CHAPTER 22 MORPHOLOGY OF THE ERYTHRON
CHAPTER 22 MORPHOLOGY OF THE ERYTHRON
BRIAN S. BULL
Erythroid Progenitor and Stimulating Factors
The Erythroblastic Island
Marrow Iron Metabolism
The Erythroblastic Series
Megaloblasts and Dyserythropoiesis
Pathology of the Reticulocyte and Erythrocyte
Structure and Shape of the Erythrocyte
Red Cell Shape and Survival in the Circulation
Nomenclature of Common Red Cell Shapes
The Normal Physiology and the Pathophysiology of Red Cell Shape
Acronyms and abbreviations that appear in this chapter include: BFU-E, burst forming unit—erythroid; CFU-E, colony forming unit—erythroid; MCHC, mean corpuscular hemoglobin concentration; RBC, red blood cell.
Collectively the progenitor and adult red cells have been termed the erythron to reinforce the idea that they function as an organ. The widely dispersed cells that make up this organ arise from undifferentiated, pluripotential stem cells. Following commitment, erythroid progenitors progress through several replicative stages, becoming more functionally specialized with maturation (Table 22-1). Eventually the reticulocyte and finally the mature, circulating erythrocyte are produced.
TABLE 22-1 ANTIGENIC DISTRIBUTION DURING ERYTHROID DIFFERENTIATION
ERYTHROID PROGENITOR AND STIMULATING FACTORS
The earliest progenitor committed to the erythroid lineage is the BFU-E. This cell is defined by its ability to create a “burst” on semisolid media, that is, a colony consisting of several hundred cells.
As maturation progresses a late progenitor, CFU-E, develops. The CFU-E is very sensitive to erythropoietin (see Chap. 14 and Table 22-1).
THE ERYTHROBLASTIC ISLAND
The anatomic unit of erythropoiesis in the normal adult is the erythroblastic island.46 It consists of one or two centrally located macrophages surrounded by maturing erythroid cells (Fig. 22-1). The adhesion between erythroid cells and macrophages occurs at the CFU-E stage of maturation.47 Phase-contrast microcinematography reveals that the macrophage is far from passive or immobile. Its pseudopodium-like cytoplasmic extensions move rapidly over cell surfaces of the surrounding wreath of erythroblasts. In scanning electron micrographs the central macrophage of the erythroblastic island is spongelike, with surface invaginations in which the erythroblasts lie. As the erythroblast matures it moves along a cytoplasmic extension of the macrophage away from the main body. When 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 (see Chap. 29). The nucleus is ejected prior to egress from the marrow, phagocytized and degraded by the marrow macrophages.
FIGURE 22-1 Erythroblastic island. (a) Erythroblastic island as seen in a Giemsa-stained marrow. (b) Erythroblastic island in the living state examined by phase-contrast microscopy. The macrophage shows dynamic movement in relation to its surrounding erythroblasts.
In addition to fibronectin48,49 there is probably a cell-cell recognition system that undergirds the formation of the erythroblastic island. Marrow macrophages express hemagglutinin ligands, sialoadhesins (Sn), and erythroblast receptors (EbR).50 This cell recognition system is also operative in vitro. Erythroblastic islands form in long-term marrow cultures with an adherent stromal cell layer. Likewise, erythroblasts grown from BFU-E in methylcellulose or in plasma clots will also form erythroid islands if the clots are lysed, permitting erythroblast-macrophage association.51,52
The erythroblastic island is, however, a fragile structure and is usually disrupted in the process of obtaining a marrow specimen by needle aspiration. Maturing erythroblasts juxtaposed to a macrophage fragment are only occasionally encountered in stained films of marrow aspirates. These cell fragments are typically rich in iron and thus more easily seen in iron-stained preparations. Only in clinical situations with accelerated erythroblastic activity, such as acute hemolytic anemia and erythroleukemia, are erythroblastic islands commonly seen in marrow films.
MARROW IRON METABOLISM
Details of iron metabolism are discussed in Chap. 24. In normal humans, the marrow macrophage plays a major role in iron conservation. Aged and damaged erythrocytes, identified and trapped within the marrow microcirculation, are phagocytosed by the macrophage. Lysosomes release their lytic enzymes into the primary phagosome of the macrophage, and within 60 min digestion of the engulfed red cell is virtually complete. The membrane is reduced to multiple myelin laminae, and erythrocyte iron is transformed into aggregates of ferritin (Fig. 22-2).
FIGURE 22-2 Ultrastructural aspects of ferritin. (a) Electron micrograph of a membrane-bound erythroblast siderosome, showing that it is composed of individual ferritin molecules. (b) Electron micrograph of negatively stained ferritin and apoferritin mixture, showing the ferritin molecule (f) with its protein coat and central dense iron core; apoferritin molecules (a) lack the central iron cores. (From Bessis and Breton-Gorius.174)
Ferritin is a 440-kDa protein consisting of 243 subunits arranged to form a hollow sphere. The central cavity may store 4500 iron atoms53 in the form of electron-dense particles of about 6 nm (60 Å) (Fig. 22-2). Microdiffraction techniques have shown that the iron cores display a hexagonal structure.54 Hemosiderin is the intracellular yellowish iron-containing pigment visible under light microscopy in iron-loaded tissues. Under electron microscopy it is largely composed of dense clusters of ferritin, most of which are membrane enclosed.55 Ferritin is converted into hemosiderin upon partial degradation of its protein shell by lysosomal enzymes.56
The outer membrane of erythroblasts possesses transferrin receptors on clathrin-coated pits. The adherence of transferrin to these portions of the cell membrane initiates a local membrane invagination, and intracytoplasmic vesicles are formed. These rapidly shed their clathrin coats and fuse with lysosomes to form endosomes.53,57 The acid pH in the endosome permits the transfer of iron from transferrin53 to mobilferrin, which in turn shuttles the iron to the sites of hemoglobin formation.58 The apotransferrin and the transferrin receptor molecules are cycled back to the cell membrane, where apotransferrin is released into the extracellular medium. Ferritin molecules are also endocytosed by coated pits on erythroblasts. This phenomenon was termed rhopheocytosis59 before it became evident that this mechanism for acquiring iron was only one example of a more general cellular mechanism for recycling receptors. Immunocytochemical labeling of the transferrin receptors has shown that the same pit may contain both the transferrin receptor and ferritin molecules.60 Calculations suggest that the coated vesicle transfers 1000 times more iron via ferritin than via transferrin.57
Despite the efficiency of this transfer mechanism, it is still not clear whether ferritin iron can support the biosynthesis of heme in mitochondria.61,62 Possibly the cytosolic ferritin in early red cell precursors is utilized for hemoglobin synthesis, while the ferritin clusters in mature erythroblasts represent storage of excess iron. The H ferritin (see Chap. 24) mRNA accumulates specifically during early erythroid differentiation.63
Uncomplexed iron together with superoxide provides a lethal mixture containing reactive hydroxyl radicals. These radicals cause lipid peroxidation, DNA strand breakage, and degradation of other biomolecules. By retaining iron in the safe, bound ferric form, extracellular transferrin and intracellular ferritin (and membrane-encapsulated iron stored as hemosiderin) serve as effective iron detoxifiers.64
THE ERYTHROBLASTIC SERIES
Numerically the BFU-E and CFU-E represent only a minute proportion of human marrow. In mice CFU-E can be generated in large numbers and then enriched by centrifugal elutriation and Percoll density gradient centrifugation. Under the electron microscope these cells show large nucleoli, abundant polyribosomes, and large mitochondria.65 Blasts from human marrow, enriched for CFU-E using a monoclonal antibody, exhibit similar ultrastructural characteristics (Fig. 22-3).66
FIGURE 22-3 Right inset: Unstained section of a normal presumptive CFU-E which was enriched by panning from marrow, using the monoclonal antibody FA-152.175 This blast has a large nucleolus (Nu). The incubation in diaminobenzidine medium reveals weak peroxidase activity in the endoplasmic reticulum (ER). In the Golgi zone, several granules appear as vacuoles (arrow). Main figure: On enlargement of the Golgi zone, a portion of the nucleus (N) can be seen surrounded by a perinuclear cistern containing weak peroxidase activity. Several granules with a pale matrix contain ferritin molecules (arrows). Left inset: High magnification of a granule showing ferritin molecules (F) of characteristic structure and density. (Adapted from Breton-Gorius, et al,66 with permission.)
On stained films the proerythroblast (Fig. 22-4) is a large cell, 20 to 25 µm in diameter, irregularly rounded or slightly oval. The nucleus occupies approximately 80 percent of its area and contains fine chromatin delicately distributed in small clumps. One or several well-defined nucleoli are present.
FIGURE 22-4 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 (Nu) 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.
Polyribosomes arranged in groups of two to six are numerous in the cytoplasm and are characteristic of the cytoplasm of proerythroblasts. It is this high concentration of polyribosomes that gives the cytoplasm of these cells its characteristic intense basophilia. At high magnification, ferritin molecules can be seen dispersed singly throughout the cytoplasm, and rhopheocytosis is easily observed at cell margins.
Three to twelve granules present in the Golgi zone also contain ferritin molecules.67 They stain for acid phosphatase, indicating their lysosomal nature, and differ from another class of small granules: the catalase-containing granules. Diffuse cytoplasmic density on sections stained for peroxidase indicates that hemoglobin is already present. Dispersed glycogen particles are present in the cytoplasm.68
The basophilic erythroblast is smaller than the proerythroblast, measuring 16 to 18 µm (Fig. 22-5). 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 linear strands. The whole arrangement often resembles wheel spokes or a clock face. The cytoplasm stains deep blue, leaving a perinuclear halo enlarged to a juxtanuclear clear zone around the Golgi apparatus.
FIGURE 22-5 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.
The cytoplasmic basophilia at this stage is due to the continued presence of polyribosomes. Microtubules are often seen connecting two erythroblasts in mitosis.
Following the second mitotic division of the erythropoietic series, the cytoplasm changes from blue to pink as hemoglobin dilutes the polyribosome content (Fig. 22-6). Cells at this stage are smaller than basophilic erythroblasts, measuring approximately 12 to 15 µm in diameter. The nucleus occupies less than half of the cell area. Its heterochromatin is in well-defined clumps spaced regularly about the nucleus, producing a checkerboard pattern. The nucleolus is lost; the perinuclear halo persists.
FIGURE 22-6 Polychromatophilic erythroblast. Phase-contrast micrograph (inset) demonstrates a diminution in the size of this cell in comparison with its precursor, with further clumping of nuclear chromatin to give 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.
Electron microscopy of the polychromatophilic erythroblast reveals increased aggregation of nuclear heterochromatin. Active rhopheocytosis is always evident, and siderosomes can be identified within the cytoplasm46 along with dispersed ferritin molecules. This normal distribution of ferritin iron in the erythroblast characterizes the normal sideroblast. Mitochondrial iron is usually not apparent, even though it is here that the iron is incorporated into protoporphyrin. The Golgi apparatus becomes quite small and may contain lysosomes.
After the final mitotic division of the erythropoietic series, the concentration of hemoglobin increases within the erythroblast. More than any of its predecessors, this cell stains like a mature erythrocyte (Fig. 22-7). However, because of the residual monoribosomes and polyribosomes it is always somewhat polychromatophilic.
FIGURE 22-7 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 are degenerating. Nuclear chromatin is clumped into large masses, and a perinuclear canal (pnc) is seen.
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; it varies from 10 to 15 µm in diameter. The nucleus occupies approximately one-fourth of the cell area and is eccentric.
Under the phase-contrast microscope a surprising motility can be appreciated. Round projections appear suddenly in different parts of the cell periphery and are just as quickly retracted. The movements are probably in preparation for ejection of the nucleus.46
The ultrastructure of the cell is characterized by irregular borders, reflecting its motile state. The nucleus is eccentric; the heterochromatin forms large masses. The cytoplasmic ribosomes are further dispersed into diribosomes and monoribosomes. Mitochondria are reduced in number and size. Hemoglobin is present within the nucleus itself.68,69
Prior to enucleation, intermediate filaments and the marginal band of microtubules disappear. Vimentin decreases in quantity throughout the cytoplasm. However, tubulin and actin become concentrated at the point where the nucleus will exit.70 These changes, accompanied by microtubular rearrangements, play a role in nuclear expulsion.71,72
The expulsion of the nucleus in vitro is by no means an instantaneous phenomenon; it requires a period of minutes.46 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 accompanied by a thin rim of hemoglobinized cytoplasm. It is this loss of a “corona” of hemoglobin with the nucleus that leads, in part, to an increase in the “early peak” of stercobilin when the rate of erythropoiesis is increased.73
In vivo, expulsion of the nucleus may take place while the erythroblast is still part of an erythroblastic island (Fig. 22-8), or the nucleus may be lost during passage through the wall of a marrow sinus. The nucleus, unable to traverse the small opening, remains in the marrow. In either case, the expelled nucleus is rapidly ingested by a macrophage.
FIGURE 22-8 Orthochromatic 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.
Two proposals have been advanced to explain how the reticulocyte exits the marrow; the precise mechanism is still unknown. The reticulocyte may actively traverse the sinus epithelium74 or more likely, since it appears incapable of directed amoeboid motion, it may be driven across by a pressure differential.75,76
As it enters the circulation the reticulocyte retains mitochondria, small numbers of ribosomes, the centriole, and remnants of the Golgi bodies. The reticulocyte contains no endoplasmic reticulum. Supravital staining with brilliant cresyl blue or new methylene blue produces aggregates of ribosomes, mitochondria, and other cytoplasmic organelles. These artifactual aggregates stain deep blue and, arranged in reticular strands, give the reticulocyte its name.
The in vitro maturation is very similar to that occurring in vivo. However, in a plasma clot the naked nuclei remain undamaged (Fig. 22-9). If the clot is lysed, the macrophages present in the culture immediately recognize and phagocytose the expelled nuclei. Maturation of the circulating reticulocyte requires from 24 to 48 h. During this period some 20 percent of the ultimate hemoglobin content will be synthesized and the final assembly of the submembrane skeleton completed. Living reticulocytes observed by phase-contrast microscopy are slightly motile, irregularly shaped cells with a characteristically puckered exterior. Examined by electron microscopy, reticulocytes are irregular in shape and contain many remnant organelles. These are grouped in the hilar region along with small smooth vesicles and an occasional centriole. In “young” reticulocytes the vast majority of ribosomes dispersed throughout the cytoplasm are in the form of polyribosomes. As protein synthesis diminishes during maturation these are gradually transformed into monoribosomes. Simultaneously, there is loss of transferrin receptors,77,78 and eventually the capacity for endocytosis disappears as well.79
FIGURE 22-9 Erythroblast maturation in an in vitro plasma clot from BFU-E. (a) At day 9 of culture, a cell resembling the in vivo proerythroblast exhibits numerous rhopheocytotic invaginations (arrows). (b) One invagination seen at high magnification. Note the numerous ferritin molecules (F) dispersed in the cytoplasm. (c) At day 12 of culture, an extruded nucleus is seen close to a reticulocyte. Insert: When the hemoglobin is emphasized by cytochemical staining, the thin rim of cytoplasm surrounding the nucleus is clearly visible. (d) In the cell suspension produced by clot lysis a macrophage phagocytoses a hemoglobin-rimmed erythroblast nucleus that has been recently extruded. (Parts c and d are adapted from Breton-Gorius, et al,52 with permission.)
MEGALOBLASTS AND DYSERYTHROPOIESIS
The morphologic abnormalities that characterize megaloblastic maturation and the dyserythropoietic anemias are described in Chap. 37.
A heterogeneous group of erythrocyte maturation disorders is accompanied by ineffective erythropoiesis and hyperferremia. These disorders include acquired idiopathic sideroblastic anemia, pyridoxine-responsive anemia, alcohol-induced sideroblastic anemia, lead intoxication, dyserythropoietic anemia, and certain hemoglobinopathies (see Chap. 35, Chap. 53, Chap. 63). All these conditions are characterized by the presence of pathologic sideroblasts. When stained for iron these cells may show small iron-containing granules arranged in a ring around the nucleus. For this reason they are commonly referred to as ringed sideroblasts.80 Iron stains of normal erythroid precursors demonstrate a few very fine granules that are difficult to see without carefully focusing up and down through the cell.
Electron microscope studies show granules in ringed sideroblasts to be iron-loaded mitochondria. Because this mitochondrial iron is distinct from ferritin antigenically, ultrastructurally, and by electron probe analysis, it has been termed ferruginous micelles.46 In hereditary sideroblastic anemia these mitochondrial iron deposits occur primarily in the late, polychromatophilic erythroblasts. In acquired sideroblastic anemia the iron overload affects the early proerythroblast.81 In cells with these iron-loaded mitochondria, many ferritin molecules are deposited between adjacent erythroblast membranes (Fig. 22-10).82
FIGURE 22-10 The pathologic sideroblast is an erythroblast characterized by the presence of mitochondrial deposits of iron-containing ferruginous micelles (arrows) between the cristae.
PATHOLOGY OF THE RETICULOCYTE AND ERYTHROCYTE
The reticulocyte may show pathologic alterations in size or staining properties. It may also contain inclusions visible by light microscopy or identifiable only on ultrastructural analysis. The majority of pathologic inclusions usually attributed to erythrocytes are actually found in reticulocytes (Table 22-2) and are nuclear or cytoplasmic remnants derived from the late-stage normoblasts.
TABLE 22-2 ERYTHROCYTE AND RETICULOCYTE INCLUSIONS
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.84 They are spherical in shape, usually no larger than 0.5 µm in diameter. Generally only one is present, but they may be numerous. In pathologic situations they appear to represent chromosomes that have been separated from the mitotic spindle during abnormal mitosis.85 More commonly, during normal maturation they arise from nuclear fragmentation (karyorrhexis) or incomplete expulsion of the nucleus.86 Howell-Jolly bodies are pitted from the reticulocytes in their passage through the interendothelial slits of the splenic sinus. They are characteristically present in the blood of splenectomized persons and in those suffering from hemolytic anemia, megaloblastic anemia, and hyposplenic states. More recently, hyposplenic states and post-splenectomy blood has been more sensitively and specifically characterized by pocked red cell counts. (See Pocked [or pitted] red cells.)
POCKED (OR PITTED) RED CELLS
When viewed under interference-phase microscopy, pocked red cells (described by Koyama in 1962)87 appear to have surface membrane “pits” or craters. The vesicles or indentations that characterize these cells represent autophagic vacuoles adjacent to the cell membrane.88 These vacuoles appear to be instrumental in the disposal of cellular debris as the erythrocyte passes through the microcirculation of the spleen.89 Within one week following splenectomy, pocked red cell counts begin to rise, reaching a plateau at 2–3 months.90 Pocked red blood cell (RBC) counts are being increasingly utilized as a test of splenic function.
The ringlike or figure-of-eight structures sometimes seen in megaloblastic anemia within reticulocytes and in an occasional, heavily stippled, late intermediate megaloblast91 are designated Cabot rings. Their exact composition is still open to question. Some have suggested that they originate from spindle material that has been mishandled during an abnormal mitosis.92 Others have found no indication of DNA or spindle filaments but have shown the rings to be associated with adherent granular material containing both arginine-rich histone and nonhemoglobin iron.93 Since both histone biosynthesis and iron metabolism/mobilization are abnormal in pernicious anemia, these structures may be a marker of “cytoplasmic currents” within the cell.46
Basophilic stippling consists of granulations of variable size and number that stain deep blue with Wright stain. Electron microscope studies have shown that punctate basophilia represents aggregated ribosomes.94 These clumps form during the course of drying and postvital staining of the cells, much as “reticulum” in reticulocytes is precipitated from ribosomes during the process of supravital staining. The clumped ribosomes may also include degenerating mitochondria and siderosomes. In conditions such as lead intoxication and thalassemia, the altered reticulocyte ribosomes have a greater propensity to aggregate. As a result, the 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 (see Chap. 53); in hereditary defects of the hexose monophosphate shunt (see Chap. 45); the thalassemias (see Chap. 46); unstable hemoglobin syndromes (see Chap. 48); or sickle cell disease.95 Not seen on ordinary Wright- or Giemsa-stained blood films, Heinz bodies are readily visible in red cells that have been stained supravitally with brilliant cresyl blue or with crystal violet. They tend to adhere to the interior of the red cell membrane, protruding into the cytoplasm. Their position in dried and stained blood films is characteristically about one-third of the distance in from the edge of the disc, where membrane curvature is at a minimum, presumably because of the membrane stiffening that they cause. Membrane stiffening also results in their removal from red cells as the cells traverse the interepithelial slits of the splenic sinus.96
HEMOGLOBIN H INCLUSIONS
Hemoglobin H is composed of b4 tetramers, indicating that b chains are present in excess as a result of impaired a-chain production. Exposure to redox dyes such as brilliant cresyl blue, methylene blue, or new methylene blue results in denaturization and precipitation of the abnormal hemoglobin.97 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.98 Most frequently seen in b-thalassemia, these changes may also be found in patients with unstable hemoglobin99 and in rare cases of erythroleukemia.100,101
SIDEROSOMES AND PAPPENHEIMER BODIES
Normal or pathologic cells containing siderosomes (“iron bodies”) are usually reticulocytes. In the pathologic state the iron granulations are larger and more numerous, and electron microscopy has shown that many of these are mitochondria containing ferruginous micelles rather than the ferritin aggregates that characterize the normal siderocyte.102 Siderosomes are usually found in the periphery of the cell, whereas basophilic stippling tends to be distributed homogeneously throughout the cell. Pappenheimer bodies are siderosomes that stain with Wright stain. Electron microscopy of these bodies shows that the iron is often contained within a lysosome, as confirmed by the presence of acid phosphate. Siderosomes may also contain degenerating mitochondria, ribosomes, and other cellular remnants.
In the presence of an intense erythropoietin response to acute anemia, or experimentally in response to large doses of exogenously administered erythropoietin, “stress” reticulocytes are released into the circulation.103 These cells may be up to twice the normal volume, with a corresponding increase in hemoglobin content. Whether this increase results from one less mitotic division during maturation or from some other process is not yet clear. In contrast, even under moderate erythropoietic stress some of the reticulocytes in the marrow pool are shifted to the circulating pool. These “shift” reticulocytes contain a higher RNA content than normal and can now be quantified. This is commonly done by applying a fluorescent stain to the aggregated ribosomal material 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.103,104,105 and 106
STRUCTURE AND SHAPE OF THE ERYTHROCYTE
The normal resting shape of the erythrocyte is a biconcave disc. 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 7.5 to 8.7 µm, which decreases slightly with cell age. They have an average volume of 90 fl107 and a surface area of approximately 136 µm2.108 The membrane is present in sufficient excess to allow the cell to swell to a sphere of approximately 150 fl or to enter a capillary of 2.8 µm in diameter. The normal erythrocyte stains reddish-brown in Wright-stained blood films and pink with Giemsa stain. The central one-third of the cell appears relatively pale compared with the periphery, reflecting its biconcave shape. Red cells on dried blood films are 0.6 µm thick, having lost about two-thirds of their normal thickness.46 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.109 Friction and surface tension involved in the preparation of the blood film produce fragmentation, “doughnut cells” or annulocytes, crescent-shaped cells, etc.109 Observed in the phase-contrast or interference microscope, the red cell shows a characteristic internal scintillation known as red cell flicker.110 This is due to thermally excited undulations of the red cell membrane. Frequency analysis of these surface undulations has provided an estimate of the curvature elastic constant and of changes in this constant due to alcohol, cholesterol loading, and exposure to cross-linking agents.111
RED CELL SHAPE AND SURVIVAL IN THE 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 it travels a distance of approximately 250 km. That it survives this long is at least partially due to the unique capacity of its membrane to “tank-tread”—rotate around the red cell contents.112 This arrangement transmits shocks from wall contact through the membrane to the viscous hemoglobin solution in the interior rather than concentrating the energy of contact in the membrane. The physical arrangement of membrane skeletal proteins in a uniform shell113 of highly folded hexagonal/pentagonal units114,115,116 permits this unusual behavior and is also responsible for the characteristic biconcave shape of the resting cell.117 Subtle differences in the discoid shape that resting cells assume are probably related to variations in the elastic properties of the submembrane skeleton.108 A deficiency in the amount of spectrin or the presence of mutant spectrin in the submembrane skeleton results in the abnormal discoid cells in hereditary spherocytosis, elliptocytosis, and pyropoikilocytosis (see Chap. 43).118 In regions of circulatory standstill or very slow flow, red cells travel in aggregates of two to a dozen cells, forming rouleaux.119 Within large vessels, aggregation is disrupted by the increased shear forces.
NOMENCLATURE OF COMMON RED CELL SHAPES
An international terminology using uniform Greek word stems has been introduced to describe cells on the basis of their three-dimensional morphology (Table 22-3).120
TABLE 22-3 NOMENCLATURE OF RED CELL SHAPES AND ASSOCIATED DISEASE STATES
The discocyte is the form that a red cell assumes when it is not subjected to external deforming stress. It is a smooth, biconcave disc. A discocyte can be reversibly and rapidly transformed by a variety of environmental agents into two other forms, the stomatocyte, a uniconcave cup-shaped cell, and the echinocyte, covered by 10 to 30 short projections evenly spaced over the cell surface. In general these changes can be superimposed on other red cell shapes, which suggests that they represent membrane energy equilibrium states.117
The acanthocyte has an irregular shape, with 2 to 10 hemispherically tipped spicules of variable length and variable diameter. The bases of the spicules on the acanthocyte are of varying girth, unlike those on echinocytes, where the spicules are of remarkably uniform dimensions.
Notwithstanding the time-honored use of the word, spherocytes are not truly spherical cells. Their thickness is greatly increased, so that the central concavity is significantly reduced and may be overlooked. On scanning electron microscopic examination the spherocyte frequently bears a small dimple or irregular area suggesting derivation from a stomatocyte.
Schizocyte refers to a red cell fragment that characteristically assumes a half-disc shape with two or three pointed extremities. Because it is produced by the sealing of two opposing membrane surfaces followed by physical cleavage or fragmentation of a red cell, it is smaller than the normal discocyte and may display one or more regions of stiffened and distorted membrane where the sealing and cleavage has occurred.
Drepanocyte (sickle cell) is a term that describes the sickle cell and a variety of shapes induced by the polymerization of sickle hemoglobin. Such cells vary in shape from bipolar, spiculated forms to cells with long, irregular spicules and holly-leaf configurations.
The elliptocyte (ovalocyte) is basically an oval biconcave disc showing varying degrees of elliptical aberration, from a slightly oval to an almost cylindrical, bipolar, elongated cell.
A relative excess of membrane in the codocyte (target cell) results in membrane recurvature in the center of the dimple. Hemoglobin accumulates where the upper and lower cell membranes separate when the cell is on a blood film, forming the central density, or “bulls eye,” of the target.
Dacryocyte refers to cells characterized by a single elongated or pointed extremity. This cell shape has previously been referred to as a teardrop, racket, or tail poikilocyte.
The leptocyte is a wafer-thin cell which is generally large in diameter and displays a thin rim of hemoglobin at the periphery with a large area of central pallor. Such a cell reflects an increased surface/volume ratio.
Keratocytes are red cells with a relatively normal cell volume that have been deformed by removal of a region of apposed and sealed membranes so that they present with two or more points.
“Bite” cells are red cells that have had one or more semicircular portions removed from the cell margin when Heinz bodies are pitted out by the splenic macrophages.121
If necessary, any shape variation of the red cell may be described precisely by the use of compound terms such as spherostomatocyte. The addition of modifiers such as micro to denote a changed volume may add to descriptive precision, as in microspherocyte or macroleptocyte.
Variability in the size of red cells is designated anisocytosis, and any type of shape abnormality is designated as poikilocytosis (see Chap. 2).
THE NORMAL PHYSIOLOGY AND THE PATHOPHYSIOLOGY OF RED CELL SHAPE
THE BICONCAVE DISC
The means by which a healthy red blood cell maintains its normal biconcave shape are still in dispute. However, most proposals can be subsumed under two headings: (1) The red cell is a reference shape into which the membrane is cast, much as a latex rubber glove is cast in the shape of a human hand,122 or (2) it is a dynamic equilibrium form controlled by the minimization of bending energy in the membrane.123 Among the observations that undermine the reference-shape hypothesis is the ability of the discocyte to withstand the relocation of the biconcavities anywhere on the membrane surface without significantly changing its shape.124 Against the minimization-of-bending-energy hypothesis are the measured values of the membrane-bending modulus. All estimates thus far are too low by one-half an order of magnitude125 to account for the observed membrane behavior.
Proposals that the submembrane skeleton behaves as an ionic gel126 and that the spectrin network functions as an entropic spring127 have served notice that the mechanical properties of the red blood cell membrane are exceedingly complex and still far from being completely understood.
The Stomatocyte-Echinocyte-Discocyte Equilibrium At physiologic pH and in the presence of normal levels of plasma proteins (particularly albumin), healthy red cells will always be smooth, biconcave discs (Fig. 22-11). As the pH is raised or the albumin concentration lowered, or in the presence of lysolecithin or anionic phenothiazine derivatives, the rim of the disc becomes bumpy. These bumps are low, are widely spaced, and involve only the membrane of the red cell rim. This form is an echinocyte I. Further environmental stress will result in transformation to echinocytes II and III. These cells bear 10 to 30 projections of surprisingly uniform dimensions, equally spaced over the entire cell surface. Should the environmental stress be sufficiently intense or of sufficient duration so that the echinocyte III becomes a spheroechinocyte I or spheroechinocyte II, the process is irreversible.
FIGURE 22-11 The discocyte-echinocyte and the discocyte-stomatocyte transformation. The upper panel schematically depicts the echinocytic transformation as induced by a rise in pH, a lack of albumin in the suspension, or exposure to an anionic phenothiazine derivative. Note particularly that the low protuberances that herald the echinocytogenic transformation appear preferentially over the rim of the biconcave disc. The lower panel schematically depicts stomatocyte formation as induced by a cationic phenothiazine derivative, a lowering of pH, or an excess of albumin in the suspending medium. Note that the intermediate form between the disc and the early cup is not a bent disc but rather a bow-tie form with very steep sides to the dimples.176 The microscopic appearance in wet preparations of stomatocytes (right), discocytes (middle), and echinocytes (left) is shown in the center panel.
Environmental stress caused by low pH, excess albumin, or cationic phenothiazine derivatives will transform the discocyte into an intermediate form with deeper biconcavities, and then into a cup-shaped cell with only a single concavity, a stomatocyte. Thus far the changes are readily reversible, but if the single deep depression on the stomatocyte surface is obliterated by membrane loss, the transformation becomes irreversible and a spherostomatocyte is the result.
In addition to pH and albumin there exists a wide array of pharmacologic agents that effect stomatocytic-echinocytic changes in red cell shape. These are thought to act by preferentially expanding the outer half of the phospholipid bilayer (echinocytogenic) or the inner half (stomatocytogenic). This explanation is sometimes referred to as the bilayer-couple hypothesis.128 While this hypothesis does account for the effects of these membrane-active pharmacologic agents on the red cell membrane, it is probably not a complete explanation for all stomatocytes or echinocytes. It is unlikely that pH and albumin are acting by directly expanding the inner or outer half of the phospholipid bilayer.117
THE AGED CELL
While there is general agreement that the reticulocyte loses membrane as it matures into a discocyte, it is less certain that membrane loss continues throughout the erythrocyte life span. The notion that erythrocyte aging is synonymous with membrane loss, increasing MCHC, and decreasing deformability is largely the result of studies on density-separated cells and the equating of dense cells with aged cells. Indeed, dense cells are dense because their MCHC is elevated, and an elevated MCHC exerts a profoundly depressant effect on red cell deformability. Thus dense cells will always be relatively nondeformable—but whether they are aged is still not settled. One thing is clear: unlike the reticulocyte, the aged red cell is not easily distinguished morphologically. Red cell aging and senescence are discussed in Chap. 29.
In the circulation the codocyte is a bell-shaped cell that assumes a target configuration when dried on a slide in the preparation of a blood film.109 On a flat surface the codocyte tends to evert its concavity into a central projection into which hemoglobin redistributes. This results in a central density (target) on the blood film. The codocyte is characterized by relative membrane excess due either to increased red cell surface area or to decreased intracellular hemoglobin. In patients with obstructive liver disease there is a depression of lecithin cholesterol acetyltransferase (LCAT) activity. This increases the cholesterol/phospholipid ratio129 and produces an absolute increase in the surface area of the red cell membrane. In contrast, the membrane excess is only relative in patients with iron-deficiency anemia and thalassemia, because of the reduced quantity of intracellular hemoglobin.
Acanthocytes are generated from normal red blood cells under conditions that alter their membrane lipid content, possibly by loss of glycerophospholipids resulting in a relative increase in sphingomyelin.130 Once produced, the shape is irreversible except in the rare McLeod syndrome, where incubation of the acanthocytic cells with phosphatidylserine or chlorpromazine will restore the discoid shape.131 A markedly increased membrane cholesterol/lecithin ratio is common to acanthocytes from patients with hepatocellular disease and abetalipoproteinemia.
THE DISCOCYTE-DREPANOCYTE TRANSFORMATION
The sickle cell, or drepanocyte, displays a characteristic variation of form on stained blood films. Most commonly encountered is the fusiform cell in the shape of a crescent with two pointed extremities. Examination by phase-contrast microscopy of deoxygenated sickle cell blood reveals varied cell forms characterized by pointed extremities in holly-leaf and poikilocytic configurations, many with multiple spicules several microns in length. The spicules are quite fragile and are easily avulsed from the cell. If sickle cell formation is observed, the earliest change with deoxygenation is the loss of flicker,132 followed by slight deformation at the border of the discocyte with displacement of the hemoglobin to one region of the cell. The cell then elongates and becomes rigid due to polymerization of hemoglobin S in rods or filaments.133 The rods are 15 to 18 nm (150 to 180 Å) in diameter and composed of monomolecular filaments of 6 to 7 nm (60 to 70 Å) intertwined into a six-stranded helix.134 In partially sickled cells such polymers display random orientation, but as polymerization increases the polymeric filaments undergo lateral reorientation into rods that are generally aligned with the long axis of the drepanocyte. Upon reoxygenation the drepanocyte resumes the discocyte form and in so doing loses membrane by microspherulation and fragmentation during the retraction of long spicules.135 There is suggestive evidence that more of the typical sickle-shaped cells form under slow deoxygenation. Thus, the cell membrane will be maximally stressed, and more of it will be lost during the unsickling cycle after slow deoxygenation.136 The unsickling process also leads to the formation of micro-Heinz bodies that adhere to the internal surface of the red cell membrane and contribute to the increased membrane rigidity and cation leak.95 With each sickle-unsickle cycle membrane damage accumulates, until the cells become incapable of reversion to the biconcave disc shape even when fully oxygenated. They thus become irreversibly sickled cells.137 These cells have an increased hemoglobin concentration and increased cation permeability, with decreased potassium and increased sodium. In addition, there is a marked decrease in membrane deformability.137 In addition to irreversibly sickled cells, the blood of patients with sickle cell anemia contains small numbers of another rigid, membrane-damaged cell—the sequestrocyte. These cells are characterized by linear zones of membrane fusion that entrap lakes of hemoglobin. In the light microscope they appear massively vacuolated. They presumably arise from a combination of physical damage from sickle-unsickle cycles and oxidative membrane damage that causes transcellular cross-bonding of the cell membrane.138
Fibrin strands in damaged blood vessels may be arrayed so that they sieve the passing red cells. Should a passing red cell fold over or otherwise attach to the strand, the bloodstream will pull on the arrested cell and stretch and eventually fragment it.139,140 If prior to rupture the two inner surfaces of the red cell membrane become approximated, the torn membranes will seal141 and the schizocyte will contain hemoglobin. The more rigid schizocytes and those with a low relative surface area are rapidly removed by the spleen; the remainder may circulate for many days.
Red cells sensitized with antibodies, complement, or immune complexes undergo loss of cholesterol and thus of surface area, displaying the increased osmotic fragility of the spherocyte.142 Heinz body formation leads to membrane depletion by fragmentation, with spherocyte formation.143 A spherogenic mechanism common to both Heinz body hemolytic anemias and immune hemolysis is partial phagocytosis of portions of the cell containing aggregates of denatured hemoglobin143 and portions of the sensitized membrane,144 respectively. Stomatocytosis is a rare form of spherocytosis.145 The anomaly is due to an abnormal ion permeability of the red cell membrane resulting in high levels of sodium and low levels of potassium in the cell interior (see Chap. 43). The cells take up water, become macrocytic and hypochromic, and show a dramatic increase in osmotic fragility. On blood films numerous spherocytic stomatocytes are present. These spherocytes, in contrast to those seen in hereditary spherocytosis, are large and hypochromic.
A spectrum of abnormal cells varying from normal discocytes to stomatocytes, spherostomatocytes, and dense microspherocytes is seen in hereditary spherocytosis.146
HEAT-INDUCED SHAPE CHANGES
Heating red cells to temperatures above 49°C will depolymerize spectrin. If the heating episode is brief and the inner surfaces of the biconcavities are in contact, these surfaces will fuse upon cooling.147 More vigorous heating causes marked spherulation of the entire cell. Microspherocytes bud from the cell surface, and the entire cell becomes transformed into small spherical fragments. Such fragments may be recovered from the blood after severe burns.
In blood films of normal subjects, elliptical or oval cells usually constitute less than one percent of the erythrocytes. In various pathologic situations, with or without anemia (thalassemia trait, folate and iron deficiency, etc.), 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 (see Chap. 43) the number of elliptical erythrocytes varies greatly, from 0 to 98 percent.148 Such fluctuations have forced hematologists to substitute a biochemical and functional (rheologic) definition of hereditary elliptocytosis for the original morphologic one.148 Both qualitative and quantitative anomalies of spectrin118,149,150 and 151 and protein 4.1,152,153 two major proteins of the membrane skeleton, are associated with hereditary elliptocytosis. As a consequence, rheologic membrane properties are impaired.149,154 A severe hemolytic anemia, however, is seen only in the homozygous form of the disease where schizocytes are typically present.
These cells are typically found in the bloodstream of patients with marrow fibrosis, often accompanied by extramedullary hemopoiesis. How these marrow changes give rise to dacryocytes is still unknown. Aspiration of red cell membrane into a micropipette of appropriate dimensions will produce a morphologically similar shape change; however, the cell usually recovers completely within minutes. Similar deformation in a reticulocyte might be permanent since it would occur while the assembly of the submembrane skeleton was still in process. A delay during egress from the marrow would provide such an opportunity.
KERATOCYTES (“HORN CELLS” OR “HELMET CELLS”)
Keratocytes are erythrocytes with one or more roughly circular bites removed from the discocyte margin. They differ from schizocytes in that their hemoglobin content is normal or only slightly lower than normal; they have not been formed by the bisection of a red cell. Rather, they appear to arise when all the hemoglobin is squeezed out of a portion near the edge of a discocytic red cell and the two opposite membrane surfaces fuse.155 This process forms a pseudovacuole which soon ruptures, probably because of stiffening of the membrane skeleton in the fused portion. The result is a notch with bordering spicules or horns. Experimentally, membrane fusion with pseudovacuole formation can be produced by heat in excess of 49°C147 and by mechanical stress.141 In vitro exposure to diamide and N-ethylmaleimide156 will produce this characteristic form.
“Bite” cells are formed when the Heinz bodies are pitted from the cells by splenic macrophages.121 Emphasis on the missing portion rather than on the horns that remain has led to the term bite cell.157 In vivo exposure to sulfonamide drugs such as dapsone and sulfasalazine and the urinary tract antiseptic phenazopyridine will result in “bite” cells158 in susceptible individuals. Bite cells are obviously a form of keratocyte. It is, however, worthwhile to distinguish them from other keratocytes because their method of formation involves removal of denatured hemoglobin (and membrane), whereas keratocytes in general appear to be formed by membrane apposition and subsequent removal of the apposed membranes.
CRYSTALS OF HEMOGLOBIN C DISEASE
In splenectomized patients with homozygous hemoglobin C disease, as many as 10 percent of the circulating cells may contain tetrahedral crystals.159 In blood films from nonsplenectomized patients crystal-containing cells are rare or absent.160 The efficiency in splenic removal may be due to spherocyte formation from the release of osmotically active particles as the hemoglobin C crystals “melt” while undergoing deoxygenation in the spleen. “Melting” upon deoxygenation occurs readily in vitro, behavior that is opposite to that of sickle hemoglobin crystals.161 In vitro dehydration of hemoglobin C–containing cells for a 24-h period between slide and coverslip162 or hypertonic dehydration of red cells in 3% NaCl buffer for 4 to 12 h readily produces crystals. In homozygous hemoglobin C disease up to 75 percent of the cells may show crystals; lower percentages occur in hemoglobin SC and other hemoglobin C variants. Molecular subunits in a tetragonal or hexagonal arrangement may be identified within the hemoglobin C crystals.163 Hemoglobin Setif, like hemoglobin C, may also precipitate as intracellular crystals when the tonicity of the suspending medium is raised.164 This takes place in oxygenated solutions and at osmolarities achieved in the renal medulla. Even so, there have been no reported clinical symptoms among heterozygous carriers of the Setif gene.
The red cell behaves as an osmometer.165 When placed into a hypertonic solution it shrinks, and the inner surfaces of the biconcavities touch over a progressively larger central region. When red cells in hypotonic solutions reach their critical hemolytic volume, holes greater than 10 nm (100 Å) in size appear166 and the hemoglobin exits. Alternatively, a large tear may develop in the red cell membrane. Following hemolysis (exit of the hemoglobin), the holes or tears close and the cell resumes its original biconcave shape.
An important determinant of the survival of a red cell in the circulation is its deformability. The deformability of the intact cell is made up of contributions from the intrinsic deformability of the membrane itself, the internal viscosity (for practical purposes, the MCHC), and the surface/volume ratio of the cell. The deformability of the intact cell can be measured by the time it takes a red cell suspension to traverse a filter of known pore size167; or the cells may be suspended in a viscous medium and exposed to a shear force, and the change in shape observed microscopically, as in the rheoscope,168 or by laser diffraction, as in the ektacytometer.169 Additional information can be obtained from ektacytometric analysis by varying the osmolarity of the suspending medium and thus changing the surface/volume ratio as well as the internal viscosity of the cells during the analytical procedure.170 Alternatively, the red cell may be folded over a spiderweb strand in the presence of rapidly flowing buffer. From the relationship between the flow rate of the buffer and the deformation of the red cell, the deformability of the membrane can be estimated.171
An increase of the MCHC by 20 percent results in an increase in the internal viscosity of about 600 percent.172 An increase of this magnitude leaves the red cell with sufficient deformability to survive in the circulation. This is not the case for erythrocytes from patients with xerocytosis or dessicocytosis. Here the erythrocytes are always perilously close to the upper limits of internal viscosity consistent with traversing the vasculature.173
In the circulation the primary cause of decreased red cell deformability is likely to be insufficient membrane (spherocytosis) rather than stiffening of the membrane. The interendothelial slits of the splenic sinus stress cells with a normal surface/volume ratio, and splenic phagocytes remove those with a ratio that is lower than normal. It is self-evident that a perfectly spherical red cell will be rigid no matter how low the MCHC or how flexible the isolated membrane might be.
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Ernest Beutler, Marshall A. Lichtman, Barry S. Coller, Thomas J. Kipps, and Uri Seligsohn