CME Bulletin Haematology 2000; 3(2):39-41
Review Articles
Diagnosis of red cell membrane disorders
Abstract
Hereditary spherocytosis and hereditary elliptocytosis
are the common inherited haemolytic anaemias known to be
associated with defective red cell cytoskeletal proteins.
Studies of patients’ red cells using biochemical and
molecular biology techniques have provided an insight in the
organisation of erythrocyte structural proteins and the
molecular basis of these disorders. In contrast, the current
laboratory tests for screening membrane disorders have been
in use for over 40 years. This article presents a brief
overview on the first- and second-generation rapid
laboratory tests for detecting red cell membrane disorders.
The second-generation assays should provide a greater degree
of confidence in discriminating between HS and other types
of haemolytic anaemia in the shortest possible time as this
becomes crucial for paediatric cases.
Keywords
Hereditary spherocytosis, hereditary elliptocytosis,
hereditary stomatocytosis, red cell cytoskeleton, inherited
haemolytic anaemia.
The diagnosis of moderate and severe forms of
hereditary spherocytosis (HS), hereditary
elliptocytosis (HE), and hereditary pyropoikilocytosis
(HPP, a more severe form of HE) can be made from
a set of parameters: red cell indices, reticulocytosis,
blood film examination, family history,
hyperbilirubinemia, low or no haptoglobin, ethnic
origin, and the exclusion of other haemolytic
conditions (namely, enzymopathy, haemoglo-
binopathy, and autoimmune haemolytic anaemia).
In many cases typical red cell morphology with or
without the family history are sufficient to establish
the diagnosis. In the others, particularly for new
cases without a family history, measuring osmotic
fragility (OF) of red cells or using one of the second
generation tests (vide infra) would be adequate as
confirmatory diagnosis of HS without resorting to
doing SDS-polyacrylamide gel electrophoresis
(SDS-PAGE) for the identification of specific
membrane defect(s).1 In some cases of HE, spectrin
analysis may be required when the blood film
appears equivocal; i.e., only few elliptocytes or
poikilocytes.2
The first-generation assays, often used to
differentiate red cell membrane disorders from other
types of haemolytic anaemia, include osmotic
39
M-J. King
fragility, acid glycerol lysis, and autohaemolysis tests
which measure the extent of or the rate of red cell
lysis over a period of time, several minutes to 24
hours, in various incubation media. Typical HS is
indicated by an increase of both MCHC and
haemoglobin distribution width (as determined by a
laser type cell counter), and confirmed by an
increased osmotic fragility. The main drawback of
the OF test is a lack of sensitivity. The OF test may
be affected by reticulocytes, which are more resistant
to lysis than normal red cells.; indeed only 66% of
the non-splenectomised HS patients were found to
have an increased osmotic fragility.3 Moreover,
increased red cell fragility can be found in immune
mediated and other haemolytic conditions. The
acidified glycerol lysis test has a higher detection rate
in asymptomatic relatives of known HS individuals
than the OF test. However, positive results are also
obtained for samples from pregnancy and renal
failure. The autohaemolysis test has limited
diagnostic value as correction of haemolysis by
added glucose indicates a possible “normal”
glycolytic pathway. An intrinsic red cell defect is
indicated when added glucose does not correct
haemolysis during incubation. In conclusion, these
laboratory tests for HS can be inf luenced by many
factors unrelated to the red cell cytoskeletal defect
which give rise to poor specificity and sensitivity.
The second generation assays, developed in
particular for their specificity for detecting HS red
cells, include hypertonic cryohemolysis test4 and the
f low cytometric method5 using a f luorescent dye
eosin-5-maleimide (EMA). These assays exploit the
physical properties of red cell membranes rather than
the surface area-to-volume ratio of spherocytic red
May-Jean King PhD
cells. The cryohemolysis test measures cell lysis after International Blood Group
lowering the incubation temperature of red cells Reference Laboratory
from 37oC to 0oC in a hypertonic medium. Its Bristol
specificity in the diagnosis of HS is 86%. In the dye
binding method, EMA binds covalently to Lys-430 Correspondence:
May-Jean King PhD
in the extracellular domain of band 3 on intact red International Blood Group
cells.6 A reduction in f luorescence intensity was Reference Laboratory
obtained for red cells from 96 out of 98 HS patients Southmead Road
while all of them have quantitative protein Bristol, BS10 5ND
deficiencies detected by SDS-PAGE. The dye test UK
e-mail:
has a sensitivity of 92.7% and a specificity of 99.1%
may-jean.king@ nbs.nhs.uk
for HS.5 Moreover, the dye method is not affected by Tel: 0117 991 2111
reticulocytosis, microcytosis, and splenectomy. Fax: 0117 959 1660
40 CME Bulletin Haematology 2000; 3(2):39-41

Diagnosis of red cell membrane disorders

Despite improved sensitivity and specificity, both
techniques are capable of producing “positive” results with
certain rare red cell abnormality. Reduced mean channel
f luorescence readings were obtained after incubation of
EMA with red cells from patients with congenital
dyserythropoiesis type II (CDAII known to have
anomalous band 3 clustering) and with Southeast Asian
ovalocytosis (carrying rigid band 3 molecules). The
cryohemolysis test could detect Melanesian type
elliptocytosis and CDAII.7
Identification of the specific red cell membrane
deficiency or qualitative abnormality always requires SDS-
PAGE.1 This is not necessary in straightforward cases but
it is indicated in more complex cases, in those with an
atypical presentation and in families with a heterogeneous
phenotype, particularly those with an unexpected severe
phenotype (Table 1). The diagnosis of HS is based on
protein phenotype with a reduction of spectrin, ankyrin,
protein 4.2 or band 3 (Figure 1). Spectrin variants weaken
tetramer formation in the red cell cytoskeleton of a
majority of non-Caucasian HE and HPP patients. Protein
4.1 deficiency is prevalent among Caucasians (Figure 2)
with HE (one case involved a Chinese family). Thus,
knowing the protein phenotype of a patient can be helpful
not only in the understanding of the varying clinical
presentations but it is also a prerequisite if defective gene
Indications for SDS-PAGE for the diagnosis
of membrane abnormalities
If the clinical phenotype is more (or less) severe than predicted
based on red cell morphology
In cases where the red cell morphology of the patient is more
severe than predicted from the parental blood films. (A modulating
effect due to low expressed allele** inherited in trans to the patho-
logical allele is suspected in the proband. In HE, compound het-
erozygosity is also a possibility.HPP is both a qualitative and a
quantitative abnormality in association with spectrin.)
In difficult cases of haemolytic anaemia where the red cell mor-
phology does not reveal the specific diagnoses- e.g. neonates,*
transfusion-dependent cases prior to splenectomy if the diagnosis is
uncertain
Table 1. This applies after the exclusion of enzymopathy,
haemoglobinopathy, iron deficiency (in the case of HE), Coombs
negative, and a positive result with one of the second generation
screening tests (in the case of HS).
*Neonatal HS- Delhommeau et al. Blood 2000; 95: 393-397. A
transient elliptopoikilocytosis is occasionally seen at birth, producing
severe haemolytic anaemia, elliptocytes, marked red cell budding,
fragmentation, and poikilocytosis. The infant can become transfusion
dependent. However, in the second year, haemolysis and
poikilocytosis decline. The proband does not require any transfusion
and presents a clinical picture of compensated mild HE similar to that
of the affected parent(s).
**Low expressed alleles in HS and HE: Dhermy, 2000 in Human
Blood Cells ed. MJ King pp 229-250. Imperial College Press,
London.

Figure 1. Schematic presentation of erythrocyte cytoskeleton- structural stabilisation through membrane protein interactions. The α spectrin
associates side-by-side in an anti-parallel manner with β spectrin to form heterodimers. This pairing process is initiated at the nucleation site.
The αLELY allele (previously known as αV/41 polymorphism) has an effect on this nucleation site (see Dhermy, 2000).
Dhermy D. (2000) Spectrin polymorphisms and low expressed spectrin alleles. In: Human Blood Cells, consequences of genetic
polymorphisms and variations. (ed M-J King) pp 229-250. Imperial College Press. London
CME Bulletin Haematology 2000; 3(2):39-41
Figure 2. Blood film of sphero-elliptocytosis (thanks to Dr. Jim
Murray, Selly Oak Hospital, Birmingham). Patient: female, aged
64yrs, presented with shortness of breath on exertion, and routine
blood count showed a microcytic anaemia and a thrombocytosis. The
blood film shows post-splenectomy change as well as spherocytes,
elliptocytes, and bizarre poikilocytes. Osmotic fragility was increased
in a pattern typical of congenital spherocytosis. Serum bilirubin was
normal. Her red cells were found to have a truncated protein 4.1,
which was demonstrated by immunoblotting of erythrocyte
membrane proteins using anti-protein 4.1 after SDS-PAGE. One of
her daughters had jaundice in childhood, and was also found to have
truncated protein 4.1.
analysis is required because HS and HE/HPP are not single
gene disorders. Although SDS-PAGE provides a precise
diagnosis of HS and HE/HPP, the analysis takes 7-10 days
to complete. Furthermore, this technique is not routinely
available in haematology laboratory.
Finally, a rare group of red cell membrane disorders is
that caused by abnormal membrane permeability to
Na+/K+ cations with varying degrees of stomatocytosis
(Figure 3).8,9 This includes hereditary overhydrated
stomatocytosis (also called hydrocytosis), hereditary
dehydrated stomatocytosis (or xerocytosis) and two rare
variants, pseudohyperkalaemia10 and cryohydrocytosis.11
Patients with xerocytosis have little or no anaemia; and
affected kindreds may have pseudohyperkalaemia and/or
References:
1. Tse WT & Lux SE; Red blood cell membrane disorders. Br J Haematol
1999; 104: 2-13.
2. Feo C. & Dhermy D.; Confirming or ruling out hereditary elliptocytosis.
Acta Haemat. 1988; 79: 116.
3. Cynober T, Mohandas N & Techernia G; Red cell abnormalities in
hereditary spherocytosis: relevance to diagnosis and understanding of the
variable expression of clinical severity. J Lab Clin Med 1996; 128: 259-269.
4. Streichman S, Gesheidt Y & Tatarsky I; Hypertonic cryohemolysis: a
diagnostic test for hereditary spherocytosis. Amer J Hematol 1990; 35:
104-109.
5. King M-J, Behrens J, Rogers C et al; Rapid f low cytometric test for
haemolytic anaemia associated with abnormal red cell membrane
cytoskeleton. Br J Haematol 1999; 105: 98. Manuscript is in press (Br J
Haematol).
6. Cobb CE & Beth AH ; Identification of the eosinyl-5-maleimide reaction
site on the human erythrocyte anion-exchange protein: overlap with the
reaction sites of other chemical probes. Biochemistry 1990; 29: 8283-8290.
7. Streichman S & Gescheidt Y; Cryohemolysis for the detection of
41
Diagnosis of red cell membrane disorders
Figure 3. Blood film of patient with hereditary stomatocytosis
(thanks to Dr. Gordon Stewart, University College Hospital,
London).
perinatal oedema.8 However, patients with hydrocytosis
have mild-moderate haemolytic anaemia and may initially
be thought to have atypical HS. A definitive diagnosis for
stomatocytosis is essential due to an increased risk of post-
splenectomy thromboembolic disease.12 In hydrocytosis
there is a marked increase in Na+ f lux, stomatocytic and
target cells on the blood film, a reduced MCHC,
macrocytosis (MCV ranges from 100-110 f l) and increased
OF. In xerocytosis there is loss of intracellular K+, red cell
dehydration, a high MCHC, target cells and bizarre red
cells on the film and a reduced OF, distinguishing it from
hydrocytosis.8,13 Determination of intracellular Na+/K+
content and monovalent cation transport are confirmatory
tests for stomatocytosis.
hereditary spherocytosis: correlation studies with osmotic fragility and
autohemolysis. Amer J Hematol 1998; 58: 206-212.
8. Delaunay J., Stewart G & Iolascon A.; Hereditary dehydrated and
overhydrated stomatocytosis: recent advances. Curr. Opin. Hematol. 1999;
6: 110-114.
9. Kanzaki A & Yawata Y; Hereditary stomatocytosis: phenotypical
expression of sodium transport and band 7 peptides in 44 cases. Br J
Haematol 1992; 82: 133-141.
10. Coles SE, Ho MM., Chetty MC et al; A variant of hereditary
stomatocytosis with marked pseudohyperkalaemia. Br J Haematol 1999;
104: 275-283.
11. Coles SE, Chetty MC, Ho MM, et al; Two British families with variants
of the “cryohydrocytosis” form of hereditary stomatocytosis. Br J Haematol
1999; 105: 1055-1065.
12. Stewart GW, Amess JAL, Eber SW et al; Thromboembolic disease after
splenectomy for hereditary stomatocytosis. Br J Haematol 1996; 93: 303-310.
13. Nolan GR; Hereditary xerocytosis: a case history and review of the
literature. Pathology 1984; 16: 151-154.