The clinical phenotypes of hereditary membrane disorders

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HS may present at birth. The functions of the spleen become mature only after birth, so severe anaemia in utero is rare. Erythropoiesis is highly active before birth but enters a phase of reduced activity in the neonatal period. Severe anaemia, developing over 5-30 days post delivery and requiring transfusion, may result from this double physiological development of reduced production and increased destruction, but the anaemia may greatly reduce during the first year of life as compensatory erythropoiesis develops. Decisions about splenectomy do not need to be taken during this time.

Molecular pathology

About 60% of HS cases result from a defect in the ankyrin-spectrin complex, with both a and P (genes SPTA1, SPTB) of the spectrin dimer or ankyrin (ANK1) being implicated in different genetic types (Table 8.2). A further 25% involve deficiency in band 3, the anion channel. In the remainder of the dominantly inherited HS families there is a deficiency of protein 4.2 or no abnormality has yet been identified. Deficiency of protein 4.2 is particularly common in Japanese families with HS (Table 8.2). These defects involving spectrin-ankyrin-band 3 interactions affect the 'vertical interactions' described by Jiri Palek and colleagues.

It will be appreciated that the genetic defect that produces the dominant form of HS affects only one of a pair of genes. The presence of one abnormal protein influences the proteinprotein interactions of these complexes, leading to partial deficiency of several proteins, even if they are not genetically disturbed. This is particularly true of spectrin. Complete loss of complex function is probably not viable, so homozygous children are not found. Double heterozygosity or inheritance of separate membrane defects does occur and is associated with usually severe haemolytic anaemia. Other recessive forms of HS are also seen in which the inheritance of one defective gene involving the spectrin subunit produces no clinical effect, whereas homozy-gosity produces a severe defect and haemolysis.

Laboratory diagnosis

The typical findings of extravascular haemolysis are present in HS (Table 8.1). The diagnosis is usually made on the basis of morphology of the blood, backed up where possible with a family history. The mean cell haemoglobin concentration (MCHC) is often increased above 35 g/L in HS, but the presence of macro-cytic reticulocytes usually results in a low normal mean cell volume (MCV) rather than true microcytosis. These changes result not only from the reduction of surface area-volume ratio but also from the slight dehydration of HS cells. A number of variants of the typical HS features have been described, usually the more severe forms that may have denser and less perfectly round cells in the peripheral blood. In infancy, the morphology may be more difficult to interpret. The effect of immature splenic function and the macrocytosis and anisocytosis of infancy combine with the HS phenotype to produce red cell appearances not typical of the developing HS. Family studies may assist in the diagnosis.

Osmotic fragility test

The osmotic fragility test measures the sensitivity of red cells to lysis in vitro to swelling caused by incubation in increasingly hypotonic saline solutions. Red cells are able to swell with increasing volume until the pressure disrupts the unstretch-able membrane and lysis occurs. In normal red cells with the biconcave disc shape, 50% lysis occurs when the saline solution reaches about 0.5% sodium chloride. The more rigid HS cells have less ability to swell and so lyse at higher concentrations - a right-shifted osmotic fragility curve. One of two patterns may be seen in HS, a generally right-shifted curve, which is the more common finding, and one where there appears to be a 'tail' of lysis-sensitive cells. Incubation of blood for 24 h at 37°C accentuates the fragility (Figure 8.5).

The acidified glycerol lysis test (AGLT) uses glycerol to slow the entry of water into the cells in vitro. The time taken for lysis to occur is a function of the osmotic resistance of the cells. HS cells lyse more rapidly than normal cells. The test is easier to perform than the osmotic fragility test.

Osmotic Fragility Curves

Figure 8.5 Osmotic fragility test in hereditary spherocytosis. Osmotic fragility is increased in the microspherocytes (right shift), but there is also a small population of resistant cells due to increased reticulocytes. After splenectomy, the microspherocytes remain but the proportion of reticulocytes is reduced to normal values and the resistant cells are not seen.

Figure 8.5 Osmotic fragility test in hereditary spherocytosis. Osmotic fragility is increased in the microspherocytes (right shift), but there is also a small population of resistant cells due to increased reticulocytes. After splenectomy, the microspherocytes remain but the proportion of reticulocytes is reduced to normal values and the resistant cells are not seen.

Autohaemolysis test

The autohaemolysis test examined the ability of red cells to withstand metabolic deprivation by incubation in vitro for 24 h with and without the addition of glucose. It is a crude, insensitive test which has generally been abandoned.

Identification of protein abnormalities or gene defects

Methods that identify the defective gene or its product are the most specific for membrane defects but are beyond the scope of most routine haematology laboratories. The original identification of membrane proteins using sodium dodecyl sulphate-solubilized polyacrylamide gel (SDS-PAGE) electrophoresis has led to the classification according to the banding system indicated in Table 8.2. The identification of specific genetic abnormalities may be important in compound haemolytic syndromes but requires specialist laboratories.

One screening test for HS makes use of the binding of eosin-labelled maleimide, in the form of eosin-5 maleimide, to lysin 430 in band 3 and cysteine molecules in surface proteins, particularly rhesus blood groups. In about 25% of HS patients there is a deficiency of band 3 and a loss of surface proteins caused by the instability of the lipid bilayer. HS red cells bind eosin-5 malei-mide less than normal cells, by about 25-30%. Even when the main defect is not in band 3, there may be sufficient loss of eosin-5 maleimide binding to indicate HS. The screening test has to be used in conjunction with morphology because South-East Asian ovalocytosis, congenital dyserythropoietic anaemia type II and cryohydrocytosis also give reduced fluorescence.

Clinical course and complications

In most kindred, the course of the disorder is similar in affected members although, as with most inherited defects, there is some variable penetrance and it is not rare to find a very mildly affected parent with more severely affected offspring.

As with all congenital haemolytic anaemias, the anaemia may be aggravated by environmental factors. This may be consequent on an increase in the red cell destruction or a decrease in production. Increased jaundice may occur during viral infections or bacterial sepsis, the anaemia also being aggravated by a decrease in production consequence on the effects of the acute-phase response or the inhibition of erythropoiesis by interferon gamma (IFN-y).

Primary infection with parvovirus 19 produces a specific and marked inhibition of erythropoiesis, often characterized as an aplastic crisis. In patients with a shortened red cell survival, severe anaemia may be produced by the inhibition, which lasts for some 4-7 days. In normal individuals with a red cell lifespan of 120 days, such an inhibition produces no clinical effect. The anaemia associated with parvovirus infection in HS may require urgent transfusion. The diagnosis is made by finding absent parvovirus antibodies with subsequent appearance of IgM antibodies. The presence of IgG antibodies at the time of the anaemia excludes the diagnosis.

Acute anaemia due to splenic sequestration is a relatively uncommon complication of HS in childhood. The pathogenesis is probably increased splenic size and activity leading to increased trapping of HS cells within the spleen. This complication may also require urgent transfusion.

Malnutrition may increase anaemia because of folate deficiency but also from increased jaundice through the effect of low-calorie input on unconjugated bilirubin levels in the blood.

The anaemia of pregnancy may aggravate a haemolytic anaemia and hence bring the condition to the attention of clinicians and patients. Classical HS is not a risk to mother or child in pregnancy.

Gallstones are an expected complication in HS as in other chronic haemolytic anaemias. Silent gallstones require no intervention. Recurrent cholecystitis or biliary colic may require cholecystectomy accompanied by splenectomy (see below). Leg ulcers are a rare but well-recognized complication of HS, as with other chronic haemolytic anaemias. Extramedullary haemopoietic masses, usually paravertebral, occur rarely in more severe HS.

Management

Patients with well-compensated haemolysis and no transfusion requirements need no treatment other than reassurance and folic acid supplements (e.g. 400 |ig daily or 5 mg weekly). For people with a well-balanced and adequate diet, folic acid supplements are probably unnecessary, but custom dictates the practice should be continued. Radiolucent gallstones, if detected by chance on ultrasound, are common and need no treatment unless complications arise. Gallstones without recurrent inflammation are not a risk factor for a carcinoma of the gall bladder. Recurrent cholecystitis or obstruction would be an indication for cholecystectomy, which would also be an indication for splenectomy.

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