CHAPTER 31

STRUCTURE AND COMPOSITION

OF THE ERYTHROCYTE
SUMMARY

Collectively, the erythroid progenitors, terminally differentiating erythroblasts (precursors), and

adult red cells are termed the erythron to reinforce the idea that they function as an organ. The
widely dispersed cells comprising this organ arise from pluripotential hematopoietic stem cells.
Following commitment to the erythroid lineage (unipotential progenitor), further maturation
gives rise to the erythroid progenitors, burst-forming unit–erythroid and, subsequently, colony-
forming unit–erythroid (CFU-E), that can be identified by their development into representative
clonal colonies of red cells in vitro. The CFU-E then undergoes terminal differentiation,
progressing through four to five morphologic stages, each having characteristic light microscopic
and ultrastructural features. During terminal erythroid differentiation there is an increasing
amount of hemoglobin synthesis accompanied by nuclear chromatin condensation and at the
final stage of differentiation there is nuclear extrusion to generate an anucleate
polychromatophilic macrocyte (reticulocyte with supravital staining). The human
polychromatophilic macrocyte (reticulocyte) matures over 2 to 3 days, first in the marrow and
then in circulation into the discoid erythrocyte. During reticulocyte maturation, cytoplasmic
inclusions including residual mitochondria and ribosomes are degraded and the reticulocyte loses
surface area to achieve the mean cell volume and surface area of a discoidal erythrocyte. Mature
erythrocytes are approximately 7 to 8 μm in diameter and undergo extensive deformation to pass
through 3-μm diameter capillaries and the 1-μm wide and 0.5-μm thick endothelial slits in the
red pulp of the spleen. The ability of the red cell to undergo extensive reversible deformation is
essential for both its function and its survival. Red cell deformability is a function of its
geometry, the viscosity of the cytoplasm, largely determined by the concentration of
hemoglobin. Decreased deformability is a feature of red

Acronyms and Abbreviations: BFU-E, burst-forming unit–erythroid; CFU-E, colony-forming

unit–erythroid; cP, centipoise; DIC, disseminated intravascular coagulation; EMP, erythroblast
macrophage protein; ICAM-4, intercellular adhesion molecule-4; IL, interleukin; MCH, mean
cell hemoglobin content; MCHC, mean corpuscular hemoglobin concentration; MCV, mean cell
volume; MDS, myelodysplastic syndrome; SA:V, surface area-to-volume ratio; TTP, thrombotic
thrombocytopenic purpura.

cells in various pathological states. The erythrocyte is unique among eukaryotic cells in that its
principal physical structure is its cell membrane, which encloses a concentrated hemoglobin
solution. Thus, all of the structural properties of this cell are in one way or another linked to the
cell membrane. In contrast to other cells, the erythrocyte has no cytoplasmic structures or
organelles. Only, red cells and platelets do not have a nucleus.

ERYTHRON

The mass of circulating erythrocytes constitutes an organ responsible for the transport of
oxygen to tissues and the removal of carbon dioxide from tissues for exhalation. Collectively, the
progenitors, precursors, and adult red cells make up an organ termed the erythron, which arises
from pluripotent hematopoietic stem cells. Following commitment to the erythroid lineage,
unipotential progenitors mature into the erythroid progenitors, the burst-forming unit–erythroid
(BFU-E) and, subsequently, the colony-forming unit–erythroid (CFU-E), which then undergoes
further maturation to generate anucleate polychromatophilic macrocytes (reticulocytes on
supravital staining). The BFU-E and CFU-E are identified by their development into
morphologically identifiable clonal colonies of red cells in vitro. The reticulocyte further
matures, first in the marrow for 2 to 3 days and, subsequently, in the circulation for
approximately 1 day, to generate discoid erythrocytes.1–5 The proerythroblast, the first
morphologically recognizable erythroid precursor cell in the marrow undergoes four to five
mitoses prior to maturation to an orthochromatic erythroblast, which then undergoes nuclear
extrusion. A feature of erythropoiesis is that following each cell division the daughter cells
advance in their state of maturation as compared to the parent cell and, ultimately, become
functional as mature erythrocytes.4 In this process, they acquire the human blood group antigens,
transport proteins, and all components of the erythrocyte membrane.4,6 In the adult stage of
development, the total number of circulating erythrocytes is in a steady state, unless perturbed by
a pathologic or environmental insult. This effect is not so during growth of the individual in
utero, particularly in the early stages of embryonic development and also during neonatal
development as the total blood volume increases markedly. Consequently, erythrocyte
production in the embryo and fetus differs markedly from that in the adult.

THE EARLIEST ERYTHRON

In the very early stages of human growth and development, there are two forms of erythroid
differentiation: primitive and definitive.7–10 Chapters 5 and 7 provide detailed descriptions of
embryonic and fetal hematopoiesis. The primitive erythron supplies the embryo with oxygen
during the phase of rapid growth before the definitive form of maturation has had a chance to
develop and seed an appropriate niche. The hallmark of this primitive erythron is the release of
nucleated erythroid precursors containing embryonic hemoglobin. Although primitive in the
sense that the cells contain nuclei when released into the circulation, this form of maturation
differs from avian and reptilian erythropoiesis in that the nucleus is eventually expelled from the
mammalian cells as they circulate. The transient presence of a nucleus in the cells of the
circulating primitive erythron can decrease the efficiency of gas exchange in the lungs and
microvasculature because the nucleus prevents the red cell from behaving as a fluid droplet.11
The definitive stage of maturation makes its appearance around week 5 of embryogenesis when
multipotential stem cells develop and seed the liver which maintains the erythron for most of
fetal life. In later fetal life, skeletal development provides marrow niches to which erythropoiesis
relocates being sustained in the form of erythroblastic islands, a central macrophage with
circumferential layers of developing erythroid cells.12 The definitive stage of erythroid
maturation predominates during the remainder of fetal development and is the only type of
erythroid maturation present through childhood and adult life. All of normal human
erythropoiesis occurs in the marrow in the form of erythroblastic islands.13

ERYTHROID PROGENITORS

Burst-Forming Unit–Erythroid

The earliest identifiable progenitor committed to the erythroid lineage is the BFU-E (see Chap.
32, Fig. 32–1). A BFU-E is defined in vitro by its ability to create a “burst” on semisolid
medium—that is, a colony consisting of several hundred to thousands of cells by 10 to 14 days of
growth, during which time smaller satellite clusters of cells form around a larger central group of
erythroid cells, giving rise to the designation of a “burst.” The generation of BFU-E from
hematopoietic stem cells requires interleukin (IL)-3, stem cell factor, and erythropoietin for
differentiation, proliferation, prevention of apoptosis, and maturation (Chap. 18).5,13

Colony-Forming Unit–Erythroid

As erythroid maturation progresses, a later progenitor, the CFU-E, derived from the BFU-E, can
be defined in vitro. The CFU-E is dependent on erythropoietin for its development and can
undergo only a few cell divisions.5,14 Thus, the CFU-E forms a smaller colony of
morphologically recognizable erythroid cells in 5 to 7 days (see Chap. 32, Fig. 32–1). Adhesion
between erythroid cells and macrophages occurs at the CFU-E stage of maturation. Using cell-
surface markers, IL-3 receptor, CD34, and CD36, highly purified populations of BFU-E and
CFU-E can be isolated from human marrow.5 Gene expression profiling show distinctive
changes in gene expression profiles in hematopoietic stem cells, BFU-E, and CFU-E.5 Some of
the marrow failure syndromes are the result of defects in differentiation of stem cells into
erythroid progenitors.

ERYTHROBLASTIC ISLAND

The anatomical unit of erythropoiesis in the normal adult is the erythroblastic island or
islet.13,16,17 The erythroblastic island consists of a centrally located macrophages surrounded
by maturing terminally differentiating erythroid cells (Fig. 31–1A). A number of binding
proteins are implicated in the cell–cell adhesions important to this process. These include α4β1
integrin, erythroblast macrophage protein (EMP), and intercellular adhesion molecule-4 (ICAM-
4) on the erythroblasts and vascular cell adhesion molecule (VCAM-1), EMP, αV integrin on
macrophages.16 Additional macrophage receptors include CD69 (sialoadhesin) and CD163, but
the counterreceptors for these on erythroblasts remains to be defined.16 Phase-contrast
microcinematography reveals that the macrophage is far from passive or immobile. Evidence
suggests that either the erythroblastic islands migrate or that erythroid precursors move from
island to island, as islands near sinusoids are composed of more mature erythroblasts while
islands more distant from the sinusoids are composed of proerythroblasts.18 The macrophage’s
pseudopodium-like cytoplasmic extensions move rapidly over cell surfaces of the surrounding
wreath of erythroblasts. On phase contrast micrographs, the central macrophage of the
erythroblastic island appears sponge-like, with surface invaginations in which the erythroblasts
lie (Fig. 31–1B). As the erythroblast matures, it moves along a cytoplasmic extension of the
macrophage away from the main body. When the erythroblast is sufficiently mature for nuclear
expulsion, the erythroblast makes contact with an endothelial cell, passes through a pore in the
cytoplasm of the endothelial cell and enters the circulation as a polychromatophilic macrocyte
(reticulocyte).19–21 The nucleus is ejected prior to egress from the marrow, phagocytized, and
degraded by marrow macrophages.22 In addition to the unique cytologic features described
above, the macrophage of the erythroblastic island is also molecularly distinct as demonstrated
by a unique immunophenotypic signature.23 In addition, the macrophage of the erythroblastic
island appears to play a stimulatory role in erythropoiesis independent of erythropoietin. The
anemia of chronic inflammation and of the myelodysplastic syndrome (MDS) may result, at least
in part, from inadequate stimulation of erythropoiesis by these macrophages (Chap. 5).

Despite the central role of erythroid islands in erythropoiesis in vivo, morphologically

normal development of erythroid cells can be recapitulated in vitro without these structures as
long as developing cells are provided with supraphysiologic concentrations of appropriate
cytokines and growth factors. Such growth, however, occurs at a much slower rate than that
observed in vivo, when erythroblasts form erythroblastic islands.24 The erythroblastic island is a
fragile structure. It is usually disrupted in the process of obtaining a marrow specimen by needle
aspiration but can be seem in marrow biopsies.

Macrophages in erythroblastic islands not only affect erythroid differentiation and/or

proliferation, but also perform other functions, including rapid phagocytosis (<10 min) of
extruded nuclei as a result of exposure of phosphatidylserine on the surface of the membrane
surrounding the nucleus.22 This phagocytosis is the reason for the inability to find extruded
nuclei in marrow aspirates in spite of the fact that 2 million nuclei are extruded every second
during steady-state erythropoiesis. A protective macrophage function linked to efficient
phagocytosis has been described. In normal mice, DNase II in macrophages degrades the
ingested nuclear DNA but in DNase II knockout mice the inability to degrade DNA results in
macrophage toxicity with resultant decrease in number of marrow macrophages and in
conjunction with severe anemia.25 Macrophages can play both positive and negative regulatory
roles in human erythropoiesis but the mechanistic basis for these regulatory processes are not
completely understood.16,24 These processes may play a role in the ineffective erythropoiesis in
disorders such as MDS, thalassemia, and malarial anemia.

Another potentially important role originally proposed for the central macrophage is
direct transfer of iron to developing erythroblasts mediated by ferritin exchange between
macrophages and erythroblasts (Chap. 42).13 Although this is an interesting concept, there is no
definitive evidence for this exchange.

ERYTHROID PROGENITORS AND PRECURSORS

Early Progenitors A “progenitor” in the hematopoietic system is defined as a marrow cell that is
a derivative of the pluripotent hematopoietic stem cell through the process of differentiation and
is antecedent to a “precursor” cell, the latter being identifiable by light microscopy by its
morphologic characteristics (see Chap. 83, Fig. 83–2). In erythropoiesis, the earliest precursor is
the proerythroblast. Erythroid progenitor cells are identified as marrow cells capable of forming
erythroid colonies in semisolid medium in vitro under conditions in which the appropriate
growth factors are present. Progenitor cells also may be identified by characteristic profiles of
surface CD antigens using flow cytometry. Numerically, erythroid progenitors, BFU-E and CFU-
E represent only a minute proportion of human marrow cells. BFU-E range from 300 to 1700 ×
106 mononuclear cells and CFU-E range from 1500 to 5000 × 106 mononuclear cells.5 In vitro
cultures using CD34+ cells from blood, cord blood, and marrow as the starting material have
identified the critical cytokines required for erythroid differentiation and maturation and enabled
the identification and isolation of pure cohorts of erythroid progenitors and erythroblasts at all
stages of terminal erythroid maturation.4,5

Precursors

Figure 31–2 shows the sequence of precursors as seen in marrow films.

Figure 31–3 shows the marrow precursors as isolated by flow cytometry.

Proerythroblasts On stained films, the proerythroblast appears as a large cell, irregularly

rounded or slightly oval.13 The nucleus occupies approximately 80 percent of the cell area and
contains fine chromatin delicately distributed in small clumps. One or several well-defined
nucleoli are present. The high concentration of polyribosomes gives the cytoplasm of these cells
its characteristic intense basophilia. At very high magnification, ferritin molecules are seen
dispersed singly throughout the cytoplasm and lining the clathrin-coated pits on the cell
membrane (see Figs. 31–2 and 31–4) Diffuse cytoplasmic density on sections stained for
peroxidase indicates hemoglobin is already present. Dispersed glycogen particles are present in
the cytoplasm. Basophilic Erythroblasts Basophilic erythroblasts are smaller than
proerythroblasts. The nucleus occupies three-fourths of the cell area and is composed of
characteristic dark violet heterochromatin interspersed with pink-staining clumps of euchromatin
linked by irregular strands.13 The whole arrangement often resembles wheel spokes or a clock
face. The cytoplasm stains deep blue, leaving a perinuclear halo that expands into a juxtanuclear
clear zone around the Golgi apparatus. Cytoplasmic basophilia at this stage results from the
continued presence of polyribosomes (see Figs. 31–2 and 31–5). Polychromatophilic
Erythroblasts Following the mitotic division of the basophilic erythroblast, the cytoplasm
changes from deep blue to gray, as hemoglobin dilutes the polyribosome content. Cells at this
stage are smaller than basophilic erythroblasts. The nucleus occupies less than half of the cell
area. The heterochromatin is located in well-defined clumps spaced regularly about the nucleus,
producing a checkerboard pattern. The nucleolus is lost, but the perinuclear halo persists.13 It is
at this point that erythroblasts lose their mitotic potential. Electron microscopy of the
polychromatophilic erythroblast reveals increased aggregation of nuclear heterochromatin.13
Active ferritin transport across the cell membrane is always evident, and siderosomes along with
dispersed ferritin molecules can be identified within the cytoplasm (see Figs. 31–2 and 31–6).

Orthochromic (syn. Orthochromatic) Erythroblasts After the final mitotic division of the
erythropoietic series, the concentration of hemoglobin increases within the erythroblast. Under
the light microscope, the nucleus appears almost completely dense and featureless. It is
measurably decreased in size. This cell is the smallest of the erythroblastic series.13 The nucleus
occupies approximately one-fourth of the cell area and is eccentric. Cell movement can be
appreciated under the phase-contrast microscope. Round projections appear suddenly in different
parts of the cell periphery and are just as quickly retracted.13 The movements probably are made
in preparation for ejection of the nucleus. The cell ultrastructure is characterized by irregular
borders, reflecting its motile state. The heterochromatin forms large masses. Mitochondria are
reduced in number and size (see Figs. 31–2, 31–7, and 31–8).

Normal Sideroblasts All normal erythroblasts are sideroblasts in that they contain iron in
structures called siderosomes, as evident by transmission electron microscopy. These structures
are essential for the transfer of iron for heme (hemoglobin) synthesis. By light microscopy, under
the usual conditions of Prussian blue staining for iron, a minority of normal erythroblasts
(approximately 15 to 20 percent) can be identified as containing siderosomes and those that can
be so identified have very few (one to four) small Prussian blue–positive granules. Pathologic
Sideroblasts A heterogeneous group of erythrocyte disorders is accompanied by ineffective
erythropoiesis, abnormal erythroblast morphology and hyperferremia. These disorders include
acquired megaloblastic anemia (Chap. 41), congenital dyserythropoietic anemias (Chap. 39),
thalassemias (Chap. 48), the inherited and acquired sideroblastic anemias, pyridoxine-responsive
anemia, alcohol-induced sideroblastic anemia, and lead intoxication (Chaps. 52 and 59). Some of
these conditions are characterized by the presence of pathologic sideroblasts.

Pathologic sideroblasts are of two types. One type is an erythroblast that has an increase
in number and size of Prussian blue– stained siderotic granules throughout the cytoplasm.
Another type is the erythroblast that shows iron-containing granules that are arranged in an arc or
a complete ring around the nucleus (Fig. 31–8). These pathologic sideroblasts are referred to as
ring or ringed sideroblasts.26,27 Electron microscopic studies show that granules in ringed
sideroblasts are iron-loaded mitochondria. In cells with iron-loaded mitochondria, many ferritin
molecules are deposited between adjacent erythroblast membranes.

RETICULOCYTE

Birth

Prior to enucleation at the late orthochromatic erythroblasts stage, intermediate filaments and the
marginal band of microtubules disappear. Enucleation is a highly dynamic process that involves
coordinated action of multiple mechanisms.28–30 Tubulin and actin become concentrated at the
point where the nucleus will exit. These changes, accompanied by microtubular rearrangements
and actin polymerization, play a role in nuclear expulsion. Expulsion of the nucleus in vitro is
not an instantaneous phenomenon; it requires a period of 6 to 8 minutes. The process begins with
several vigorous contractions around the midportion of the cell, followed by a division of the cell
into unequal portions.

The smaller portion consists of the expelled nucleus surrounded by a thin ring of hemoglobin and
plasma membrane (Fig. 31–9). In vivo, expulsion of the nucleus may occur while the
erythroblast is still part of an erythroblastic island or the nucleus may be lost during passage
through the wall of a marrow sinus as the nucleus, which cannot traverse the small opening,
remains in the marrow. The outer leaflet of the bilaminar membrane surrounding the expelled
nucleus is high in phosphatidylserine, a signal for macrophage ingestion (Fig. 31–10).22 It is not
clear what fraction of the expelled nuclei is ingested by the macrophage of the erythroblastic
island or by other macrophages resident in marrow. Two hypotheses have been proposed to
explain how the reticulocyte exits the marrow.19–21 The reticulocyte may actively traverse the
sinus epithelium to enter the lumen. More likely, however, the reticulocyte may be driven across
by a pressure differential because it appears incapable of directed amoeboid motion. The precise
mechanism is yet to be defined.

Maturation

Following nuclear extrusion, the reticulocyte retains mitochondria, small numbers of ribosomes,
the centriole, and remnants of the Golgi apparatus. It contains no endoplasmic reticulum.
Supravital staining with brilliant cresyl blue or new methylene blue produces aggregates of
ribosomes, mitochondria, and other cytoplasmic organelles. These aggregates stain deep blue
and, arranged in reticular strands, give the reticulocyte its name. Maturation of the reticulocyte
requires 48 to 72 hours. During this period, approximately 20 percent of the membrane surface
area is lost and cell volume decreases by 10 to 15 percent and the final assembly of the
membrane skeleton is completed.31–33 Living reticulocytes observed by phase-contrast
microscopy are irregularly shaped cells with a characteristically puckered exterior and a motile
membrane. Examined by electron microscopy, reticulocytes are irregularly shaped and contain
many remnant organelles.13 The organelles, small smooth vesicles, and an occasional centriole
are grouped in the region of the cell where the nucleus is expelled. In “young” reticulocytes, the
vast majority of ribosomes dispersed throughout the cytoplasm are in the form of polyribosomes.
As protein synthesis diminishes during maturation, the polyribosomes gradually transform into
monoribosomes. During reticulocyte maturation there is significant remodeling of the membrane,
including loss of membrane proteins that include transferrin receptors, Na-K adenosine
triphosphatase (ATPase), and adhesion molecules, as well as loss of tubulin and cytoplasmic
actin.33 During the remodeling process the membrane becomes more elastic and acquires
increased membrane mechanical stability.32

Macroreticulocytes “Stress” reticulocytes are released into the circulation during an intense
erythropoietin response to acute anemia or experimentally in response to large doses of
exogenously administered erythropoietin.34 These cells may be twice the normal volume, with a
corresponding increase in mean cell hemoglobin (MCH) content. Whether the increase results
from one less mitotic division during maturation or from some other process such as changes in
cell cycle is not clear. It is interesting to note that mice do not have the ability to produce stress
reticulocytes with increased mean cell volume (MCV) and MCH. In contrast, even under
moderate erythropoietic stress, some reticulocytes in the marrow pool shift to the circulating
pool. These “shift” reticulocytes with normal MCH contain a higher-than-normal RNA content
and now can be quantified. Quantification is commonly performed by applying a fluorescent
stain to tag RNA and then dividing reticulocytes into high-, medium-, and low-fluorescence
categories using a fluorescence-sensitive flow cytometer. The “stress” reticulocytes of the older
literature likely fall in the high- and medium-fluorescence categories. Unfortunately, at present
little attention is being paid to discriminate stress and shift reticulocytes.

Pathology of the Reticulocyte

The reticulocyte may show pathologic alterations in size or staining properties. The reticulocyte
may contain inclusions visible by light microscopy or identifiable only on ultrastructural
analysis. Most pathologic inclusions usually attributed to erythrocytes are actually found within
reticulocytes and are nuclear or cytoplasmic remnants derived from late-stage erythroblasts. In
splenectomized patients, they may also be found in mature erythrocytes.

RED CELL INCLUSIONS

See Fig. 31–11 for images of red cell inclusions.

Howell-Jolly Bodies

Howell-Jolly bodies are small nuclear remnants that have the color of a pyknotic nucleus on
Wright-stained films and give a positive Feulgen reaction for DNA.35,36 They are spherically
shaped, randomly distributed in the red cell and usually no larger than 0.5 μm in diameter.
HowellJolly bodies may be numerous, although generally only one is present. In pathologic
situations, they appear to represent chromosomes that have separated from the mitotic spindle
during abnormal mitosis, and contain a high proportion of centromeric material along with
heterochromatin. More commonly, during normal maturation they arise from nuclear
fragmentation or incomplete expulsion of the nucleus. Howell-Jolly bodies are pitted from the
reticulocytes during their transit through the interendothelial slits of the splenic sinus. They are
characteristically present in the blood of splenectomized persons and in patients suffering from
megaloblastic anemia, and hyposplenic states.

Pocked (or Pitted) Red Cells

When viewed by interference-phase microscopy, pocked red cells appear to have surface
membrane “pits” or craters.37–39 The vesicles or indentations characterizing these cells
represent autophagic vacuoles adjacent to the cell membrane. The vacuoles appear to be
instrumental in disposal of cellular debris as the erythrocyte passes through the microcirculation
of the spleen. Within 1 week following splenectomy, pocked red cell counts begin to rise,
reaching a plateau at 2 to 3 months. Pocked red blood cell counts sometimes are used as a
surrogate test for splenic function.

Cabot Rings

The ring-like or figure-of-eight structures sometimes seen in megaloblastic anemia within

reticulocytes and in an occasional, heavily stippled, late-intermediate megaloblast are designated
Cabot rings.40,41 Their composition is nuclear. Some investigators have suggested that Cabot
rings originate from spindle material that was mishandled during abnormal mitosis. Others have
found no indication of DNA or spindle filaments but have shown the rings are associated with
adherent granular material containing arginine-rich histone and nonhemoglobin iron.

Basophilic Stippling

Basophilic stippling consists of granulations of variable size and number that stain deep blue
with Wright stain. Electron microscopic studies have shown that punctate basophilia represents
aggregated ribosomes.42 Clumps form during the course of drying and postvital staining of the
cells, much as “reticulum” in reticulocytes precipitates from ribosomes during supravital
staining. The clumped ribosomes may include degenerating mitochondria and siderosomes. In
conditions such as lead intoxication (Chap. 52), pyrimidine 5′-nucleotidase deficiency (Chap.
47), and thalassemia (Chap. 48), the altered reticulocyte ribosomes have a greater propensity to
aggregate. As a result, basophilic granulation appears larger and is referred to as coarse
basophilic stippling.

Heinz Bodies

Heinz bodies are composed of denatured proteins, primarily hemoglobin, that form in red cells as
a result of chemical insult; in hereditary defects of the hexose monophosphate shunt; in the
thalassemias (Chap. 48); and in unstable hemoglobin syndromes (Chap. 49).43Heinz bodies are
not seen on ordinary Wright- or Giemsa-stained blood films. Heinz bodies are readily visible in
red cells stained supravitally with brilliant cresyl blue or crystal violet and are eliminated as red
cells traverse the endothelial slits of the splenic sinus.

Hemoglobin H Inclusions

Hemoglobin H is composed of β4 tetramers, indicating that β chains are present in excess as a

result of impaired α-chain production (Chap. 48). Exposure to redox dyes such as brilliant cresyl
blue, methylene blue, or new methylene blue, results in denaturation and precipitation of
abnormal hemoglobin.44–46 Brilliant cresyl blue causes the formation of a large number of
small membrane-bound inclusions, giving the cell a characteristic “golf ball–like” appearance
when viewed by light microscopy. Methylene blue and new methylene blue generate a smaller
number of variably sized membrane-bound and floating inclusions. These changes are seen most
frequently in α-thalassemia but also can be found in patients with unstable hemoglobin (Chap.
49) and in rare patients with primary myelofibrosis who develop acquired hemoglobin H disease.

Siderosomes and Pappenheimer Bodies

Normal or pathologic red cells in blood containing siderosomes (“iron bodies”) usually are
reticulocytes. The iron granulations are larger and more numerous in the pathologic state (Chap.
59). Electron microscopy shows that many of these bodies are mitochondria containing
ferruginous micelles rather than the ferritin aggregates characterizing normal siderocytes.47
Siderosomes usually are found in the cell periphery, whereas basophilic stippling tends to be
distributed homogeneously throughout the cell. Pappenheimer bodies are siderosomes that stain
with Wright stain. Electron microscopy of Pappenheimer bodies shows that the iron often is
contained within a lysosome, as confirmed by the presence of acid phosphatase. Siderosomes
may contain degenerating mitochondria, ribosomes, and other cellular remnants.

STRUCTURE AND SHAPE OF ERYTHROCYTES

The normal resting shape of the erythrocyte is a biconcave disc (Fig. 31–12). Variations in the
shape and dimensions of the red cell are usefulin the differential diagnosis of anemias. Normal
human red cells have a diameter of 7 to 8 μm, and the diameter decreases slightly with cell age.
The size decrease likely results from loss of membrane surface area during erythrocyte life span
by spleen-facilitated vesiculation. The cells have an average volume of approximately 90 fL and
a surface area of approximately 140 μm.2 The membrane is present in sufficient excess to allow
the cell to swell to a sphere of approximately 150 fL or to deform so as to enter a capillary with a
diameter of 2.8 μm. The normal erythrocyte stains reddish-brown with Wright-stained blood
films and pink with Giemsa stain. The central third of the cell appears relatively pale compared
with the periphery, reflecting its biconcave shape. Many artifacts can be produced in the
preparation of the blood film. They may result from contamination of the glass slide or coverslip
with traces of fat, detergent, or other impurities. Friction and surface tension involved in the
preparation of the blood film produce fragmentation, “doughnut cells” or anulocytes, and
crescent-shaped cells. Observed under the phasecontrast or interference microscope, the red cell
shows a characteristic internal scintillation known as red cell flicker.48 The scintillation results
from thermally excited undulations of the red cell membrane. Frequency analysis of the surface
undulations has provided an estimate of the membrane curvature elastic constant and of changes
in this constant resulting from alcohol, cholesterol loading, and exposure to cross-linking agents.

RED CELL SHAPE AND SURVIVAL IN CIRCULATION

The red cell spends most of its circulatory life within the capillary channels of the
microcirculation. During its 100- to 120-day life span, the red cell travels approximately 250 km
and loses approximately 15 to 20 percent of its cell surface area. The long survival of the red cell
is at least partially a result of the unique capacity of its membrane to “tank tread”—that is, to
rotate around the red cell contents and thereby facilitate more efficient oxygen delivery. The
physical arrangement of membrane skeletal proteins in a uniform shell of highly folded
hexagonal spectrin lattice permits this unusual behavior.49–51 The arrangement also is
responsible for the characteristic biconcave shape of the resting cell. Red cells must also be able
to withstand large shear forces and must be able to undergo extensive reversible deformation
during transit through the microvasculature and in transiting from the splenic red cell pulp back
into circulation. The resiliency and fluidity of the membrane to deformation is regulated by the
spectrin-based membrane skeleton.49 A deficiency in the amount of spectrin or the presence of
mutant spectrin in the submembrane skeleton results in abnormally shaped cells in hereditary
spherocytosis, elliptocytosis, and pyropoikilocytosis (Chap. 46).49 In regions of circulatory
standstill or very slow flow, red cells travel in aggregates of two to 12 cells, forming rouleaux.
Within large vessels, increased shear forces disrupt this aggregation.

RED CELL COMPOSITION

The erythrocyte is a complex cell. The membrane is composed of lipids and proteins, and the
interior of the cell contains metabolic machinery designed to sustain the cell through its 120-day
life span and maintain the integrity of hemoglobin function. Each component of red blood cells
may be expressed as a function of red cell volume, grams of hemoglobin, or square centimeters
of cell surface. These expressions are usually interchangeable, but under certain circumstances
each may have specific advantages. However, because disease may produce changes in the
average red cell size, hemoglobin content, or surface area, the use of any of these measurements
individually may, at times, be misleading. For convenience and uniformity, data in the
accompanying tables (Tables 31–1 through 31–6) are expressed in terms of cell constituent per
milliliter of red cell and per gram of hemoglobin. In many instances, this process required
recalculation of published data. These recalculations assume a hematocrit value of 45 percent
and 33 g of hemoglobin per deciliter of red cells. To obtain concentration per gram of
hemoglobin, the concentration per milliliter red blood cell can be multiplied by 3.03. The tables
list only some of the most commonly referred to constituents of the erythrocyte. The reference on
which each value is based is the first number presented in the last column of each table. Where
applicable, additional confirmatory references are given. In some instances, only the percentage
of the total of the type of constituent present is given. Chapter 46 discusses the detailed protein
composition of the red cell membrane and its various protein constituents.

ERYTHROCYTE DEFORMABILITY

During its 120-day life span, the erythrocyte must undergo extensive passive deformation and
must be mechanically stable to resist fragmentation and cellular deformability is an important
determinant of red cell survival in the circulation. Red cell deformability is influenced by
threedistinct cellular components: (1) cell shape or cell geometry, which determines the ratio of
cell surface area to cell volume (SA:V); higher values of SA:V facilitate deformation; (2)
cytoplasmic viscosity, which is primarily regulated by the mean corpuscular hemoglobin
concentration (MCHC) and is therefore influenced by alterations in cell volume; and (3)
membrane deformability and mechanical stability, which are regulated by multiple membrane
properties, which include elastic shear modulus, bending modulus, and yield stress.52–55 Either
directly or indirectly, membrane components and their organization play an important role in
regulating each of the factors that influence cellular deformability.

The biconcave disc shape of the normal red cell creates an advantageous SA:V
relationship, allowing the red cell to undergo marked deformation while maintaining a constant
surface area. The normal human adult red cell has a volume of 90 fL and a surface area of 140
μm.2 If the red cell were a sphere of identical volume, it would have a surface area of only 98
μm.2 Thus, the discoid shape provides approximately 40 μm2 of excess surface area, or an extra
43 percent, that enables the red cell to undergo extensive deformation. Most deformations
occurring in vivo and in vitro involve no increase in surface area. This is important because the
normal red cell can undergo large linear extensions of up to 230 percent of its original dimension
while maintaining its surface area, but an increase of even 3 to 4 percent in surface area results in
cell lysis. Either membrane loss, leading to a reduction in surface area, or an increase in cell
water content, leading to an increase in cell volume, will create a more spherical shape with less
redundant surface area. This loss of surface area redundancy results in reduced cellular
deformability, compromised red cell function, and diminished survival as a result of splenic
sequestration of spherocytic red cells. A 17-percent reduction in surface area results in rapid
removal of red cells by the human spleen.56

Cytoplasmic viscosity, another regulatory component of red cell deformability, is largely

determined by the MCHC, which is determined in large part by cell water content. As the
hemoglobin concentration rises from 27 to 35 g/dL (the normal range for red blood cells), the
viscosity of hemoglobin solution increases from 5 to 15 centipoise (cP), 5 to 15 times that of
water. At these levels, the contribution of cytoplasmic viscosity to cellular deformability is
negligible. However, viscosity increases exponentially at hemoglobin concentrations greater than
37 g/ dL, reaching 45 cP at 40 g/dL, 170 cP at 45 g/dL, and 650 cP at 50 g/dL. At these levels,
cytoplasmic viscosity may become the primary determinant of cellular deformability. Thus,
cellular dehydration, usually caused by the failure of normal volume homeostasis mechanisms,
can severely impair cellular deformability and thus decrease optimal oxygen delivery by
impairing the ability of red cells to undergo rapid deformation necessary for passage through the
microvasculature. As examples, cellular dehydration reduces red cell deformability in hereditary
xerocytosis, sickle cell anemia, hemoglobin CC, and β-thalassemia.55,57,58 However, changes
in cellular dehydration by itself have little influence on red cell survival.

The property of membrane deformability determines the extent of membrane deformation

that can be induced by a defined level of applied force. The more deformable the membrane, the
less the force required for the cell to pass through the capillaries and other narrow openings, such
as fenestrations in the splenic cords. The property of membrane mechanical stability is defined as
the maximum extent of deformation that a membrane can undergo, beyond which it cannot
completely recover its initial shape. This is the point at which the membrane fails. Normal
membrane stability allows human red cells to circulate for 100 to 120 days without fragmenting,
while decreased stability leads to cell fragmentation under normal circulating stresses. Both
membrane deformability and membrane mechanical stability are regulated by structural
organization of membrane proteins.54 While decreased membrane deformability can reduce
effective tissue oxygen delivery it appears to have little effect on red cell survival since
Southeast Asian ovalocytes with marked reductions in membrane deformability have near-
normal red cell survival. Loss of membrane mechanical stability leading to membrane
fragmentation and consequent reduction in SA:V ratio on the other hand compromises red cell
survival as in hemolytic hereditary elliptocytosis.49

RED CELL SENESCENCE

The reticulocyte loses membrane as it matures into a discocyte and membrane loss by
vesiculation continues throughout the erythrocyte life span. The notion that erythrocyte aging is
synonymous with membrane loss, increasing MCHC, and decreasing deformability largely
results from studies on density-separated cells and the equating of dense cells with aged cells
(Chap. 33). Although it is clear that loss of membrane surface area and decreased cell volume is
a feature of normal red cell senescence and that cell density increases with cell age, there is no
direct relationship between cell age and cell density since there is a large heterogeneity in cell
densities of reticulocytes as they enter circulation. What is clear is that the densest 1 percent of
circulating red cells are the most aged—they have the highest levels of glycated hemoglobin
(HbA1C), a very good marker of cell age. The loss of membrane surface area of the senescent
red cells appears to be a result of membrane oxidation-induced band 3 clustering and consequent
membrane vesiculation and the resultant critical decrease in SA:V ratio leads to their removal
from circulation59,60
PATHOPHYSIOLOGY OF ERYTHROCYTE SHAPES

Chapter 46 discusses erythrocytes in greater detail.

See Table 31–7 and Fig. 31–13 for scanning and blood film appearance of pathologically shaped
red cells.

Spherocytes and Stomatocytes

Spherocytes (Chap. 46) represent red cells, with the most decreased SA:V ratio seen in
hereditary spherocytosis, immune hemolytic anemia, stored blood, Heinz body hemolytic
anemia, and caused by cell fragmentation.49,61 Stomatocytes are seen in hereditary
stomatocytosis, as well as in hereditary spherocytosis, alcoholism, cirrhosis, obstructive liver
disease, and erythrocyte sodium pump defects.49,62,63 Red cells sensitized with antibodies,
complement, or immune complexes lose cholesterol and surface area. As a result, they are less
deformable and more osmotically fragile. Heinz body formation leads to membrane depletion by
fragmentation, with spherocyte formation. A spherogenic mechanism common to Heinz body
hemolytic anemias and immune hemolysis is partial phagocytosis of portions of the cell
containing aggregates of denatured hemoglobin and portions of the sensitized membrane,
respectively.

Stomatocytosis appear to be an intermediate form in the generation of spherocytosis with varying

extents of decreased SA:V ratio as a result of loss of membrane surface area or increased cell
volume. Stomatocytosis is a feature of hereditary hydrocytosis caused by increased cell volume
and consequent decrease in SA:V ratio. A spectrum of abnormal cells varying from normal
discocytes to stomatocytes, spherostomatocytes, and dense microspherocytes is seen in
hereditary spherocytosis.

Elliptocytes

Elliptocytes are seen in hereditary elliptocytosis (Chap. 46) as well as in thalassemia (Chap. 48),
iron deficiency (Chap. 43), and megaloblastic anemia (Chap. 41).49 In blood films of normal
subjects, elliptical or oval cells usually constitute less than 1 percent of the erythrocytes. In
various pathologic situations, with or without anemia (thalassemia trait, folate, and iron
deficiency), the number of elliptocytes can increase to 10 percent. Exceptionally, as in
dyserythropoiesis, the proportion can be as high as 50 percent. In hereditary elliptocytosis, the
number of elliptical erythrocytes varies greatly, from 1 to 98 percent. Qualitative and
quantitative anomalies of spectrin and protein 4.1, the major proteins of the membrane skeleton,
are associated with hereditary elliptocytosis.49,64 Severe hemolytic anemia is seen only in the
homozygous or compound heterozygotes form of the disease (hereditary pyropoikilocytosis)
where extensive cell fragmentation produces pyropoikilocytes with marked decreases in SA:V
ratio.
Acanthocytes

The acanthocyte (Chap. 46) is irregularly shaped, with two to 10 hemispherically tipped spicules
of variable length and diameter. The bases of the spicules on the acanthocyte are of varying girth,
unlike the spicules on echinocytes, which have remarkably uniform dimensions. Acanthocytes
are seen in neuroacanthocytosis and in abetalipoproteinemia.65 The lack of anemia in these
conditions suggests that these cells have near normal life span in circulation.

Target Cells (Codocytes)

A relative excess of membrane surface area or decreased cell volume leading to increased SA:V
ratio results in target cells.66 Target cells may be seen in obstructive liver disease,
hemoglobinopathies (S and C), thalassemia, iron deficiency, postsplenectomy, and lecithin
cholesterol acetyltransferase deficiency. In patients with obstructive liver disease, lecithin
cholesterol acetyltransferase activity is depressed. This increases the cholesterol-to-phospholipid
ratio and produces an absolute increase in the surface area of the red cell membrane. In contrast,
membrane excess is only relative in patients with iron-deficiency anemia and thalassemia
because of the reduced cell volume. In contrast to spherocytes which exhibit increased osmotic
fragility, target red cells are osmotically resistant.

Sickle Cells (Drepanocytes)

The sickle cell (Chap. 49) displays a characteristic variation of form on stained blood films. The
fusiform cell in the crescent shape with two pointed extremities is encountered most commonly
in deoxygenated blood samples as a result of polymerization of sickle hemoglobin. If sickle cell
formation is observed by phase-contrast microscopy, the earliest change with deoxygenation is
loss of flicker, followed by slight deformation at the discocyte border with displacement of the
hemoglobin to one region of the cell. The cell then elongates and becomes rigid as a result of
polymerization of hemoglobin S. Upon reoxygenation, the sickle cell resumes the discocyte form
and, in so doing, loses membrane by microspherulation and fragmentation during retraction of
long spicules.67 Evidence suggests that the more typical sickle-shaped cells form under slow
deoxygenation. With each sickling–unsickling cycle, membrane damage accumulates resulting in
the formation of irreversibly sickled cells (ISCs).68,69 These cells are incapable of reversion to
the biconcave disc shape, even when fully oxygenated. They have an increased hemoglobin
concentration, increased cation permeability, decreased potassium, and increased sodium.

Fragmented Cells (Schistocytes)

Schistocytes (Chap. 51) are seen in microangiopathic hemolytic anemias (thrombotic

thrombocytopenic purpura [TTP], disseminated intravascular coagulation [DIC], vasculitis,
glomerulonephritis, renal graft rejection), carcinomatosis, heart valve hemolysis (prosthetic or
pathologic valves), severe burns, and march hemoglobinuria (Chap. 51). Fibrin strands in
damaged blood vessels can be arrayed so that they sieve the passing red cells. If a passing red
cell folds over or otherwise attaches to the strand, the bloodstream pulls on the arrested cell,
stretches it, and eventually fragments it.70 The spleen rapidly removes the schistocytes with a
low relative SA:V ratio; the remainder may circulate for many days.