Classification of protein abnormalities in the erythrocyte membrane that result in vertical or horizontal interaction defects and cause hemolytic anemia

• Vertical interaction defects (Produces spherocytic anemia)
  • Primary spectrin deficiency
  • Secondary spectrin deficiency due to defects or deficiencies inProtein 4.2, Ankyrin and Band 3
• Horizontal interaction defects (Produce elliptocytosis& other bizarre forms)
  • Actin
  • Protein 4.1
  • Adductin

Hereditary Spherocytosis

Introduction:

• Hereditary spherocytosis is an autosomal dominant condition, occurring due to defect in vertical interaction.

Epidemiology:

• Highest prevalence is seen in north European population (1 in 2000 births)

Etio-pathogenesis:

Deficiency of spectrin, ankyrin, band 3, protein 4.2 etc.

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Reduced membrane stability and spontaneous loss of cell membranedue to rapid vesciculation

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Reduction of cell surface to volume ratio

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RBC is forced to assume smallest possible diameter for a given volume i.e. sphere.

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As spherocytes have reduced flexibility, they are trapped in splenic sinusoids

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Extravascularhemolysis

(Complete absence of spectrin is not viable, so homozygous children are not found)

Classification:

Clinical Features:

• Varying presentation- Asymptomatic to marked hemolysis leading to hydropsfetalis. Severity depends on extent of loss of spectrin. Severity is relatively uniform in a given family.
• Anemia- usually 5-30 days after birth as functioning of spleen becomes mature only after birth. But diagnosis may be made at any age depending on severity.
• Splenomegaly- mild to moderate
• Gall stones
• Acholuric jaundice- Jaundice with absence of bile in urine.
• 2 types of crisis may be seen in hereditary spherocytosis
  • Hyperhemolytic crisis
    • Triggered by infection
    • Presents with fever, abdominal pain, Jaundice, hypotension and shock
  • Aplastic crisis
    • Triggered by parvovirus, trauma, surgery, pregnancy
    • Presents as sudden worsening anemia
• Skin lesions- Ulcerations, gouty tophi, chronic leg dermatitis, (related to decreased RBC deformability)
• Extramedullary erythropoiesis- Masses may form in spine, kidney etc
• Poor growth, deformities of hands and skull
• Predisposition to malignancy- MPN, myeloma etc
• Iron overload- May be seen even in untransfused patients due to associated HFE mutation
• Angioid streaks in optic fundi
• VTE and pulmonary hypertension
• Rare non-hematological manifestations:
  • Neuromuscular abnormalities (as ankyrin and spectrin are expressed in neural tissues)-slowly progressive spinocerebellar degenerative disease, spinal cord dysfunction, movement disorders, cardiomyopathy
  • Distal renal tubular acidosis is seen with defects with band 3.

Investigations:

• Hemogram:
  • Hemoglobin content- Normal/decreased
  • High quality smears are needed as spherocytes may appear as artefacts
  • Spherocytes- Abnormally small in sized RBCs which have uniform shape and lack central 1/3^rd of pallor
  • Polychromatophilic cells and nucleated RBCs may be seen.
  • Pincered erythrocytes are seen in Band 3 deficiency.
  • Spherocyticacanthocytes are seen in beta spectrin deficiency
  • Spherocytes and stomatocytes may be seen in Japanese protein 4.2 deficiency
  • WBC and platelets – Normal
  • MCV- Normal/decreased
  • MCH – Normal
  • MCHC – Increased (>36g/dL)- It is the only anemia where MCHC is increased. Laser based counters provide histograms with hyperdenseerythrocytes (MCHC >40) which detects all cases of HS. Hence it is a best screening tool.
  • RDW- <14%
  • HS ratio- MCHC/MCV- If more than 0.36, it indicates presence of HS.

• Reticulocyte count- >8%

• Osmotic fragility test
• Increased fragility
• Hemolysis is complete between 0.5-0.4% NaCl
• Normal test does not rule out HS.
• Osmotic gradient ektacytometry- Measures deformability of whole RBC
• Serum bilirubin-Raised
• Fecal urobilinogen- Raised
• LDH – Raised
• Haptoglobin-Decreased
• DCT and ICT- Negative
• Parental blood testing for HS
• Autohemolysis test
  • It has value in differentiating various types of congenital nonspherocytic hemolytic anemias
  • It measures the degree of spontaneous hemolysis of blood incubated at 37^o C
  • Degree of hemolysis depends on integrity of cell membrane & adequacy of cell enzymes involved in glycolysis.
  • Normal – 0.2 – 2% hemolysis after 24- 48 hours.
  • Abnormalities

• Acidified glycerol lysis test
  • Glycerol slows down entry of water into cells in vitro.
  • The time taken for lysis to occur is a function of the osmotic resistance of the cells
  • Hereditoryspherocytosis cells lyses more rapidly than normal cells
  • Test is easier to perform than osmotic fragility test.
• Sodium dodecylsulphatesolubilizedpolyacrylamide gel (SDS-PAGE) electrophoresis 
  • Used to quantify proteins in the membrane of RBCs 
  • This is the confirmatory test for HS.
  • Results are expressed as ratios of individual RBC membrane proteins to Band 3.
• Flow cytometry:
  • Binding of eosin labeled maleimide (EMA binding test)
  • Cells deficient in band 3 fail to bind to EMA
  • Other conditions giving reduced fluorescence: South east asian ovalocytosis, Congenital dyserythropoietic anemia Type-II, Hereditary pyropoikilocytosis and Cryohydrocytosis
• Identification of gene defects- Involves sequencing of relevant genes to identify mutations in them.

Treatment:

• Transfuse PRBCs as and when required
• Folic acid supplementation- Life long
• Phototherapy +/- exchange transfusion for neonates presenting with severe jaundice
• Splenectomy: 
  • Restores the life-span of red cells to normal and hence cures hemolysis&hyperbilirubinemia.
  • Should be done after 5 years of age except in patients with severe anemia
  • Dehydrated hereditary stomatocytosis is a contraindication for splenectomy, hence this must be ruled out.
  • Indications: 
    • Marked hemolysis requiring multiple transfusions
    • Growth retardation/ skeletal changes
    • Anemia induced end organ damage
    • Development of leg ulcers
    • Appearance of extramedullary hematopoietic tumors
  • Laporoscopic is more feasible and safe.
  • Cholecystectomy may be performed at the same time, if there is cholelithiasis

Hereditary Elliptocytosis

(Hereditary Ovalocytosis)

• Autosomal dominant condition
• Gene affected is closely related to Rh locus on chromosome 1
• Classification

• RBCs fail to recover their biconcave shape after they acquire elliptical shape in microcirculation where they are subjected to shear stress.
• Defect involves horizontal membrane protein interactions
  • Actin
  • Protein 4.1
  • Adductin
  • Glycophorin C

Clinical features

• 90% do not have hemolysis
• Asymptomatic HE patients may experience hemolysis in association with infections, hypersplenism, Vitamin B12 deficiency or microangiopathic disorders.
• Some can have chronic hemolysis

Investigations:

• Hemogram
  • Hemoglobin- Usually >12g/dL
  • Prominent elliptocytosis>25% RBCs & usually >60%(<25% are seen with megaloblastic anemia and iron deficiency anemia)
  • Pseudoelliptocytes (Artifacts during smear preparation)- They are seen only near the tail. Long axis of all pseudo elliptocytes are parallel.
• Reticulocyte count – Mild elevation – >4%
• In hemolytic hereditary elliptocytosis, lab findings due to hemolysis are seen
• Osmotic fragility test- Abnormal only in severe HE

Treatment

• Splenectomy for hemolytic variant (Otherwise no treatment is needed)

Note: Elliptocytes are resistant to malarial infection due to abnormal rigidity of membrane

Hereditary Pyropoikilocytosis

• Autosomal recessive
• Presents in infancy as severe hemolytic anemia with extreme poikilocytosis
• Cells fragment when heated to 45^o-46 ^o C (Normal – 50 ^o C)
• They disintegrate when incubated at 37 ^o C for more than 6 hours.

Mutant spectrin- Prevents self association of heterodimers of spectrins

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Disruption of membrane skeletal lattice & Membrane cell destabilization

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Erythrocyte fragmentation &poikilocytosis

• Peripheral smear: Morphologic abnormalities of RBCs- Budding, fragments, microspherocytes, elliptocytestriangulocytes.
• MCV – Decreased  (25-55fL)
• Auto hemolysis- Increased and not corrected by glucose.

Hereditary Stomatocytosis

• Autosomal dominant with defect in stomatin gene

Deficiency of band 7 protein (Abnormality in RBC lipids)

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RBC membrane is abnormally permeable to both Na+ & K+

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Net gain of sodium is greater than net loss of potassium

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Over hydration of cells

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Hydrocytes look like stomatocytes in dried a stained blood films

(Slit mouth like area of pallor)

Wet smear – Appear uniconcave& bowl shaped.

• Usually asymptomatic, rarely cause hemolytic anemia
• 4 variants depending on intracellular sodium concentration (Normal- 5-10 mmol/l)
  • OverhydratedHst- MCHC decreased- Na- >60mmol/L
  • Dehydrated Hst- MCHC increased- Na- 12-18mmol/L
  • Cryohydrocytosis (Temperature sensitive leak) - Na- 20-50 mmol/L
  • Pseudohyperkalemia- Na- Normal
• Peripheral smear shows stomatocytes (10% to 50%), macrocytosis
• Osmotic fragility- Increased
• Autohemolysis- increased(Partially corrected by ATP & glucose)
• Treatment:
  • Folate supplements
  • Splenectomy in selected cases- Should not be done in dehydrated variant as it associated with marked thrombotic tendencies.
• Acquired causes of stomatocytosis
  • Acute alcoholism
  • Liver disease
  • Cardiovascular disease

Hereditary Xerocytosis

• Autosomal dominant condition

Net loss of intracellular K+ exceeds the passive Na+ influx

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Net Na+ gain

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Dehydration of cells

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Target cell/ contracted & speculated cell

MCHC Increased – after >37% cytoplasmic viscosity increases

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Decreased cellular deformability

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Trapping in spleen

• Clinical features
  • Compensated hemolytic anemia- Jaundice, splenomegaly, gall stones
  • Sometimes- Recurrent fetal losses, hydropsfetalis, neonatal hepatitis, familial pseudo-hyperkalemia
• Treatment
  • Folate supplementation
  • No benefit of splenectomy

Infantile Pyknocytosis

• Preterm infants presenting with transient hemolytic anemia (at 6-9 months of age) reticulocytosis and hyperbilirubinemia
• Peripheral smear- Pyknocytes- Distorted, densely stained RBCs
• Transfused RBCs also may become distorted
• No intervention needed

Rh Deficiency Syndrome

• Absence of Rh AG complex including Rh, RHAG, LW, Glycophorin beta, CD47 and protein 4.2
• Present with mild to moderate hemolytic anemia
• Peripheral smear- Stomatocytes, occasional spherocytes
• Treatment- Splenectomy- if hemolysis is severe.

Familial deficiency of HDL

• Accumulation of cholesterol in various tissues
• Large orange tonsils
• Hepatosplenomegaly
• Hemolytic anemia with stomatocytosis

Sitosterolemia (Phytosterolemia)

• Autosomal recessive
• Mutations in transporters- ABCG5 or ABCG8 leading to increased absorption of plant sterols
• Plasma levels of plant sterols is increased
• Diagnosis confirmed by genetic tests
• Clinical features:
  • Extreme phenotypic heterogeneity
  • Xanthomatosis
  • Early onset premature cardiovascular disease
  • Hemolytic anemia with stomatocytosis, macrothrombocytopenia, splenomegaly and abnormal bleeding
  • Splenomegaly
• Treatment
  • Avoid diet rich in plant based fats
  • Bile acid sequestrants: Cholestyramine
  • Ezetimibe (selective cholesterol absorption inhibitor) - 10mg/day
  • No use of statins as HMG CoA reductase activity is already inhibited
  • Splenectomy if there is persistent anemia

Lecithin Cholesterol AcylTransferase Deficiency

• Catalyses transfer of fatty acids from phosphotidylcholine to cholesterol
• Autosomal dominant
• Features include
  • Hyperlipidemia
  • Premature atherosclerosis
  • Corneal opacities
  • Chronic nephritis
  • Proteinuria
  • Mild anemia due to mild hemolysis
• Target cells are seen in peripheral smear

Abnormalities of Membrane Lipids

Acanthocytosis/ Spur cell anemia

• Spur cells are formed in severe liver disease
• They undergo lysis in spleen as they are less deformable
• Present with progressive hemolytic anemia, splenomegaly, hepatic derangement
• Peripheral smear- RBCs show prominent, regular projection from surface.
• They are formed when the outer lipid layer of the membrane acquires additional lipid
• Splenectomy is potentially dangerous procedure in critically ill patients, hence is not recommended.
• Differential diagnosis- Zieves syndrome- Hyperlipoproteinemia, jaundice, spherocytic hemolytic anemia in alcoholic patients with liver disease

Abetalipoproteinemia

• Autosomal recessive

Defect in apolipoprotien B gene.

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Failure to synthesize/ secrete apolipoprotein

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No triglycerrides carried from intestines

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Decreased plasma triglyceride, cholesterol and phospholipid levels

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Increased sphingomyelin in outer membrane of RBCs

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Formation of acanthocytes/ burr cells

• It is often associated with
  • Mild hemolysis
  • Retinitis pigmentosa
  • Progressive ataxic neurological disease with intentional tremors
  • Fat malabsorption- Steatorrhea at 1 month of life
  • Hepatic Encephalpathy
• Investigations:
  • Peripheral smear:Acanthocytes are seen, Increased nucleated RBCs
  • Reticulocytes are increased and they have normal shape
  • Intestinal biopsy: Engorged mucosal cells with lipid droplets
• Treatment
  • Dietary restriction of triglycerrides
  • Supplement high doses of Vitamin A, D, E and K

Other neuro-acanthosis syndromes:

• Chorea acanthosis syndrome:
  • Autosomal recessive
  • Normolipoproteinemicacanthosis
  • Neurological features include:
    • Progressive orofacialdyskinesis with tics, limb chorea, lip and tongue biting
    • Neurologic muscle hypotonia and atrophy
    • Absent or diminished reflexes
  • Increased CPK
  • MRI- Abnormalities of putamen and head of caudate
  • No anemia as RBC survival is mildly decreased
  • Mechanism of acathocyte formation is not clearly known
• McLeod Phenotype
  • Acanthosis together with decreased expression of Kell antigen
  • Defective gene on Xp 21(Close to genes of Duchene muscular dystrophy and retinitis pigmentosa)
  • Gene codes for Kx protein that carries Kell blood group protein 
  • Mild compensated hemolytic anemia
  • May be associated with chronic granulomatous disease
  • Often have myopathy/ chorea
• Huntington disease like 2
  • Mutations in junctophilin-3
  • Associated with tics, parkinsonism, motor neuron disease
• Pantothenate kinase associated neurodegeneration (Hallervorden-Spatz syndrome)
  • Progressive dementia, dystonia, spasticity, pallidial and retinal degeneration
  • Allelic variant- HARP syndrome
  • Hypobetalipoproteinemia
  • Acanthosis
  • Retinitis pigmentosa
  • Pallidal degeneration
  • Both are due to mutations in pantothenate kinase-2 gene

Figures:

Figure 8.19.1- Hereditary spherocytosis- Peripheral smear

Figure 8.19.2- Hereditary elliptocytosis- Peripheral smear