Three nomenclatures are used for naming the erythroid precursors (Table 8-1). The erythroblast terminology is used primarily in Europe. Like the normoblastic terminology used more often in the United States, it has the advantage of being descriptive of the appearance of the cells. Some prefer the rubriblast terminology because it parallels the nomenclature used for white blood cell development. Normoblastic terminology is used in this chapter.

TABLE 8-1

Three Erythroid Precursor Nomenclature Systems

Nomenclature	Maturation Stages
Normoblastic	Pronormoblast Basophilic normoblast Polychromatic (polychromatophilic) normoblast Orthochromic normoblast Polychromatic (polychromatophilic) erythrocyte Erythrocyte
Rubriblastic	Rubriblast Prorubricyte Rubricyte Metarubricyte Polychromatic (polychromatophilic) erythrocyte Erythrocyte
Erythroblastic	Proerythroblast Basophilic erythroblast Polychromic (polychromatophilic) erythroblast Orthochromic erythroblast Polychromatic (polychromatophilic) erythrocyte Erythrocyte

Maturation Process

Erythroid Progenitors

As described in Chapter 7, the morphologically identifiable erythrocyte precursors develop from two functionally identifiable progenitors, burst-forming unit–erythroid (BFU-E) and colony-forming unit–erythroid (CFU-E), committed to the erythroid cell line. Estimates of time spent at each stage suggest that it takes about 1 week for the BFU-E to mature to the CFU-E and another week for the CFU-E to become a pronormoblast,¹ which is the first morphologically identifiable RBC precursor. While at the CFU-E stage, the cell completes approximately three to five divisions before maturing further.¹ As seen later, it takes approximately another 6 days for the precursors to become mature cells ready to enter the circulation, so approximately 18 to 21 days are required to produce a mature RBC from the BFU-E.

Erythroid Precursors

Normoblastic proliferation, similar to the proliferation of other cell lines, is a process encompassing replication (i.e., division) to increase cell numbers and development from immature to mature cell stages (Figure 8-1). The earliest morphologically recognizable erythrocyte precursor, the pronormoblast, is derived via the BFU-E and CFU-E from the multipotential stem cells as discussed in Chapter 7. The pronormoblast is able to divide, with each daughter cell maturing to the next stage of development, the basophilic normoblast. Each of these cells can divide, with each of its daughter cells maturing to the next stage, the polychromatophilic normoblast. Each of these cells also can divide and mature. In the erythrocyte cell line, there are typically three and occasionally as many as five divisions² with subsequent nuclear and cytoplasmic maturation of the daughter cells, so that from a single pronormoblast, eight mature RBCs usually result. The conditions under which the number of divisions can be increased or reduced are discussed later.

Figure 8-1 Typical production of erythrocytes from two pronormoblasts illustrating three mitotic divisions. *BM,* Bone marrow; *PB,* peripheral blood circulation.

Criteria Used in Identification of the Erythroid Precursors

Morphologic identification of blood cells depends on a well-stained peripheral blood film or bone marrow smear (see Chapter 16). In hematology, a modified Romanowsky stain, such as Wright or Wright-Giemsa, is commonly used. The descriptions that follow are based on the use of these types of stains.

The stage of maturation of any blood cell is determined by careful examination of the nucleus and the cytoplasm. The qualities of greatest importance in identification of RBCs are the nuclear chromatin pattern (texture, density, homogeneity), nuclear diameter, nucleus/cytoplasm (N : C) ratio (Box 8-1), presence or absence of nucleoli, and cytoplasmic color.

BOX 8-1 Nucleus-to-Cytoplasm (N : C) Ratio

The nucleus-to-cytoplasm (N : C) ratio is a morphologic feature used to identify and stage red blood cell and white blood cell precursors. The ratio is a visual estimate of what area of the cell is occupied by the nucleus compared with the cytoplasm. If the areas of each are approximately equal, the N : C ratio is 1 : 1. Although not mathematically proper, it is common for ratios other than 1 : 1 to be referred to as if they were fractions. If the nucleus takes up less than 50% of the area of the cell, the proportion of nucleus is lower, and the ratio is lower (e.g., 1 : 5 or less than 1). If the nucleus takes up more than 50% of the area of the cell, the ratio is higher (e.g., 3 : 1 or 3). In the red blood cell line, the proportion of nucleus shrinks as the cell matures and the cytoplasm increases proportionately, although the overall cell diameter grows smaller. In short, the N : C ratio decreases.

As RBCs mature, several general trends affect their appearance. Figure 8-2 graphically represents these trends.

Figure 8-2 General trends affecting the morphology of red blood cells during the developmental process. A, Cell diameter decreases and cytoplasm changes from blue to salmon pink. B, Nuclear diameter decreases and color changes from purplish red to dark blue. C, Nuclear chromatin becomes coarser, clumped, and condensed. D, Composite of changes during developmental process. (Modified from Diggs LW, Sturm D, Bell A: *The morphology of human blood cells,* ed 5, Abbott Park, Ill, 1985, Abbott Laboratories.)

1. The overall diameter of the cell decreases.

2. The diameter of the nucleus decreases more rapidly than does the size of the cell. As a result, the N : C ratio also decreases.

3. The nuclear chromatin pattern becomes coarser, clumped, and condensed. The nuclear chromatin of RBCs is inherently coarser than that of myeloid precursors, as if it is made of rope rather than yarn. It becomes even coarser and more clumped as the cell matures, developing a raspberry-like appearance, in which the dark staining of the chromatin is distinct from the almost white appearance of the parachromatin. This chromatin/parachromatin distinction is more dramatic than in myeloid cells. Ultimately, the nucleus becomes quite condensed, with no parachromatin evident at all, and the nucleus is said to be pyknotic.

4. Nucleoli disappear. Nucleoli represent areas where the ribosomes are formed and are seen early as cells begin actively synthesizing proteins. As RBCs mature, the nucleoli disappear, which precedes the ultimate cessation of protein synthesis.

5. The cytoplasm changes from blue to gray to pink. Blueness or basophilia is due to acidic components that attract the basic stain, such as methylene blue. The degree of cytoplasmic basophilia correlates with the amount of ribosomal RNA. These organelles decline over the life of the developing RBC, and the blueness fades. Pinkness or eosinophilia is due to more basic components that attract the acid stain eosin. Eosinophilia of erythrocyte cytoplasm correlates with the accumulation of hemoglobin as the cell matures. Thus the cell starts out being active in protein production on the ribosomes that make the cytoplasm quite basophilic, transitions through a period in which the red of hemoglobin begins to mix with that blue, and ultimately ends with a thoroughly pink-red color when the ribosomes are gone and only hemoglobin remains.

Maturation Sequence

Table 8-2 lists the stages of RBC development in order and provides a convenient comparison. The listing makes it appear that these stages are clearly distinct and easily identifiable. Similar to the maturing of children into adults, however, the process of cell maturation is a gradual process, with changes occurring in a generally predictable sequence but with some variation for each individual. The identification of a given cell’s stage depends on the preponderance of characteristics, although the cell may not possess all the features of the archetypal descriptions that follow. Essential features of each stage are in italics in the following descriptions. The cellular functions described subsequently also are summarized in Figure 8-3.

TABLE 8-2

Normoblastic Series: Summary of Stage Morphology

Cell or Stage	Diameter	Nucleus-to-Cytoplasm Ratio	Nucleoli	% in Bone Marrow	Bone Marrow Transit Time
Pronormoblast	12-20 µm	8 : 1	1-2	1%	24 hr
Basophilic normoblast	10-15 µm	6 : 1	0-1	1-4%	24 hr
Polychromatic normoblast	10-12 µm	4 : 1	0	10-20%	30 hr
Orthochromic normoblast	8-10 µm	1 : 2	0	5-10%	48 hr
Shift (stress) reticulocyte (polychromatic erythrocyte)	8-10 µm	No nucleus	0	1%	48-72 hr*
Polychromatic erythrocyte	8.0-8.5 µm	No nucleus	0	1%	24-48 hr*

^*Transit time in peripheral blood.

Figure 8-3 Timeline of cellular diameter, RNA and DNA synthesis, and hemoglobin concentration during erythropoiesis. (Modified from Granick S, Levere RD: Heme synthesis in erythroid cells. In Moore CV, Brown EB, editors: *Progress in hematology,* New York, 1964, Grune & Stratton.)

Pronormoblast (Rubriblast)

Figure 8-4 shows the pronormoblast.

Basophilic Normoblast (Prorubricyte)

Figure 8-5 shows the basophilic normoblast.

Nucleus

The chromatin begins to condense, revealing clumps along the periphery of the nuclear membrane and a few in the interior. As the chromatin condenses, the parachromatin areas become larger and sharper, and the N : C ratio decreases to about 6 : 1. The staining reaction is one of a deep purple-red. Nucleoli may be present early in the stage but disappear later.

Cytoplasm

When stained, the cytoplasm may be a deeper, richer blue than in the pronormoblast—hence the name basophilic for this stage.

Division

The basophilic normoblast undergoes mitosis, giving rise to two daughter cells. More than one division is possible before the daughter cells mature into polychromatic normoblasts.

Location

The basophilic normoblast is typically present only in the bone marrow.

Cellular Activity

Detectable hemoglobin synthesis occurs,³ but the large number of cytoplasmic organelles, including ribosomes and a substantial amount of messenger ribonucleic acid (RNA; chiefly for hemoglobin production), completely mask the minute amount of hemoglobin pigmentation.

Length of Time in This Stage

This stage lasts slightly more than 24 hours.³

Polychromatic (Polychromatophilic) Normoblast (Rubricyte)

Figure 8-6 shows the polychromatic normoblast.

Figure 8-6 A, Polychromatic normoblast (rubricyte), bone marrow (Wright stain, ×1000). B, Electron micrograph of polychromatic normoblast (×15,575). (B from Carr JH, Rodak BF: *Clinical hematology atlas,* ed 3, Philadelphia, 2009, Saunders.)

Nucleus

The chromatin pattern varies during this stage of development, showing some openness early in the stage but becoming condensed by the end. The condensation of chromatin reduces the diameter of the nucleus considerably, so that the N : C ratio is about 4 : 1. Notably, no nucleoli are present.

Cytoplasm

This is the first stage in which the redness associated with stained hemoglobin can be seen. The stained color reflects the accumulation of hemoglobin pigmentation over time and concurrent decreasing amounts of RNA. The color produced is a mixture of pink and blue resulting in a murky gray-blue. The stage’s name refers to this combination of multiple colors, because polychromatophilic means “many color loving.”

Division

This is the last stage in which the cell is capable of undergoing mitosis, although likely only early in the stage. The polychromatic normoblast goes through mitosis, producing daughter cells that mature and develop into orthochromic normoblasts.

Location

The polychromatic normoblast is typically present only in the bone marrow.

Cellular Activity

Hemoglobin synthesis is increasing and the accumulation begins to be visible in the color of the cytoplasm. Cellular organelles are still present, particularly ribosomes, which contribute a blue aspect to the cytoplasm. The progressive condensation of the nucleus and disappearance of nucleoli are evidence of progressive decline in transcription of deoxyribonucleic acid (DNA).

Length of Time in This Stage

This stage lasts approximately 24 hours.³

Orthochromic Normoblast (Metarubricyte)

Figure 8-7 shows the orthochromic normoblast.

Nucleus

The nucleus is completely condensed (i.e., pyknotic) or nearly so. As a result, the N : C ratio is quite low.

Cytoplasm

The pink-orange color of the cytoplasm reflects nearly complete hemoglobin production. The residual ribosomes react with the basic component of the stain and contribute a slightly bluish hue to the cell, but that fades toward the end of the stage as the organelles are degraded. The prefix ortho means “the same” and refers to the fact that the cell’s color is the same color as the eosin stain, which is red.

Division

The orthochromic normoblast is not capable of division due to the condensation of the chromatin.

Location

The orthochromic normoblast is typically present only in the bone marrow.

Cellular Activity

Hemoglobin production continues on the remaining ribosomes using messenger RNA produced earlier. Late in this stage, the nucleus is ejected from the cell. The cell develops pseudopod-like projections into which the nucleus squeezes. Ultimately, the projection separates from the cell, with the nucleus enveloped by cell membrane.⁴ The extruded nucleus is then engulfed by splenic macrophages. Often, small fragments of nucleus are left behind if the projection is pinched off before the entire nucleus is enveloped. These fragments are called Howell-Jolly bodies when seen in peripheral blood cells (see Table 18-3) and are typically removed from the circulating cells by the splenic pitting process when the cell enters the circulation.

Length of Time in This Stage

This stage lasts approximately 48 hours.³

Polychromatic (Polychromatophilic) Erythrocyte

Figure 8-8 shows the polychromatic erythrocyte.

Figure 8-8 A, Polychromatic erythrocyte (shift reticulocyte), peripheral blood (Wright stain, ×1000). B, Scanning electron micrograph of polychromatic erythrocyte (×5000). (B from Carr JH, Rodak BF: *Clinical hematology atlas,* ed 3, Philadelphia, 2009, Saunders.)

Nucleus

Beginning at the polychromatic erythrocyte stage, there is no nucleus. The polychromatic erythrocyte is a good example of the statement that a cell may not have all the classic features described but may be staged by the preponderance of features. In particular, when a cell loses its nucleus, regardless of cytoplasmic appearance, it is a polychromatic erythrocyte.

Cytoplasm

The cytoplasm can be compared easily with that of the orthochromic normoblast in that the predominant color is that of hemoglobin. By the end of the polychromatic erythrocyte stage, the cell is the same color as a mature RBC, salmon pink. It remains larger than a mature cell, however. The shape of the cell is not the mature biconcave disc but is irregular in electron micrographs, like a lumpy potato (Figure 8-8, B). It requires the polishing activity of the spleen to assist the RBC into its final discoid shape.

Division

Lacking a nucleus, the polychromatic erythrocyte cannot divide.

Location

The polychromatic erythrocyte resides in the marrow for 1 day or longer and then moves into the peripheral blood, where it circulates about 1 day. During the first several days after exiting the marrow, the polychromatic erythrocyte is retained in the spleen for pitting and polishing by splenic macrophages, which results in the biconcave discoid mature RBC.⁵

Cellular Activity

The polychromatic erythrocyte completes production of hemoglobin from residual messenger RNA using the remaining ribosomes. The cytoplasmic protein production machinery is simultaneously being dismantled. Endoribonuclease, in particular, digests the ribosomes. The acidic components that attract the basophilic stain decline over this stage to the point that the polychromatophilia is not readily evident in the polychromatic erythrocytes on a normal peripheral blood smear stained with Wright stain. A small amount of residual ribosomal RNA is present, however, and can be visualized with a vital stain such as new methylene blue, so called because the cells are stained while alive in suspension (i.e., vital), before the smear is made (Box 8-2). The residual ribosomes appear as a mesh of small blue strands, a reticulum, or, when more fully digested, merely blue dots (Figure 8-9). When so stained, the polychromatic erythrocyte is renamed the reticulocyte.

BOX 8-2 Cellular Basophilia

Diffuse and Punctate

The reticulum of a polychromatic erythrocyte (reticulocyte) is not seen using Wright stain. The residual RNA imparts the bluish tinge to the cytoplasm seen in Figure 8-8, A. Based on the Wright-stained appearance, the reticulocyte is called a polychromatic erythrocyte because it lacks a nucleus and is no longer an erythroblast but has a bluish tinge. When polychromatic erythrocytes are prominent on a peripheral blood film, the examiner uses the comment polychromasia or polychromatophilia. Wright-stained polychromatic erythrocytes are also called diffusely basophilic erythrocytes for their regular bluish tinge. This term distinguishes polychromatic erythrocytes from red blood cells with punctate basophilia, in which the blue appears in distinct dots throughout the cytoplasm. More commonly known as basophilic stippling (see Table 18-3), punctate basophilia is associated with some anemias. Similar to the basophilia of polychromatic erythrocytes, punctate basophilia is due to residual ribosomal RNA, but the RNA is degenerate and stains deeply with Wright stain.

Figure 8-9 Reticulocyte, peripheral blood (new methylene blue stain, ×1000).

A second functional change in polychromatic erythrocytes is the reduced production of receptors for the adhesive molecules that hold developing RBCs in the marrow (see details later).6–8 As these receptors decline, the cells are freed to leave the marrow.

Length of Time in This Stage

The cell typically remains a polychromatic erythrocyte for about 2 days,³ with the first day spent in the marrow and the second spent in the peripheral blood, although possibly sequestered in the spleen.

Erythrocyte

Figure 8-10 shows the erythrocyte.

Nucleus

No nucleus is present in mature RBCs.

Cytoplasm

The mature circulating erythrocyte is a biconcave disc measuring 7 to 8 µm in diameter with a thickness of about 1.5 to 2.5 µm. On a stained blood smear, it appears as a salmon pink- or red-staining cell with a central pale area that corresponds to the concavity. The pallor is about one third the diameter of the cell.

Division

The erythrocyte cannot divide.

Location and Length of Time in This Stage

Mature RBCs remain active in the circulation for approximately 120 days.⁹ Aging leads to their removal by the spleen as described subsequently.

Cellular Activity

The mature erythrocyte delivers oxygen to tissues, releases it, and returns to the lung to be reoxygenated. The dynamics of this process are discussed in detail in Chapter 10. The interior of the erythrocyte contains mostly hemoglobin, the oxygen-carrying component, and a small proportion of water. It has a surface-to-volume ratio and shape that enable optimal gas exchange to occur. If the cell were to be spherical, it would have hemoglobin at the center of the cell that would be relatively distant from the membrane and would not be readily oxygenated and deoxygenated. With the biconcave shape, even hemoglobin molecules that are toward the center of the cell are not distant from the membrane and are able to exchange oxygen.

The cell’s main function of oxygen delivery throughout the body requires a membrane that is flexible and deformable. The interaction of various membrane components described in Chapter 9 creates these properties. RBCs must squeeze through small spaces such as the basement membrane of the marrow venous sinus. Similarly, when a cell enters the red pulp of the spleen, it must squeeze between epithelial cells to move into the venous outflow. Flexibility is crucial for RBCs to enter and subsequently remain in the circulation.

Erythrokinetics

Erythrokinetics is the term describing the dynamics of RBC production and destruction. To understand erythrokinetics, it is helpful to appreciate the concept of the erythron. Erythron is the name given to the collection of all stages of erythrocytes throughout the body: the developing precursors in the bone marrow and the circulating erythrocytes in the peripheral blood and the vascular spaces within specific organs such as the spleen. When the term erythron is used, it conveys the concept of a unified functional tissue. The erythron is distinguished from the RBC mass. The erythron is the entirety of erythroid cells in the body, whereas the RBC mass refers only to the cells in circulation. This discussion of erythrokinetics begins by looking at the erythrocytes in the bone marrow and the factors that affect their numbers, their progressive development, and their ultimate release into the blood.

Hypoxia—the Stimulus to Red Blood Cell Production

As mentioned previously, the role of RBCs is to carry oxygen. To regulate the production of RBCs for that purpose, the body requires a mechanism for sensing whether there is enough oxygen being carried to the tissues. If not, RBC production and the functional efficiency of existing cells must be enhanced. Thus a second feature of the oxygen-sensing system must be a mechanism for influencing the production of RBCs.

The primary oxygen-sensing system of the body is located in peritubular interstitial cells of the kidney.10,11 Hypoxia, too little tissue oxygen, is detected by the peritubular cells, which produce erythropoietin (EPO), the major stimulatory cytokine for RBCs. Under normal circumstances, the amount of EPO produced is fairly consistent, maintaining a level of RBC production that is sufficient to replace the approximately 1% of RBCs that normally die each day (see section on erythrocyte destruction). When there is hemorrhage, increased RBC destruction, or other factors that diminish the oxygen-carrying capacity of the blood (Box 8-3), the production of EPO can be increased.

BOX 8-3 Hypoxia and Red Blood Cell Production

Teleologically speaking, the location of the body’s hypoxia sensor in the kidney is practical,¹ because the kidney receives approximately 20% of the cardiac output² with little loss of oxygen. The location provides early detection when oxygen levels decline. Making the hypoxia sensor the cell that is able to stimulate red blood cell (RBC) production also is practical, because regardless of the cause of hypoxia, having more RBCs should help to overcome it. The hypoxia might result from decreased RBC numbers, as with hemorrhage.

Decreased RBC number is only one cause of hypoxia, however. Another cause is the failure of each RBC to carry as much oxygen as it should. This can occur because the hemoglobin (Hb) is defective or because there is not enough Hb in each cell. The hypoxia may be unrelated to the RBCs in any way; poor lung function resulting in diminished oxygenation of existing RBCs is an example.

The kidney’s hypoxia sensor cannot know why there is hypoxia, but it does not matter. Even when there are plenty of RBCs compared with normal reference values, if there is still hypoxia, stimulation of RBC production is warranted, because the numbers present are not meeting the oxygen need. An elevation of RBC numbers above the normal reference values, erythrocytosis, is seen in conditions such as lung disease and cardiac disease in which the blood is not being well oxygenated. Newborns have higher numbers of RBCs because the fetal Hb in their cells does not unload oxygen to the tissues readily, and so newborns are slightly hypoxic compared with adults. To compensate, they make more RBCs.

1. Donnelly S: Why is erythropoietin made in the kidney? The kidney functions as a “critmeter” to regulate the hematocrit, Adv Exp Med Biol 543:73–87, 2003.

2. Stewart P: Physiology of the kidney, Update in Anaesthesia (9):1–3, 1998. Available at: http://www.nda.ox.ac.uk/wfsa/html/u09/u09_016.htm. Accessed May 23, 2010.

The mechanism by which hypoxia increases EPO production in peritubular cells is mainly transcriptional regulation. There is a hypoxia-sensitive region of the 3′ regulatory portion of the EPO gene that promotes gene transcription.¹² A hypoxia-inducible factor, a transcription factor, is assembled in the cytoplasm when oxygen tension in the cells is decreased.¹³ It migrates to the nucleus and interacts with the 3′ enhancer for the EPO gene and upregulates the gene expression. With hypoxia, more messenger RNA molecules are produced, which results in more EPO production.

Erythropoietin

Structure

EPO is a thermostable, nondialyzable, glycoprotein hormone with a molecular weight of 34 kD.¹⁴ It consists of a carbohydrate unit that reacts specifically with RBC receptors and a terminal sialic acid unit, which is necessary for biologic activity in vivo.¹⁵ On desialation, EPO activity ceases.¹⁶

Action

EPO is a true hormone, being produced at one location (the kidney) and acting at a distant location (the bone marrow). It is a growth factor (or cytokine) that initiates an intracellular message to the developing RBCs; this process is called signal transduction. EPO must bind to its receptor on the surface of cells to initiate the signal or message. Relatively few receptors require binding to initiate intracellular signaling.¹⁷ The EPO-responsive cells vary in their sensitivity to EPO.¹⁴ Some are able to respond to low levels of EPO, whereas others require higher levels. In healthy circumstances when RBC production needs to proceed at a modest but regular rate, the cells requiring only low levels of EPO respond. If EPO levels rise secondary to hypoxia, however, a larger population of EPO-sensitive cells are able to respond.

The binding of EPO, the ligand, to its receptor on erythrocyte progenitors initiates a cascade of intracellular events that ultimately leads to more RBCs entering the circulation in a given time. EPO’s effects are mediated by intracellular Janus tyrosine kinase signal transducers that in turn affect gene activities in the RBC nucleus.¹⁸ The resulting nuclear activities have two major effects: allowing early release of reticulocytes from the bone marrow and preventing apoptotic cell death. In addition, EPO can reduce the time needed for cells to mature in the bone marrow. These processes are described in detail later. The essence is that EPO puts more RBCs into the circulation at a faster rate than occurs without its stimulation.

Early release of reticulocytes

EPO promotes early release of developing erythroid precursors from the marrow. Part of that results from EPO-induced changes in the adventitial cell layer of the marrow/sinus membrane.¹⁹ Merely increasing the width of the spaces through which cells might move is insufficient for cells to leave the marrow, however. RBCs are held in the marrow because they possess membrane receptors for adhesive molecules. EPO downregulates the production of the receptors on the RBC surface so that cells can exit the marrow earlier than they normally would.^6–8 The result is the presence in the circulation of reticulocytes that are still very basophilic, because they have not spent as much time in the marrow as they normally would. These may be called shift reticulocytes because they have been shifted from the bone marrow early (see Figure 8-8, A). Even nucleated RBCs (i.e., normoblasts) can be released early in cases of extreme anemia when the demand for RBCs in the peripheral circulation is great. Releasing cells from the marrow early is a quick fix, so to speak; it is limited in effectiveness because the available precursors in the marrow are depleted within several days and still may not be enough to meet the need in the peripheral blood for more cells. A more sustained response is required in times of increased need for RBCs in the circulation.

Inhibition of apoptosis

A second, and probably more important, mechanism by which EPO increases the number of circulating RBCs is by increasing the number of cells that will be able to mature into circulating erythrocytes. It does this by decreasing apoptosis, the programmed death of RBC progenitors.20,21 To understand this process, an overview of apoptosis in general is helpful.

Apoptosis: programmed cell death

As noted previously, it takes about 18 days to produce an RBC from stimulation of the earliest erythroid progenitor to release from the bone marrow. In times of increased need for RBCs, such as when there is loss from the circulation during hemorrhage, this time lag would be a significant problem. One way to prepare for such a need would be to maintain a store of mature RBCs somewhere in the body for emergencies. RBCs cannot be stored in the body for this sort of eventuality, however, because they have a limited life span. Instead of storing cells for emergencies, the body produces cells more rapidly when needed. It does so by overproduction of RBC progenitors at all times. If they are not needed, which is normal, the extra progenitors are allowed to die. If they are needed, however, they have about an 8- to 10-day head start in the production process. It is like a fast food restaurant preparing extra hamburger patties for the lunch rush just to be sure they have plenty. If the hamburgers are not sold, they are tossed in the garbage, but if they are needed, just the condiments and buns must be added to make them ready to sell, which significantly reduces the time needed to meet the demand. The process of intentional wastage of cells occurs by apoptosis, and it is part of the cell’s genetic program.

Process of apoptosis

Apoptosis is a sequential process characterized by, among other things, the degradation of chromatin into large fragments of 5 to 300 kilobases that are degraded further into smaller monomers of 200 bases; protein clustering; and activation of transglutamase. In contrast to necrosis, in which cell injury causes swelling and lysing with release of cytoplasmic contents that stimulate an inflammatory response, apoptosis is not associated with inflammation.²²

During the sequential process of apoptosis, the following morphologic changes can be seen: (1) condensation of the nucleus, causing increased basophilic staining of the chromatin; (2) nucleolar disintegration; and (3) shrinkage of cell volume with concomitant increase in cell density and compaction of cytoplasmic organelles while mitochondria remain normal.²³ This is followed by a partition of cytoplasm and nucleus into membrane-bound apoptotic bodies that contain varying amounts of ribosomes, organelles, and nuclear material. The last stage of degradation produces nuclear DNA consisting of multimers of 180 base pairs. Characteristic blebbing of the plasma membrane is observed. The apoptotic cell contents remain membrane-bound and are ingested by macrophages, which avoids an inflammatory reaction.

Evasion of apoptosis by erythroid progenitors and precursors

Apoptosis of RBCs is a cellular process that depends on a signal from either the inside or outside of the cell. Among the crucial molecules in the messaging system are the death receptor on the earliest RBC precursors, Fas, and its ligand, FasL, which is expressed by more mature RBCs.23,24

When EPO levels are low, cell production should be at a low rate, because hypoxia is not present. The excess early erythroid precursors should undergo apoptosis. This occurs when the older FasL-bearing erythroid precursors, such as polychromatic normoblasts, cross-link with Fas-marked immature erythroid precursors, such as pronormoblasts, which are then stimulated to undergo apoptosis.²³ As long as the more mature cells with FasL are present in the marrow, erythropoiesis is subdued. If the FasL-bearing cells are depleted, as when EPO-stimulated early release occurs, the younger Fas-positive precursors are allowed to develop, which increases the overall output of RBCs from the marrow.

A second mechanism for escaping apoptosis exists for RBC progenitors: EPO rescue. This is the major way in which EPO is able to increase RBC production. When EPO binds to its receptor, one of the effects is to stimulate antiapoptotic molecules, which allows the cell to survive and mature.²⁵ The cell that has the most EPO receptors and is most sensitive to EPO is the CFU-E, although the late BFU-E and early pronormoblast have some receptors.²⁶ Without EPO, the CFU-E would not survive.²⁷

EPO’s effect is mediated by the transcription factor GATA-1.²⁸ The two molecules cooperate to promote the production of the anti-apoptotic molecule bcl-xL.²⁵ EPO-stimulated cells develop this molecule on their mitochondrial membranes and are able to resist the FasL activation of apoptosis.

Reduced marrow transit time

Apoptosis rescue is the major way in which EPO increases RBC mass, by increasing the number of erythroid cells that survive and mature to enter the circulation. The other effect of EPO is to increase the rate at which the surviving precursors can enter the circulation. This is like a military recruiter who recruits more young people to service and then puts them through a shortened boot camp training so that they are on the battlefield sooner. This is accomplished by two means: increased rate of cellular processes and decreased cell cycle times.

EPO stimulates the synthesis of RBC RNA and effectively increases the rate of many processes, especially hemoglobin production.²⁹ The accumulation of hemoglobin is a factor that may control other cellular processes.³⁰ RBCs will continue to cycle through division and maturation until a critical concentration of hemoglobin is achieved,³⁰ typically after three to five divisions. Under the influence of EPO, the messenger RNA increases and the critical concentration of hemoglobin is achieved in two, rather than three, divisions. The cell is released from the marrow earlier than usual. Such cells are larger, due to a lost mitotic division, and do not have time before entering the circulation to dismantle the protein production machinery that gives the bluish tinge to the cytoplasm. These cells are true shift reticulocytes similar to those in Figure 8-8, A, recognizable in the stained peripheral blood film as especially large, bluish cells lacking central pallor. They also are called stress reticulocytes because they exit the marrow early during conditions of bone marrow stress, such as certain anemias. Conversely, if hemoglobin accumulates slowly, a cell may divide more times before achieving the critical hemoglobin concentration to shut off division (Box 8-4).

BOX 8-4 Why Are Some Red Blood Cells Small or Large?

The decreased volume and diameter of erythrocytes seen in iron deficiency anemia and the thalassemias can be explained by the role of hemoglobin (Hb) in controlling cellular processes. The cell continues the process of division and maturation until a critical concentration of Hb is reached.¹ In those anemias in which Hb production is impaired and Hb accumulates more slowly, there is time for more divisions to occur before the critical concentration is reached. More divisions result in smaller cells. Anemias in which this occurs include iron deficiency anemia in which the iron deficit prevents production of the heme component of Hb and thalassemias in which globin production is impaired. The converse also would be true—when cells undergo fewer than three divisions, as is the case under the influence of increased levels of erythropoietin, larger cells result. This is consistent with what is seen as patients recover from hemorrhage and larger cells emerge from the bone marrow. Larger cells also result when DNA production is impaired, leading to fewer than normal numbers of divisions. This is the case with megaloblastic anemia.

Stohlman F: Kinetics of erythropoiesis. In Gordon AS, editor: Regulation of hematopoiesis, vol 1, New York, 1970, Appleton-Century-Crofts, pp 318–326.

The process by which hemoglobin exerts this influence is probably as a transcription factor. The presence of hemoglobin in the nucleus has been recognized for decades,³¹ and its role as a transcription factor was hypothesized even before the concept of such influential molecules had been fully deduced.³⁰ It seems that hemoglobin is transferred to the nucleus in proportion to its accumulation in the cytoplasm,³¹ and there it influences other cellular processes.

EPO also can reduce the time it takes for cells to mature in the marrow by reducing individual cell cycle time, specifically the length of time that cells spend between mitoses.³² This effect is only about a 20% reduction, however, so that the normal transit time in the marrow of approximately 6 days from pronormoblast to erythrocyte can be shortened by only about 1 day by this effect.

With the decreased cell cycle time and fewer mitotic divisions, the time it takes from pronormoblast to reticulocyte can be shortened by about 2 days. If the reticulocyte leaves the marrow early, another day can be saved, and the typical 6-day transit time is reduced to fewer than 4 days under the influence of increased EPO.

Measurement of Erythropoietin

Quantitative measurements of EPO are performed on plasma and other body fluids. EPO can be measured by immunoassays of various types, including radioimmunoassay and chemiluminescence. The reference range is 5 to 30 mUnits/mL.¹ Increased amounts of EPO are expected in the urine of most patients with anemia, with the exception of patients with anemia caused by renal disease.

Other Stimuli to Erythropoiesis

In addition to tissue hypoxia, other factors influence RBC production to a modest extent. It is well documented that testosterone directly stimulates erythropoiesis, which partially explains the higher hemoglobin concentration in men than in women.³³ Also, pituitary³⁴ and thyroid³⁵ hormones have been shown to affect the production of EPO and so have indirect effects on erythropoiesis.

Microenvironment of the Bone Marrow

The microenvironment of the bone marrow is described in Chapter 7, and the cytokines essential to hematopoiesis are discussed there. Here, the details pertinent to erythropoiesis (i.e., the erythropoietic inductive microenvironment) are emphasized, including the locale and arrangement of erythroid cells and the anchoring molecules involved.

Hematopoiesis occurs in marrow cords, essentially a loose arrangement of cells in a dilated sinus area between the arterioles that feed the bone and the venous sinus that returns blood to efferent veins. Erythropoiesis typically occurs in what are called erythroid islands (see Figures 7-5 and 7-6). These are macrophages surrounded by erythroid precursors in various stages of development. It was previously believed that these macrophages provided iron directly to the normoblasts for the synthesis of hemoglobin. This was termed the suckling pig phenomenon. Although there is some evidence that macrophages may secrete some ferritin that normoblasts acquire,³⁶ developing RBCs probably obtain most iron via transferrin (see Chapter 11), and no direct contact with macrophages is needed for this. Because macrophages do release ferritin, it is logical to assume that this could account for the intimate arrangement. However, a more credible explanation of the cellular arrangement has been established. Macrophages are now known to elaborate cytokines that are vital to the maturation process of the RBCs.^37,38 RBC precursors would not survive without macrophage support via such stimulation.

A second role for macrophages in erythropoiesis also has been identified. Although movement of cells through the marrow cords is sluggish, developing cells would exit the marrow prematurely in the outflow were it not for an anchoring system within the marrow that holds them there until development is complete. There are three components to the anchoring system: (1) a stable matrix of stromal cells to which normoblasts can attach, (2) bridging (adhesive) molecules for that attachment, and (3) receptors on the erythrocyte membrane. The system is analogous to anchoring a ship, with the anchor corresponding to a stromal cell, the anchor cable corresponding to the adhesive molecules, and the cable post on the ship corresponding to the receptor.

The major cellular anchor for the RBCs is the macrophage. Several systems of adhesive molecules and RBC receptors tie the developing RBCs to the macrophages.³⁶ At the same time, RBCs are anchored to the extracellular matrix of the bone marrow, chiefly by fibronectin.⁷

When it comes time for the RBCs to leave the marrow, they cease production of the receptors for the adhesive molecules.⁷ Without the receptor, the cells are free to move from the marrow into the venous sinus. Fibronectin appears to play an important role in the directional migration of red blood cells toward the sinus.⁸ Entering the venous sinus requires the RBC to traverse the barrier created by the adventitial cells on the cord side, the basement membrane, and the epithelial cells lining the sinus. Egress through this barrier occurs between adventitial cells, through holes (fenestrations) in the basement membrane, and through pores in the endothelial cells¹⁹ (Figure 8-11).^39,40