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RED CELLS
From the Institute of Biochemistry, Swiss Federal Institute of
Technology, Zurich; Department of Internal Medicine, Kantonsspital
Chur, and Children's Hospital, University of Zurich, Switzerland;
Division of Hematology, IRCCS Ospedale Maggiore, Milan; Pediatric
Clinic, Department of Pediatrics, Second University of Naples;
Institute for Pediatrics, University of Foggia and CEINGE-Advanced
Biotechnology Naples, Italy; and Department of Hematology, Centro
Hospitalar de Coimbra, Coimbra, Portugal.
Red cell (RBC) deformability and membrane-bound immunoglobulin G
(IgG) were studied to better understand premature clearance of
erythrocytes in hereditary spherocytosis. Averaged deformability profiles from cells having comparable cell age revealed that
splenectomy was more beneficial for spectrin/ankyrin-deficient than for
band 3-deficient RBCs. Splenectomy prevented an early loss of young cells in both types of deficiencies. It had an additional beneficial effect on spectrin/ankyrin-deficient but not band 3-deficient RBCs. It
prolonged the survival of mature spectrin/ankyrin-deficient RBCs such
that they lost their deformability more slowly than RBCs from patients
who had not undergone splenectomy. Band 3-deficient RBCs lost their
deformability at the same rate before and after splenectomy. In HS
patients with band 3 deficiency who underwent splenectomy, RBC
deformability inversely correlated with the number of RBC-bound IgG (up
to 140 molecules per cell). In spectrin/ankyrin deficiency,
RBC-bound IgG remained at control levels (60 IgG or less per cell). It
appears that spectrin/ankyrin-deficient RBCs escaped opsonization by
releasing band 3-containing vesicles because their band 3 content and
deformability dropped in parallel with increasing cell age. Band
3-deficient RBCs did not lose band 3 with increasing cell age. Hence,
it is possible that band 3 clusters required for bivalent binding of
low-affinity-IgG, naturally occurring antibodies were retained in band
3-deficient RBCs with a relative excess of skeletal proteins but were
released from spectrin/ankyrin-deficient RBCs, in which vesicle budding
was facilitated by an impaired skeleton.
(Blood. 2002;100:2208-2215) Hereditary spherocytosis (HS) is characterized by
the presence of spherocytes in peripheral blood smears with varying
degrees of hemolysis and splenomegaly (for review see references 1 and 2). HS is caused by inherited family-specific mutations. These involve
5 proteins that link the membrane skeleton to the overlaying lipid
bilayer: Little is known about the membrane alterations occurring in the 2 groups of HS,18,19 and even less is known about the
mechanisms by which the altered HS RBCs are prematurely cleared. One
reason is that the fate of HS RBCs is governed not only by their
membrane defect but also by the spleen that aggravates the inherent
defect through what is called splenic conditioning. Although HS RBCs have an increased Na+ level and a decreased K+
content,20 RBCs from the splenic pulp have particularly
diminished K+ and water contents21 and are
more fragile and spherical than those from the
circulation.22 Another reason for the limited knowledge
about membrane alterations in HS is that RBC properties are difficult
to compare,23 when determined on whole RBC populations, because the populations differ in cell age distribution between healthy
controls and HS patients, among different HS types, and before
and after splenectomy. Based on these premises we studied membrane
deformability and RBC-bound IgG of the 2 groups of HS RBCs with
virtually normal integral (spectrin/ankyrin deficient) or normal
cytoskeletal protein content (band 3 deficient) on density-fractionated HS RBCs as a function of their absolute cell age.24 We
compared membrane properties in family members who did and did not
undergo splenectomy and focused on the HS RBCs of those who did in
studying cell-bound IgG and deformability.
Patient material
Blood collection and filtration
RBCs from unrelated controls To obtain sufficient amounts of normal RBCs with a low band 4.1a/4.1b ratio, larger volumes of normal RBCs had to be fractionated. In this case, whole human control blood (0 Rh+) collected in CPD-adenine (Swiss Red Cross; Blood Bank, Zurich) was filtered through cellulose to remove white blood cells25 as described.26RBC density separation on self-forming Percoll gradients Filtered blood was centrifuged at 478g for 10 minutes, and the supernate was discarded. Pelleted cells were added to a Percoll buffer (854 g/L Percoll (Amersham Pharmacia Biotech AB, Uppsala, Sweden), 10 mM NaKHPO4, 144 mM NaCl, 0.5 mM EDTA (ethylenediaminetetraacetic acid), 5 g/L D-glucose, 30 µg/mL phenylmethylsulfonyl fluoride, osmolality 320 mOsm/kg, pH 7.4) to a hematocrit of 10% as described elsewhere.26 The suspension was mixed and centrifuged at 33 000g for 30 minutes in an angle rotor. The banding pattern of RBC density gradients was photographed. Density-separated RBCs were fractionated into 4 to 6 portions from the top to the bottom of the tube.26 Fractionated RBCs were diluted with glucose-supplemented phosphate-buffered saline (PBS-G), centrifuged at 478g for 10 minutes, and washed 3 times with 5 to 10 vol PBS-G by gentle RBC mixing and recentrifugation.Measurement of erythrocyte deformability by ektacytometry RBC deformability was measured using a laser diffraction technique with an Ektacytometer (Technikon Products, Bayer, Germany).27 RBCs were suspended in a viscous solution containing Dextran T70 (Amersham Pharmacia Biotech AB). Dextran T70 initially dissolved in bi-distilled water at a concentration of 20% was diluted to 18% by supplementing with 10 mM NaKHPO4 (pH 7.45), 1 g/L D-glucose, and 0.08% sodium azide. The concentration of NaCl was adjusted to give the desired final osmolality. Red cells (3-4 × 108 RBC/mL) were suspended in 4 mL isotonic dextran solution (300-320 mOsm/kg) immediately before they were subjected to constant shear stress (150 rpm, 159 dyne/cm2) over an osmolality gradient from 120 to 650 mOsm/kg. Osmoscans of RBCs yielded the deformability (elongation index [EI]) as a function of the applied osmolality. Averaged osmoscans were obtained by determining the values of the EI maximum, the osmolality values at the minima in the hypotonic and hypertonic arms of the curve, and the minimal and maximal osmolality values, where EI was maximal. Means of these data points were calculated for RBCs of comparable cell age from spectrin/ankyrin- and band 3-deficient unsplenectomized and splenectomized patients.Radioiodination of protein G Recombinant protein G (Sigma, St Louis, MO) was 125I-iodinated (Na125I, Amersham, Little Chalfont, England) by using chloramine T as an oxidant.28 Unreacted free 125I was removed by passage through a desalting column with Sephadex G-25 and was equilibrated with PBS containing 1 mM NaI, 1 mM EDTA, and 0.005% gelatin. Specific activities of the recovered protein ranged from 15 to 40 × 106 cpm/µg. Labeled protein was stored in aliquots at 20°C. Before use, labeled protein G was supplemented
with ovalbumin (Sigma) to a final concentration of 10 mg/mL, dialyzed
against PBS, and adjusted to a protein G concentration of 3 to 6 µg/mL with unlabeled protein G.
Radioimmunoassay for protein G binding to RBC Binding assays were performed by a phthalate oil separation method described elsewhere.29 The assay was performed in PBS-G buffer. Washed RBCs were adjusted to a cell number of 2 to 3 × 109 RBC/mL. RBC suspensions of 25 µL were added to an equal volume of 125I-labeled protein G (4 × 107 cpm/mL, 3-6 µg/mL) containing 10 mg/mL ovalbumin, mixed, and incubated on ice for 1 hour. The reaction was stopped by adding 150 µL PBS-G (4-fold dilution) and centrifuging an aliquot of 150 µL through 200 µL of a precooled phthalate oil mixture (70% dibutyl phthalate and 30% di-isonyl phthalate) in 400 µL polyethylene centrifuge tubes for 4 minutes at maximal speed in a Beckman Mikrofuge. Tubes were frozen on dry ice, tips containing pelleted cells were cut, and RBC-bound radioactivity was determined in a -counter (Kontron MR 480; Zurich, Switzerland).
Isolation of RBC membranes RBC membranes were prepared from washed cells by lysis with 30 vol cold hemolysis buffer (5 mM NaKHPO4, 1 mM EDTA, pH 7.4). Pelleted membranes were resuspended and washed twice with 30 vol cold hemolysis buffer, which in the first wash was supplemented by 0.8 mM di-isopropylfluorophosphate. Membranes were diluted with hemolysis buffer to the initial volume of packed cells. They were solubilized and alkylated by adding sodium dodecyl sulfate (SDS) and NEM to final concentrations of 1% and 5 mM, respectively. Aliquots were stored at70°C.
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to a modified Neville procedure.30 Samples were heated for 3 minutes in a boiling water bath in sample buffer (60 mM Tris-HCl, pH 6.8, 10 mM EDTA, 5% glycerin, 2% SDS, 0.002% bromphenol blue [Sigma], 40 mM dithiothreitol) and then were alkylated with 50 mM NEM. Gels containing 7% to 8% total acrylamide (0.5-mm-thick minigels) were run for 45 to 90 minutes at 50 mA in a BioRad gel electrophoresis apparatus (BioRad, Hercules, CA), stained with 0.28 g/L Coomassie brilliant blue R-250 (Sigma) in 50% methanol and 7% acetic acid, and destained in 20% methanol and 10% acetic acid. Absolute cell age parameter and densitometric quantification of proteins on SDS-PAGE Band 4.1 appears in 2 bands (4.1a and 4.1b) on SDS-PAGE.24,31,32 Chemical conversion of 4.1b to 4.1a occurs in a time-dependent manner24,32 because of deamidation at Asn 502.24 Deamidation at this single amino acid residue alters the electrophoretic mobility of the protein so that type 4.1a can be separated from type 4.1b on SDS-PAGE. The ratio of the amount of band 4.1a to band 4.1b is considered to be an excellent absolute red cell age parameter.26,33,34 The ratios of 4.1a/4.1b and of band 3/spectrin+ankyrin were quantified as follows: wet SDS polyacrylamide gels, stained with Coomassie brilliant blue R-250, were scanned with a Personal densitometer (Molecular Dynamics, Sunnyvale, CA). Digitized pictures were quantified with the Image Quant program (Molecular Dynamics) using magnified images of the gels. Background signals were subtracted.
Osmotic deformability profiles of whole cell populations from HS RBCs with band 3 or spectrin/ankyrin deficiencies Osmotic deformability profiles (osmoscans)27 of whole RBC populations from HS patients were compressed compared with those of normal RBCs (Figure 1). The EI maxima of HS RBCs from unsplenectomized patients were lower in RBCs with spectrin/ankyrin deficiency (AZ15) than in RBCs with band 3 deficiency (LR1). Deformability of the whole RBC population was higher in splenectomized than in unsplenectomized HS patients with either type of deficiency. The EI maxima, however, increased more in spectrin/ankyrin than in band 3 deficiency. Although this suggested a more pronounced beneficial effect of splenectomy on HS RBC deformability in spectrin/ankyrin than in band 3 deficiency, comparisons of whole cell population properties remain incomplete because these populations differ greatly in cell age distribution and reticulocyte numbers. Reticulocyte numbers from unsplenectomized patients were higher in those with spectrin/ankyrin deficiency than with band 3 deficiency (Table 2). Hence, osmoscans from whole RBC populations should be compared at the same extent of reticulocytosis, a criterion that was only fulfilled for postsplenectomy populations with 1.4% ± 0.7% and 1.5% ± 0.5% reticulocytes in the 2 types of deficiencies. To distinguish between abnormalities in the 2 types of deficiencies and to study the effect of splenectomy, ideally cells of the same age had to be compared.
Splenectomy improved deformability of cell age-matched RBCs to a larger extent in spectrin/ankyrin than in band 3 deficiency RBCs of a given population can be separated according to density, but not according to cell age.26 Given that density increases differently with cell age in control and patient RBC populations, RBCs from the various populations were fractionated by density and their properties were related to their absolute cell age as determined by the band 4.1a/4.1b ratio. RBCs from unsplenectomized HS patients banded primarily at high densities (Figure 2). Cells from splenectomized family members distributed at lower densities. The splenectomy-mediated shift of RBCs from higher to lower densities was observed with both deficiencies but was more pronounced for RBCs with a spectrin/ankyrin deficiency (Figure 2).
RBCs from 4 to 6 density fractions were collected, washed, and studied
by ektacytometry. Membranes from fractionated RBCs were prepared and
analyzed for their absolute cell age. To construct an average
deformability profile, the profiles of RBCs with comparable 4.1.a/4.1.b
ratios were averaged for spectrin/ankyrin or band 3 deficiency from
splenectomized and unsplenectomized family members (Figure
3). These profiles demonstrate that RBCs
from patients with both types of deficiencies profited from splenectomy
by having considerably improved deformability. The maximal RBC
deformability was 3 times higher after splenectomy in spectrin/ankyrin
deficiency. In band 3 deficiency, maximal RBC deformability of cell
age-matched RBCs was only 2 times higher in patients who underwent
splenectomy. The increase in maximal deformability in spectrin/ankyrin
deficiency was underestimated because RBCs of unsplenectomized patients
were younger (average, 4.1a/4.1b = 0.55; n = 2) than those of
splenectomized patients (average, 4.1a/4.1b = 0.9 ± 0.012;
n = 4). It was not possible to compare RBCs of exactly the same cell
age because RBCs with a band 4.1a/4.1b ratio higher than 0.55 were
already eliminated in vivo in unsplenectomized HS patients with
spectrin/ankyrin deficiency. It was furthermore not possible to collect
enough material from similarly young cells (4.1a/4.1b
Splenectomy prolonged red cell age of mature spectrin/ankyrin-deficient RBCs Additional information on the 2 types of deficiency was obtained by analyzing the band 4.1a/4.1b ratio on membrane proteins from density-fractionated RBCs. The band 4.1a/4.1b ratio was low and did not significantly increase with density in membranes from unsplenectomized patients, implying that RBCs were young and did not age considerably (Figure 4). Splenectomy altered the band 4.1a/4.1b ratio in all types of deficiencies. In band 3 deficiency, the band 4.1a/4.1b ratio was higher by 0.3 to 0.4 units but did not significantly increase with RBC density (Figure 4). In spectrin/ankyrin deficiency, the lightest fraction (fraction 1) revealed a similar shift in the band 4.1a/4.1b ratio compared with that of the paired sample. In addition, splenectomy had a unique effect on RBCs with spectrin/ankyrin deficiency it significantly increased the ratio of band 4.1a to 4.1.b
in cells with higher densities. Hence, splenectomy increased their
absolute cell age and thus their in vivo survival.
In some of the patients, we had sufficient amounts of
density-fractionated RBCs to measure deformability and correlate
maximum deformability with absolute cell age. Maximum deformability of RBCs from unsplenectomized patients decreased steeply within a narrow
range of cell age for band 3- and spectrin/ankyrin-deficient RBCs
(Figure 5, open rectangles and open
triangles). In contrast, maximum deformability of normal RBCs decreased
little with cell age (Figure 5, open circles). Splenectomy resulted in
a shift of the starting points of these curves to a higher cell age.
The extent of this shift was comparable for both types of deficiencies (Figure 5, closed rectangles and closed triangles). The finding implies
that cells with similarly high deformability were significantly older
in all splenectomized patients (Figures 4, 5). The 2 groups of
deficiencies differed, however, in the loss of deformability with cell
age. RBCs with spectrin/ankyrin deficiency lost their deformability at
a reduced rate with increasing cell age than RBCs from unsplenectomized
patients. Half-maximum deformability (EI = 0.4) compared with control
cells was reached at a band 4.1a/4.1b ratio of approximately 0.5 in
unsplenectomized and approximately 1.5 in splenectomized patients,
respectively. A similarly slow decrease of deformability with cell age
was found for 2 splenectomized HS patients with an established
spectrin/ankyrin deficiency (no unsplenectomized family members
available) (Figure 5, filled diamonds).
Band 3-deficient RBCs responded differently to splenectomy. Splenectomy increased the cell age of all fractions to a comparable extent (Figure 4). Hence, band 3-deficient RBCs from splenectomized patients lost their deformability at a rate similar to those from unsplenectomized patients (Figure 5). Half-maximum deformability (EI = 0.4) compared with control cells was reached at a band 4.1a/4.1b ratio of approximately 0.8 in unsplenectomized and approximately 1.2 in splenectomized patients, respectively. Thus, the increment in cell age units was less than half that found for spectrin/ankyrin-deficient RBCs. Firmly bound IgG on HS RBCs from splenectomized patients To study whether the premature clearance of HS RBCs was related to IgG opsonization, we studied in vivo IgG opsonization on density-fractionated RBCs from splenectomized patients. Binding of labeled protein G was used to assess RBC-bound IgG. Its amount varied among control cells and only slightly increased within the cell age range studied, which did not include the very old RBCs (Figure 6C). In contrast to this, protein G binding was high and increased with cell age in band 3-deficient patients, reaching 2 to 5 times the number recorded for control cells (P < .0001) (Figure 6A). On RBCs with a spectrin/ankyrin deficiency, however, bound protein G was comparable to that in control cells and did not increase with cell age (Figure 6B). Thus, only RBCs from splenectomized patients with band 3 deficiency showed increased opsonization with cell age that paralleled their rapid loss of deformability (Figure 5).
In vivo IgG binding to whole populations of unspecified HS RBCs had earlier been determined by agglutination. It was low before and higher after splenectomy but remained within the normal range.35 Given that HS is usually caused by spectrin/ankyrin deficiencies, the earlier finding appears to be in agreement with our results on spectrin/ankyrin-deficient RBCs from splenectomized patients. The high binding of IgG, naturally occurring autoantibodies (NAbs) to band 3-deficient RBCs in this study was clearly not due to hypergammaglobulinemia because none of the patients had more than 16 mg IgG/mL and 2 had values below 10 mg/mL (not shown). Spectrin/ankyrin-deficient, but not band 3-deficient, RBCs released band 3 HS RBCs with common spectrin/ankyrin deficiency lose surface in the form of vesicles.36 The loss of vesicles may deprive the cells of integral membrane proteins. Thus, we measured the relative content of band 3 by determining the band 3/spectrin+ankyrin ratio in membranes of density-separated RBCs from splenectomized patients. The band 3/spectrin+ankyrin ratio was inherently low in band 3-deficient RBCs and remained at the same level with increasing cell age or slightly increased (Figure 7A). In spectrin/ankyrin-deficient RBCs, the band 3/spectrin+ankyrin ratio was high because of the low content of skeletal proteins. This ratio decreased significantly from values as high as 1.13 to 0.8 or 0.9 to 0.6 (Figure 7B). Thus, spectrin/ankyrin-deficient RBCs released band 3 with increasing cell age.
Splenectomy had 2 beneficial effects in HS. First, it prevented the loss of a substantial fraction of the youngest cells in both deficiencies. This effect of splenectomy illustrates our interpretation of the finding that the lightest RBC after splenectomy had deformability similar to that of corresponding fractions of RBCs before splenectomy but a cell age that was higher by 0.3 to 0.4 units of the band 4.1a/4.1b ratio. This implies that the functional impairment of RBCs was significantly delayed after splenectomy, irrespective of the type of defect. Such an explanation is likely because reticulocytes and young RBCs are known to have the highest KCl cotransport potential that is rapidly activated by swelling, low pH,37 and oxidative damage.38 Low pH and oxidative damage may occur in splenic cords. Because only a fraction of the blood passes at any time through the splenic sinuses, it is possible that the portion of young cells that traversed the spleen might have suffered considerably more from splenic conditioning than cells that entered the spleen at a higher cell age. Second, as presumed, splenectomy prolonged the survival of mature RBCs, but only in spectrin/ankyrin and not significantly in band 3 deficiency. Band 3-deficient RBCs lost their deformability with cell age as rapidly in splenectomized as in unsplenectomized patients. In contrast to this, maximum deformability decreased at a considerably slower pace with cell age in splenectomized patients with spectrin/ankyrin deficiency. Hence, these RBCs reached a higher cell age than those from patients with band 3 deficiency, implying that the mechanisms leading to premature RBC clearance differed between the 2 types of deficiencies. The pronounced age-dependent loss of band 3 protein from spectrin/ankyrin-deficient RBCs and the complete retention of band 3 in band 3 deficiency represent the molecular hallmarks of the different clearance mechanisms. In spectrin/ankyrin deficiency, the cytoskeleton that normally forms a nearly monomolecular submembranous layer on the inner surface39 has a decreased density. As a result, areas of the plasma membrane that are not attached to the skeleton are more abundant than in normal cells and are prone to be released from the cells in the form of vesicles.40,41 Selective loss of band 3 from spectrin/ankyrin-deficient RBCs suggests that the released vesicles contained band 3, lacked skeletal proteins, and therefore might have had properties similar to those of skeleton-free vesicles released from adenosine triphosphate (ATP)-depleted normal RBCs.42 These vesicles had their band 3 protein clustered (clustered means forming interdimeric and intertetrameric oligomers rather than pre-existing dimers and tetramers) because the extent of exoplasmic chemical cross-linking was up to 10-fold higher than in intact normal RBCs.43 In support of this analogy, lateral mobility of band 3, which is a prerequisite for cluster formation, was found to be greater in RBCs from patients with spectrin/ankyrin-deficient RBCs than in control cells.44 Thus, the cell-age dependent loss of band 3 from spectrin/ankyrin-deficient RBCs might indeed be attributed to impaired skeletal constraints on the apparent excess of band 3 molecules, which allowed band 3 cluster formation and the release of band 3-containing vesicles. Band 3-deficient RBCs, with their reduced number of band 3 molecules
in the healthy cytoskeleton, must have faced a different scenario. Band
3 cluster formation might have occurred to a lower extent, but all
clusters were retained by an excess of skeleton because no band 3 was
released. The presence of band 3 clusters has recently been established
on RBCs from HS patients who underwent splenectomy.45
Hence, complete retention of band 3 and its clusters in aging band
3-deficient RBCs may explain their in vivo IgG opsonization (Figure
8A). Opsonization was significantly
higher than to control cells and reached an extent comparable to that
determined on biotinylated, in vivo, aged dog RBCs.46
Opsonization might have occurred by pre-existing NAbs, of which some
bind to RBC membrane proteins, such as anti-band 3.47
Anti-band 3 NAbs have a low affinity, and their firm bivalent binding
requires the availability of band 3 clusters48 that are
generated by lateral diffusion49 and are fixed in vivo by
hemichrome binding50,51 or oxidative damage from within
the cell.52 Thus, accelerated clearance of HS RBCs with
band 3 deficiency may require opsonization by autologous IgG, as was
found for phagocytosis of RBCs with hemoglobin
defects53,54 and oxidatively stressed
RBCs.52,55
Spectrin/ankyrin-deficient RBCs did not carry more IgG than control cells. Thus, it appears as if the release of band 3-containing vesicles from these RBCs was sufficient to prevent IgG opsonization of the remaining spherocytes. The release of membrane areas containing IgG-opsonized band 3 clusters might have allowed these cells to escape long-term opsonization (Figure 8B). Although we have no direct evidence for the involvement of anti-band 3 NAbs in this in vivo opsonization, their participation is likely given that analogous vesicles released from ATP-depleted RBCs and carrying band 3 clusters bound amounts of autologous IgG 14 times higher than those of RBCs from which they were released.56
We thank the patients who volunteered and gave their consent to study their blood. We also thank Baxter (Switzerland) for providing the sterile mini blood bags and filters required for blood collection and filtration.
Submitted November 5, 2001; accepted May 9, 2002.
Supported by grant no. 0-20423-97 from the Swiss Federal Institute of Technology, Zurich.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Hans U. Lutz, Institute of Biochemistry, Swiss Federal Institute of Technology, ETH-Hönggerberg HPM D14.2, CH 8093 Zurich; e-mail: hlutz{at}bc.biol.ethz.ch.
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© 2002 by The American Society of Hematology.
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  M. Olsson and P.-A. Oldenborg CD47 on experimentally senescent murine RBCs inhibits phagocytosis following Fc{gamma} receptor-mediated but not scavenger receptor-mediated recognition by macrophages Blood, November 15, 2008; 112(10): 4259 - 4267. [Abstract] [Full Text] [PDF]
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 M. Mariani, W. Barcellini, C. Vercellati, A. P. Marcello, E. Fermo, P. Pedotti, C. Boschetti, and A. Zanella Clinical and hematologic features of 300 patients affected by hereditary spherocytosis grouped according to the type of the membrane protein defect Haematologica, September 1, 2008; 93(9): 1310 - 1317. [Abstract] [Full Text] [PDF]
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 A. Luginbuhl, M. Nikolic, H. P. Beck, M. Wahlgren, and H. U. Lutz Complement Factor D, Albumin, and Immunoglobulin G Anti-Band 3 Protein Antibodies Mimic Serum in Promoting Rosetting of Malaria-Infected Red Blood Cells Infect. Immun., April 1, 2007; 75(4): 1771 - 1777. [Abstract] [Full Text] [PDF]
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I. Theurl, V. Mattle, M. Seifert, M. Mariani, C. Marth, and G. Weiss Dysregulated monocyte iron homeostasis and erythropoietin formation in patients with anemia of chronic disease Blood, May 15, 2006; 107(10): 4142 - 4148. [Abstract] [Full Text] [PDF]
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 P. G. Gallagher, D. G. Nilson, C. Wong, J. L. Weisbein, L. J. Garrett-Beal, S. W. Eber, and D. M. Bodine A dinucleotide deletion in the ankyrin promoter alters gene expression, transcription initiation and TFIID complex formation in hereditary spherocytosis Hum. Mol. Genet., September 1, 2005; 14(17): 2501 - 2509. [Abstract] [Full Text] [PDF]
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 P. G. Gallagher Red Cell Membrane Disorders Hematology, January 1, 2005; 2005(1): 13 - 18. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
and 
spectrin,
0.5) from
controls and splenectomized patients because such cells comprised a
small portion of the whole cell populations. All other RBC fractions compared in Figure 
