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Chapter 45 Red Blood Cell Membrane Disorders 639

spectrin: In normal RBCs, spectrin is denatured and RBCs fragment patients the percentage of elliptocytes is not greater than 5%, although
at 50°C. HPP RBCs fragment and their spectrin denatures at 41°C. in earlier reports it was listed as high as 15%. Previous diagnostic
However, the diagnostic value of this test is limited, because thermal criteria of HE, based on the percentage of elliptocytes, such as 25%,
instability of RBCs is also noted in HE RBCs containing mutant 33%, or 40%, and their axial ratio, do not appear useful. The most
spectrin. In contrast, an occasional patient with otherwise typical reliable differentiation of HE from the other conditions mentioned
HPP may have normal thermal stability of RBCs and spectrin. RBCs is based on a positive family history rather than on the percentage of
in common HE have unstable membranes and membrane skeletons elliptocytes.
when subjected to shear stress.
Therapy and Prognosis
Electrophoretic Separation of Solubilized
Membrane Proteins As in the case of HS, RBCs from patients with more severe forms of
HE are retained by the spleen, producing a marked engorgement of
In HE and HPP, SDS-PAGE can reveal proteins of abnormal mobil- splenic pulp. Consequently, patients with symptomatic hemolysis
ity, the origin of which can be subsequently identified by Western benefit from splenectomy. This procedure is virtually never indicated
blotting (e.g., truncated α- or β-spectrins in HE and HPP, or elon- in heterozygotes with autosomal dominant HE because most do not
gated or truncated forms of the 4.1R protein, and a partial or, rarely, have clinically significant hemolytic anemia. If hemolysis is still active
complete deficiency of the 4.1R protein in HE). In HPP, SDS-PAGE after splenectomy, folate should be administered daily. Recommenda-
reveals a partial deficiency of spectrin, as indicated by a decreased tions for antibiotic prophylaxis, immunizations, and monitoring for
spectrin/band 3 ratio. Spectrin deficiency, in conjunction with an intercurrent illnesses are similar to those noted earlier for HS patients
elliptocytogenic spectrin mutation affecting the spectrin heterodimer before and after splenectomy. Serial interval ultrasonographic inves-
contact, is invariably found in cases of HPP. tigations to detect gallstones should be performed in patients with
significant hemolysis.
Nondenaturing Gel Electrophoresis of Low-Ionic-
Strength Spectrin Extract Spherocytic Elliptocytosis

Analysis of the ratio of tetrameric and dimeric spectrin in the low- Spherocytic elliptocytosis, which shares features of HS and HE, has
ionic-strength extracts reveals the most common functional abnor- been designated spherocytic HE, HE with spherocytosis, or heredi-
mality in HE (i.e., weakened self-association of spectrin heterodimers tary hemolytic ovalocytosis. The diagnosis is based on the simultane-
into tetramers). Because the spectrin dimer-tetramer interconversion ous presence of elliptical RBCs and spherocytes or “fat,” round
has a high activation energy, it is kinetically immobilized at near 0°C. sphero-ovalocytes in the peripheral blood film. In contrast to common
Consequently, the percentage of spectrin dimers and tetramers in the HE, cells of other shapes, such as rod-shaped cells, poikilocytes, and
0°C crude spectrin extract reflects the relative distribution of these fragments, are absent. Importantly, hemolysis, despite relatively mild
species in the RBC membrane in situ. Mutations of α- or α-spectrin alterations in RBC morphologic features, and increased osmotic
residing within or near the αβ-spectrin heterodimer self-association fragility are the main diagnostic features distinguishing this disorder
site invariably lead to an increase in the fraction of dimeric spectrin from common HE.
in the crude 0°C spectrin extract. The molecular basis of spherocytic HE is unknown. However,
patients with mutations, particularly truncations at the C-terminal
Tryptic Peptide Mapping of Spectrin and the of β-spectrin, have many of the clinical features of spherocytic HE
and probably represent an example of this disorder. Patients who lack
Detection of the Underlying DNA Defect GPC have rounded, smooth elliptocytes and could be classified as
having a mild, recessively inherited variant of spherocytic HE. Finally,
Tryptic digestion of spectrin followed by electrophoretic separation some patients with recessively inherited defects of protein 4.2 can
gives rise to highly reproducible tryptic peptide patterns. Among display some features of spherocytic HE, particularly mild
these peptides, the 80-kDa αI domain peptide representing the self- ovalostomatocytosis.
association site of the normal α-spectrin is among the most promi-
nent. Nearly all α- or β-spectrin mutations reported are associated
with a formation of tryptic peptides of abnormal size and mobility Southeast Asian Ovalocytosis
that are generated from the normal 80-kDa αI domain peptide. The
cleavage sites of the most common abnormal tryptic peptides are SAO is characterized by the presence of oval RBCs, many containing
found in the third helix of a given triple-helical repetitive segment. one or two transverse ridges or a longitudinal slit. The condition is
The reported mutations reside in the vicinity of these cleavage sites widespread in certain ethnic groups of Malaysia, Papua New Guinea,
either in the same helix or, less commonly, in helix 1 or 2 of a given the Philippines, and Indonesia. Numerous functional abnormalities
repetitive segment. Consequently, tryptic peptide mapping remains of ovalocytes have been reported, including increased RBC rigidity,
a powerful tool with which to map the site of the underlying spectrin decreased osmotic fragility, increased thermal stability, resistance to
mutation, which can be subsequently defined by polymerase chain shape change by echinocytogenic agents, and a reduced expression of
reaction amplification and sequencing of the respective region of the many RBC antigens. A remarkable feature of ovalocytes is their
genomic DNA or complementary DNA (cDNA). resistance to in vitro invasion by several strains of malaria parasites,
including P. falciparum and Plasmodium knowlesi. In areas of endemic
malaria, SAO individuals have reduced numbers of intracellular para-
Differential Diagnosis sites in vivo, with decreased prevalence and disease severity of malaria
in SAO patients compared with controls.
Various acquired and inherited conditions can be associated with SAO individuals are heterozygotes for two band 3 gene mutations
elliptocytosis and poikilocytosis, including iron deficiency, thalasse- in cis: the deletion of nine codons encoding amino acids 400 through
mias, megaloblastic anemias, myelofibrosis, myelophthisic anemias, 408 from the boundary of the cytoplasmic and membrane domains
myelodysplastic syndromes, and pyruvate kinase deficiency. The of band 3, and the 56 Lys to Glu substitution. The 56 Lys to Glu
percentage of elliptocytes in these conditions is seldom greater than substitution represents an asymptomatic polymorphism known as
60%. However, this is not diagnostically useful, because some HE band 3 Memphis. The SAO phenotype is associated with a tighter
patients can have a relatively low percentage of elliptocytes. In normal binding of band 3 to ankyrin, increased tyrosine phosphorylation of