Haemolytic Anaemia II: Membrane Disorders

• The presentation covers Haemolytic Anaemia II, focusing on membrane disorders

Red Blood Cells

• The primary function of red blood cells is to deliver oxygen to tissues and transport deoxygenated blood back to the lungs
• The structure of red blood cells should be flexible and reversibly deformable for micro vascular circulation.
• Red cell survival depends on a normal red cell membrane, hemoglobin structure and function, and normal red cell metabolism
• Normal red blood cells have a biconcave shape, with a diameter of 7-8µm and a thickness of 2-3µm.

Red Cell Membrane Defects (Membranopathies)

• These are disorders affecting the red cell membrane, are heterogeneous hemolytic disorders
• Most are inherited, some are acquired
• Membrane defects lead to extravascular hemolysis

Red Cell Membrane Structure

• It has lipid bilayer structure.
• Proteins, glycolipids, and specialized molecules stabilize the cell membrane.
• The lipid bilayer consists of about 60% phospholipids, 30% cholesterol and 10% glycolipids.
• Phospholipids constitute the outer and inner surfaces of the bilayer.
• Phospatidylcholine and sphingomyelin mainly makeup the outer leaflet of the lipid bilayer
• phosphatidylethanolamine and phosphatidylserine mainly make up the inner leaflet
• The interior of the membrane involves hydrophobic bonding of acyl chains and cholesterol
• Requires ATP energy from the glycolytic pathway to maintain proper function.
• Integral and peripheral proteins determine the biconcave shape and function through interactions with lipids.

Integral and Peripheral Proteins

• Integral proteins have hydrophobic domains and associate with the bilayer's hydrophobic part.
• They are transmembrane proteins that span the membrane, providing plasma-interior channels.
• They interact with each other and cytoskeleton peripheral proteins, also known as vertical connections
• Band 3, Protein 4.2 (pallidin), and ankyrin form a major vertical pathway that binds to the spectrin tetramer’s ꞵ-chain
• Mutations or loss in this complex can lead to outer leaflet lipid membrane loss and spherocytosis.
• Peripheral membrane proteins form the RBC membrane skeleton, and they laminate the inner membrane surface.
• The skeleton is composed of spectrin, actin, tropomyosin, tropomodulin, adducin, dematin, protein 4.1, and ankyrin, also known as horizontal connections
• The membrane skeleton will support the lipid bilayer and adds durability and flexibility for survival during circulation.
• Spectrin mutations or deficiencies impact horizontal interactions, causing elliptocytosis.

Clinical sequelae of abnormalities

• Hereditary spherocytosis (HS)
• Hereditary elliptocytosis (HE)
• Hereditary pyropoikilocytosis (HPP)
• South-East Asian ovalocytosis (SAO)
• Hereditary acanthocytosis (HA)
• Hereditary stomatocytosis (HSt)

Hereditary Spherocytosis (HS)

• Inherited hemolytic anemia with spherical red cells
• Red cells appear as round with smaller diameter
• HS is defined by spherocytosis, increased osmotic fragility, and dominant inheritance
• It is caused by mutations that lead to defects in red blood cell (RBC) membrane proteins

Pathogenesis of HS

• Marrow produces red cells with a normal biconcave shape
• Intrinsic defects in erythrocyte membrane proteins cause Red cell cytoskeleton instability
• Defects affect proteins in vertical interactions between the membrane skeleton and the lipid bilayer
• This results in membrane loss from releasing lipid bilayer parts unsupported by the skeleton and leads to spherical RBC production (spherocytes).
• Spherocytes cannot pass through splenic microcirculation and die prematurely, causing extravascular hemolysis in the spleen, leading to splenomegaly

Molecular Basis of Hereditary Spherocytosis

• Ankyrin deficiency or abnormalities
• Alpha or Beta Spectrin deficiency or abnormalities
• Band 3 abnormalities
• Protein 4.2 (Pallidin) abnormalities
• Genes coding for the proteins in the vertical connections are present on specific chromosomes:
  • a-Spectrin deficiency: chromosome 1
  • ꞵ-Spectrin deficiency chromosome 14
  • Ankyrin deficiency: chromosome 8
  • Protein 4.2 (Pallidin) abnormalities: chromosome 15 (long arm)
  • Band 3 abnormalities: chromosome 17
• Spectrin deficiency is the most common defect, whose degree correlates with the extent of spherocytosis, abnormality on osmotic fragility, and severity of hemolysis.
• Genetic abnormalities range from mutations and deletions to frame shifts, resulting in the absence of certain proteins
• Complete absence of the protein is rare, therefore homozygous children are not found, as it's incompatible with life
• Double heterozygosity or inheritance of different membranes defects is associated with severe hemolytic anaemia.
• 60% of HS cases result from a defect in the ankyrin-spectrin complex.
• 25% has deficiency in band 3 (the anion channel).
• 15% have a deficiency of protein 4.2 or abnormality.

Clinical features of HS

• Autosomal dominant variable expression
• A few cases are autosomal recessive
• Normal clinical picture often contains moderate Haemolytic anaemia
• Possible asymptomatic hemolysis cases
• The anaemia presents at any age
• Rare with severe neonatal hemolysis with kernicterus (hyperbilirubinaemia)
• Associated with fluctuating jaundice
• Gilbert's Jaundice is a possible defect, leading to liver disfunction
• Splenomegaly is usually the palpated rule for 75%
• Pigment gallstones (Cholelithiasis) are frequent
• Sudden anaemia severity (aplastic crises) is typically by parvovirus infection.
• Jaundice, with moderate splenomegaly, and anemia are the most consistent findings.
• Usually, 1/3 of patients show well-compensated hemolysis, hemoglobin levels may range in the normal parameters
• HS patients are described as 'more yellow than sick'

Hematological Findings in HS

• Diagnosis is blood morphology and family history.
• Typical signs include polychromatic cells, spherocytes and microspherocytes that are densely staining smaller than the average red cells
• Spherocytes greater than 1-2% per field suggests a significant finding
• There's often high Reticulocyte count (5-20%)
• Increased mean cell hemoglobin above 35g/L

Investigations for HS

• Osmotic Fragility Test shows red cells as excessively fragile in dilute saline solutions
• Classic finding: increased osmotic fragility
• A rapid flow analysis of eosin-maleimide (EMA) is used to test
• EMA has replaced osmotic fragility test
• Abnormalities may be seen after 24-hour incubation at 37°C.
• Direct antiglobulin is usually normal
• Useful to distinguish between steady states from intermittent episodic changes

Osmotic Fragility Test in HS

• Measures in vitro red cell lysis
• Red cells lyse due to swelling by incubation in hypotonic saline solutions.
• The red cells swell until the pressure ruptures the membrane
• A normal red cell will show 50% lysis when the saline solution reaches a 0.5% sodium chloride solution
• Rigid HS cells have less ability to swell so they lyse at higher concentrations
• The Osmotic fragility curve is shifted to the right .

Acidified Glycerol Lysis Test

• Uses glycerol to slow water entry in vivo
• Time taken forlysis to become present os related to to osmotic resistance.
• HS cells show more rapid lysis

Auto Haemolysis

• Measures red cell ability to resist metabolic deprivation by incubation for 24 hours with or without glucose.
• Depends on the glucose present in serum
• Normal blood contains than 2% red cells
• HS is seen in 0.5% if glucose is added
• HS has high metabolism, therefore autohamolysis is high without glucose
• glucose correction is seen when glucose is added
• This insensitive test which has generally been abandoned

HS Clinical course and complications

• Patients are subjected to various crisis
• Haemolytic
• Aplastic
• Acute Sequestration
• Megaloblastic.
• Haemolytic is most common, with increased cell destruction (hyperhaemolysis). during which an increase of jaundice by viral or bacterial sepsis could accur
• Aplastic related to Parvovirus B19 infecting BFU-E which results in inhibition of erythropoiesis
• Megaloblastic: develops when folic acid becomes inadequate
• Acute Splenic Sequestration: increased splenic size causing an increase of trapped HS cells which results in severe anaemia
• Requiring immediate blood transfusions

Other rarer complications of HS

• Gallstones are common
• Silent gallstones need no intervention
• Cholecystitis/Biliary Colic leads to cholecystectomy
• Indolent leg ulcer
• Chronic erythematous dermatitis on the legs
• Gout.

Management of HS

• Well-compensated haemolysis involves reassurance and folic acid supplements (400µg dly / 5mg weekly).
• Splenectomy is the main management (Role of spleen)
• Splenectomy restores the life span, therefore curing haemolysis
• Produces haemoglobin level to normal
• Note the risks vs benefits of side effects in patients of splenectomy
• Performed unless clinical indication/severe anaemia or gallstones
• Hazards: Susceptibility to severe infection (encapsulated organisms)
• Complications can be treated accordingly

Hereditary elliptocytosis (HE)

• Hereditary disorder characterized by the presence of a large number of elliptoid red cells in peripheral blood
• The red cells are called elliptocytes.
• Similar manifests and pathophysiology as HS
• Milder disorder asymptomatic
• Symptoms not clearly visible , possibly discoverd by chance
• Pathognomonic finding has large elliptocytes
• Splenectomy can happen.

HE Clinical Subtypes

• Common HE (Most prevalent):
  • Silent carriers, mild HE, and HE with Infantile Poikilocytes
• Common HE with haemolysis:
  • Including HE with dyserythropoiesis, Hereditary Pyropoikilocytosis (HPP)
• Spherocytic HE
• South-East Asian ovalocytosis (band 3 deletion)
• Deficiency with spectrin tetramers can caused spectrum ranging from compensated haemoliss to lie threatening anaemia.
• Erythrocyte is long, flexible, composed with spectrin dimer parallel
• The heterodimers association has heterotetramers defect
• There’s genetic mutations
• Patients with homozygous or doubly heterozygous elliptocytosis are vulnerable to haemolytic with poikilocytes splenomegaly (hereditary pyropoikilocytosis)

The molecules of hereditary elliptocytosis

• Alpha or Beta spectrin dimer
• Alpha or Beta spectrin-ankyrin
• Protein 4.1 (Pallidin) deficiencies
• Homozygous results include anaemic, bizarre cells, and bizarre cells

Treatment of HE

• Folic acids
• Splenectomy

Characteristics of South-East Asian ovalocytosis

• Autosomal dominant inheritance
• Found in southern: East Asia has falciparum malaria
• Cell has stoma-like slits which resist invasion by malarial parasites
• Asymptomatic, sometimes may have mild anaemia
• Can present a deletion of band 3

Anomalies with Lipids

• Can be inherited or acquired

Inherited lipids

• Familial Lecithin Cholesterol Acyltransferase (LCAT) Deficiency
• Rare in autosomal
• Excesses of tissue cholesterol are moved by liver
• Abnormal membranes lipid, forming target cells

A-ß-lipoproteinaemia (Inherited lipid)

• Mutation of apolipoprotein with low cholesterol
• Sphingomyelin produces athanycytes
• Retinitis pigmentosa/fat malabsorption/hepatic-phalothy

Acquired

• Echinocytosis (Burr cells) Echinocytes of renal failure are Spiculated cells of acquired
• Acanthocytosis or spur cells
• Is a disorder of liver disease that has lipid plasma interaction

Conclusion

• Red cell membranes can cause extravascular
• Common types are found with HS and HE
• Use with smear ,fragility, flow cytrometric
• Support splenectomy if needed

Explore Haemolytic Anaemia II with a focus on red blood cell membrane disorders. Learn about the critical role of red blood cells in oxygen transport and the impact of membrane defects on hemolysis. Understand the structure and function of the red cell membrane and its associated proteins and lipids.