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.1,2,3,4,5 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.
In the very early stages of human growth and development, there are two forms of erythroid differentiation: primitive and definitive.7,8,9,10 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
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
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.
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).19,20,21 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).
Erythroblastic island. A. Erythroblastic island as seen in Wright-Giemsa–stained marrow. Note central macrophage surrounded by a cohort of attached erythroblasts. B. Erythroblastic island in the living state examined by phase-contrast microscopy. The macrophage shows dynamic movement in relation to its surrounding erythroblasts. (A, reproduced with permission from Lichtman's Atlas of Hematology, www.accessmedicine.com.)
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
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
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.
Human erythrocyte precursors. Light microscopic appearance. Marrow films stained with Wright stain. There are five stages of erythroblast development recognizable by light microscopy. A. Proerythroblasts. Two are present in this field. They are the largest red cell precursor, with a fine nuclear chromatin pattern, nucleoli, basophilic cytoplasm, and often a clear area at the site of the Golgi apparatus. B. Basophilic erythroblast. The cell is smaller than the proerythroblast, the nuclear chromatin is slightly more condensed and cytoplasm is basophilic. C. Polychromatophilic erythroblasts. The cell is smaller on average than its precursors. The nuclear chromatin is more condensed with a checkerboard pattern developing. Nucleoli are not apparent, usually. The cytoplasm is gray, reflecting the staining modulation induced by hemoglobin synthesis, which adds cytoplasmic content that takes an eosinophilic stain, admixed with the residual basophilia of the fading protein synthetic apparatus. D. Orthochromic normoblast. Smaller on average than its precursor, increased condensation of nuclear chromatin, with homogeneous cytoplasmic coloration approaching that of a red cell. E. Late orthochromatic erythroblasts (asterisks). The orthochromatic erythroblast to the right is undergoing apparent enucleation. The other three mononuclear cells are lymphocytes. A degenerating four-lobed neutrophil is also present. (Reproduced with permission from Lichtman's Atlas of Hematology, www.accessmedicine.com.)
Human erythroblast precursors as isolated by cell flow cytometry. Images of populations of human erythroblast precursors at stages of erythroid maturation when sorted from human marrow by flow cytometry. A and B. Proerythroblasts and early basophilic erythroblasts; (C) polychromatic erythroblasts; and (D) orthochromatic erythroblasts.
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.
Proerythroblast. Phase-contrast micrograph (inset) of a proerythroblast showing the immature nucleus with nucleoli and finely dispersed nuclear chromatin. The centrosome (juxtanuclear clear zone) is apparent with its dense accumulation of mitochondria. Electron microscopic section of the proerythroblast shows nucleoli (n) in contact with the nuclear membrane. Chromatin is finely dispersed and forms small aggregates in the fixed nuclear membrane. The perinuclear canal is narrow but well defined. Polyribosome groups, many in helical configuration, are dispersed throughout the cytoplasm. The Golgi apparatus (g) is well developed, and regions of endoplasmic reticulum (arrows) are seen.
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).
Basophilic erythroblast. Phase-contrast photomicrograph (inset) shows increased clumping of the nuclear chromatin and further rounding of the cell, with aggregation of the mitochondria and centrosome into the regions of nuclear indentation. Electron microscopic section shows clumping of the nuclear chromatin, nuclear pores (p), organization of the nucleoli, increased density of polyribosomes (pr), well-developed Golgi apparatus (g), and a decrease in smooth endoplasmic reticulum.
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).
Polychromatophilic erythroblast. Phase-contrast micrograph (inset) demonstrates diminished size of this cell compared with its precursor. Further clumping of nuclear chromatin gives the nucleus a checkerboard appearance. The centrosome is condensed, and a perinuclear halo has developed. Electron microscopic section demonstrates relative reduction of the density of polyribosomes and dilution by the moderately osmiophilic hemoglobin in the cytoplasm. Nuclear chromatin shows a marked increase in clumping, and nuclear pores (P) are enlarged.
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).
Orthochromic erythroblast. Phase-contrast appearance of this cell in the living state (inset) shows the irregular borders indicative of its characteristic motility, the eccentric nucleus making contact with the plasmalemma, further pyknosis of the nuclear chromatin, and condensation of the centrosome. Electron microscopic section shows further dilution of polyribosomes, some of which appear to be disintegrating into monoribosomes, by the increasing hemoglobin. The number of mitochondria is decreased, and some mitochondria are degenerating. Nuclear chromatin is clumped into large masses, and a perinuclear canal (pnc) is seen.
Pathologic sideroblast is an erythroblast characterized by the presence of mitochondrial deposits of iron-containing ferruginous micelles (arrows) between the cristae.
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.
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.
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.28,29,30 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.19,20,21 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.
Morphology of cells during reticulocyte maturation. A. Orthochromatic erythroblast extruding its nucleus. B. Multilobular, motile reticulocyte generated following nuclear extrusion. C. The cup-shaped, nonmotile reticulocyte at a later stage of maturation. D. Mature discoid red cell.
Orthochromic erythroblast ejecting its nucleus. A thin rim of cytoplasm surrounds the nucleus. In the cytoplasm, a single centriole (c) is partially encircled by some Golgi saccules.
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.31,32,33 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
“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.
See Fig. 31–11 for images of red cell inclusions.
Red cell inclusions. Blood films. A. Red cells with Howell-Jolly bodies (arrows) postsplenectomy. The crisp circular border, dark blue color, and peripheral location are characteristic. B. Basophilic stippling. These basophilic inclusions may be fine or coarse. In this case, the cell contains coarse stippling seen in lead poisoning (arrow). C. Siderocyte. These cells contain purple granules when stained with Wright stain (Pappenheimer bodies). Compared to basophilic stippling, siderotic granules are usually fewer in number and sometimes clustered. These Prussian blue–stained cells confirm that the granules contain iron (blue reaction product). Arrow points to two siderocytes. D. Cabot ring. Rare red cell inclusion (arrow). See text for further description. E. Heinz bodies. These cells from a patient with glucose-6-phosphate dehydrogenase deficiency were incubated with a supravital dye, which stains the denatured globin precipitates. F. Red cells from a patient with hemoglobin H disease (α-thalassemia). The hemoglobin precipitates are stained with brilliant cresyl blue. (Reproduced with permission from Lichtman's Atlas of Hematology, www.accessmedicine.com.)
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. Howell-Jolly 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.37,38,39 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.
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 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 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).43 Heinz 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 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.44,45,46 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 useful in 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 phase-contrast 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.
Scanning electron micrographs of distinct red cell morphologies. Discoid normal red cells (top left panel). Elliptocytes and fragmented red cells (top right panel). Oxygenated sickle red cells (middle left panel) and deoxygenated sickle red cells (middle right panel). Stomatocytic red cells (bottom left panel). Acanthocyte (bottom right panel).
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.49,50,51 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.
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, 31–2, 31–3, 31–4, 31–5, and 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.
Table 31–1.Human Erythrocyte Protein and Water Content ||Download (.pdf) Table 31–1. Human Erythrocyte Protein and Water Content
|Component ||mg/mL RBC ||Reference(s) |
|Water ||721 ± 17.3 ||71 |
|Total protein ||371 ||71 |
|Nonhemoglobin protein ||9.2 ||71,72 |
|Insoluble stroma protein ||6.3 ||72 |
|Enzyme proteins ||2.9 ||72 |
|Extensive study by proteomic methods || ||73,74 |
Table 31–2.Human Erythrocyte Phospholipids ||Download (.pdf) Table 31–2. Human Erythrocyte Phospholipids
|Lipid ||Amount ||Reference |
|Total phospholipids ||2.98 ± 0.20 mg/mL RBC ||75 |
|Cephalin ||1.17 (0.38–1.91) mg/mL RBC ||75 |
|Ethanolamine phosphoglyceride ||29% of total phospholipid ||75 |
|Mean plasmalogen content ||67% of ethanolamine phosphoglyceride ||75 |
|Serine phosphoglyceride ||10% of total phospholipid ||75 |
|Mean plasmalogen content ||8% of serine phosphoglyceride ||75 |
|Lecithin ||0.32 (0.03–0.95) mg/mL ||76 |
|Sphingomyelin ||0.12–1.13 mg/mL ||76 |
|Lysolecithin ||1.82% of total phospholipids ||77 |
Table 31–3.Human Erythrocyte Coenzyme and Vitamins ||Download (.pdf) Table 31–3. Human Erythrocyte Coenzyme and Vitamins
|Compound ||μmol/mL RBC ||Reference |
|Ascorbic acid ||0.02892 ± 0.00431 ||78 |
|Choline free ||Trace ||79 |
|Cocarboxylase ||0.00021 ||80 |
|Coenzyme A ||0.0027 ||81 |
|Nicotinic acid ||0.105 ||82 |
|Pantothenic acid ||0.001 ± 0.00028 ||83 |
|Pyridoxal phosphate ||20∞10−6 ± 2∞10−6 ||84 |
|Pyridoxal ||11∞10−6 ± 3∞10−6 ||84 |
|Total vitamin B6 aldehydes ||30∞10−6 ± 8 ∞10−6 ||84 |
|Pyridoxamine phosphate ||8∞10−6 ± 8 ∞10−6 ||84 |
|4-Pyridoxic acid ||4∞10−6 ± 4 ∞10−6 ||84 |
|Riboflavin ||0.00059 ± 0.00021 ||85 |
|Flavin adenine dinucleotide ||0.000398 ± 0.000042 ||86 |
|Thiamine ||0.00027 ||87 |
Table 31–4.Nucleotides ||Download (.pdf) Table 31–4. Nucleotides
|Compound ||μmol/mL RBC ||Reference(s) |
|Adenosine monophosphate ||0.021 ± 0.003 ||88,89,90,91 |
|Adenosine diphosphate ||0.216 ± 0.036 ||88,89,90,91 |
|Adenosine triphosphate ||1.35 ± 0.035 ||90,91,92,93,94 |
|Cyclic adenosine monophosphate ||0.015 ± 0.0024 ||95 |
|Cyclic guanosine monophosphate ||0.013 ± 0.0042 ||95 |
|Guanosine diphosphate ||0.018 ± 0.005 ||90 |
|Guanosine triphosphate ||0.052 ± 0.012 ||89,90 |
|Inosine monophosphate ||0.031 ± 0.005 ||90,91,92 |
|Nicotinamide adenine dinucleotide || ||96,97 |
| Reduced ||0.0018 ± 0.001 ||96,97 |
| Oxidized ||0.049 ± 0.006 |
|Nicotinamide adenine dinucleotide phosphate || ||96,97 |
| Reduced ||0.032 ± 0.002 || |
| Oxidized ||0.0014 ± 0.0011 || |
|S-adenosylmethionine ||0.005 ||98 |
|Total nucleotide ||1.534 ± 0.033 ||99 |
|Uridine diphosphoglucose ||0.031 ± 0.005 ||90,100 |
|Uridine diphosphate N-acetyl glucosamine ||0.018 ||100 |
Table 31–5.Human Erythrocyte Carbohydrates, Organic Acids, and Metabolites ||Download (.pdf) Table 31–5. Human Erythrocyte Carbohydrates, Organic Acids, and Metabolites
|Compound ||μmol/mL RBC ||Reference(s) |
|Dihydroxyacetone phosphate ||0.0094 ± 0.0028 ||88 |
|2,3-Diphosphoglycerate ||4.171 ± 0.636 ||88,94 |
|Fructose ||0.000354 ± 0.0000191 ||101 |
|Fructose 6-phosphate ||0.0093 ± 0.002 ||88,91,94,102 |
|Fructose 3-phosphate ||0.013 ± 0.001 ||103,104 |
|Fructose 2,6-diphosphate* ||48 ± 13 ||105 |
|Fructose 1,6-diphosphate ||0.0019 ± 0.0006 ||88,91,94,102 |
|Glucuronic acid ||Trace ||106 |
|Glucose ||In equilibrium with plasma ||107,108 |
|Glucose 6-phosphate ||0.0278 ± 0.0075 ||88,91,94,102 |
|Glucose 1,6-diphosphate ||0.18–0.30 ||91,109 |
|Glyceraldehyde 3-phosphate ||Not detectable ||88 |
|Lactic acid ||0.932 ± 0.211 ||72,88,110 |
|Mannose 1,6-diphosphate ||0.150 ||109 |
|Octulose 1,8-diphosphate ||Trace ||111 |
|Pyruvate ||0.0533 ± 0.0215 ||88 |
|3-Phosphoglycerate ||0.0449 ± 0.0051 ||88,94 |
|2-Phosphoglycerate ||0.0073 ± 0.0025 ||88,94 |
|Phosphoenol pyruvate ||0.0122 ± 0.0022 ||88 |
|Ribonucleic acid ||1.355 mg ||112 |
|Ribose 1,5-diphosphate ||<0.02 ||113,114 |
|Ribulose 5-phosphate ||Trace ||115 |
|Sedoheptulose 7-phosphate ||Trace ||115 |
|Sedoheptulose diphosphate ||Trace ||116 |
|Sialic acid ||0.825 ± 0.028 ||113 |
|Sorbitol ||31.1 ± 5.3 ||101,103 |
|Sorbitol 3-phosphate ||0.013 ± 0.001 ||104 |
Table 31–6.Human Erythrocyte Electrolytes ||Download (.pdf) Table 31–6. Human Erythrocyte Electrolytes
|Electrolyte ||μmol/mL RBC ||Reference |
|Aluminum ||0.0026 ||117 |
|Bromide ||0.1225 ||118,119 |
|Calcium ||0.0089 ± 0.0030 ||119,120,121 |
|Chloride ||78 ||119,122 |
|Chromium ||0.0004 ||123 |
|Cobalt ||0.0002 ||119,124 |
|Copper ||0.018 ||123,125,126 |
|Fluoride ||0.0131 ||127 |
|Iodine, protein-bound ||0.0013 ||128 |
|Lead ||0.0082 ||117,119,125,129 |
|Magnesium ||3.06 ||123,130,131,132 |
|Manganese ||0.0034 ||117,133 |
|Nickel ||0.0009 ||123 |
|Phosphorus (acid soluble): || || |
| Total P ||13.2 ||134 |
| Inorganic P ||0.466 ||134 |
| Lipid P ||3.840 ||135 |
| Unidentified P ||0.955 ||134 |
|Potassium ||102.4 ± 3.9 ||130,136,137,138,139,140 |
|Rubidium ||0.054 ||119 |
|Silicon ||0.036–0.060* ||141 |
|Silver ||Trace ||117 |
|Sodium ||6.2 ± 0.8 ||136,137,138 |
|Sulfur ||0.0044 ||142 |
|Tin ||0.0022 ||117 |
|Zinc ||0.153 ||123,143,144 |
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 three distinct 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.52,53,54,55 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
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.
Table 31–7.Nomenclature of Red Cell Shapes and Associated Disease States ||Download (.pdf) Table 31–7. Nomenclature of Red Cell Shapes and Associated Disease States
|Terminology (Greek Meaning) ||Old Terms, Synonyms ||Description ||Micrograph ||Associated Disease States |
|Discocyte (disc) ||Biconcave disc ||Biconcave disc form of RBC || || |
|Echinocyte (I–III) (sea urchin) ||“Burr cell,” crenated cell, “berry cell” ||Spiculated RBC with short, equally spaced projections over entire surface; progressing from the “crenated disc” (echinocyte I) to the crenated sphere (echinocyte IV—not shown) with nearly complete loss of spicules || || |
Uremia, liver disease
Low-potassium red cells
Immediately posttransfusion with aged or metabolically depleted blood
Carcinoma of stomach and bleeding peptic ulcers
|Acanthocyte (spike) ||“Spur cell,” acanthoid cell, acanthrocyte ||Irregularly spiculated RBC with projections of varying length and position || || |
Alcoholic liver disease
|Stomatocyte (I–III) (mouth) ||Mouth cell, cup form, mushroom cap, uniconcave disc, microspherocyte ||Bowled-shaped RBC with single concavity; progressing from shallow bowl (I) to near sphere with small dimple (seen as mouth-shaped form in peripheral film) || || |
Alcoholism, cirrhosis, obstructive liver disease
Erythrocyte sodium-pump defect
|Spherostomatocyte (sphere) ||Spherocyte, prelytic sphere, microspherocyte ||Spherical RBC with dense hemoglobin content; scanning electron microscopy shows a persistent minimal dimple || || |
Hereditary spherocytosis (cells actually spherostomatocytes)
Immune hemolytic anemia
Heinz body hemolytic anemia
|Schizocyte (cut) ||Schistocyte, helmet cell, fragmented cell ||Split RBC, often showing half-disc shape with two or three pointed extremities; may be small, irregular fragment || || |
Microangiopathic hemolytic anemia (TTP, DIC, vasculitis, glomerulonephritis, renal graft rejection)
Heart-valve hemolysis (prosthetic or pathologic valves)
|Elliptocyte (oval) ||Ovalocyte ||Oval to elongated ellipsoid RBC (with polarization of hemoglobin) || || |
|Drepanocyte (sickle) ||Sickle cell ||RBC containing polymerized hemoglobin S; showing varying shapes from bipolar, spiculated forms to holly-leaf and irregularly spiculated forms || || |
Sickle cell disorders (SS, S trait, SC, SD, S thalassemia, etc.)
|Codocyte (bell) ||Target cell ||Bell-shaped RBC that assumes a target shape on dried films of blood || || |
Obstructive liver disease
Hemoglobinopathies (S, C)
Lecithin cholesterol acetyltransferase deficiency
|Dacryocyte (tear) ||Teardrop cell ||RBC with a single elongated or pointed extremity || || |
|Leptocyte (thin) ||Thin cell, wafer cell ||Thin, flat RBC with hemoglobin at periphery || || |
Obstructive liver disease (± iron deficiency)
|Keratocyte (horn) ||Horn cell ||RBC with spicules resulting from ruptured vacuole; cell appears half-moon shaped or spindle shaped || ||DIC or vascular prosthesis |
A. Normal blood. Arrow points to a normochromic-normocytic discocyte. B. Stomatocytes. The double arrow points to the two morphologic types of stomatocyte: upper cell with a slit-shaped pale area and lower cell with a small central circular pale area. C. Echinocytes. The field has several such cells. The arrow points to one example with evenly distributed, blunt, short, circumferentially positioned, projections. D. Acanthocytes. The arrow points to one example with a few spike-shaped projections, unevenly distributed and of varying lengths. E. Spherocytes. Small, circular, densely-staining (hyperchromic) cells which, when fully developed, show no central pallor. F. Schizocytes (schistocytes, helmet cells, fragmented red cells). These microcytic cell fragments may assume varied shapes. The arrow points to a triangular shape, but two others of different shape are also present in the field. Despite being damaged and very small, they frequently maintain a biconcave appearance as witnessed by their central pallor. G. Sickle cells (Drepanocytes). Numerous sickle cells are shown. Two are in the classic shape of the blade on the agricultural sickle (arrow). Many red cells that have undergone the transformation to a “sickle” cell take the slightly less extreme form of elliptical cells with a very narrow diameter with condensed hemoglobin in the center (para-crystallization). About eight such cells are in the field. H. Elliptocytes and ovalocytes. The lower arrow points to an elliptocyte (cigar-shaped). The upper arrow points to an ovalocyte (football-shaped). Because both forms may be seen together in a case of inherited disease (same gene mutation resulting in both shapes), as shown here, it has been proposed that all such shapes be called elliptocytes with a Roman numeral to designate the severity of the shape change toward the elliptical, that is, elliptocytes I, II, and III. I. Target cells (Codocytes). The arrow points to one characteristic example among several in the field. The hemoglobin concentration corralled by membrane recurvature in the center of the cell gives it the appearance of an archery target. J. Tear-drop–shaped cells (Dacryocytes). Three dacryocytes are in this field. One example is indicated by the arrow. K. Horn cell (Keratocyte). Several examples are in the field. The arrow points to a typical such cell with two sharp projections. (Reproduced with permission from Lichtman's Atlas of Hematology, www.accessmedicine.com.)
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 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.
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.
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.