Updated on: 10.05.25

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 in Protein 4.2, Ankyrin and Band 3
• Horizontal interaction defects (Produce elliptocytosis & other bizarre forms)
  • Actin
  • Protein 4.1
  • Adductin

Detailed structure of RBC membrane is available at: https://howitreat.in/hematology/red-blood-cell

Hereditary Spherocytosis

Introduction:

• Hereditary spherocytosis is an autosomal dominant condition, occurring due to defect in vertical interaction, characterized by abnormal shape and flexibility of RBCs.

Epidemiology:

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

Etiology:

• Mutation of genes encoding:
  • Ankyrin- ANK 1
  • Band 3- SLC4A1
  • Beta spectrin- SPTB1
  • Biallelic defects in band 4.2- EPB42
  • Biallelic defects in Alpha spectrin- SPTA1

Pathogenesis:

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

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Weakening of the vertical interaction

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Reduced membrane stability and spontaneous loss of cell membrane due to rapid vesciculation (process by which small, membrane-bound sacs i.e. vesicles are formed and bud off from the cell membrane)

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Reduction of cell surface to volume ratio 

(Surface becomes less while volume remains same)

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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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Extravascular hemolysis

Note:

• Complete absence of spectrin is not viable, so homozygous children are not found. 
• Clinical severity depends on degree of spectrin deficiency

Classification:

Clinical Features:

• Varying presentation- Asymptomatic (compensated hemolysis) to marked hemolysis leading to hydrops fetalis. 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, may manifest with early satiety or left upper quadrant fullness.
• Gall stones- Pigmented type
• Acholuric jaundice- Jaundice with absence of bile in urine. Usually present since birth.
• Family history: Found in 75% cases
• 3 types of crisis may be seen in hereditary spherocytosis
  • Hyperhemolytic crisis
    • Triggered by infection
    • Presents with fever, abdominal pain, splenic enlargement, Jaundice, hypotension and shock
  • Aplastic crisis
    • Triggered by parvovirus, trauma, surgery, pregnancy
    • Presents as sudden worsening anemia
  • Megaloblastic crisis
    • Occurs due to acquired folic acid deficiency, usually in high demand situations such as pregnancy.
• Skin lesions- Leg ulcers, 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 (Ferritin >500mg/l and transferin saturation of >60% indicates iron overload)
• Angioid streaks in optic fundi
• Venous thromboembolism 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:

(Findings are not very typical in newborns except high MCHC, hence better to rescheduled until the sixth month of age or later)

• Hemogram:
  • Hemoglobin content- Normal/decreased
  • High quality smears are needed as spherocytes may appear as artifacts
  • 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.
  • Spherocytic acanthocytes are seen in beta spectrin deficiency
  • Spherocytes and stomatocytes may be seen in Japanese protein 4.2 deficiency
  • MCV- Normal/decreased
  • MCH – Normal
  • MCHC – Increased due to cellular dehydration (>36g/dL)- It is the only anemia where MCHC is increased. Laser based counters provide histograms with hyperdense erythrocytes (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.
  • WBC and platelets – Normal

• Reticulocyte count- >8%
• Mean reticulocyte volume: Decreased
• Reticulocyte count/ immature reticulocyte fraction ratio: Mild cases: More than 19 and severe cases More than 7.7.

• Osmotic fragility test
• Increased fragility
• Hemolysis is complete between 0.5-0.4% NaCl
• Normal test does not rule out HS.
• False negative test is found in newborns due to the high level of fetal hemoglobin
• Positive test is also seen in hereditary elliptocytosis, and autoimmune hemolytic anemia
• Osmotic gradient ektacytometry- Measures deformability of whole RBC
• Serum bilirubin-Raised (Mostly indirect)
• Fecal urobilinogen- Raised
• LDH – Raised
• Haptoglobin-Decreased
• DCT and ICT- Negative (needed, as spherocytes are seen in AIHA as well)
• Peripheral smear of family members 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

• Cryohemolysis test:
  • HS RBCs have a high susceptibility for lysis when rapidly cooled from 37 degree C to 0 degree celcius.
• 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
  • Hereditory spherocytosis cells lyses more rapidly than normal cells
  • Test is easier to perform than osmotic fragility test.
• Sodium dodecylsulphate solubilized polyacrylamide 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.
  • Done only in specialized diagnostic centres and is time-consuming
• Flow cytometry:
  • Binding of eosin labeled maleimide (EMA binding test)
  • Cells deficient in band 3 fail to bind to EMA
  • Most accurate screening test, with a relatively short time-around-time, high sensitivity, economical, and feasible. It can be done even in patients who have been transfused.
  • Other conditions giving reduced fluorescence: South east asian ovalocytosis, Congenital dyserythropoietic anemia Type-II, Hereditary pyropoikilocytosis and Cryohydrocytosis
  • Interpretation (Based on grade of fluorescence):
    • Positive for HS- Fluorescence is > 21%
    • Negative- Fluorescence is < 16% 
    • Equivocal- If between 16% and 21%
• Identification of gene defects by NGS- 
  • Involves sequencing of relevant genes to identify mutations in them.
  • Widely available, cost effective and highly reliable.

Criteria for diagnosis:

• History (including family history) and clinical findings (splenomegaly) suggestive of HS.
• Presence of spherocytes and reticulocytosis
• Negative direct antiglobulin test
• If diagnosis is equivocal/ otherwise also: Preferable to do genetic testing (by NGS) for specific genetic mutations.

Treatment:

• Folic acid supplementation- Life long for moderate and severe HS.
• Transfuse PRBCs as and when required
• Severe neonatal jaundice:
  • Phototherapy +/- exchange transfusion 
  • Some children may need transfusion support for almost a year due to inability to mount an adequate erythropoietic response. Erythropoietin may be used until age of 9 months.
• Splenectomy: 
  • Restores the life-span of red cells to normal and hence cures hemolysis & hyperbilirubinemia.
  • Should be done after 6 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 (Severe HS)
    • Growth retardation/ skeletal changes
    • Anemia induced end organ damage
    • Development of leg ulcers
    • Appearance of extramedullary hematopoietic tumors
  • Laporoscopic is more feasible and safe. Any splenunculi (accessory splenic tissue) must be removed to prevent recurrence of symptoms.
  • Routine postoperative thromboprophylaxis should be given for adults.
  • Vaccinations (Pneumococcus, Hemophiluc influenza type b and meningococcus) must be given before procedure.
  • Prophylactic penicillin must be given for several years.
  • Cholecystectomy may be performed at the same time, if there is cholelithiasis (even if asymptomatic).
• Chelation therapy/ phlebotomies- if there is iron overload.
• Counseling to potential parents regarding possibility of neonatal jaundice and need for exchange transfusion

Hereditary Elliptocytosis

(Hereditary Ovalocytosis)

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

• Defect involves horizontal membrane protein interactions
  • Actin
  • Protein 4.1
  • Adductin
  • Glycophorin C

•  RBCs fail to recover their biconcave shape after they acquire elliptical shape in microcirculation where they are subjected to shear stress.

Clinical features

• 90% do not have hemolysis
• Asymptomatic HE patients may experience hemolysis in association with infections, hypersplenism, Vitamin B12 deficiency or microangiopathic disorders.
• Stomatocytic HE Melanesian/ South east Asian ovalocytosis children can present with neonatal jaundice, but hemolysis resolves by 3 years of age. Homozygous status results in hydrops fetalis.
• 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 axes 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.
• Pathogenesis: 

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 condition
• 4 variants depending on intracellular sodium concentration (Normal- 5-10 mmol/l)
  • Dehydrated Hst- MCHC increased- Na- 12-18mmol/L
  • Overhydrated Hst- MCHC decreased- Na- >60mmol/L
  • Cryohydrocytosis (Temperature sensitive leak) - Na- 20-50 mmol/L
  • Pseudohyperkalemia- Na- Normal
• Normal:

Shear stress and pressure on RBC in narrow capillaries

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Activation of PIEZO1

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Influx of Ca 2+ into the cytoplasm of RBC

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Activation of KCNN4

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Increased efflux of K+ out of RBC

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Drawing of water out of RBC

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Reduced volume of RBC

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Easy passage through capillary which is 1/3^rd of RBC size

• In dehydrated hereditary stomatocytosis:

Mutation in PIEZO1 and KCNN4 genes

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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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Intravascular hemolysis

• In overhydrated hereditary stomatocytosis:

Defect in stomatin gene

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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)

• Clinical features: 
  • Compensated hemolytic anemia- Jaundice, splenomegaly, gall stones
  • Sometimes- Recurrent fetal losses, hydrops fetalis, neonatal hepatitis, familial pseudo-hyperkalemia
• Investigations:
  • Wet smear – Appear uniconcave & bowl shaped.
  • 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 is associated with marked thrombotic tendencies.
• Acquired causes of stomatocytosis
  • Acute alcoholism
  • Liver disease
  • Cardiovascular disease
  • Artifacts in peripheral smear
• Syndromic forms include:
  • Stomatin deficient cryohydrocytosis with mental retardation, seizures, spastic rigidity, ataxia and hepatosplenomegaly (Mutation in SLC2A1 gene)
  • Phytosterolemia nonleaky stomatocytosis with macrothrombocytopenia (Mutation in ABCG5 or ABCG8 genes): Discussed below

Sitosterolemia (Phytosterolemia)

• Autosomal recessive
• Mutations in genes encoding transporter proteins- ABCG5 or ABCG8 
• Pathogenesis:

Increased absorption and decreased excretion of sterols

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Plasma levels of plant sterols is increased

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Accumulation of plant sterols in cell membranes

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Hemolytic anemia

• Diagnosis confirmed by genetic tests
• Clinical features:
  • Extreme phenotypic heterogeneity
  • Xanthomatosis with severe hypercholesterolemia
  • Early onset premature cardiovascular disease due to accelerated atherosclerosis
  • 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

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

Lecithin Cholesterol Acyl Transferase 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

• Greek: Acantha means thorn
• Spur cells are RBCs with irregular projections that vary in width, length and surface distribution.
• Cause: Severe liver disease due to any cause
• 2 step process:
  • Outer lipid layer of the RBC membrane acquires additional nonesterified cholesterol from abnormal plasma lipoproteins
  • Remodelling of abnormally shaped RBCs within spleen
• 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.
• 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 codition
• Pathogenesis:

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 fat soluble vitamins (Vitamin A, D, E and K) and essential fatty acids.

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 (Kx protein)
  • 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 
  • Males are affected
  • Mild compensated hemolytic anemia
  • May be associated with chronic granulomatous disease
  • Often have myopathy/ chorea
  • Patients may develop severe alloimmunization if transfused with Kell positive blood.
• 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