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PLENARY PAPER
From the Laboratoire d'hématologie, AP-HP,
Faculté de Médecine Paris XI, INSERM U473, Hôpital de
Bicêtre, Le Kremlin Bicêtre, France; Bayer
Diagnostics, Tarrytown, NY; Laboratoire d'hématologie,
Hôpital Bichat- Claude Bernard, Paris, France; and
Lawrence Berkeley National Laboratory, Berkeley, CA.
Spherocytic red cells with reduced membrane surface area are a
feature of hereditary spherocytosis (HS) and some forms of autoimmune
hemolytic anemia (AIHA). It is generally assumed that membrane loss in
spherocytic red cells occurs during their sojourn in circulation. The
structural basis for membrane loss in HS is improper assembly of
membrane proteins, whereas in AIHA it is due to partial
phagocytosis of circulating red cells by macrophages. A hypothesis was formed that these different mechanisms should lead to temporal differences in surface area loss during red cell genesis and during sojourn in circulation in these 2 spherocytic syndromes. It was proposed that cell surface loss could begin at the
reticulocyte stage in HS, whereas surface area loss in AIHA involves
only circulating mature red cells. The validity of this hypothesis was
established by documenting differences in cellular features of
reticulocytes in HS and AIHA. Using a novel technique to quantitate
cell surface area, the decreased membrane surface area of both
reticulocytes and mature red cells in HS compared with normal
cells was documented. In contrast, in AIHA only mature red
cells but not reticulocytes exhibited decreased membrane surface area.
These data imply that surface area loss in HS, but not in AIHA, is
already present at the circulating reticulocyte stage. These findings
imply that loss of cell surface area is an early event during genesis
of HS red cells and challenge the existing concepts that surface
area loss in HS occurs predominantly during the sojourn of mature red
cells in circulation.
(Blood. 2001;98:2894-2899) Spherocytic red cells result from a loss of
membrane surface area and, consequently, exhibit increased
cell sphericity and reduced cellular deformability.1,2
Increased splenic sequestration of these indeformable spherocytic red
cells results in anemia. Hemolytic anemia characterized by circulating
spherocytic red cells can stem from various etiologies, the principal
ones being hereditary spherocytosis (HS),3 an inherited
disease, and autoimmune hemolytic anemia (AIHA) due to warm antibodies,
an acquired disorder.4,5 Although spherocytic red cells in
these 2 settings are morphologically indistinguishable, the mechanistic
basis for their generation is very different. In HS, mutations in genes
coding for various membrane proteins (including ankyrin, band 3, protein 4.2, The aim of our study was to explore the temporal differences
between HS and AIHA regarding the occurrence of surface area loss
during red cell genesis and maturation. We propose that cell surface
loss can exist in HS at the reticulocyte stage, whereas in AIHA it
involves only mature red cells. The validity of this hypothesis rests
on documenting differences in cellular features of reticulocytes in HS
and AIHA. Using a novel technique12 to quantitate the
surface area and volume of both reticulocytes and mature red cells, we
documented decreased membrane surface area of both reticulocytes and
mature red cells in HS compared with normal cells. Importantly,
splenectomy had no effect on the extent of reticulocyte or mature red
cell surface area loss in HS. In marked contrast, in AIHA only mature
red cells but not reticulocytes exhibited decreased membrane surface
area. These data imply that surface area loss in HS, but not in AIHA,
is manifested at the circulating reticulocyte stage and challenge the
existing concepts that membrane loss in HS occurs predominantly during
the sojourn of mature red cells in circulation. Furthermore, we have
shown that although the spleen plays a major role in sequestering
spherical cells, its contribution to the generation of spherocytic red
cells in HS is much less important than previously thought. We also noted that compared with either AIHA or normal reticulocytes, the
volume of HS reticulocytes was reduced while cell hemoglobin concentration was increased. These findings further suggest that reticulocyte parameters could be potentially useful in the differential diagnosis of spherocytes of HS and AIHA.
Subjects
Methods
Cell surface area.
A new strategy that applies a simple geometric model to measure values
of critical hemolytic volumes of reticulocytes and red cells was
developed to derive the surface area of reticulocytes and red
cells.12 Briefly, a series of 16 different solutions of
varying osmolalities ranging from 290 and 110 mosm/kg were prepared by adding appropriate amounts of sodium chloride to 10 mM
phosphate buffer solution (pH 7.4) containing the sphering agent (Bayer
Diagnostics, France) and the reticulocyte-specific dye oxazine 750. Freshly drawn blood samples were diluted (1:1000) in the various
osmotic solutions, equilibrated for 30 minutes, and analyzed in
duplicate within 2 minutes using the flow cytometry-based hematology
analyzer H*3 (Bayer Diagnostics, France). The percentage of
reticulocytes in each of the 16 solutions remained the same. Red cell
and reticulocyte volumes at different osmolarities were recorded. With
decreasing suspending medium osmolality, there is a progressive
increase in cell volume until at a defined osmolality the cells begin
to hemolyze, resulting in an abrupt decrease in cell volume (Figure
1A). The maximal spherical volume is
attained just prior to hemolysis. The MCV measured at the osmolality
preceding the osmolality where cells begin to lyse is defined as the
critical hemolytic volume (Vc). Since Vc represents the maximum
spherical volume of the cell for a defined surface area, the cell
surface area is calculated as
Statistical analysis. Statistical analysis was performed using the unpaired Student t test and the nonparametric Mann-Whitney test.
Red cell morphology, hemoglobin values, and reticulocyte counts Morphologic examination of peripheral blood smears showed that spherocytic red cells are a distinguishing feature of red cell morphology in all patients with either HS or AIHA. In nonsplenectomized patients with HS, the mean hemoglobin value (Hb) was decreased compared with controls (P < .0001), but as previously reported,2 Hb values were either normal or higher than normal in splenectomized patients with HS (Table 1). The reticulocyte count was significantly increased in nonsplenectomized patients with HS compared with controls (P < .0001), whereas it was only marginally elevated in splenectomized patients with HS (Table 1). In patients with AIHA, the mean Hb value was significantly decreased (P < .0001), and the reticulocyte count was increased as compared with controls (P < .0001). The magnitude of increase in the reticulocyte counts in patients with AIHA was similar to that seen in nonsplenectomized patients with HS (P = .74).
Red cell deformability studies A representative set of osmotic gradient deformability profiles of red cells from patients with HS or AIHA are shown in Figure 2. A decrease in DImax value, indicative of decreased red cell surface area, was a distinguishing feature of all HS and AIHA deformability profiles. Consistent with previous data,2 the mean DImax was 0.28 (range 0.18-0.35) for 48 patients with HS compared with the mean DImax of 0.40 for healthy controls (P < .0001). The extent of decrease in surface area was similar in splenectomized and nonsplenectomized patients with HS. DImax values were similarly decreased in patients with AIHA (mean 0.28; range, 0.14-0.36). A shift to the left of O' in the hypertonic region of the deformability profile, reflecting red cell dehydration, was noted in 31 of 48 patients with HS and in 8 of 13 patients with AIHA. A shift to the right of Omin in the hypotonic region of the deformability profile, reflecting a decreased surface-to-volume ratio and increased osmotic fragility, was noted in 33 of 48 patients with HS and in 8 of 13 patients with AIHA. Thus, red cells from a subset of patients with either HS or AIHA may exhibit normal osmotic fragility in the face of decreased surface area, due to a cell dehydration-induced decrease in cell volume.
Surface area of reticulocytes and mature red cells We employed a new experimental strategy to quantitate the surface area of both reticulocytes and mature red cells. In order to validate the new method we measured the surface area of density-fractionated normal red cells. With progressive increase in red cell density, the cell surface area decreased and mean cell hemoglobin concentration increased (Figure 1B). The surface area of the least dense red cells was 145 µm2 and the surface area of the most dense cells was 117 µm.2 These measured values are in agreement with those derived previously using the micropipette method (135 µm2 ± 12 µm2 for the least dense cells and 112 µm2 ± 9 µm2 for most dense cells).18 Having validated the method, we measured surface area of reticulocytes and mature red cells in 12 healthy individuals (Figure 3). The mean surface area of normal reticulocytes is 142.4 µm2 ± 2.0 µm2 (range, 139.6 µm2 -145.9 µm2) and the mean surface area of normal mature red cells is 133.6 µm2 ± 3.0 (range, 128.6 µm2 -138.1 µm2). Interestingly, in 3 individuals who had been splenectomized for reasons unrelated to a red cell membrane abnormality, the surface area of mature red cells was significantly higher than in nonsplenectomized normals (139.4 µm2 ± 1.4 µm2 vs 133.6 µm2 ± 3.0 µm2), whereas no difference was noted in the surface area of reticulocytes (143.7 µm2 ± 3.1 µm2 vs 142.4 µm2 ± 2.0 µm2). These findings are consistent with a major role of the spleen in membrane remodeling during the maturation of normal circulating reticulocytes into mature red cells.
In 12 patients with HS (7 nonsplenectomized and 5 splenectomized), the surface area of both reticulocytes and mature red cells (Figure 3) was significantly reduced compared with normal cells (P < .0001). The surface area of HS reticulocytes was 130.8 µm2 ± 3.1 µm2 (range, 124.2 µm2 -134.8 µm2) and that of mature HS red cells was 121.9 µm2 ± 3.9 µm2 (range, 114.6 µm2 -130.6 µm2). No noticeable differences in surface area could be documented between cells from nonsplenectomized and splenectomized individuals with HS. It is noteworthy that the difference in surface area between reticulocytes and mature red cells is of very similar magnitude in HS cells and in normal cells (Figure 3). These data imply that surface area loss in HS is manifested at the circulating reticulocyte stage and that the extent of splenic remodeling is similar in patients with HS and in normal controls. In marked contrast to HS, the surface area of reticulocytes in 7 patients with AIHA was similar to that of normal reticulocytes (Figure 3). However, the surface area of mature red cells in AIHA was decreased when compared with normal red cells (Figure 3). The surface area of reticulocytes in AIHA is 143.6 µm2 ± 1.8 µm2 (range, 140.1 µm2 -145.3 µm2) and that of mature red cells is 128.1 µm2 ± 4.6 µm2 (range, 122.6 µm2 -136.0 µm2). The difference in surface area between reticulocytes and mature red cells is larger in AIHA when compared with either normal cells or to HS (Figure 3). The finding that in AIHA only mature red cells, but not reticulocytes, exhibited decreased membrane surface area implies that surface area loss in AIHA occurs only in mature red cells. Volume and hemoglobin concentration of reticulocytes and red cells As red cell dehydration has been previously documented in HS,2,13,19,20 we explored if cell dehydration is also a feature of AIHA. Indeed, increased percentage of hyperdense cells (cells with a hemoglobin concentration of > 41.0 g/dL) was a feature of red cells in all cases of HS and in all AIHA cases except one (Table 2). Similarly, mean values of corpuscular hemoglobin concentration of mature red cells (CHCMmRBC) was elevated in all patients with HS and in 12 of the 13 patients with AIHA (Table 2).
To assess if the observed cell dehydration is related to the loss of
membrane surface area, we analyzed the hemoglobin concentration and
volume histograms of reticulocytes and red cells in HS and AIHA (Figure
4). It can be seen that the hemoglobin
concentration histogram of HS reticulocytes is shifted toward higher
values compared with AIHA reticulocytes. Indeed, mean corpuscular
hemoglobin concentration of HS reticulocytes (CHCMr), but not that of
AIHA reticulocytes, is significantly increased compared with normal reticulocytes (P < .0001 vs P = .06) (Table
2). Cell dehydration and decreased surface area are thus features of
both reticulocytes and mature red cells in HS whereas these features
are exhibited only by mature red cells in AIHA.
Similarly, it can be seen that the MCVr in patients with HS is
shifted toward lower values when compared with AIHA (Figures 4 and 5)
and normal control reticulocytes (P < .0001) (Figure 5). The MCVr in nonsplenectomized
patients with HS is 99.7 fL ± 7.3 fL (range, 86.3 fL-110.9
fL) and in splenectomized patients with HS it is 97.0 fL ± 5.4 fL
(range, 87.7 fL-109.4 fL) compared with normal MCVr of 109.8 fL ± 6.1 fL (range, 98.0 fL-123.4 fL). In marked contrast, MCVr in
AIHA was found to be modestly increased from normal cells
(P = .03). As already observed,2 the MCV of
mature HS red cells in both nonsplenectomized and splenectomized patients is also decreased compared with that of normal red cells (P < .0001) (Figure 5) whereas the MCV of AIHA mature red
cells was not significantly different than that of normal red cells (P = .12) (Figure 5).
Reversal of red cell abnormalities in AIHA following treatment To obtain further support for the potential relationship between surface area loss and cell dehydration, we monitored various red cell parameters following steroid therapy and normalization of the antiglobulin test (DAT) in 3 patients with AIHA. Representative data from one of these individuals is shown in Figure 6. Following treatment, the osmotic deformability profile showed a normal DImax value, implying normal cell surface area. The hemoglobin concentration histogram showed marked reduction in the percentage of dense red cells, implying decreased cell dehydration.
By maintaining a favorable surface area to volume ratio, the normal red cell is able to undergo the extensive passive deformations1,2,16,18,21-24 needed to traverse the microvasculature for optimal oxygen delivery. Loss of cell surface area resulting in increased sphericity18,23,24 is detrimental for red cell deformability and there is experimental evidence that these spherical cells are rapidly sequestered from circulation by the spleen.5,9,11,25-30 Hemolytic anemia characterized by spherocytic red cells with reduced membrane surface area is a feature of HS1,2,19 and of some forms of AIHA.4 In HS, the spherocyte formation has long been attributed to the increase propensity of abnormal red cells to shed membrane during their sojourn in circulation due to improper assembly of membrane proteins6-8 whereas in AIHA it is due to partial phagocytosis4 of the circulating red cell membrane bound antibodies complexes by macrophages.4,5,10,11,28 We surmised that these different mechanisms could result in temporal differences in surface area loss in these 2 spherocytic syndromes. Specifically, we proposed that cell surface loss could occur during erythroid maturation in the bone marrow in HS whereas surface area loss in AIHA might only involve circulating mature red cells. Using a novel technique12 to quantitate cell surface area, we indeed were able to document developmentally related temporal differences in membrane loss in HS and AIHA. Specifically decreased membrane surface area is a feature of HS reticulocytes but not of AIHA reticulocytes. On the other hand, mature red cells of both HS and AIHA exhibited decreased surface area. These data imply that surface area loss in HS, but not in AIHA, is already present at the circulating reticulocyte stage. In fact, much of the observed difference in cell surface area of HS and normal red cells can be attributed to decreased surface area at the reticulocyte stage. These findings have enabled us to unequivocally establish that loss of cell surface area is an early event during genesis of HS red cells. Moreover, the findings raise interesting issues regarding how disordered membrane assembly during erythropoiesis can lead to membrane loss.8,31,32 It is also intriguing to note that the average difference in the surface area of reticulocytes and mature red cells in HS is identical to that seen in normal cells. In marked contrast, cell surface loss in AIHA occurs during the life span of the mature red cells in circulation: the average difference in the surface areas of reticulocytes and mature red cells in AIHA is 15.6 µm2 compared with 8.9 µm2 in both normal cells and HS. These findings imply an extensive membrane loss involving mature circulating AIHA red cells, as a result of partial phagocytosis of the antibody-coated membrane,4,5,10 while normal and HS red cells experience similar extents of membrane loss during their life span. Progressive decrease in red cell surface area and cell volume has been shown to occur during their life span in circulation.18,23,24,33 As a consequence of decreased cell volume, red cells with decreased membrane surface area have elevated cell hemoglobin concentration. It is unclear, however, if a direct relationship exists between membrane loss and decreased cell volume. In a recent study, Waugh and colleagues23 showed that following reinfusion of red cells with decreased membrane surface area and increased sphericity, the surviving fraction of spherocytic red cells progressively reduced their cell volume to attain a more favorable surface area to volume ratio and decreased cell sphericity index. These findings strongly suggest that following membrane loss, normal red cells can initiate volume regulatory pathways that enable them to reduce their volume and thereby reduce their sphericity, which in turn prolongs their life span. HS reticulocytes from the very beginning have decreased cell volume and increased cell hemoglobin concentration, which probably result from an early attempt to counterbalance membrane loss. It would be interesting in future studies to determine which of the various volume regulatory pathways contribute to the tight coupling between cell surface area and cell volume. One practical consequence of the tight coupling between volume and surface area loss in HS is that HS reticulocyte volume is consistently lower and hemoglobin concentration consistently higher than those of either normal or AIHA reticulocytes. The filtration function of the spleen is well known. The spherocytes are trapped in "red" pulp of the cords of Billroth5,9,26,27 because they are less deformable1,2,21,22 than normal red cells and thus not able to traverse through the endothelium of splenic sinuses. The trapped cells are subsequently destroyed by the macrophages.5 Support for this thesis comes from the observation that the life span of red cells in HS is significantly increased following splenectomy. Life span of spherocytic red cells has been shown to negatively correlate with the mean intrasplenic red cell transit, in HS and AIHA.24,25,34,35 In addition to removing damaged or senescent red cells from the circulation, the spleen also plays a role in reticulocyte membrane remodeling.36-38 Remodeling of reticulocytes into mature red cells involves loss of lipid membrane,31,39,40 loss of surface area,12,19,41 and decrease in cell volume.41-43 Our finding that the surface area of mature red cells from individuals splenectomized for reasons unrelated to a blood cell disorder was significantly higher than in nonsplenectomized healthy controls in the absence of increased surface area of reticulocytes supports a key role for the spleen in inducing selective membrane loss during maturation of the normal reticulocyte into a mature red cell. However, our finding that the striking reduction in cell surface area can be observed as early as the reticulocyte stage in both nonsplenectomized and splenectomized patients with HS makes it unlikely that excessive remodeling of HS reticulocytes during the splenic passage is responsible for their decreased surface area. We have thus obtained an important insight into the function of the spleen in HS: although the spleen plays a major role in sequestering spherical cells from the circulation, accounting for their reduced survival, the contribution of this organ to the generation of spherocytic HS red cells with reduced surface area is much less important than previously implied. The reduced surface area of HS reticulocytes that we documented is likely to arise precociously during membrane assembly during erythropoiesis,8,31,32,44 enucleation,45-47 or reticulocyte egress from the bone marrow.48,49 Further, in vivo and in vitro studies are necessary to define the contributions of these different mechanisms to the reduced surface area of HS reticulocytes. As such, documentation of the presence of small, dehydrated reticulocytes in the context of spherocytosis could be potentially useful in distinguishing spherocytes of HS from those of AIHA.
We thank Joel-Anne Chasis and Frédéric Bouquet for their insightful comments regarding the manuscript.
Submitted June 5, 2001; accepted July 25, 2001.
Supported in part by la Direction de la Recherche Clinique Assistance Publique-Hopitaux de Paris (CRC96082) and by a National Institutes of Health grant DK26263.
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: Narla Mohandas, Lawrence Berkeley National Laboratory, Mailstop 74-157, 1 Cyclotron Rd, Berkeley, CA 94720; e-mail: mnarla{at}lbl.gov.
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Abstract
Introduction
- or 
-spectrin)
SA) was similar for HS and
controls (right panel). In contrast, the difference in surface area
between reticulocytes and mature red cells is larger in AIHA when
compared with either normal cells (P = .0022) or
with HS cells (P = .0084).
