Hereditary spherocytosis
Epidemiology § 1:5000 births (1:2000 if subclinical forms included) § Northern Europeans / Americans principally § Also Algeria, Tunisia, Egypt, Japan, N India, Brazil § Rare in black population
Pathology § 7 Important proteins, , , , , and § Vertical connections § Link plasma surface to cytoskeleton eg. Band 3, glcophorin § HS § Horizontal connections § Inner network of cell membrane eg. Spectrin § HE § HS – 60% are due to defects in ankyrin-spectrin complex, 25% involve band 3 § HE – deficiency is in the spectrin tetramer § Severity determines phenotype of HE or HPP
AD HS is often caused by mutations in three genes: 1. Alpha-spectrin (SPTA gene) § Alpha and beta spectrin sub units fold into triple helices § Beta-spectrin binds to ankyrin, protein 4.1 and actin § Vertical attachment to the lipid bilayer occurs through binding to band 3 and glycophorin C 2. Band 3 (AE1 gene) § Band 3 exists as dimers and tetramers on the red cell membrane. § Binds beta-spectrin via ankyrin and protein 4.1 § Band 3 also binds ankyrin via protein 4.2 which provides additional stability in the red cell membrane 3. Ankyrin (ANK1 gene) § Binds Band 3 and beta spectrin together
These result in secondary protein deficiencies which can be detected by SDS-PAGE
Clinical features § Ususally diagnosed in childhood / early adulthood but can be any point in life.
Presentations: 1. Neonatal jaundice and anaemia § May need transfusions for the first year of life (can then become transfusion independent since erythropoietic drive poor during first year of life). § Epo may be of benefit 2. Anaemia, jaundice, splenomegaly with family history in early childhood § May be associated with neuromuscular conditions / eye disease (angiod streaks) – particularly when ankyrin gene involved on chromosome 8. 3. Aplastic crisis § Parvoviurs B19 or influenza 4. Just spherocytes and reticulocytosis § Normal Hb and bilirubin § Mild HS may be exacerbated by illness such as EBV which cause spenomegaly 5. Gall stones and splenomegaly 6. Hydrops fetalis § If fetus inherits defects from both parents
Severe disease § Growth failure (increased metabolic burden of marrow turn over) § Marrow expansion and skeletal deformities § Leg ulcers
Investigations § Need to be careful to distinguish from hereditary stomatocytosis (where there is abnormal permeability of the red cell membrane to sodium and potassium ions) since splenectomy in these conditions leads to a high risk of thrombosis.
FBC § Hb reduced § MCV reduced § MCHC increased (>35) § % hyperdense cells increased § RDW increased § Retics increased
Film § Freshly made film as features of stomatocytosis can be obscured in old films § Spherocytes § Acanthocytes (8-15% in beta-spectrin gene mutations) § Mushroom shaped cells § Irregular contour (cells resembling hereditary pyropoikilocytosis) in combined ankyrin and spectrin deficiency § Ovalostomatocytosis in Japanese patients with protein 4.2 deficiency
DAT § Important to exclude AIHA § Sometimes difficult to distinguish DAT-negative haemolytic anaemia (particularly IgA) § Can be resolved by flow cytometric assessment of red cell Ig density
Osmotic fragility § Measures sensitivity of red cells to lysis by incubation in increasingly hypotonic saline § More rigid HS cells have less ability to swell § Normal cells around 50% lyse at NaCl 0.5% § May be normal in 10-20% of cases of HS § Also false positives (eg in immune spherocytosis) § Recommended purely as a supportive investigation § Acidified glycerol lysis test is similar but easier to perform
Cryohaemolysis and EMA binding § Both recommended as screening tests § No reports of positives in immune or non-membrane-associated disorders § Not specific – will be positive in § Rare membrane disorders such as aberrant band 3 proteins (eg CDAII and SAO) § Altered intracellular viscosity (eg sickle) § Temperature sensitive cation transport eg cryohydrosis § Can be difficult to interpret in mild and atypical cases
Cryohaemolysis § Packed cells in tube – measure % haemolysis after transfer from 37 to 0 centigrade for 10min
EMA (eosin-5-maleimide) binding § Measure fluorescence using flow cytometry § Distinct histograms for HS (93 sensitivity / 99% specificity) § Reduced fluorescence in CDAII, cryohydrocytosis and SAO.
Osmotic gradient extacytometry § Laser diffraction sicometer that measures red cell deformability at constant shear stress as a function of suspending osmolality. § Gives distinct curves for HS, HE, HPP, Stomatocytosis and sickle
SDS-PAGE § Allows quantification of membrane proteins § Not necessary for the majority of cases § Recommended when: a) Clinical phenotype is more severe than the red cell morphology b) Morphology more severe than the parents blood film when they are known to have HS c) When the diagnosis is not clear prior to splenectomy
Molecular genetics § Not possible in the UK § Not currently of clinical use
Suggested investigation pathway a) If typical clinical features (splenomegaly) and FH with spherocytes, raised MCHC and retics do not need further investigation b) If diagnosis equivocal then perform either cryohaemolysis test or EMA binding c) If still uncertainty perform SDS-PAGE
Management 1. Folate therapy § Probably only required in severe and some moderate cases (dose 2.5mg od up to age 5 then 5mg od) NB associated with poverty. § Pregnancy § Should measure if not being supplemented
2. Clinic visits § Annual visit for mild to moderate haemolysis § Debatable whether adults with mild disease need follow up § Assess general health, exercise tolerance, growth, spleen size § Information on Parvovirus B19 § Folate status if not being supplemented § US for biliary stones every 3-5 years from age of 5 § Post splenectomy vaccinations / antibiotics § Fasting iron status in non splenectomised – chronic anaemia enhances iron absorption and coinheritance of haemochromatosis gene (C282Y) will lead to severe iron overload
§ Severe require haematological supervision during viral infections
3. Transfusion § Children who are regularly transfused should receive genotyped leukodepleted blood to reduce the risk of alloimmunization.
4. Splenectomy § No evidence that laparoscopic is superior to open Indications a) Severe disease should try and defer until age >6 as risk of sepsis if possible (partial splenectomy can be performed in those <6 in attempt to preserve some splenic function). b) Moderate disease considered (reduced exercise tolerance, metabolic burden from increased marrow turnover) c) In those undergoing cholecystectomy for gall-stones (and cholecystectomy should be considered in those undergoing splenectomy who have gall stones but once the spleen is removed pigment stones do not develop and cholecystectomy may be associated with an increased risk of ca colon) d) Visible jaundice but no stones for cosmetic reasons – difficult group
Post op develop thrombocytosis – often plts > 1000 – prophylactic clexane
5. Post splenectomy care § Risk of pneumococcal bacteraemia and meningitis 280 times normal in one study § Vaccination against haemophilus influenzae B, Meningitis C and Pneumococcus (needs repeating every 5 years) § Prophylactic penicillin V (or erythromycin) standard practice but little evidence of efficacy § Splenectomy card should be issued
Blood film
Hereditary membrane disorders
Hereditary Elliptocytosis
§ 3 main groups 1. Hereditary elliptocytosis 2. Spherocytic HE 3. South East Asian Ovalocytosis
Hereditary Elliptocytosis § AD § Mutations in alpha or beta spectrin or mutations in protein 4.1 or absence of glycophorin C § 1:2500 N Europeans (Also W Africa) § Most patients asymptomatic § Well compensated haemolysis § Homozygotes are more severely affected
Diagnosis § Family history § Elliptical / oval cells on blood film § Anaemia, raised LDH, raised bilirubin, with reduced haptoglobins and negative DAT § Osmotic fragility study normal (compared to hereditary spherocytosis)
Complications § Gall stones § Folate deficiency
Treatment § Folic A 5mg od § Severe cases may need splenectomy
Hereditary pyropoikilocytosis § Often occurs in the same familes as HE § Much more distorted forms – fragmentation occurs at lower temperatures compared to normal cells
Spherocytic HE § Elliptical and spherical ‘sphero-ovalocytes’ in peripheral blood § Haemolysis and increased osmotic fragility distinguish it from common hereditary elliptocytosis § Molecular defect unknown
South-east Asian Ovalocytosis § AD § Asymptomatic of haemolysis § Rigid and hyperstable red cells – tiger cells (double stoma) § 25% or more ovalocytes § SDS-PAGE detects slower migrating Band 3
Stomatocytosis
Overhydrated hereditary stomatocytosis (hydrocytosis) § AD § 1:1,000,000 births § Clinical features identical to HS § Stomatocytes on film (up to 30%) § Decreased MCHC and macrocytosis § Stomatin absent (band 7.2 in SDS-PAGE) § Intracellular Na >60mM
Dehydrated hereditary stomatocytosis (xerocytosis) § AD § 1:10,000 § Heterogenous clinical features § Combined with pseudohyperkalaemia and/or perinatal oedema § Lack red cell deformity § Some stomatocutes and target cells § Increased MCHC § Normal stomatin on SDS-PAGE § Abnormal intracellular [Na] / [K]
Familial pseudohyperkalaemia § AD § Non-haemolytic § Normal red cell morphology § Normal intracellular [Na] § Net loss of intracellular [K] when stored at room temperature (apparently normal cation fluxes at 37C)
Cryohydrocytosis § AD § Rare, mild stomatocytic haemolytic state with hyperbilirubinaemia and gall stones § Red cell lysis when stored at 4C § Diagnosed by measurement of intracellular [Na] and [K]
CDAII (HEMPAS) § AR § Mild and intermittent jaundice and splenomegaly. § Hepatomegaly and gallstones are less frequent § Anaemia, cirrhosis § Secondary siderosis § Bi and multinucleated erythroblasts (usually 15-30%). § Mild to moderate Hb level, normal to mild elevated MCV § Low reticulocyte count § SDS-PAGE shows a more compact and faster migrating band 3 (due to incomplete N-glycosyation) § Diagnosed by electron microscopy of bone marrow
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