Margaret C. Chetty

A variant of hereditary stomatocytosis with marked pseudohyperkalaemia

A family with an unusual form of hereditary stomatocytosis is described. The affected members showed a mild, dominantly‐inherited, haemolytic anaemia with intracellular Na and K levels of 41–48 and 44–53 mmol/(l cells) respectively. This anaemia was associated with marked ‘pseudohyperkalaemia’: that is, loss of K from red cells on storage at room temperature. At 37°C, ‘leak’ tracer flux rates (assessed as the ouabain + bumetanide‐resistant K fluxes) showed a roughly 5‐fold acceleration compared to normal, and an abnormal temperature dependence with a shallow slope between 37 and 20°C (mean Q10 (ratio of reaction rates at temperature T and T − 10) over this interval, 1.6; normal 2.2). The pseudohyperkalaemia could be attributed to the disparity between pump and leak at 20°C. This is an identical mechanism to that previously shown for the haematologically trivial condition, ‘familial pseudohyperkalaemia’. No protein or lipid abnormality was found in the membrane of these cells.

Four new cases of stomatin-deficient hereditary stomatocytosis syndrome: association of the stomatin-deficient cryohydrocytosis variant with neurological dysfunction

This report concerns congenitally Na+–K+ leaky red cells of the ‘hereditary stomatocytosis’ class. Three new isolated cases and one new pedigree are described, and one previously reported case is expanded. In all cases, Western blotting of red cell membranes revealed a deficiency in the 32 kDa membrane protein, stomatin. All showed pronounced cation leaks at 37°C with markedly abnormal intracellular Na+ and K+ concentrations, like all other such stomatin‐deficient cases. Consistent with recent findings in two previously described British pedigrees, immunocytochemistry demonstrated that the deficiency of stomatin was not complete. On typical blood films, some red cells showed positive stomatin immunoreactivity, while most were negative, although in one case only a minority were negative. All platelets and neutrophils were stomatin positive. The cases differed markedly between themselves with regard to the temperature dependence of the passive leak to K+. Three showed a simple monotonic temperature dependence, while two showed a minimum at around 20–25°C, such that the cells were extremely leaky at 0°C, giving the phenotype known as ‘cryohydrocytosis’. These patients are the only two known cases of stomatin‐deficient cryohydrocytosis. Both showed a congenital syndrome of mental retardation, seizures, cataracts and massive hepatosplenomegaly, probably defining a new haemato‐neurological syndrome.

Two British families with variants of the 'cryohydrocytosis' form of hereditary stomatocytosis

We describe two British families with similar, dominantly‐inherited, temperature‐related variants of hereditary stomatocytosis, consistent with the original description of ‘cryohydrocytosis’. The cells show a 5–6‐fold increase in passive permeability at 37°C with abnormal intracellular Na and K levels at 15–20 and 60–65 mmol/(l cells) respectively. Marked temperature effects were evident: lysis of red cells on storage in the cold was blatant and when whole heparinized blood was stored at room temperature, K accumulated in the plasma, producing ‘pseudohyperkalaemia’. Studies of the temperature dependence of passive permeability showed that the minimum in the passive permeability, which is seen in normal cells at 8–10°C, was shifted up to 23°C in these abnormal cells, such that the permeability at 0°C exceeded that at 37°C. The abnormal temperature dependence in these genetically abnormal red cells strongly resembles that seen in normal cells when suspended in media in which either Na or Cl has been replaced by an organic cation or anion: it could be said these cells had a genetic mutation that somehow rendered the cell resistant to the stabilizing action of NaCl at low temperatures.

Two further British families with the 'cryohydrocytosis' form of hereditary stomatocytosis.

We describe two families with the ‘cryohydrocytosis’ form of stomatocytosis. Both show a mild stomatocytic anaemia with Hb levels of 12–16 g/dl and reticulocyte counts of 4·3–24%, with very marked autohaemolysis at refrigerator temperatures and pseudohyperkalaemia as a result of loss of K from red cells on storage at room temperature. The ouabain + bumetanide‐insensitive ‘passive leak’ K influx showed a ‘U’‐shaped temperature dependence, with a minimum at 23°C. In one family, there was consistent variation in haematological severity within the pedigree. In the other, the parents of the proposita were normal, but all three of her children were affected, consistent with a new mutation of a dominant condition. Cold storage of the red cells led to a very marked increase in osmotic fragility and macrospherocytosis, explaining why a diagnosis of ‘hereditary spherocytosis’ can easily be reached in these pedigrees.

Familial pseudohyperkalaemia Chiswick: a novel congenital thermotropic variant of K and Na transport across the human red cell membrane

Two families with inherited abnormalities in Na and K transport across the red cell membrane are described. Both presented with ‘pseudohyperkalaemia’ as a result of loss of K from the red cells on storage at room temperature. Routine haematology was essentially normal, except for macrocytosis in one family. Studies of the temperature dependence of the passive leak to K showed a novel shoulder pattern with a minimum at 25°C, a maximum at 10°C, followed by a further fall. As in other cases of red cell‐based pseudohyperkalaemia, the abnormal temperature dependence of this ‘leak’ flux could be held to account for the loss of K from the cells at room temperature. These cases represent a novel variant of the temperature dependence of the passive leak of K and Na across the red cell membrane, and can be classified as a mild, non‐haemolytic form of the group known as the hereditary stomatocytosis and allied disorders’.

Familial pseudohyperkalaemia Cardiff: a mild version of cryohydrocytosis.

Summary.  We have investigated a Welsh pedigree showing the ‘familial pseudohyperkalaemia’ phenotype of dominantly inherited, red‐cell‐based, temperature‐dependent pseudohyperkalaemia associated with normal haematology. The ‘passive leak’ to K across the membrane of these abnormal red cells showed a ‘U‐shaped’ temperature dependence, with a minimum at about 23°C, qualitatively similar to that seen in the frankly haemolytic ‘cryohydrocytosis’ variant of the hereditary stomatocytosis group. Like three previous pedigrees with cryohydrocytosis, these patients show an excess of ether lipids in the membrane. However, these patients differ from other ‘familial pseudohyperkalaemia’ pedigrees, in which the leak showed different temperature profiles.

Cold storage of ‘cryohydrocytosis’ red cells: the osmotic susceptibility of the cold-stored erythrocyte

Summary. ‘Cryohydrocytosis’ is an unusual human haemolytic anaemia of the ‘hereditary stomatocytosis’ group, in which the red cell membrane is abnormally permeable to Na and K+ at both body and (even more prominently) refrigerator temperatures. If whole cryohydrocytosis blood is anticoagulated in heparin or EDTA and stored on ice overnight, about 50% of the cells will lyse. Citrate phosphate dextrose adenine (CPDa) anticoagulant, empirically verified as an optimal anticoagulant for storage of normal blood before transfusion, very markedly ameliorated this overnight lysis, suggesting that these cells might form an informative model in which cold storage of the red cell could be studied in a short time scale. Accordingly, we conducted studies of ion flux, cell swelling and lysis in different media used historically for blood preservation and compared the experimental data with an ‘integrated red cell model’, which seeks mathematically to model the osmotic behaviour of red cells under different conditions. Upon experiment, lysis in these cells was reduced by additives that could be regarded as impermeant extracellular solutes (citrate, mannitol) and by low pH, but not by those agents that are regarded as protecting the cell against energy depletion or oxidation (adenine, glucose, nicotinic acid). The protective effects of these extracellular additives were all reproduced by the computer simulation, confirming the validity of this model, although the effect of pH could be simulated only semi‐quantitatively, possibly because Na+ permeability itself depends on pH.

ATP-dependent vesiculation in red cell membranes from different hereditary stomatocytosis variants

Summary. The hereditary stomatocytoses are a group of dominant haemolytic anaemias that show two main features: invaginated, ‘stomatocytic’ morphology; and a membrane leak to the univalent cations Na and K. A patient with the most severe variant of these conditions was reported to show a defect in an in vitro process of ATP‐dependent endocytic vesiculation (ADEV), which is found in normal red cells. We have examined this endocytosis process in 11 leaky red cell pedigrees available to us in the UK. ADEV in broken membranes was absent only in the two most severely affected, ‘overhydrated’ pedigrees studied, both of which showed a deficiency in the membrane raft protein, stomatin. The process was present, although typically diminished by about 10–20% compared with normal red cells, in all others. The cross‐linker dimethyl adipimate (DMA), which could correct the cation leak in some of these patients, also corrected the ADEV defect in the same patients. In those patients in whom DMA had no effect on the ion leak, ADEV was not absent. In normal cells, this process of vesiculation was inhibited by inhibitors of membrane ‘raft’ function, by an antistomatin antibody and by vanadate and N‐ethyl maleimide, but not by inhibitors of a number of kinases. These data highlight the heterogeneity of these conditions. A mechanism is discussed by which a defect in raft‐based endocytosis could lead to the exaggerated surface exposure of an ion channel, which could then function constitutively, i.e. ‘leak’.

Four pedigrees of the cation-leaky hereditary stomatocytosis class presenting with pseudohyperkalaemia. Novel profile of temperature dependence of Na +-K + leak in a xerocytic form

We report four pedigrees of the group of Na+–K+‐leaky red cell disorders of the ‘hereditary stomatocytosis’ class. Each showed pseudohyperkalaemia because of temperature‐dependent loss of K+ from red cells on storage of whole blood at room temperature. All pedigrees showed an abnormality in the temperature dependence of the ‘passive leak’ of the membrane to K+. Two pedigrees, both of which showed a compensated haemolytic state with dehydrated red cells and target cells on the blood film, showed a novel pattern, in which the profile was flat between 37°C and about 32°C then dropped as the temperature was reduced to zero. The third showed the ‘shallow slope’ profile, with stomatocytes on the blood film and very markedly abnormal intracellular Na+ and K+ levels. Minimal haemolysis was present. The fourth pedigree, of Asian origin, showed the shoulder pattern (minimum at 32°C, maximum at 12°C) with essentially normal haematology. Both of these latter two forms have previously been seen in other pedigrees. The first variant represents a novel kind of temperature dependence of the passive leak found in these pedigrees presenting with pseudohyperkalaemia.

Dehydrated hereditary stomatocytosis is associated with neonatal hepatitis

Dehydrated hereditary stomatocytosis (DHSt) is an inherited haemolytic anaemia associated with increased red cell membrane permeability to Na+ and K+. It is increasingly recognized that a syndrome of self‐limiting perinatal ascites can accompany the haemolysis. The cause of the perinatal ascites is unknown, and it has been argued that this could be due to cardiovascular, hepatic or lymphatic problems. We describe the case of a 16‐year‐old girl who presented neonatally with abnormal liver function tests and ascites. She was extensively investigated at that time. A liver biopsy showed hepatitis and fatty changes. Her ascites resolved within 6 months. At the age of 15 years, she developed an episode of acute haemolysis and was re‐investigated. A diagnosis of DHSt was made. Pseudohyperkalaemia, due to ex vivo loss of K+ from red cells, was present. This study confirms the previously noted association of DHSt, pseudohyperkalaemia and perinatal ascites, and suggests that the latter is of predominantly hepatic origin.