Cell membrane biology in haemolytic anaemias: advances in diagnosis and treatment
Submitting InstitutionUniversity College London
Unit of AssessmentClinical Medicine
Summary Impact TypeHealth
Research Subject Area(s)
Biological Sciences: Biochemistry and Cell Biology
Medical and Health Sciences: Cardiorespiratory Medicine and Haematology, Clinical Sciences
Summary of the impact
Research at UCL on human haemolytic anaemias known as the `hereditary
stomatocytoses' has improved diagnosis of these conditions, meaning that
patients now avoid unnecessary and potentially life-threatening
splenectomies, and inappropriate investigation and treatment for raised
potassium levels. Identification of a common single nucleotide
polymorphism that causes apparently normal red blood cells to leak salt
when cooled (as is normal procedure with donated blood) has raised
awareness of this issue in the NHS Blood and Transfusion service, with the
result that individuals with this condition have been identified among
existing donors, and work is underway to develop a screening method to
exclude such individuals from donating blood that cannot be stored safely.
Finally, the research has facilitated diagnosis of the recessive metabolic
disorder phytosterolaemia by blood count, allowing these individuals to be
given appropriate dietary treatment to control their cholesterol levels.
The hereditary stomatocytoses are a class of human haemolytic anaemias in
which the membrane of the red blood cell `leaks' the salt atoms sodium and
potassium. These `leaky' cells have a tendency to swell and burst in the
circulation. Research over the last 20 years by Professor Gordon Stewart
at the UCL Division of Medicine has made novel contributions to the
molecular understanding of these diseases, resulting in advances in
diagnosis and clinical management.
Prior to work by Stewart, patients with stomatocytosis were commonly
diagnosed with a related condition, hereditary spherocytosis. Stewart
identified that a crucial feature in diagnosis is the dependence of the
increased sodium-potassium permeability as temperature falls. This
discovery allowed the clear distinction of a series of phenotypic variants
and discriminated each of these variants from hereditary spherocytosis .
In that condition, removal of the spleen is beneficial; Stewart was the
first to demonstrate that splenectomised patients with hereditary
stomatocytosis, however, have an increased risk of venous thromboembolism
(deep vein thrombosis, DVT), with potentially fatal embolism of clot to
the lungs .
The research also demonstrated that many of these patients were also
being misdiagnosed with hyperkalaemia (high plasma potassium). True
hyperkalaemia is a medical emergency, but in these patients the plasma
potassium was normal in vivo. The high measured potassium was an artefact
of blood sample processing. If a freshly taken blood sample was allowed to
cool towards room temperature prior to separation of red cells from plasma
during routine blood sampling, potassium leaked from the red cells and
gave rise to factitious or `pseudohyperkalaemia'. These patients were at
risk of inappropriate referral to hospital and erroneous emergency
treatment for the raised potassium.
In collaboration with Dr Lesley Bruce (NHS Blood and Transplant) Stewart
showed that these diseases can be caused by mutations in at least three
different genes: SLC4A1, coding for the band 3 anion exchanger ;
RhAG, coding for the so-called rhesus-associated glycoprotein, a
gas channel ; and SLC2A1, coding for the GLUT1
facilitative glucose transporter . Salt-leaky mutations in
GLUT1 cause recognisable haematological-neurological-ophthalmological
In the course of this work, Stewart (in collaboration with Dr David Rees,
King's College London) also showed that the rare hypercholesterolaemic
metabolic condition phytosterolaemia can readily and inexpensively be
diagnosed via a specific haematological presentation . This is
an important diagnosis, because the condition does not respond to statins,
the mainstay of treatment of raised cholesterol.
Among the many families studied by Stewart, that found in Cardiff 
is emerging as important. That family's asymptomatic non-haemolytic
condition is caused by a gene change that is already recognised as a
single nucleotide polymorphism, present at a frequency of 0.1% in the
caucasian population. Red cells from affected donors show a major salt
leak at refrigerator temperatures only, and the cells are unsuitable for
storage in blood transfusion, as the blood is likely to cause true
hyperkalaemia when administered to a recipient.
References to the research
 Bruce LJ, Robinson HC, Guizouarn H, Borgese F, Harrison P, King MJ,
Goede JS, Coles SE, Gore DM, Lutz HU, Ficarella R, Layton DM, Iolascon A,
Ellory JC, Stewart GW. Monovalent cation leaks in human red cells caused
by single amino-acid substitutions in the transport domain of the band 3
chloride-bicarbonate exchanger, AE1. Nat Genet. 2005 Nov;37(11):1258-63. http://dx.doi.org/10.1038/ng1656
 Bruce LJ, Guizouarn H, Burton NM, Gabillat N, Poole J, Flatt JF,
Brady RL, Borgese F, Delaunay J, Stewart GW. The monovalent cation leak in
overhydrated stomatocytic red blood cells results from amino acid
substitutions in the Rh-associated glycoprotein. Blood. 2009 Feb
 Flatt JF, Guizouarn H, Burton NM, Borgese F, Tomlinson RJ, Forsyth
RJ, Baldwin SA, Levinson BE, Quittet P, Aguilar-Martinez P, Delaunay J,
Stewart GW, Bruce LJ. Stomatin-deficient cryohydrocytosis results from
mutations in SLC2A1: a novel form of GLUT1 deficiency syndrome. Blood.
2011 Nov 10;118(19):5267-77. http://dx.doi.org/10.1182/blood-2010-12-326645.
 Bawazir WM, Gevers EF, Flatt JF, Ang AL, Jacobs B, Oren C, Grunewald
S, Dattani M, Bruce LJ, Stewart GW. An infant with pseudohyperkalemia,
hemolysis, and seizures: cation-leaky GLUT1-deficiency syndrome due to a
SLC2A1 mutation. J Clin Endocrinol Metab. 2012 Jun;97(6):E987-93. http://dx.doi.org/10.1210/jc.2012-1399.
 Rees DC, Iolascon A, Carella M, O'marcaigh AS, Kendra JR, Jowitt SN,
Wales JK, Vora A, Makris M, Manning N, Nicolaou A, Fisher J, Mann A,
Machin SJ, Clayton PT, Gasparini P, Stewart GW. Stomatocytic haemolysis
and macrothrombocytopenia (Mediterranean
stomatocytosis/macrothrombocytopenia) is the haematological presentation
of phytosterolaemia. Br J Haematol. 2005 Jul;130(2):297-309. http://dx.doi.org/10.1111/j.1365-2141.2005.05599.x
Details of the impact
The underpinning research described above has improved the diagnosis of
the hereditary stomatocytoses, avoiding potentially harmful splenectomies
in these patients, and inappropriate investigation of artefactual
hyperkalaemia. This work has thus established a standard of care for this
Since 2008, Stewart's team have analysed ~500 blood samples from Europe
and North America, successfully identifying hereditary stomatocytoses in
17 pedigrees. The impact of this on patient care and management has been
paramount, facilitating an accurate diagnosis, enabling counselling and
avoiding splenectomy for at least 30 individuals in the UK. One patient
described the impact of these findings on her family as follows,
contrasting those individuals who underwent splenectomy, with those who
were able to avoid it as a result of Stewart's research:
"Over the years a number of the family have had their spleens removed
either here or in Northern Ireland. Our own mother had a splenectomy and
she died in 2004 with problems which were associated with blood clotting
and Crohn's Disease. My elder sister has had the most problems including
a blockage in a vein leading to the liver after her splenectomy and now
has problems with bleeding from the gullet ... I have an aunt over in
Northern Ireland who had her spleen removed and she had constant
problems with her breathing. Those of us who have not had our spleens
removed (which includes myself and my younger brother) are well with
little problem or complication from DHS [dehydrated hereditary
stomatocytosis]. We have another aunt over in Northern Ireland who did
not have her spleen removed. She had 12 children without any major
problems. We have anaemia, and we need occasional transfusions (not all
of us), but none of the breathing problems or other complications which
other family members have experienced" [a].
A further impact of accurate diagnosis is the correct understanding of
high plasma potassium levels. In about half of the cases, high plasma
potassium levels have been explained on the basis of leaky red cells,
removing the need for repeated urgent hospital attendances for repeat
potassium measurements. A patient described the impact on her family as
"High plasma potassium levels... were a bother to us all as, after any
blood test we would be recalled to hospital as an emergency... One thing
that has changed our lives is the recognition that our habitually very
high plasma potassium levels are due to the abnormality in the red cell
membrane. This new understanding has saved us countless late-night
hospital visits for urgent repeat potassium measurements" [b].
This work has been widely cited and included in standard textbooks on
this subject [c].
As described above, Stewart's research identified a common single
nucleotide polymorphism (SNP) in the Caucasian population (`Cardiff' [ref
7, above]) which causes a milder non-haemolytic anaemia. When blood from
these individuals is cooled, the effect on the red cells is substantial,
resulting in major potassium leaks. As blood taken from donors is
routinely cooled, this raises the potassium in the donated blood from
these individuals. When administered to patients this risks unexpected and
potentially harmful true hyperkalaemia and the transfusion of damaged or
partly lysed red cells, making the transfusion less efficacious. As a
result of work by Stewart and Bruce, blood from such donors has been
identified in the existing pool of donated blood at the National Blood
Transfusion Service (NBTS). In reaction to this finding, the NBTS is now
in the process of implementing a screening strategy for this polymorphism
on all donated blood in order to avoid this complication. This will have
an impact at a national and potentially international level. The work is
the subject of a current patent application [d].
Finally, the underpinning research has improved the diagnosis of the rare
metabolic condition phytosterolaemia (sitosterolaemia), in which
absorption of both dietary cholesterol and plant sterols is unrestricted.
This can now be identified quickly by a blood count rather than mass
spectroscopy of lipids, enabling diagnosis of the condition to take place
via routine haematology, without expensive mass spectrometry. As a result
of this work, a number of individuals and families have been identified in
the UK (five families in Stewart's study, [ref 6, above]), in China [e]
and in the US [f]. The impact of the correct diagnosis is to
enable effective treatment of the condition. Patients with the condition
do not respond to the HMG CoA reductase inhibitors (statins), which are
currently the mainstay in the treatment of hypercholesterolaemia.
Individuals instead must be treated with a diet low in cholesterol as well
as plant sterols including sitosterol. With correct dietary treatment,
however, cholesterol remains low [g].
Sources to corroborate the impact
[a] Testimony from Patient CM. Copy available on request.
[b] Testimony from Patient SB. Copy available on request.
[c] For example: Handin R, Lux S, Stossel T, editors. Blood: Principles
and Practice of Hematology. Philadelphia: JB Lippincott; 2003. See chapter
on Disorders of the red blood cell membrane, p.1709-1858. Copy available
[d] Statement provided by Senior Research Scientist, Bristol Institute
for Transfusion Sciences, NHS Blood and Transplant, corroborating effects
of cooling on blood from donors; the identification of such individuals
among existing donors, and the ongoing development work. Copy available on
[e] Wang G, Wang Z, Liang J, Cao L, Bai X, Ruan C. A phytosterolemia
patient presenting exclusively with macrothrombocytopenia and stomatocytic
hemolysis. Acta Haematol. 2011;126(2):95-8. http://dx.doi.org/10.1159/000327248.
[f] Neff AT. Sitosterolemia's stomatocytosis and macrothrombocytopenia.
Blood. 2012 Nov 22;120(22):4283. http://dx.doi.org/10.1182/blood-2012-06-429449
[g] Testimony from patient PP. Copy available on request.