyrimidine salvage cycle
• defects in glycolysis can be due to either: hexokinase (HK) deficiency or pyruvate kinase (PK) deficiency - both of which are enzymes involved in the stages to create ATP
• PK catalyses the reaction of PEP+ADP to pyruvate+ATP and a deficiency of PK causes impaired formation of ATP - causing anaemia and shortened red cell life span. it is inherited
• the defect in the pentose phosphate pathway is a deficiency of glucose-6-phosphate dehydrogenase (G6PD). it catalyses the reaction of glucose-6-phosphate + NADP to 6-phosphogluconate + NADPH